PROTECTION
L AGENCY
EPA-600/3-76-044 DALLAS, TEXAS
April 1S76
THE OF GREAT LAKES CLADQPHQM
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
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RESEARCH REPORTING SERIES
Research reports o! the Qtiue- o! Reseatch ano Development U S Environmental
Protection Agency have- been grouped ink five series These- five bioaa
categories were established to facilitate further devennniem and application o'
e-fivifutimerital technoiog\ Elimination 01 iradrtioiiai grouping was consciously
planned to foster technology tra'>sfe< and a rriaxiniuiT: interface, in relatea fieifis
The five series art-
1 Environmental Health Effects Research
2 Environmental Protection Technology
3 Ecological Research
A Environmental Monitoring
5 Socioeconomic Environmental Stuaies
This repon has been assigned to the ECOLOGICAL RESEARCH series This series
describes research on the effects of pollution on humans plant and animal
species and materials Problems are assessed for their long- and short-term
influences Investigations include formation, transport, and pathway studies to
determine the fate of pollutants arid then effects This wor> provides the technical
basis fo' setting standards to minimize undesirable changes ir, living organisms
in the aquatic, terrestrial, and atmospheric environments
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c
EPA-600/3-76-044
April 1976
THE NUTRITION OF GREAT LAKES CLADOPHORA
by
Gerald C. Gerloff
Department of Botany
and
George P. Fitzgerald
Water Resources Center
University of Wisconsin
Madison, Wisconsin 53706
Grant No. R-802464
Project Officer
Nelson A. Thomas
Large Lakes Research Station
Environmental Research Laboratory-Duluth
Grosse He, Michigan 48138
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL RESEARCH LABORATORY
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection Agency, nor
does mention of trade names or commercial products constitute endorse-
ment or recommendation for use.
11
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ABSTRACT
A synthetic culture medium was developed for the laboratory culture of
Great Lakes Cladophora sp. In initial phases of the study, Cladophora
could be maintained in culture only when a sewage effluent or lake
water supplement was added to the medium. With recognition that
C ladophora has an unusual requirement, among freshwater green algae,
for vitamins BI and 612, the effluent or lake water supplement could
be omitted from the culture medium. Cladophora was isolated from
each of the Great Lakes with most of the detailed nutritional studies
carried out on an isolate from Lake Michigan identified as Cladophora
glomerata.
Systematic manipulations and modifications of the components of the
initial culture medium led to considerable yield increase in a specific
time interval. In the optimum culture medium that evolved, C_. glomerata
routinely produced 500 mg oven-dry algae in a 21-day growth period.
The quantitative requirements of C_. glomerata for essential inorganic
nutrients were expressed primarily as critical cell concentrations,
that is minimum cell concentrations which permit maximum yield. Crit-
ical concentrations were established for most of the essential elements.
gloroerata can be characterized as having relatively low critical N
and P concentrations (1.1% and 0.06%, respectively) but very high boron
(110 ppm) and sulfur (.150%) critical concentrations. Ortho-, pyro-,
meta, and tripoly-P can be utilized in the growth of C_. glomerata as
can NHit and NOa-N. Phosphite-P is relatively unavailable.
Nutrient supplies and limiting nutrients for the growth of Cladophora sp.
in the Great Lakes were evaluated by two plant bioassays, primarily
plant analysis and to some extent the Fitzgerald tests. The plant
analysis bioassay evaluates available nutrient supplies by comparisons
of essential element concentrations in Great Lakes Cladophora samples
and critical concentrations established in laboratory studies. Plant
analysis assays for phosphorus in each of the Great Lakes showed that
phosphorus concentrations in C 1 adophora correlated closely with the
recognized pollution of the areas sampled. Furthermore, the
111
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assays indicated phosphorus to be limiting or close to limiting in
several relatively unpolluted areas. Nevertheless, there were indica-
tions that elements other than phosphorus, particularly boron and to
some extent zinc, at times limited Cladophora growth. There was some
evidence that vitamin Bi supply might limit Cladophora growth in the
Great Lakes.
This report was submitted in fulfillment of Grant R-802464 by Gerald
C. Gerloff and George P. Fitzgerald under the sponsorship of the
Environmental Protection Agency. Work was completed as of October 15, 1975,
IV
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CONTENTS
Sections Page
I Conclusions 1
II Recommendations 3
III Introduction 5
IV Isolation and Initial Culture of Great Lakes 8
Cladophora sp.
V Laboratory Culture of Great Lakes Cladophora glomerata 14
in a Defined Medium
VI Critical Concentrations of Essential Elements for 25
Cladophora glomerata
VII Recommended Culture Medium for Cladophora glomerata 42
VIII Cladophora glomerata Response to Several Physical 55
Environmental Factors
IX Calibration of Fitzgerald Tests for Field Evaluations of 60
Nutrient Supplies
X Evaluation of Nutrient Supplies in the Great Lakes for 76
Cladophora sp. Growth
XI Reliability of Sampling and Analytical Methods 103
XII References 108
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FIGURES
No.
1 Effect of Vitamin BI on the Growth of Cladophora 18
glomerata (Ind. 1484) in Different Dilutions of
Gorham's Medium Major Elements and Sewage Effluent
2 Apparatus Used in the Routine Laboratory Culture 26
of Cladophora sp. in Nutritional Experiments
3 The Relationship Between Oven-dry Yield and P 30
Concentration in Cladophora glomerata after
Culturing in Solutions Varying in P Concentration
4 The Relationship Between Oven-dry Yield and Total 32
N Content of Cladophora glomerata after Culturing
in Solutions Varying in NOs-N Concentration
5 Comparison of Yield and Total P Concentration of 61
Lake Michigan Cladophora sp. after a 17-day Culture
Period in Solutions Varying in P Concentration
6 Comparison of Yield and Extracted POi^-P of Lake 62
Michigan Cladophora sp. after a 17-day Culture Period in
Solutions Varying in P concentration
7 Comparison of Yield, Total N in the Algae, and Rate of 68
NHi^-N Uptake in the Dark when Cladophora sp. was Grown
at Different Solution Concentrations of NOs-N
8 Green Bay and Lake Michigan Area 95
9 Green Bay and Lake Michigan Sampling Area and Total 98
P (%) of Cladophora sp.
VI
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TABLES
No. Page
1 Stimulatory Effect of Sewage Effluent and Lake Water 10
on the Growth of Cladophora sp. in Nutrient Cultures
2 Quantitative Response of Cladophora sp. to the 10
Addition of Lake Mendota Water to the Culture Medium
3 Response of Cladophora glomerata to Several Concen- 15
trations of the Essential Trace Elements
4 Destruction by Ashing of Sewage Effluent and Lake 17
Water Factors Stimulatory to Cladophora glomerata
Growth
5 Response of Cladophora glomerata to Various Vitamins 19
and Organic Growth Factors When Grown in Culture
Medium
6 Quantitative Response of Cladophora glomerata to 20
Various Concentrations of Vitamin BI in the Culture
Medium
7 Quantitative Response of Cladophora glomerata to 21
Vitamin 612 in the Culture Medium
8 The Vitamin BI Requirement of Great Lakes Cladophora sp. 22
9 The Vitamin 612 Requirement of Cladophora sp. 22
10 Response of Cladophora glomerata to Additions of Plant 24
Hormones to the Culture Medium
11 Yield and Total P Concentration of Cladophora glomerata 29
Grown in Culture Medium Differing in P Concentration
12 Yield and Total N Concentration of Cladophora glomerata 31
Grown in Culture Medium Differing in NOa-N Concentration
13 Yield and K Concentration of Cladophora glomerata Grown 33
in Culture Medium Differing in K Concentration
14 Yield and Ca Concentration of Cladophora glomerata Grown 34
in Culture Medium Differing in Ca Concentration
15 Yield and Mg Concentration of Cladophora glomerata Grown 35
in Culture Medium Differing in Mg Concentration
16 Yield and S Concentration in Cladophora glomerata Grown 36
in Culture Medium Differing in S Concentration
VII
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17 Yield and Fe Concentration of Cladophora glomerata Grown 37
in Culture Medium Differing in Fe Concentration
18 Yield and Mn Concentration of Cladophora glomerata Grown 38
in Culture Medium Differing in Mn Concentration
19 Yield and Zn Concentration of Cladophora glomerata Grown 39
in Culture Medium Differing in Zn Concentration
20 Yield and Cu Concentration of Cladophora glomerata Grown 39
in Culture Medium Differing in Cu Concentration
21 Yield and Mo Concentration of Cladophora glomerata Grown 40
in Culture Medium Differing in Mo Concentration
22 Yield and B Concentration of Cladophora glomerata Grown 40
in Culture Medium Differing in B Concentration
23 Critical Concentrations and the Range of Concentrations 41
of Essential Nutrient Elements in Cladophora glomerata
24 Variations in the Concentrations of Inorganic Constituents 43
to Improve the Culture Medium for Cladophora glomerata
25 Yield of Cladophora glomerata Grown in Culture Medium 47
Containing Several Concentrations of Potentially Essential
Micronutrients
26 Response of Cladophora glomerata to Several Combinations 48
of Vitamins Added to an Improved Culture Medium
27 Response of Cladophora glomerata to Various Concentrations 49
of Vitamin BI in Culture Medium II
28 Response of Cladophora glomerata to Additions to the 51
Culture Medium of Hot Water Extracts of Oven-dried C_.
glomerata
29 Comparison of Cladophora glomerata Yields Grown in 52
Cladophora Medium II and in Lake Water Fortified with
Components of the Medium
30 Composition of Recommended Synthetic Nutrient Medium 53
(Medium II) for the Culture of Cladophora glomerata
31 The Growth Response of Cladophora glomerata to Three 56
Ranges of Temperature
32 The Growth Response of Cladophora glomerata to Four 56
Levels of Light Intensity
Vlll
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33 The Effect of Medium Volume and Aeration on the Growth 58
of Cladophora glomerata
34 Effect of "Etching" on Cladophora glomerata Growth 59
35 The Response of Cladophora sp. Isolated From Each of 63
the Great Lakes to Different P Concentrations
36 The Relative Availability of Different Sources of P 65
to P-limited Lake Wingra Cladophora sp.
37 The Availability to Lake Michigan Cladophora sp. of P 65
From Different Sources When Measured in Growth Tests
38 The Availability of the P of Littoral Lake Muds to 66
Lake Michigan Cladophora sp.
39 The Availability of Different Sources of Nitrogen to 70
Lake Michigan Cladophora sp. When Measured in Growth
Experiments
40 Comparison of the Effectiveness of Several Sources of 71
Fe for Cladophora sp. Growth in Nutrient Cultures
41 The Availability of the Iron of Littoral Muds for the 72
Growth of Cladophora sp.
42 The Absorption of Phosphorus by Phosphorus-limited 73
Cladophora sp. from Lake Michigan
43 The Effect of Ammonium and Nitrate Nitrogen on the 74
NHit-N Absorption Rates of Nitrogen-limited Cladophora sp.
From Lake Mendota
44 The Effect of Incubation with Added Iron on the Iron 74
Content of Cladophora sp. From Lake Mendota
45 Inorganic Analysis of Cladophora sp. Collected From 78
Lake Michigan at Various Sites and at Various Times
During the Summer
46 Inorganic Analysis of Cladophora sp. Collected From 81
Lakes Erie, Ontario, and Huron at Various Sites and at
Various Times During the Summer
47 Concentrations of Several Non-essential Elements in 82
Cladophora sp. Collected From Lake Michigan
48 Concentrations of Several Non-essential Elements in 84
Cladophora sp. Collected From Lakes Erie, Ontario,
and Huron.
IX
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49 Total P and Total Fe Analyses of Several Algae Species 86
Collected in Lake Superior and in Northern Lakes Michigan
and Huron
50 Total P and Fe Analyses of Cladophora sp. Collected From 89
the Milwaukee Area of Lake Michigan in 1974
51 Fitzgerald Tests for N, P, and Fe Supplies to Cladophora 91
sp. Collected From the Milwaukee Harbor Area of Lake
Michigan, 1973
52 Total P and Fe Concentrations in Cladophora sp. Collected 92
From Western Lake Erie
53 Total P and Fe Concentrations in Cladophora sp. Collected 93
From Eastern Lake Erie
54 Total P and Fe Concentrations in Cladophora sp. Collected 94
From Lake Ontario
55 Nitrogen and Phosphorus Inputs to the Lower Fox River 96
56 Summary of Analyses of Cladophora sp. Collected During 99
September, 1974 From Green Bay and Lake Michigan
57 Agreement in Replicate Analyses for Total and Extractable 104
P in Field Samples of Cladophora sp.
58 Agreement in Replicate Analyses for P, N, Ca, and Fe in 105
Samples of Cladophora sp.
59 Agreement in the Total P and Total Fe Concentrations in 107
Samples of Cladophora sp. Collected From Within a Five-
foot Radius
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ACKNOWLEDGMENTS
The role of Vic Muth, Judy Armstrong, John Devereux, Corinne Scofield,
Mrs. Selma Faust, Leslie Fleming, and Leslie Pratt both in planning
and performing the experiments reported and in assisting in preparation
of the manuscript is gratefully acknowledged.
We thank Renee Hundrieser and Vicki Pengelly for their patience and
endurance when typing the manuscript.
Appreciation is expressed for the assistance and interest of the Grant
Project Officer, Mr. Nelson Thomas.
XI
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SECTION I
CONCLUSIONS
There were two primary aspects to these studies on Cladophora sp., a
primary nuisance green alga in the Great Lakes. One was concerned
with developing suitable procedures for the laboratory culture of
C ladophora and establishing its qualitative and quantitative nutrient
requirements. The second aspect involved evaluations of nutritional
factors that are critical in controlling growth of Cladophora in the
Great Lakes.
The primary conclusions for this project can be summarized as follows:
1. Unlike most other algae found in midwestern lakes, Cladophora
glomerata must be provided with external supplies of vitamins BI and
Bi2. It appears that C_. glomerata is unable to synthesize required
amounts of these vitamins.
2. With vitamins BI and 812 added to the culture medium, C_. glomerata
can be satisfactorily cultured in a completely defined medium and under
environmental conditions similar to those used in the culture of other
freshwater algae. Techniques now are available which make laboratory
experimentation with Cladophora sp. relatively simple and easy.
3. When evaluated as critical cell concentrations (minimum concen-
trations which permit optimum growth) , C_. glomerata is indicated to
have several unusual nutrient requirements. Compared to other fresh-
water algae, the requirements for nitrogen and phosphorus are relatively
low. In comparison with plants in general, C_. glomerata has very high
requirements for boron and sulfur. Supplies of these elements in
relation to C_. glomerata requirements should be carefully considered
in evaluating the nutrition of the alga in the Great Lakes.
4. Various forms of phosphorus (ortho-, pyro-, meta-, and tripoly-P)
can be utilized by Cladophora sp., phosphite-P is relatively unavailable.
Cladophora sp. can utilize both NHit and NOa-N for growth.
5. Phosphorus concentrations in Cladophora from all of the Great Lakes
correlate well with the recognized pollution of specific areas. Furthermore,
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plant analysis bioassays indicate that phosphorus supply is limiting,
or close to limiting, in areas with relatively little pollution. Al-
though more data are needed, reduction in phosphorus supply probably
is a practical means of reducing nuisance growths of Cladophora in
the Great Lakes.
6. Nitrogen supply does not seem to limit Cladophora growth in the
Great Lakes.
7. Bioassays by plant analysis indicate that supplies of elements
other than phosphorus at times become limiting for Cladophora growth
in the Great Lakes. This is particularly true for the trace element
boron and perhaps for zinc. The possibility that vitamin BI supply
limits Cladophora growth in the Great Lakes should not be disregarded
and needs further study.
8. Plant bioassays (both plant analysis and the Fitzgerald tests) are
recommended for evaluating nutrient supplies and growth-limiting
nutrients for Cladophora in natural environments.
9. The sampling and analytical procedures used in the studies reported
were sufficiently reliable so that in plant analysis bioassays signifi-
cance could be attached to differences in nutrient concentrations in
lake samples of Cladophora.
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SECTION II
RECOMMENDATIONS
The development of plans and measures for the control of nuisance
Cladophora sp. growths in the Great Lakes will be aided by (1) know-
ledge of the qualitative and quantitative nutritional requirements
of the organisms, (2) recognition of unusual nutritional features
of the Cladophora sp., and (3) the availability of simple procedures
for accurate evaluation of nutrient supplies and growth-limiting
nutrients in the lakes and streams where Cladophora occurs. This
project has contributed to all of these requirements. The primary
recommendations for the use and further development of the infor-
mation obtained are:
1. As a result of the development of satisfactory procedures for the
culture of Cladophora sp. in the laboratory, additional and confirmatory
laboratory experiments on the qualitative and quantitative nutritional
requirements of the Cladophora should be carried out.
2. Plant bioassays, demonstrated to be relatively simple and useful
assay procedures, should be used in further tests of nutrient supplies
and growth-limiting nutrients in the Great Lakes, particularly in areas
known to vary markedly in degree of pollution.
3. The plant analysis bioassay should be further developed and refined
for use with Cladophora. This should include more precise definitions
of trace element critical cell concentrations, studies of factors
which may cause critical concentration values to vary, and comparisons
of the results of nutrient assays by plant analysis and other commonly
used assay procedures.
4. Because of indications in the present study that phosphorus supplies
were limiting or close to limiting in several Great Lakes areas from
which Cladophora was collected, emphasis in further studies probably
should be in clarifying the role of phosphorus as a growth-limiting
element. Nevertheless, the possibility that nutrients other than
phosphorus, both essential inorganic elements and vitamins, can become
critical in nuisance Cladophora growths should be recognized and tested for.
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5. The aggressiveness and effectiveness of Cladophora in competing
for and absorbing nutrients at lake water concentrations should be
established and compared with comparable data for other organisms
with which Cladophora grows and competes in the Great Lakes.
6. The data and information from this and other similar studies
should be utilized in developing plans for reducing nuisance growths
of Cladophora in the Great Lakes.
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SECTION III
INTRODUCTION
Green algae in the genus Cladophora are responsible for some of the most
troublesome nuisance plant growths in the Great Lakes. Eutrophication
and pollution have increased these nuisance growths. Identification of
specific pollution factors which are primarily responsible for nuisance
Cladophora growths and the development of plans for reducing these
conditions should be based on data on the quantitative and qualitative
nutritional requirements of the Cladophora.
The organisms most abundant in freshwater environments might be expected
to have simple nutritional requirements and to be easy to culture in the
laboratory. This has not been true. Bloom-producing blue-green algae,
for example, were relatively difficult to bring into laboratory culture
(Gerloff, Fitzgerald, and Skoog, 1950). In spite of their abundance in
certain lakes, Cladophora sp. also have been difficult to culture in
synthetic media. As a result, their specific nutritional requirements
have been poorly understood.
The primary objectives of this study were (1) to culture nuisance
Cladophora sp. from the Great Lakes in defined nutrient media, (2) to
study under laboratory conditions the qualitative and quantitative
nutritional requirements for optimum Cladophora sp. growth and (3) to
evaluate the nutrition of Cladophora sp. in the Great Lakes and deter-
mine nutritional factors critical in controlling the growth of the
algae under field conditions.
Requirements for both organic and inorganic factors must be determined
when developing a culture medium for a specific alga and in evaluating
its nutrition. As with other chlorophyll-containing organisms, most
freshwater algae do not require external supplies of organic growth
factors such as vitamins and amino acids. They are able to synthesize
needed quantities of these compounds.
Both the qualitative and quantitative requirements of an alga for
various mineral nutrients must be established. The qualitative
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nutrient requirements are concerned with the essentiality of specific
elements for an organism. For normal growth, most plants require the
16 chemical elements routinely made available in synthetic culture
media. A number of algae have unusual, specific additional require-
ments. For example, blue-green algae require sodium and cobalt, the
green alga Scenedesmus quadricauda requires vanadium, and diatoms grow
only in the presence of silicon. Quantitative inorganic nutrient
requirements are concerned with the specific amounts and the balance
of various elements necessary for optimum organism growth.
The successful culture of an alga requires consideration of other
nutrient solution factors, such as pH and the total salt concentration.
Optimum growth also usually occurs only within a narrow range of tempera-
ture and light with the optima differing for various organisms.
The quantitative requirements of an organism for essential inorganic
nutrients can be expressed in several ways. Probably the most common
expression is in terms of solution concentrations. These are by no
means specific, and therefore are of limited value, because the volume
of solution to which an organism is exposed also is a factor determining
the total amount of each element available to an organism. The require-
ment for elements also can be expressed in terms of the critical
concentrations, that is the minimum concentration of each element in a
plant or plant part necessary for maximum yield, a concept discussed
by Gerioff and Krombholz (1966), Gerloff (1969), and Gerloff and
Fishbeck (1973). One of the primary goals of this project was to
establish the critical concentration in Cladophora of all essential
chemical elements likely to limit growth in the Great Lakes.
Evaluation of nutrient supplies and of the most likely growth-limiting
nutrients in natural environments can be made by chemical analyses of
water samples or by various bioassays. Several suggested advantages
of the bioassay approach (Gerloff, 1969) were responsible for the
emphasis of bioassays in evaluating the nutrition of Cladophora under
field conditions on this project. One type of bioassay used is known
as plant analysis. It involves comparison of the concentrations of
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key elements in field collected Cladophora sp. with the critical con-
centrations of those elements established in the laboratory. A non-
limiting supply of an element at sampling time is indicated if the
concentration in an algae field sample is above the critical concen-
tration; a concentration of the element at or below the critical
concentration indicates the element had become growth-limiting in the
environment (Gerloff and Krombholz, 1966; Gerloff, 1973). The second
type of bioassay used is based on the rate of NH^-N by algae in the
dark as a measure of N supply and on the extraction of P from the
algae as a measure of P availability (Fitzgerald, 1969).
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SECTION IV
ISOLATION AND INITIAL CULTURE OF GREAT LAKES Cladophora sp.
To isolate Great Lakes Cladophora sp. for laboratory nutritional studies,
it was necessary to have available a culture medium in which the algae
would grow, or at least could be maintained. Unpurified Cladophora did
not grow well in a medium generally satisfactory in the culture of many
green algae, blue-green algae, and diatoms. This was a solution con-
taining the concentrations of major essential elements specified by
Hughes, Gorham, and Zehnder (1958) in a solution hereafter referred to
as Gorham's Medium, and one-tenth the concentrations of trace elements
in a culture solution developed by Johnson, Stout, Broyer, and Carlton
(1957) hereafter referred to as Johnson's trace elements. Cladophora sp.
did not grow satisfactorily in a more dilute medium developed by Gerloff,
Fitzgerald, and Skoog (1950) in studies on the nutrition of blue-green
algae.
Several unsuccessful attempts were made to grow Lake Michigan Cladophora
sp. in Zuraw's C Medium (1969) reported satisfactory for the culture of
Cladophora glomerata. Lake Michigan Cladophora sp. also failed to make
significant growth in Zuraw's medium with 2 ppm vitamin Bi added and with
the iron (Fe) source increased to 1.12 ppm as FeEDTA. Growth did in-
crease somewhat when lake water was added to Zuraw's solution and
increased further when the sodium chloride specified was omitted.
INITIAL CULTURE MEDIUM
Cladophora sp. grew reasonably well in a solution containing the con-
centrations of the major essential elements specified in Gorham's
Medium fortified with effluent (10 to 25% by volume) from the Madison,
Wisconsin, Sewage Disposal Plant. It also was established that
Cladophora sp. growth was improved considerably by the addition of
2 ppm vitamin BI. This combination became the basic medium in ini-
tial efforts to isolate and study Great Lakes Cladophora sp.
The stimulation of Cladophora growth resulting from additions of 25%
sewage effluent to the culture medium in three different experiments
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is shown in Table 1. The sewage effluent increased yields by 300-400%'.
In further experiments, it was established that additions of water
from several local lakes (25-50%) had the same stimulatory effect
as sewage effluent (Tables 1 and 2). Lake water rather than sewage
effluent was added to the culture medium in most subsequent experiments.
ISOLATION OF UNIALGAL CULTURES
For detailed nutritional studies, it was essential to work with cul-
tures in which only a single species of Cladophora was utilizing the
nutrients provided. To meet this requirement, Cladophora sp. col-
lected from six lakes were isolated in unialgal condition by the
following general procedure.
Small (approximately 1 mg dry weight) clumps of Cladophora which had
been well rinsed in lake water at the sampling site were placed in
25 to 200 ml of sterile culture medium, usually Gorham's Medium
diluted with 25% secondary sewage effluent. The algae clumps were
thoroughly rinsed in the culture medium and several subcultures were
transferred to fresh medium. As soon as long filaments of Cladophora
developed from the original clump, they were isolated, washed in
sterile medium, and subcultured. The resulting growth was periodi-
cally checked for algal contamination. All potentially unialgal
cultures were transferred to streaked or poured plates of agar med-
ium (1% agar in Gorham's Medium plus 25% sewage effluent). Growths
on the agar plates were checked for algal contaminants. All uni-
algal cultures were transferred to sterile medium for maintenance as
stock cultures. Approximately 150 cultures were initially inoculated
in isolating unialgal Cladophora sp. from each of six lakes, Mendota
(Madison, Wis.)> Superior, Michigan, Huron, Erie, and Ontario.
ISOLATION OF BACTERIA-FREE Cladophora
It was necessary to separate the Cladophora sp. from the contaminating
bacteria to demonstrate possible roles of bacteria in the algae's
growth, for example, in producing stimulatory organic growth factors.
Previous work (Fitzgerald and Faust, 1967; Fitzgerald, 1969) showed
that selective use of some chemicals, such as quaternary ammonium
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Table 1. STIMULATORY EFFECT OF SEWAGE EFFLUENT AND LAKE WATER
ON THE GROWTH OF Cladophora sp. IN NUTRIENT CULTURES
Experiment
I
I
II
II
II
III
III
III
Supplement to
basal medium
None
25% Sewage effluent
None
25% Sewage effluent
25% Lake Wingra water
None
25% Sewage effluent
100% Lake Mendota water
Ave. algae dry-wt
yield, b mg/1
74
276
125
352
228
62
215
344
The nutrient medium contained Gorham's Medium major elements plus
vitamin B! (2 mg/1).
""Average of triplicate cultures in each treatment.
Table 2. QUANTITATIVE RESPONSE OF Cladophora sp. TO THE ADDITION
OF LAKE MENDOTA WATER TO THE CULTURE MEDIUM
0
Lake water,
%
0
5
10
25
50
90
pH
at inoculation
8.9
9.0
8.9
9.0
8.7
8.9
PH
at harvest
8.2
8.3
8.5
8.8
9.2
9.6
Ave. algae dry-wt
yield, b mg/1
83
77
114
157
295
283
Lake water collected Jan. 8, 1974; nutrient medium as specified in
Table 1.
Average of triplicate cultures in each treatment.
10
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compounds or chlorine, would decrease the number of bacteria in
algal cultures. Preliminary experiments with Lake Michigan Cladophora
sp. indicated that free available chlorine killed bacteria associated
with the algae without destroying the Cladophora. Isolates of
Cladophora sp. from each of the five Great Lakes were treated with
chlorine in an effort to free them of bacteria. The procedure
employed was to wash a small amount (approximately 10 mg dry weight)
of Cladophora in two changes of sterile culture medium and then to
mix the algae in a sterile Waring Blender, for approximately one
minute. This usually was sufficient to produce a suspension of
sections of Cladophora filaments. Aliquots of the blended Cladophora
were transferred by a sterile inoculating loop to culture media con-
taining 2.5, 5, or 10 mg of chlorine/1. At each of ten different
treatment times, varying from 5 to 120 minutes, a loopful of
Cladophora filaments was transferred to sterile medium for incuba-
tion. After one to four weeks of incubation, those cultures that
showed Cladophora growth were tested for the presence of bacteria
by plating a portion of the Cladophora on tryptone, glucose, extract
agar (TGE agar). The remaining algae were transferred to sterile
culture medium. Cladophora cultures which seemed uncontaminated
were again tested in the TGE agar and, finally, those cultures which
appeared to be bacteria-free were dragged across TGE agar and cul-
tured in sterile medium. The difference in the technique for iso-
lating bacteria-free Cladophora and those previously used for other
algae resulted from the failure of Cladophora to grow on the organic
medium used to test for bacteria. Therefore, portions of the Cladophora
cultures had to be sacrificed in testing for bacterial contaminants.
The general technique described resulted in the isolation of multiple
bacteria-free Cladophora sp. cultures from Lakes Superior, Michigan,
and Erie. Samples from Lakes Huron and Ontario were initially iso-
lated in bacteria-free condition, but after several transfers were
found to be contaminated with bacteria again and were discarded. The
bacteria-free isolates from Lakes Superior, Michigan, and Erie sur-
vived at least three subcultures over a four-month period, and were
11
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in a healthy condition in the apparent absence of bacteria.
Some time later, to further check for contamination, portions of the
Lake Michigan isolates were transferred to small amounts of a sterile
medium containing 10 g trytone, 10 g dextrous anhydrous and 5 g yeast
extract added to one liter Cladophora Medium I (see Section V). This
procedure indicated that all of the Lake Michigan isolates had become
contaminated. Maintenance of bacteria-free algae cultures in inorgan-
ic media is difficult. Efforts now are underway to re-establish
bacteria-free Cladophora sp. using the procedure described above and
also treatments with mixtures of antibiotics.
SPECIES IDENTIFICATION
Attempts have been made to establish the species of Cladophora iso-
lated from Lake Michigan and used in most of the nutrition experiments.
According to Whitton (1970), studies on Cladophora in the Great Lakes
have been mostly with C_. glomerata and C_. fracta. Unfortunately, the
only available key for Cladophora is for European species (Van Den Hoek,
1963). This key leaves little doubt that the Cladophora sp. is either
C_. glomerata or C_. fracta which are rather difficult to distinguish.
The diameter of the apical cells is one distinguishing feature.
Cladophora fracta apical cells usually are 19-24 y in diameter;
C_. glomerata are 30-40 u. Measurements on apical cells from a young
culture averaged 32.4 u; the average from an old culture was 36.7 y.
All of the additional morphological and ecological characteristics
were consistent with identification as C_. glomerata, except the forma-
tion of zoospores which occurs in both C_. glomerata and C_. fracta.
Germlings which had the size and appearance to indicate origins from
zoospores, swollen apical cells containing spore-like bodies, and
structures among the filaments which resemble zoospores have been ob-
served. Actual zoospores have not been detected. The species used
in most of these studies was considered to be Cladophora glomerata.
MORPHOLOGY
Cladophora exhibits several morphological variations obvious to the
eye and under the microscope. In laboratory cultures, the most common
12
-------
forms were:
a. Loosely tangled masses of filaments which can be highly
or slightly branched and which vary considerably in size,
depending on their age.
b. "Knots" consisting of large filaments tangled and inter-
twined; usually akinetes with thick walls can be observed.
c. Germlings which are attached to the walls of culture
flasks; they usually are unbranched and range from small,
spore-like cells to filaments many cells in length.
Preliminary experiments have been carried out to determine if the rate
of C_. glomerata culture varies with the morphological form of the
inoculum, if the morphological form of the inoculum persists through
a subsequent transfer, and if agitation of cultures during an incu-
bation period influences the morphology of the resulting growth. Re-
sults of the limited experiments were not sufficiently conclusive to
justify presentation and discussion of the data.
13
-------
SECTION V
LABORATORY CULTURE OF GREAT LAKES Cladophora glomerata
IN A DEFINED NUTRIENT MEDIUM
Following isolation and identification of Cladophora glomerata from
Lake Michigan and its maintenance in unialgal culture in a non-defined
medium, studies could be initiated to define the specific nutritional
requirements of the alga. The first effort was to identify the
stimulatory factors in sewage effluent and lake water.
Sewage effluent and lake water could be beneficial for several reasons.
They could satisfy a C_. glomerata requirement for an organic growth
factor other than vitamin Bj. They also could provide an essential
inorganic trace element, either a trace element presently unrecog-
nized or an additional amount of a recognized essential element to
satisfy a relatively high requirement. In addition, a favorable
balance of major essential elements (for example, a high Ca-Mg envir-
onment) could be provided by effluent and lake water. They could
affect the pH of the medium, influence the availability of Fe, or
provide N or another element in a form preferred by C_. glomerata.
RESULTS
Major Variations in Inorganic Components
The data in Table 3 show the response of C_. glomerata to several levels
of the essential trace elements in the presence and absence of lake
water in the culture medium. Average yields with no lake water in the
medium (Treatments A-D) were only 60% of yields with lake water added
(Treatments E-H). The addition of trace elements did not increase
yields either in the absence or presence of lake water.
Even though there was no response to trace elements, they were added
to the medium in most subsequent nutrition experiments at 0.02x the
concentration specified by Johnson, et al. (1957) for higher plants.
This was to insure that any responses in subsequent experiments on
organic growth factors would not be limited by inadequate trace
elements. There was no indication that the concentrations of the
14
-------
Table 3. RESPONSE OF Cladophora glomerata TO SEVERAL
CONCENTRATIONS OF THE ESSENTIAL TRACE ELEMENTS
Designation
A
B
C
D
E
F
G
H
pH at
inoc.
9.2
9.1
8.8
8.8
8.8
8.9
8.8
8.9
pH at
harvest
8.2
8.3
8.3
8.4
8.6
8.6
8.6
8.5
Lake
water,
%
0
0
0
0
25
25
25
25
Trace element
a
concentration
O.Olx
O.lx
0.2x
O.Olx
O.lx
0.2x
Ave. algae ,
dry-wt yield,
mg/1
165
141
141
132
261
263
238
210
a"x" refers to the concentration of the trace elements Mn, Zn, Cu, B,
Mo, and Cl in Johnson's Medium.
Average of triplicate cultures in each treatment.
trace elements added were toxic to C_. glomerata (Table 3).
The possibility of a very high requirement for one or several trace
elements also was considered. Analyses of C_. glomerata collected
from Lake Michigan and of C_. glomerata grown in fortified lake water
in the laboratory showed that the alga contained relatively high
concentrations of Mn and B. Additions of these elements in relatively
high concentrations did not increase yields and did not duplicate the
beneficial effects of sewage effluent and lake water.
The pH values of cultures receiving lake water (Treatments E-G, Table 3)
were slightly higher at harvest than in cultures without lake water.
This effect of lake water also was apparent in the data of Table 2.
These minor pH differences probably reflect variations in C_. glomerata
growth rather than a significant influence of the lake water on the
medium. The pH of the nutrient medium increases as a result of algae growth.
15
-------
Past experience indicated that N and Fe sources in the culture medium
could have marked effects on C_. glomerata growth. For example, some
organisms prefer NH^-N rather than NOa-N. Several N concentrations
were provided as NaNOa, NHitNOs, and urea. Unless sewage effluent or
lake water was provided, growth was poor in all cultures. There was
no preference for a specific form of N. Because of the relatively
high pH of the culture medium (8.0-9.5), maintaining adequate Fe in
an available form could be difficult. For the culture of macrophytes
under similar conditions (Gerloff, 1973), FeEDDHA, a chelate of Fe with
ethylene-diamine di-(o-hydroxyphenylacetate), was found to be a more
effective iron source than FeEDTA, a chelate with ethylene-diamine
tetraacetic acid. An experiment involving various iron sources and
concentrations gave no indications that Fe concentrations higher
than in Gorham's Medium were beneficial or that FeEDDHA was a better
iron source than FeEDTA either with or without the addition of sewage
effluent.
Both sewage effluent and lake water also provided relatively high
concentrations of Ca and Mg in the medium. Increased Ca and Mg
concentrations in Gorham's medium did not duplicate the beneficial
effects of the effluent and lake water.
Organic Growth Factor Requirements
The data in Table 4 show the results of two experiments to establish
whether the beneficial effects of lake water and sewage effluent were
associated with organic or inorganic factors. Volumes of sewage
effluent and lake water equivalent to the volumes added to cultures
were evaporated to dryness, the residues were ashed in a muffle
furnace at 450°C for 6 hours and then were added to C_. glomerata
cultures. Destruction of organic constituents by ashing markedly
reduced Cladophora glomerata growth in both experiments.
Vitamins, plant hormones, and amino acids seemed the most likely
trace organic factors destroyed by ashing sewage effluent and lake
water. A requirement for vitamin BI was initially established when
C. glomerata (Ind. 1484, Indiana Culture collection) was grown for
16
-------
Table 4. DESTRUCTION BY ASHING OF SEWAGE EFFLUENT AND LAKE WATER
FACTORS STIMULATORY TO Cladophora glomerata GROWTH
Experiment
Ia
I
IIb
II
II
Treatment
25% sewage effluent
As A, except ashed
No lake water added
25% Lake Mendota water
As C, except ashed
pH at
inoc.
9.3
9.2
8.8
8.8
9.2
pH at
harvest
8.3
8.6
8.6
Ave. algae dry-wt
yield, mg/1
230
152
141
238
ISO
aThe nutrient medium contained Gorham's Medium major elements plus
vitamin BI (2 ppm).
The nutrient medium contained Gorham's Medium major elements, 1/2
Johnson's trace elements, and vitamin BI (2 ppm).
£
Average of triplicate cultures in each treatment.
15 days in a solution containing Gorham's Medium major elements and
different proportions of secondary sewage effluent with and without
vitamin BI. The results are shown in Figure 1.
It is evident that the cultures to which 2 mg/1 of vitamin BI were
added produced greater yields than those lacking vitamin BI. Further-
more, adding vitamin BI, after 9 days, to a culture containing sewage
effluent stimulated growth when measured after an additional six days.
The positive response to vitamin BI suggested the need to determine
whether C_. glomerata required additional vitamins. After preliminary
experiments showed a response to vitamin 612, the experiment reported
in Table 5 was carried out. The nutrient media contained 25% lake
water. Because of possible destruction during autoclaving at the
alkaline pH of the medium, all vitamins were individually sterilized
by millipore filtration and added to the autoclaved medium. The results
clearly show a requirement of C_. glomerata for both vitamin Bj and 612.
17
-------
160
VITAMIN B,
O
VITAMIN B,
FOR 6 DAYS
10 20 30
SEWAGE EFFLUENT (%)
Figure 1. Effect of vitamin B! on the growth of Cladophora glomerata
(Ind. 1484) in different dilutions of Gorham's Medium major elements
and sewage effluent; 15-day culture period.
With only vitamin BI or B12 added, yields were 53 and 58 mg/1 respectively;
with both vitamins added, yields increased to 229 mg/1. Additional
vitamins did not result in significant yield increases.
The quantitative requirements of Cladophora glomerata isolated from
Lake Michigan for vitamins BI and 612 were established in the experi-
ments reported in Tables 6 and 7, respectively. There was a positive
response to each concentration of vitamin BI tested, including the
maximum concentration of 2.0 mg/1. Maximum yield was obtained with
18
-------
Table 5. RESPONSE OF Cladophora glomerata TO VARIOUS VITAMINS AND
ORGANIC GROWTH FACTORS WHEN GROWN IN CULTURE MEDIUM
Designation
A
B
C
D
E
F
G
H
I
J
K
L
Organic growth
factor additions
None
Bi
Bl2
BI, Bia
BI, B12, inositol
BI, 612, nicotinic acid
BI, 612, p-aminobenzoic acid
BI, 612, pyridoxine-HCl
BI, 612, thymine
BI, 612, folic acid
BI, 612, DL-Ca-pantothenate
All above vitamins
Ave. algae dry-wt
yield, b mg/1
38 a
53 a
58 a
229 b
256 b
260 b
263 b
244 b
290 b
258 b
225 b
287 b
nutrient medium contained Gorham's Medium major elements, 1/50
Johnson's trace elements and 25% lake water.
Average of triplicate cultures in each treatment; yields with a
common letter are not significantly different at the 5% level using
Duncan's multiple range test.
19
-------
Table 6. QUANTITATIVE RESPONSE OF Cladophora glomerata TO VARIOUS
CONCENTRATIONS OF VITAMIN BI IN THE CULTURE MEDIUM3
Cone, of vitamin BI, mg/1
0
0.01
0.05
0.25
1.0
2.0
Ave. algae dry-wt
36
58
96
136
168
258
yield, mg/1
aThe nutrient medium contained Gorham's Medium major elements plus
vitamin 612 (20 yg/1); 28 day culture period.
Average of triplicate cultures in each treatment.
0.50 yg/1 and higher concentrations of vitamin Bi2 in Experiment I of
Table 7 and with 0.25 yg/1 and higher concentrations in Experiment II.
The concentrations of vitamin Bj and B12 to add to C_. glomerata
culture media were established as 2.0 mg/1 and 0.5 yg/1, respectively.
An experiment also was carried out to determine if the Cladophora sp.
isolated from each of the Great Lakes required vitamin BI. Table 8
summarizes the results. There was a limited amount of growth in the
Gorham's major element medium plus 10% lake water, but the addition
of vitamin BI doubled or nearly doubled the yield of all five
Cladophora isolates. The requirement of Cladophora sp. for vitamin
BI seems to be general.
To determine if all eight Cladophora isolates cultured on this pro-
ject required vitamin 612, small quantities of each source of
Cladophora were initially grown in Gorham's major element medium
plus vitamin BI and then approximately 0.1 mg samples were transferred
to cultures with and without 20 yg/1 of vitamin Bi2. After a 32-day
growing period the cultures were harvested and the average yields
compared (Table 9).
20
-------
Table 7. QUANTITATIVE RESPONSE OF Cladophora glomerata TO VITAMIN Bi2
(CYANOCOBALAMIN) IN THE CULTURE MEDIUM
Experiment
Ia
nb
Concentration of
vitamin B12, ug/1
0
0.05
0.10
0.50
1.0
0
0.13
0.25
0.50
1.00
2.00
4.00
8.00
Ave. algae dry-wt
yield, mg/1
59
1'21
182
224
203
151
230
258
257
211
226
246
194
athe nutrient medium contained Gorham's Medium major elements plus
vitamin BI (2 mg/1); 22-day culture period.
rhe nutrient medium contained Gorham's Medium major elements, 1/50
Johnson's trace elements, vitamin BI (2 mg/1) and 25% lake water;
21-day culture period.
f*
Average of triplicate cultures in each treatment.
21
-------
Table 8. THE VITAMIN BI REQUIREMENT OF GREAT LAKES Cladophora sp.'
Cladophora source
Lake Michigan
Lake Superior
Lake Huron
Lake Erie
Lake Ontario
Ave. algae dry-wt yield, mg/1
Without vit. BI
74
47
73
41
14
With 2 mg/1 vit. BI
150
100
123
96
136
dThe nutrient medium contained Gorham's Medium major elements,
vitamin 612 (20 yg/1) and 10% Lake Mendota (Madison, Wis.) water;
14-day culture period.
Average of triplicate cultures in each treatment.
Table 9. THE VITAMIN Bi2 REQUIREMENT OF Cladophora sp,
Ave. algae dry-wt yield,0 mg/1
Cladophora isolate
Cladophora glomerata
(Ind. 1484)
Cladophora glomerata
(Ind. 1488)
Lake Mendota
Lake Superior
Lake Michigan
Lake Huron
Lake Erie
Lake Ontario
Without vit. BJ2
28
8
5
79
34
56
19
100
With 20 yg/1 vit. Bi2
100
350
210
310
270
150
120
360
aThe nutrient medium contained Gorham's Medium major elements plus
vitamin B! (2 mg/1); 32-day culture period.
Inoculum for this experiment obtained from Cladophora isolates grown
in nutrient medium minus vitamin Bi2.
CAverage of triplicate cultures in each treatment.
22
-------
It is evident that all eight isolates of Gladophora sp. responded to
and required vitamin Bi2.
The data in Table 10 show the response of C_. glomerata to compounds
representing the three major types of plant hormones (auxins,
giberellins, and cytokinins). There was no response to the several
concentrations of the three compounds tested. Cladophora glomerata
apparently is able to synthesize required quantities of these com-
pounds .
DISCUSSION
With the establishment of the vitamin BI and 612 requirements of
Cladophora glomerata, it was possible to produce 200-350 mg oven-
dry algae in a liter of defined culture medium after a 21-day culture
period. Supplements of lake water and sewage effluent could be
omitted.
The demonstrated requirements of Cladophora glomerata for vitamins
BI and Bia are unusual for a freshwater green alga, and probably are
the critical factor in the successful laboratory culture of C_. glomerata
in defined media. At this point in the study, there were no indications
that the qualitative or quantitative requirements of C_. glomerata for
the inorganic components of nutrient media could be considered unusual.
It grew satisfactorily at the high pH and with the nutrient concen-
trations that have been satisfactory for the culture of many other
green algae, blue-green algae, and diatoms.
The culture medium for C_. glomerata developed in the work described
in this section contained the Hughes, Gorham, Zehnder concentrations
of the major essential elements (1958), 0.02x the concentrations of
the essential trace elements specified by Johnson et al. (1957),
2.0 mg/1 vitamin BI, and 0.5 yg/1 vitamin Bi2. Supplemental trace
elements also were recommended to maximize the chances for response
to other factors. The trace element supplements were derived from
two solutions of salts that provided V, Cr, Ni, Co, W, Ti, and Sn
(each element at a concentration of 0.001 ppm in the culture solution)
23
-------
Table 10. RESPONSE OF Cladophora glomerata TO ADDITIONS OF
PLANT HORMONES TO THE CULTURE MEDIUM*1
Designation
A
B
C
D
E
F
G
H
I
Hormone addition
None
None, 25% lake water
IAA, 10 ]M
GA3, 1 yM
BAP, 1 yM
IAA, 1 yM
GA3, 0.1 yM
BAP, 0.1 yM
IAA, 1 yM; GA3, 0.1 yM; BAP, 0.1 yM
Ave. algae dry-wt
yield, mg/1
128
221
104
93
120
136
123
128
127
aThe nutrient medium contained Gorham's Medium major elements (except
2x Ca), 1/50 Johnson trace elements, B? and Ci3 solutions, and
vitamins BI and 612.
IAA (indole acetic acid); GA3 (giberellic acid); BAP (benzylamino-
purine).
Q
Average of triplicate cultures in each treatment.
and Al, As, Cd, Sr, Hg, Pb, Li, Rb, Br, I, F, Se and Be (each at a
concentration of 0.0005 ppm) (Arnon, 1938). This culture medium was
used extensively in establishing critical element concentrations in
Cladophora glomerata. It will be referred to as Cladophora Medium I.
-------
SECTION VI
CRITICAL CONCENTRATIONS OF ESSENTIAL
ELEMENTS FOR Cladophora glomerata
Development of a defined culture medium made possible experiments to
establish the critical concentrations of various elements for C_.
glomerata. Establishing the critical concentrations was important
for several reasons: 1) the nutritional features of C_. glomerata
could be characterized by comparisons of the alga's critical con-
centrations with critical concentrations for other plants; 2) evalu-
ations of nutrient supplies and potential growth-limiting nutrients
by the plant analysis technique would be possible and 3) modifications
to improve the culture medium could be suggested because critical
concentrations are established by systematic variation of the compo-
nents of the medium.
EXPERIMENTAL PROCEDURES
The general experimental procedures used in establishing critical
concentrations of elements in aquatic organisms have been described
by Gerloff and Krombholtz (1966) and Gerloff (1973). To establish
the critical concentration of a specific element in C_. glomerata,
the alga was grown at concentrations of that element varying from
less than adequate to more than adequate for optimal growth. All
other components of the culture medium were held constant at the
concentration specified in Cladophora Medium I. After a 21-day
culture period, the algae were harvested, oven-dried, weighed and
then analyzed for the element under study. The critical concentration
(the concentration of the element associated with approximately 5%
less than maximum yield) was established from a curve obtained by
plotting algae yield vs. concentration of the element in the algae.
From experience in the initial studies, the following techniques and
procedures were developed for culturing and harvesting C_. glomerata.
Algae were grown in 500 ml flasks containing 200 ml culture medium.
In each flask, gentle aeration was provided from a pyrex tube extending
through a rubber stopper (Figure 2). Prior to passage through the
25
-------
AIR OUTLET WITH
SMALL TEST TUBE
LATEX SURGICAL
TUBING (3/16 in)
PYREX TUBING (4mm)
COTTON PLUG 8
INJECTION NEEDLE
(20 go)
(TO AIR SUPPLY)
500 ml ERLENMEYER
Figure 2. Apparatus used in the routine laboratory culture of Cladophora
sp. in nutritional experiments.
cultures, air was filtered through a polyester fiberfil trap and then
through a double-distilled water trap.
Double-distilled water (second distillation from an all-glass still)
was used in preparing nutrient media. All glassware was washed,
rinsed in distilled water, soaked in approximately 4N_HN03, and then
thoroughly rinsed in distilled water and double-distilled water.
26
-------
Following preparation, all media and transfer apparatus were autoclaved
at 15 psi for 15 minutes.
To inoculate a series of cultures, the main mass of a 3 to 4 week old
C. glomerata stock culture was transferred with an inoculating loop
to a petri dish containing sterile double-distilled water. After
rinsing, the filaments were transferred to a second petri dish. Using
a sterile forceps and scissors small aliquots (approximately 6-8 mg
dry weight) of the alga were removed from the main mass and distri-
buted on a Coors spot-plate. As inoculum, uniform aliquots were
selected and randomly distributed to the 500 ml flasks containing
medium (one per flask). Sterile technique and instruments were used
throughout this procedure. Inoculum used in trace element experiments
(Mn, Zn, B, Cu, and Mo) was derived from algae grown in culture medium
from which the trace element to be studied had been omitted.
The algae were cultured at 23°C ± 1° under continuous fluorescent
light (T-12 cool-white bulbs) of 350-450 foot-candles intensity.
Supplemental incandescent light was not provided.
The algae were harvested by filtering cultures through 270 mesh nylon
net supported in a Buchner funnel in a suction flask. Considerable
effort often was required to loosen algae filaments from the sides of
the flasks. After harvest, algae were dried for 48 hours in a forced-
draft oven at 63-65°C, cooled in a desiccator, and weighed. Entire
harvests from individual flasks, or combined harvests from several
flasks, could be used as individual samples in chemical analyses.
Grinding of samples was not required.
Element concentrations in C_. glomerata were determined by quantitative
procedures in general use for plant analysis. Total N analyses were
by a semi-micro Kjeldahl procedure. Phorphorus was determined by a
vanado-molybdate yellow-complex procedure following dry ashing of
oven-dried algae at 550°C (Jackson, 1958). Potassium analysis was
by emission flame photometry of IN^ ammonium acetate extracts of tissue
samples. Tissues were prepared for Fe analysis by dry ashing for 6
hours at 660°C. Iron then was determined as a complex with o-phenanthroline.
27
-------
Following dry ashing and acid solution of the residues, Ca, Mg, Zn, and
Mn were determined by atomic absorption using a Jarrell-Ash instrument.
Boron was measured as a curcumin complex (Johnson and Ulrich, 1959)
and Mo as a complex with thiocyanate following stannous reduction
(Johnson and Arkeley, 1954). Both analyses were on dry-ashed tissues.
Analyses for S were by turbidemetric measurements of BaSOi* precipitated
in HNOa-HClOit digests of plant tissues (Blanchar, Rehm, and Caldwell,
1965). Copper analyses and B analyses on some samples were run by
the Wisconsin Alumni Research Foundation Laboratories. Copper was
determined by atomic absorption and B by a Jarrell-Ash Multichannel
Emission Spectrometer.
RESULTS
Critical N and P Concentrations
The data presented in Table 11 and graphed in Figure 3 are from an
experiment to establish the critical concentration of P in Cladophora
glomerata. Nutrient solution P concentrations in 8 treatments varied
from 0.049 to 7.0 ppm. Yields at these P concentrations ranged from
148 to 361 mg dry-weight/I; the P concentrations in the algae varied
from 0.04 to 0.54%. Below approximately 0.06% P, C_. glomerata yield
was less than maximum; above 0.06%, yield was at a constant maximum,
even though the P concentration in the algae continued to increase to
0.54%. The highest P concentrations were somewhat inhibitory. From
the data presented, the critical P concentration was established at
0.06%. This value was verified in a second experiment.
The data presented in Table 12 and plotted in Figure 4 are from an
experiment to establish the critical N concentration for C_. glomerata.
The N concentration in C_. glomerata varied from 0.83 to 4.89%. Below
approximately 1.1%, yield was less than maximum and was directly related
to the amount of N provided in the culture medium. The critical N
concentration was established as 1.1%, a value verified in a second
experiment.
Critical Concentrations of Other Elements
Critical concentrations for most of the other essential elements were
28
-------
Table 11. YIELD AND TOTAL P CONCENTRATION OF Cladophora glomerata
GROWN IN CULTURE MEDIUM DIFFERING IN P CONCENTRATION
P added to
culture soln, ppm
0.049
0.07
0.10
0.14
0.28
0.56
1.40
7.00
Ave. algae dry-wt
yield, b mg/1
148 a
174 ab
226 b
315 c
361 c
336 c
351 c
305 c
Ave . P cone .
in algae, %
0.04
0.03
0.04
0.04
0.06
0.14
0.32
0.54
aThe nutrient medium contained Gorham's Medium major elements (-P), 1/50
Johnson's trace elements, By and Cia solutions, and vitamins BI and
812.
Average of six replicates in each treatment; yields with a common
letter are not significantly different at the 5% level using Duncan's
multiple range test.
determined in the experiments summarized in Tables 13-22. The critical
concentrations established from all the experiments and the range in
concentration of each element in the C_. glomerata are summarized in
Table 23.
The critical concentrations for Zn, Cu, and Mo are reported in Table
23 as equal to or less than specific concentrations. These concen-
trations were obtained by analyzing C_. glomerata grown in basal medium
to which the trace element under study was not added. Yields were
not reduced by omission of these three elements, so the values presented
may not be quite the minimum which will result in maximum yield. How-
ever, to obtain more specific critical concentrations would have
involved considerable effort and time in purifying culture media of
Cu, Zn, and Mo to the point that deficiency could be demonstrated.
29
-------
400
300
200
LJ
O
100
I
I
I
I
O.I
0.2 0.3 0.4 0.5
TISSUE CONTENT OF P (%)
0.6
Figure 3. The relationship between oven-dry yield and P concentration
in Cladophora glomerata after culturing in solutions varying in P
concentration.
Because a considerable part of the project would have been required for
just these experiments, the above procedure seemed justified. It also
seemed justified to conclude that supplies of certain elements were
unlikely to limit growth if the concentrations of those elements in
Cladophora sp. samples from the Great Lakes considerably exceeded the
concentrations in the algae when grown in unpurified culture media
30
-------
Table 12. YIELD AND TOTAL N CONCENTRATION OF Cladophora glomerata
GROWN IN CULTURE MEDIUM DIFFERING IN N03-N CONCENTRATION
N added to
culture soln, ppm
1.0
1.6
2.5
3.3
4.1
5.6
8.2
10.7
21.0
82.0
Ave. algae dry-wt
yield, b mg/1
74 a.
183 be
222 cd
225 cd
242 d
230 cd
176 be
184 be
162 b
158 b
Ave. N cone.
in algae, %
0.93
0.83
0.96
1.32
1.52
2.35
3.87
4.30
4.89
4.51
aThe nutrient medium contained Gorham's Medium major elements (-N), 1/50
Johnson's trace elements, By and Cia solutions, and vitamins BI and
Biz.
Average of four replicates in each treatment; yields with a common
letter are not significantly different at the 5% level using Duncan's
multiple range test.
which supported maximum growth.
DISCUSSION
As will be discussed in Section X, the critical concentrations estab-
lished for Cladophora glomerata were used in evaluating nutrient supplies
and growth-limiting nutrients in the Great Lakes. In addition, com-
parisons of these critical concentrations with those established for
other species, particularly other algae and aquatic organisms, permit
evaluations of the inorganic nutrient requirements of C_. glomerata.
The 1.1% critical N and the 0.06% critical P concentrations, and
consequently the total requirements for these elements, in C_. glomerata
are relatively low. For comparison, the critical N concentration in
31
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300
o>
200
cc
o
i 100
UJ
o
I
I
I
1.0 2.0 3.0 4.0
TISSUE CONTENT OF N (%)
5.0
Figure 4. The relationship between oven-dry yield and total N content
of Cladophora glomerata after culturing in solutions varying in N03-N
concentration.
Draparnaldia plumosa, another filamentous green alga, was 2.30% and
the critical P concentration was 0.18% (Gerloff, 1975). Critical N
and P concentrations in the bloom-producing blue-green alga Microcystis
aeruginosa were 4.00% and 0.12%, respectively (Gerloff and Skoog, 1954).
In some macrophytes, the critical N and P concentrations exceeded the
C. glomerata values. However, those for Myriophyllum spicatum (0.75% N
32
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Table 13. YIELD AND K CONCENTRATION OF Cladophora glomerata GROWN
IN CULTURE MEDIUM DIFFERING IN K CONCENTRATION
K added to
o
culture so In, ppm
0.045
0.09
0.18
0.90
1.8
2.7
3.6
5.4
9.0
18.0
Ave. algae dry-wt
yield, mg/1
43 b
51 b
51 b
104 b
160 c
185 c
187 c
197 c
246 c
195 c
aThe nutrient medium contained Gorham's Medium major e
Ave. K cone.
in algae, %
1.09
0.99
1.10
1.22
1.30
1.70
2.12
2.84
3.23
4.74
.ements (except
-K and 8.2 ppm N), 1/50 Johnson's trace elements, 87 and Cis solutions,
and vitamins BI and 612.
Average of ten replicates in each treatment (needed to obtain adequate
material for K analysis).
"Average of four replicates in each treatment.
and 0.07% P) were comparable to the C_. glomerata critical concentrations
(Gerloff, 1975).
Algae characteristically have relatively low Ca requirements as shown
by variations in critical concentrations from 0.00 to 0.06% in six
species studied by Gerloff and Fishbeck (1969). In comparison, the
0.30% for C_. glomerata seems relatively high. The 0.15% Mg critical
concentration is comparable to the average value of 0.20% for the six
algae species in the same study. The critical K concentration in the
six algae varied considerably, from 0.25 to 2.40%. The 1.30% value for
C_. glomerata approximates the average of 1.04% for the six species.
33
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Table 14. YIELD AND Ca CONCENTRATION OF Cladophora glomerata GROWN
IN CULTURE MEDIUM DIFFERING IN Ca CONCENTRATION
Ca added to
culture soln, ppm
0.49
0.97
2.4
4.9
7.3
9.7
19.4
29.1
Ave. algae dry-wt
yield, b mg/1
11 a
108 b
130 b
227 c
270 c
236 c
237 c
240 c
Ave. Ca cone.
in algae, %
0.20
0.25
0.30
0.35
0.39
0.56
0.83
1.06
aThe nutrient medium contained Gorham's Medium major elements (-Ca),
1/50 Johnson's trace elements, B? and Cis solutions and vitamins BI
and Bia.
Average of four replicates in each treatment; yields with a common
letter are not significantly different at the 5% level using Duncan's
multiple range test.
Several C_. glomerata critical concentrations were high, relatively,
and can be considered very unusual. This is true of the 1.50% value
for S. Sulfur critical concentrations and requirements in plants
usually are comparable to or somewhat below P requirements. The
critical S concentration in Elodea occidentalis, the only aquatic
plant for which information is available, was 0.08% (Gerloff, 1975).
Sulfur concentrations in deficient agricultural and horticultural
crops generally were less than 0.20% (Chapman, 1966).
The 110 ppm B critical concentration in C_. glomerata is tremendously
high in comparison with critical concentrations in other organisms.
Gerloff (1968) in studies on the B nutrition of algae found that B
was neither taken up nor required by the three species of green algae
tested, both unicellular and filamentous forms; in contrast, B was an
34
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Table 15. YIELD AND Mg CONCENTRATION OF Cladophora glomerata GROWN
IN CULTURE MEDIUM DIFFERING IN Mg CONCENTRATION
Mg added to
culture soln, ppm
0.08
0.15
0.30
0.60
0.75
1.50
7.50
Ave. algae dry-wt
yield, rag/1
187 a
266 ab
328 bed
319 be
392 bed
429 cd
454 d
Ave. Mg cone.
in algae, %
0.05
0.08
0.14
0.18
0.15
0.26
0.37
aThe nutrient medium contained Gorham's Medium major elements (except
-Mg, 16.4 ppm N and 2,8 ppm P), 1/5 Johnson's trace elements, B? and
Cis solutions and vitamins BI and 612.
Average of six replicates in each treatment; yields with a common
letter are not significantly different at the 5% level using Duncan's
multiple range test.
essential element for three species of blue-green algae and the element
was accumulated by cells to relatively high concentrations. Critical B
concentrations for agricultural and horticultural plants vary from
approximately 4.0 to 30.0 ppm (Chapman, 1966); dicotyledonous plants
in general have higher requirements than monocotyledons. The very
high B requirement of C_. glomerata not only contrasts with the lack
of its essentiality for other green algae that have been tested but
seems to be considerably higher than reported for any other plant
species.
To check on the very high B critical concentration established by
analysis of the algae with a colorimetric procedure, additional B
deficient C_. glomerata were grown and submitted to the Wisconsin
Alumni Research Foundation Laboratories for analysis with a Jarrell-
Ash Multichannel Emission Spectrometer. The values were lower than
35
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Table 16. YIELD AND S CONCENTRATION IN Cladophora glomerata GROWN
IN CULTURE MEDIUM DIFFERING IN S CONCENTRATION
S added to
culture soln, ppra
0.97
1.21
1.90
2.43
2.90
4.85
9.70
14.55
19.40
Ave. algae dry-wt
yield, b mg/1
Exp. I
59 a
146 b
244 c
436 d
;
Exp . I I
93 a
192 b
316 c
429 d
330 cd
393 cd
Ave. S cone.
in algae, %
Exp. I
1.84
1.42
1.22
1.77
Exp. II
1.58
1.27
1.40
1.67
2.30
2.32
aThe nutrient medium contained Gorham's Medium major elements (-S), 1/5
Johnson's trace elements, B7 and Cis solutions, and vitamins Bj and B
Average of six replicates in each treatment; yields with a common
letter are not significantly different at the 5% level using Duncan's
multiple range test.
in the reported experiment but still far above concentrations reported
in B-deficient plants of other species.
Cladophora glomerata requirements for Fe and the trace elements Mn, Cu,
Zn, and Mo were quite comparable to those for the macrophyte Elodea
occidentalis (Gerloff, 1975) and for economic plants (Chapman, 1966).
However, the 15 ppm critical Mn concentration is approximately 4x the
value established in Elodea occidentalis.
Nitrogen and phosphorus usually are given primary consideration in
evaluating possible growth-limiting nutrients in lakes and streams.
Knowledge of the critical concentrations makes this possible by the
plant analysis technique. The very high and unusual critical con-
centrations for S and B suggest that supplies of these elements in
36
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Table 17. YIELD AND Fe CONCENTRATION OF Cladophora glomerata GROWN
IN CULTURE MEDIUM DIFFERING IN Fe CONCENTRATION
Fe added to
culture so In, ppm
0.00224
0.0056
0.0112
0.056
1.12
Ave. algae dry-wt
yield, b mg/1
140 a
193 ab
245 b
351 c
371 c
Ave. Fe cone.
in algae, ppm
55
37
36
93
1110
aThe nutrient medium contained Gorham's Medium major elements (except
-Fe, and 8.2 ppm N) , 1/50 Johnson's trace elements, B? and Cis solu-
tions, and vitamins BI and
Average of eight replicates in each treatment; yields with a common
letter are not significantly different at the 5% level using Duncan's
multiple range test.
relation to C_. glomerata requirements also must be carefully considered
in evaluating the nutrition of the alga in the Great Lakes.
The systematic variations of nutrients in the critical concentration
experiments suggested modifications of the initial culture medium
would be desirable. Those modifications which became a part of the
general culture technique are indicated as footnotes to the appropriate
tables.
37
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Table 18. YIELD AND Mn CONCENTRATION OF Cladophora glomerata GROWN
IN CULTURE MEDIUM DIFFERING IN Mn CONCENTRATION
Mn added to
culture soln, ppm
0.00
0.00054
0.0027
0.0054
0.027
0.27
2.70
Ave. algae dry-wt
yield, b mg/1
225
273
264
293
479
453
129
Ave. Mn cone.
in algae, ppm
7.8
7.2
14.7
13.7
17.4
180
4690
aThe nutrient medium contained Gorham's Medium major elements (except
8.2 ppm N), 1/50 Johnson's trace elements (-Mn), 67 and Cis solutions,
and vitamins BI and 612.
The data were obtained from two separate experiments. Some of the
treatments were not repeated in both experiments. There were eight
replicates of each treatment in each experiment.
38
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Table 19. YIELD AND Zn CONCENTRATION OF Cladophora glomerata GROWN
IN CULTURE MEDIUM DIFFERING IN Zn CONCENTRATION
Zn added to
culture so In, ppm
0.00
0.0026
0.13
0.65
Ave. algae dry-wt
yield, b mg/1
556 a
563 a
517 a
245 b
Ave. Zn cone.
in algae, ppm
5.3
5.6
38.2
1039
The nutrient medium contained Gorham's Medium major elements (except
8.2 ppm N) , 1/50 Johnson's trace elements (-Zn) , B7 and Cis solutions
and vitamins BI and
Average of twelve replicates in each treatment; yields with a common
letter are not significantly different at the 5% level using Duncan's
multiple range test.
Table 20. YIELD AND Cu CONCENTRATION OF Cladophora glomerata GROWN
IN CULTURE MEDIUM DIFFERING IN Cu CONCENTRATION
Cu added to
culture soln, ppm
0.00
0.00006
0.0006
0.03
0.30
3.00
Ave. algae dry-wt
yield, mg/1
359 a
358 a
372 a
350 a
23 b
Insignificant growth
Ave. Cu cone.
in algae, ppm
8.6
4.9
8.5
14.6
aThe nutrient medium contained Gorham's Medium major elements, 1/50
Johnson's trace elements (-Cu), B? and Cia solutions and vitamins
BI and 612.
Average of eight replicates in each treatment; yields with a common
letter are not significantly different at the 5% level using Duncan's
multiple range test.
39
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Table 21. YIELD AND Mo CONCENTRATION OF Cladophora glomerata GROWN
IN CULTURE MEDIUM DIFFERING IN Mo CONCENTRATION
Mo added to
culture soln, ppm
0.00
0.00002
0.0002
0.002
0.01
1.00
Ave. algae dry-wt
yield, " mg/1
584 a
524 a
584 a
546 a
619 a
538 a
Ave. Mo cone.
in algae, ppm
1.39
0.78
0.90
3.6
14.0
1360
aThe nutrient medium contained Gorham's Medium major elements(except
8.2 ppm N), 1/50 Johnson's trace elements (-Mo), 87 and Cis solutions,
and vitamins BI and 812.
Average of eight replicates in each treatment; yields with a common
letter are not significantly different at the 5% level using Duncan's
multiple range test.
Table 22. YIELD AND B CONCENTRATION OF Cladophora glomerata GROWN
IN CULTURE MEDIUM DIFFERING IN B CONCENTRATION
B added to
o
culture soln, ppm
0.00
0.00054
0.0054
0.054
0.27
Ave. algae dry-wt
yield, mg/1
127 a
144 a
335 b
470 c
492 c
Ave. B cone.
in algae, ppm
211
178
116
110
180
aThe nutrient medium contained Gorham's Medium major elements (except
8.2 ppm N), 1/50 Johnson's trace elements (-B), B7 and Ci3 solutions,
and vitamins BI and Bia. Culture flasks were low-boron, alkali-
resistant glassware.
Average of eight replicates in each treatment; yields with a common
letter are not significantly different at the 5% level using Duncan's
multiple range test.
40
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Table 23. CRITICAL CONCENTRATIONS AND THE RANGE OF CONCENTRATIONS
OF ESSENTIAL NUTRIENT ELEMENTS IN Cladophora glomerata
Element
N
P
Ca
Mg
K
S
Fe
Zn
Mn
Cu
Mo
B
Critical concentration
1.10%
0.06%
0.30%
0.15%
1.30%
1.50%
45 ppm
< 5.0 ppm
16 ppm
< 6.0 ppm
< 0.7 ppm
110 ppm
Range in concentration
0.83 - 4.89%
0.04 - 0.54%
0.20 - 1.06%
0.05 - 0.37%
0.99 - 4.74%
1.22 - 2.32%
36 - 1110 ppm
5.3 - 1039 ppm
7.2 - 4690 ppm
4.9 - 14.6 ppm
0.7 - 1360 ppm
110 - 211 ppm
41
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SECTION VII
RECOMMENDED CULTURE MEDIUM FOR Cladophora glomerata
Systematic variation of element levels in the critical concentration
experiments indicated the optimum concentration of each essential
element for maximum Cladophora glomerata growth during the 21-day
culture period. The experiments described in this section were con-
cerned primarily with improving the Cladophora glomerata culture
medium through modifications suggested by the data on optimal con-
centrations of individual elements. Because of the increased growth
in this revised medium, it also was necessary to determine if further
modifications of the organic and buffering components would be
desirable. For example, vitamin concentrations that had been ade-
quate to produce 250 mg oven-dry algae per liter (Section V) might
be inadequate for twice that yield. From these efforts, a recommended
culture medium for Cladophora glomerata was derived.
The techniques used in growing, harvesting, and analyzing C_. glomerata
were in general those described in Section VI.
RESULTS
Inorganic Constituents
The critical concentration experiments indicated that several elements
were present in Cladophora Medium I at much higher concentrations
than required for the yields obtained. For example, yields actually
were decreased by the specified concentrations of N and P. In addition,
the unusually high requirements of C_. glomerata for several elements
suggested that the concentrations present in Cladophora Medium I might
be inadequate. The data in Table 24 show C_. glomerata response to
variations in the inorganic components of the culture medium suggested
by the critical concentration experiments.
Lowering the N concentration from 82 ppm to 8.2 ppm and the P from
7.0 to 1.4 ppm in Experiment I did not significantly reduce the
yield of C_. glomerata. This was as anticipated because the 1.1%
critical N concentration indicates 8.2 ppm N in the culture medium
42
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Table 24. VARIATIONS IN THE CONCENTRATIONS OF INORGANIC CONSTITUENTS
TO IMPROVE THE CULTURE MEDIUM FOR Cladophora glomerata
Experiment
I
II
III
IV
V
Treatment
designation
A
B
C
A
B
C
D
A
B
C
D
A
B
C
A
B
C
D
E
Medium modification
Cladophora Medium I,
8.2 ppm N, 7.0 ppm P
As A, but 8.2 ppm N,
1 . 4 ppm P
As A, but 16.4 ppm N,
2.8 ppm P
Cladophora Medium I
As A, but
10 x trace elements
As A, but 2x Ca,
2x Mg, 2x K, 2x S
As A, but 2x Ca, x/2 Mg,
x/2 K, x/2 S
Cladophora Medium I, but
lOx trace elements
As A, but
2x Ca, 2x Mg, 2x K
As A, but 2x S
As A, but 2x Ca,
2x Mg, 2x K, 2x S
Cladophora Medium II,
9.7 ppm Ca, 16.4 ppm N
As A, but 23.4 ppm Ca,
16.4 ppm N as Ca(N03)2
As A, but 23.4 ppm Ca
as CaCl2
Cladophora Medium I,
Na2C03 & Na2Si03
As A, minus Na2C03
As A, minus Na2Si03
As A, minus
Na2C03 § Na2Si03
As A, but 2x Na2C03,
minus Na2Si03
Ave. algae dry-wt
yield, mg/1
245 a
234 a
322 a
353 a
438 a
365 a
223 b
466 a
437 a
528 a
493 a
338 a
454 a
374 a
362 a
326 a
324 a
165 b
274 ab
aYields within each experiment with a common letter are not signifi-
cantly different at the 5% level using Duncan's multiple range test.
43
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would provide adequate N for approximately 750 mg C_. glomerata oven-dry
yield per liter of medium; the 1.4 ppm P would be adequate for 2,300 mg
algae at the 0.06% critical concentration. However, the algae were
greenish-yellow rather than green at the reduced N and P concentrations.
Therefore, the N concentration in the recommended medium was increased
to 16.4 ppm; P was increased to 2.8 ppm.
The high requirements of C_. glomerata for B and Mn suggested that
culture medium concentrations of those elements should be increased.
When concentrations of all the trace elements were increased by lOx
(Treatment B, Experiment II), average yield was increased from 353
to 438 mg/1. This difference was not statistically significant,
probably due to the low number of replicates within treatments. How-
ever, in the absence of toxic effects, the recommended concentration
of all the essential elements was increased by lOx. The level would
insure that responses due to variations in other constituents would
not be inhibited by trace element deficiencies.
The effects of varying the concentrations of the major essential
elements other than N and P are shown in data from Experiments II and
III. Doubling the concentrations of Ca, Mg, K, and S specified in
Cladophora Medium I did not significantly increase yields; halving
the concentration of Mg, K, and S significantly lowered yields (Treat-
ments C and D, Experiment II). Reference to the S critical concen-
tration experiment suggests the decreased yield probably was due to
inadequate S. The results from Experiment III show that with the
higher level of trace elements, doubling the concentration of Ca, Mg, K
and S did not result in a significant yield increase in any treatment.
As a result, Gorham's concentrations of Mg, K, and S are specified
in Cladophora Medium II. Based on critical concentrations, these
amounts are adequate for considerably greater yields than the 500 mg/1
currently obtained but are not inhibitory.
To keep the total salt concentration low, it is desirable to utilize
salts in a culture medium which individually provide two essential
elements. This is a factor that determines the concentrations of
44
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some elements in culture media. For example, because C_. glomerata
has a relatively high S requirement and MgSOij is the S source, the
amount of Mg provided also is relatively high. The desire to reduce
the total salt concentration also accounts for the increase in the
Ca concentration from 9.7 ppm in Cladophora Medium I to 23.4 ppm in
Medium II. In the Hughes, Gorham, and Zehnder Medium (1958) from
which Cladophora Medium I was derived, Ca is obtained from CaCl2 and
N from NaNOa. An alternative which is used in many culture media is
to derive both Ca and N from Ca(NOa)2. A test of this modification
(Experiment IV, Table 24) indicated yield was not reduced when 16.4 ppm
N was provided as Ca(NOa)2 in Treatment B and Ca, as a result, was
increased to 23.4 ppm. Increasing Ca to 23.4 ppm as CaCl2 (Treatment
C) also was not detrimental. As a result, Ca(NOa)2 is the specified
source of both Ca and N in the revised medium.
Buffer Components
Sodium carbonate and sodium silicate are added to C_. glomerata culture
media primarily to provide alkaline buffering. It seemed desirable to
confirm the beneficial effects of these salts, because they provide
relatively high concentrations of Na which could be toxic to the algae.
In addition, in trace element experiments, purification of the culture
medium might be less difficult if Na2COa, Na2SiOa, or both could be
omitted.
The data from Experiment V show that either NaaCOs or NaaSiOa can be
omitted from the medium without reduction in yields (Treatments A, B,
and C), but that omission of both salts reduced yield to approximately
50% of maximum (Treatment D). Doubling the amount of NaaCOa and
omitting NaaSiOa (Treatment E) did not significantly affect yields.
These results have been verified in several experiments.
Unrecognized Essential Trace Elements
In studying the culture requirements of any algae, it must be recognized
that the organism might require one or even several trace elements
currently not recognized as generally essential for plants. For this
reason, the effects on C. glomerata of elements which have been shown
45
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essential for only specific organisms or which have been suggested
but not proven as essential were determined. Mixtures of these
elements are commonly designated as A-Z solutions (Hewitt, 1966).
Furthermore, the components of A-Z solutions commonly are divided
into several subgroups to facilitate identification of any elements
which give positive growth responses.
The results of two experiments on the response of C_. glomerata to
trace element supplements are presented in Table 25. The results
from the two experiments are not in complete agreement. The data
from Experiment I show that an element in each of the B7 and the
Ci3 solutions, when provided at the proper concentration, signifi-
cantly increased C^. glomerata growth (Treatments A, B, E, and F).
Experiment II verified a stimulatory effect of the Cia solution
but not the By (Treatments A and E).
The results with supplemental trace elements justify routine addi-
tion of these solutions to the culture medium until further studies
positively establish the response and permit identification of the
specific element or elements involved. These studies will require
considerable time and effort, because they should involve purifi-
cation of the culture medium and environment by the techniques used
in the most sensitive trace element studies (Hewitt, 1966).
Organic Growth Factors
Because of increases in Cladophora glomerata yields resulting from
manipulations of the inorganic components of the medium, it seemed
desirable to determine if the specific vitamins and the concentrations
of those vitamins established as adequate in early experiments were
adequate in the new medium.
As in other experiments, growth was stimulated tremendously by
vitamin Bj (Treatments A and B, Table 26). The additional yield
increase resulting from vitamin B12 (Treatment C) was not statisti-
cally significant. The critical point of this experiment was the
lack of a significant response from additional vitamins when auto-
claved separately from the medium to minimize breakdown.
46
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Table 25. YIELD OF Cladophora glomerata GROWN IN CULTURE MEDIUM
CONTAINING SEVERAL CONCENTRATIONS OF POTENTIALLY
ESSENTIAL MICRONUTRIENTS
Designation
A
B
C
D
E
F
G
Treatment
Cladophora Medium I
As A, plus B7 (0.01 ppm)
As A, plus B7 (0.002 ppm)
As A, plus Ci3C (0.005 ppm)
As A, plus Cis (0.001 ppm)
As A, plus B7 (0.01 ppm) £
Ci3 (0.005 ppm)
As A, plus B7 (0.01 ppm) §
Cis (0.001 ppm)
Ave. algae dry-wt
yield, mg/1
Exp I
255 a
332 b
313 a
311 a
386 b
379 b
Exp II
352 a
321 a
445 b
393 a
Average of ten replicates in each treatment; yields with a common
letter are not significantly different at the 5% level using Duncan's
multiple range test.
The B7 trace element mixture contains the following elements: V, Cr,
Ni, Co, W, Ti, and Sn.
"The Cis trace element mixture contains the following elements: Al, As,
Cd, Sr, Hg, Pb, Li, Rb, Br, I, F, Se, and Be.
The lack of a statistically significant response of C_. glomerata to
vitamin B12 (Treatments B and C) contrasts with the positive responses
to the vitamin in experiments from initial stages of developing a
culture medium for the organism (Section V). The difference probably
is associated with a failure to obtain inoculum for the experiment
reported in Table 26 from a C_. glomerata culture to which vitamin 612
was not added. Considerable 612 may have been carried along with
the inoculum. A difference in the bacteria, which produce vitamin
612, associated with the algae over a period of time is a less likely
47
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Table 26. RESPONSE OF Cladophora glomerata TO SEVERAL COMBINATIONS
OF VITAMINS ADDED TO AN IMPROVED CULTURE MEDIUM
Designation'
Vitamin supplement
Ave. algae dry-wt
yield,b mg/1
A
B
C
D
None
As A, plus vitamin
As A, plus B! and
As A, BI and Bia plus
additional vitamins
autoclaved separately
140 a
414 b
529 b
450 b
alnoculum for this experiment was not cultured in medium lacking in
vitamin B12.
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.
°For a listing of the additional vitamins see Table 5.
but possible explanation. The vitamin Bia requirement of Lake Michigan
C_. glomerata can be more positively established when a bacteria-free
culture is obtained. General experience on this project indicates that
C_. glomerata growth is improved by vitamin 612. Until additional in-
formation indicates otherwise, the addition of vitamin Bia to the cul-
ture medium is recommended.
In studies reported in Section V on the quantitative requirement of
£. glomerata for vitamin BI, the alga responded positively to the
highest concentration tested (2 mg/1). In an additional test reported
in Table 27, the response to vitamin concentration above 2 mg/1 was
determined. Although yield differences at 2 and 4 mg/1 were not statis-
tically significant (Treatments D and E), yields were in general greater
in the treatments providing more than 2 mg/1 of vitamin 612. The yield
at 2 mg/1 (Treatment D) was 321 mg of C_. glomerata; the average yield
48
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Table 27. RESPONSE OF Cladophora glomerata TO VARIOUS CONCENTRATIONS
OF VITAMIN BI IN CULTURE MEDIUM II
Designation
A
B
C
D
E
F
G
H
Cone, of BI ,
mg/1
0.0
0.5
1.0
2.0
4.0
6.0
8.0
10.0
Ave. algae dry-wt
yield, mg/1
110 a
280 ab
295 ab
321 b
442 b
308 ab
465 b
476 b
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.
at higher BI concentrations (Treatments E, F, G, and H) was 423 rag.
Because of this response and the absence of adverse effects at con-
centrations as high as 10 mg/1, the recommended concentration of
vitamin BI in Cladophora Medium II was increased to 4 mg/1. Require-
ments may be lower if vitamin BI is sterilized separately from the
medium. Autoclaving of vitamin BI in Medium II is recommended be-
cause of convenience.
Unrecognized Growth Factors
In initial efforts at culturing C_. glomerata, both lake water and
extracts of dried algae provided factors that stimulated C_. glomerata
growth. As a final aspect of developing an improved culture medium,
it seemed desirable to determine if the stimulatory effects of lake
water and hot water algae extracts still would be observed when added
to the improved medium.
49
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The data in Table 28 show the yield responses from adding several
concentrations of a hot-water extract of oven-dried C_. glomerata
to C_. glomerata cultures. Yields increased with increasing con-
centrations of extract (Treatments A-D), with the 50 ml addition
showing statistical significance. The most significant aspect of
the results seems to be that when volumes of extract equivalent to
the volumes in Treatments B-D were ashed and the residues were added
to the culture medium, a significant yield response was retained at
the two highest levels of the extract (Treatment F and G). Similar
results were obtained when this experiment was repeated.
Table 29 presents the results of an experiment to compare the growth
of C_. glomerata in Cladophora Medium II and in water from Lake Michigan
to which Medium II constituents were added. There was a significantly
greater yield when Medium II constituents were added to Lake Michigan
water rather than distilled water (Treatments A and B). An unidenti-
fied factor in the lake water stimulated the algae. When all inor-
ganic constituents were added to the lake water but vitamin BI was
omitted, yields were severely reduced (Treatment C) to only 34% of
maximum. The reduction in yield to 29% of maximum when only N, P,
and Fe and vitamins were added to the Lake Michigan water in Treatment
D indicates that one or more inorganic elements other than N, P, or
Fe were present in relatively low concentrations in the lake water
sample and might limit C_. glomerata growth under field conditions.
Treatments E, F, and G in which P, N or Fe were omitted indicate the
relative availability of those elements in the sample of Lake Michigan
water used. As anticipated, yields were sharply reduced when N, P,
or Fe was omitted from the medium. However, surprisingly growth
with P omitted (Treatment E) or Fe omitted (Treatment G) was not
significantly reduced below Treatment F to which N, P, Fe and vitamins
were added but not the other essential elements. This supports the
evidence from Treatments B and D that an essential element other
than N, P, or Fe limited growth in the water sample. The yield in
Treatment F lacking N was significantly less than in any other
50
-------
Table 28. RESPONSE OF Cladophora glomerata TO ADDITIONS TO THE
CULTURE MEDIUM OF HOT WATER EXTRACTS OF OVEN-DRIED
C. glomerata
Designation
A
B
C
D
E
F
G
Treatment
Cladophora Medium II
As A, plus 1 ml algae extract /I
As A, plus 5 ml extract/1
As A, plus 50 ml extract/1
As A, plus 1 ml ashed extract/1
As A, plus 5 ml ashed extract/1
As A, plus 50 ml ashed extract/1
Ave, algae dry-wt
yield, mg/1
467 a
465 ab
591 ac
614 c
494 ab
532 be
578 be
aAverage of six replicates in each treatment; yields with a common
letter are not significantly different at the 5% level using Duncan's
multiple range test.
Algae extract prepared by heating 2.5 g oven-dry C_. glomerata in 250
ml distilled water for 60 minutes in Arnold sterilizer.
treatment suggesting N was the primary limiting nutrient in the Lake
Michigan sample.
DISCUSSION
The nutrient medium recommended for the culture of Cladophora glomerata
as a result of the present studies is presented in Table 30. Cladophora
glomerata yields of 500-600 mg dry wt/1 are routinely obtained in this
medium after a 21-day culture period. Yields in this range are com-
parable to the growth of other green algae under similar conditions
(Gerloff and Fishbeck, 1969) and are approximately double the C. glomerata
yields obtained in early phases of this project. With the evolution of
Cladophora Medium II, a primary goal of this project was achieved. A
defined culture medium had been developed which was suitable for quanti-
tative laboratory experiments with Cladophora glomerata.
51
-------
Table 29. COMPARISON OF Cladophora glomerata YIELDS GROWN IN
Cladophora MEDIUM II AND IN LAKE WATER FORTIFIED
WITH COMPONENTS OF THE MEDIUM
Designation
A
B
C
D
E
F
G
Treatment
Cladophora Medium II
Lake water + all
Medium II constituents
As B, except
lacking vitamin BI
Lake water + N + P + Fe
+ vitamins BI, Bja
Lake water + N + Fe (-P)
+ vitamins BI, 612
Lake water + P + Fe (-N)
+ vitamins BI, 612
Lake water + P + N (-Fe)
+ vitamins BI, 812
Average
algae dry-wt
yield, mg/1
420 c
644 d
216 b
188 b
245 b
83 a
248 b '
Proportion
of maximum
yield, %
64
100
34
29
38
13
39
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.
Lake water collected 7-3-75 at Green Bay, University of Wisconsin
campus.
It probably is incorrect to describe any nutrient solution as an
"optimal medium". Unanticipated responses to inorganic and/or or-
ganic factors may lead to further yield increases and culture medium
modifications. In fact, the results presented suggest that Medium
II is not optimal; Cladophora glomerata would benefit from further
modifications. It seems particularly interesting that several ex-
periments suggested an additional inorganic factor would improve
£ .gloroerata yields. This was indicated by the positive response
to unidentified trace elements (Table 25), by the beneficial effect
52
-------
Table 30. COMPOSITION OF RECOMMENDED SYNTHETIC NUTRIENT MEDIUM
(MEDIUM II) FOR THE CULTURE OF Cladophora glomerata
Salt
Ca(N03)2*4H20
Na2HPOi,
KC1
MgSOit'7H20
Na2Si03-5H20
Na2C03
o
Fe citrate-Citric acid
EDTA
b
KC1
b
H3B03
b
MnS(VH20
ZnSOit«7H2Ob
CuS0lt-5H2Ob
(NHlt)6Mo702^4H2Ob
Q
B7 trace elements
d
Ci3 trace elements
Vitamin BI
Vitamin Bi2
Stock
solution,
g/1
13.82
1.28
3.44
7.50
4.30
2.00
0.60 (of each)
0.10
3.728
1.546
0.845
0.575
0.125
0.0184
ml stock/1
final soln.
10
10
10
10
10
10
10
10
.2
.2
.2
.2
.2
.2
1
1
10
5
Cone, in
final solution,
ppm
N -
P -
K -
Mg -
S -
Ca -
Fe -
Cl -
B -
Mn -
Zn -
Cu -
Mo -
(Each) -
(Each) -
16.4
2.8
18
7.5
9.7
23.4
1.12
cr
0.35^
0.054
0.054
0.026
0.006
0.002
0.001
0.0005
4
.0005
aStock solution heated to 80 C, then stored in refrigerator.
1.2 ml of a mixture of equal volumes of the six stock solutions are
added per liter final solution.
Q
The 87 trace element mixture contains the following elements: V, Cr,
Ni, Co, W, Ti, and Sn.
The Cis trace element mixture contains the following elements: Al,
As, Cd, Sr, Hg, Pb, Li, Rb, Br, I, F, Se, and Be.
Q
The vitamin BI stock solution is prepared fresh, just prior to medium
preparation. (40 mg of thiamine'HCl powder per 100 ml d.d. H20).
53
-------
f
A 0.1 mg/1 stock solution of vitamin 612 is filtered through a sterile
0.22 y millipore filter apparatus, stored in the freezer, then thawed
and added aseptically to the autoclaved medium.
a
Cl is also made available in KC1 stock solution; the total Cl con-
centration in the final solution is much greater than provided in
the trace element mixture.
of Lake Michigan water (Table 29), the response to ashed extracts of
dried C_. glomerata (Table 28), and the lack of a response to additional
vitamins (Table 26).
Several aspects of the experiment in which components of Cladophora
Medium II were added to Lake Michigan water (Table 29) seem of in-
terest in relation to limiting factors for-C_. glomerata under lake
conditions. First, yield was very poor unless vitamin Bj was in-
cluded among the supplements added to lake water. In addition,
maximum yield was obtained only when inorganic nutrients in addition
to N, P, and Fe were made available. This contrasts with the results
of comparable experiments with Microcystis aeruginosa, a bloom-pro-
ducing blue-green alga. Adding only N, P, and Fe to cultures of this
organism in lake water resulted in maximum yield (Gerloff and Skoog,
1957). Finally, N rather than P was the primary growth limiting
nutrient in the Lake Michigan sample. This was consistent with the
results from the comparable test with Microcystis aeruginosa.
As discussed by Gerloff (1969), caution must be exercised in making
conclusions on growth-limiting nutrients from experiments on small
water samples withdrawn from lakes for laboratory experiments. With
this procedure, opportunities for renewal of nutrient supplies in the
water are eliminated. Nevertheless, this approach does provide some
information on relative supplies of various nutrients in lakes. The
data obtained suggest that additional similar experiments with water
samples obtained at various sites and at various times of the year
would be desirable.
54
-------
SECTION VIII
Cladophora glomerata RESPONSE TO SEVERAL
PHYSICAL ENVIRONMENTAL FACTORS
Non-nutritional factors must be considered in determining optimal
conditions for the laboratory culture of an organism. The results of
experiments on the response of C_. jglomerata to variations in tempera-
ture, light, and several other physical factors are presented in this
section.
Unless indicated, culture techniques and growth conditions were as
described in Section VI.
RESULTS AND DISCUSSION
Light and Temperature
Experiments were carried out to determine if Cladophora glomerata
showed unusual responses to light or temperature and if the alga
was being grown in the laboratory under conditions close to optimal.
Table 31 shows that the temperature (22-23°C) under which C_. glomerata
has been routinely cultured on the project is optimum or close to
optimum. The algae seemed relatively insensitive to high temperature
with optimum yields maintained at 30-31°C. This contrasts sharply
with the results of Wojick (1972) in which yields of several green
algae were severely inhibited at 30°C. Growth of C_. glomerata was
reduced to only 63% of optimum at temperatures of 16-17.5°C.
The results in Table 32 indicate that C_. glomerata is relatively
insensitive to variations in light intensity. The different in-
tensities tested were achieved within one growth cabinet by place-
ment of the cultures and by covering some cultures with one or two
layers of cheesecloth. Yields at the four light intensities tested
(ranging from approximately 200 to 1100 ft. candles) varied from only
363 to 457 mg/1. The differences were small but statistically signi-
ficant. Maximum yield was under the 630-750 ft. candle treatment which
is slightly above the 400-500 ft. candles which had been used for
routine culture of C. glomerata. Both the highest (1000-1100 ft. candles)
55
-------
Table 31. THE GROWTH OF Cladophora glomerata TO THREE RANGES OF
TEMPERATURE
Designation
A
B
C
Temperature, °C
22-23
16-17.5
30-31
Ave. algae dry-wt
506 b
318 a
502 b
yield, mg/1
aAverage of six replicates in each treatment; yields with a common
letter are not significantly different at the 5% level, using Duncan's
multiple range test. The nutrient medium contained Cladophora
Medium II elements except 2 ppm vitamin BI and 9.7 ppm Ca as
Table 32. THE GROWTH RESPONSE OF Cladophora glomerata TO FOUR
LEVELS OF LIGHT INTENSITY
Designation
A
B
C
D
Light intensity, f.c.
1000-1100
630-750
310-430
160-260
Ave. algae dry-wt
yield, mg/1
410 c
457 d
384 b
363 a
Color
Yellow
Yellow-green
Green
Dark green
aAverage of six replicates in each treatment; yields with a common
letter are not significantly different at the 5% level, using Duncan's
multiple range test. The nutrient medium contained Cladophora Medium
II elements except 2 ppm vitamin BI and 9.7 ppm Ca as CaCl2-
and the lowest (160-260 foot candles) intensities tested resulted in
some yield decreases.
There were marked variations in algae color under the different light
intensities. The cultures which were darkest green (Treatments D and
C) did not produce the highest yields. Cultures grown at 1000-1100
foot candles were yellow, and yield was also below maximum.
56
-------
Volume and Aeration of Culture Medium
The primary source of carbon for algae growth in culture media is
C02 dissolved from the air above the cultures. Most media contain
only limited amounts of added bicarbonates or carbonates (15 to 20
mg/1) as a means of buffering the pH to alkaline values. The im-
portance of the C02 supplied from the air is demonstrated by the
slight growth that develops in rubber-stoppered flasks as compared
with the growth in flasks with cotton or plastic-foam closures.
The rate at which COa can be replaced in an algal culture depends
on the ratio of surface area to total volume of the medium in the
flask. As the volume of medium in a culture is increased and the
surface area remains the same or is decreased, replacement of
utilized CC>2 within the medium is slowed.
Aeration or shaking of cultures does compensate to some extent for
the effects of increased solution volumes as shown in Table 33. In
this experiment, C_. glomerata was grown in different volumes of medium
which were either aerated or quiet (non-aerated). The growth of
C. glomerata decreased in both aerated and non-aerated cultures as
the volume of the medium increased. When harvested after 14 days
of growth, an increase in the amount of medium from 20% of flask
volume (100 ml) to 60% (300 ml) resulted in a yield decrease from
170 to 70 mg in the non-aerated cultures and from 245 to 115 mg in
the aerated cultures. There was considerably more growth in all
of the aerated cultures. When non-aerated cultures from another ex-
periment were harvested after a 24 day period, unreported data, there
was only a 35% yield decrease when the medium was increased to 60%
of flask volume. These results were similar to those from aerated
cultures harvested after a longer culture period. In these studies,
the rate of C02 supply seems to regulate the rate of C^. glomerata
growth.
In additional experiments to determine the effect of CC>2 supply on
rate of C_. glomerata growth, algae were grown in tubes (38 x 300 mm)
containing 150 ml of medium and were aerated either above the medium
57
-------
Table 33. THE EFFECT OF MEDIUM VOLUME AND AERATION
ON THE GROWTH OF Cladophora glomerata
Medium
volume,
ml/500 ml
Erlenmeyer
100
200
300
Ave. algae dry-wt
yield, b mg/1
Non-aerated
170
115
70
Aerated
245
200
150
Effect of volume on
yield, % decrease
Non-aerated
0
32
59
Aerated
0
18
39
Effect of
aeration
on yield,
% increase
45
75
115
The nutrient medium contained Gorham's Medium major elements plus 10%
lake water.
Average of triplicate cultures; 14-day culture period.
or by extending a tube to 25 or 60 mm depths in the medium. Air was
provided at approximately 200 ml/minute. Increased aeration did not
affect the final culture pH. After a 10 day growing period, average
yields of 4 cultures in each treatment were 20, 85 and 140 mg/1,
respectively, indicating definite increases in rate of C_. glomerata
growth with increased C02 supply
Growth Surface
One of the most troublesome aspects of laboratory experimentation
with Cladophora glomerata has been the poor agreement in yields from
replicate cultures within a treatment. As a result, unusually large
numbers of replicates have been required to establish statistically
significant differences among the treatments of an experiment.
Filaments of C_. glomerata often grow attached to the sides of pyrex
culture flasks. The amount of attached growth has been observed to
vary considerably. It was considered possible that algae growth
to a degree was determined by the amount of attached growth which
in turn was related to differences in the interior surfaces of
flasks resulting from use and repeated washing. The algae would
more readily attach to rough surfaces.
58
-------
Table 34. EFFECT OF "ETCHING" ON Cladophora glomerata GROWTH
Experiment0
"Treatment"
Ave. algae dry-wt
yield, mg/1
Range of yield and
standard deviation,
mg/1
I
I liter
pyrex beaker,
normal
1 liter
pyrex beaker,
etched
500 ml
Erlenmeyer,
normal
500 ml
Erlenmeyer,
etched
206.8
320.0
385.9
572.4
140.8-329.2; 106.0
282.6-343.6; 32.7
200.0-610.5; 155.6
403.5-669.5; 105.9
aThe nutrient medium contained Cladophora Medium II elements except
2 ppm vitamin BI and 9.7 ppm Ca as CaCl2.
Average of three replicates in each treatment.
Average of eight replicates in each treatment.
The data in Table 34 are from two experiments in which C_. glomerata
growth in new glassware and in glassware artificially scratched with
emery cloth or a grinding stone was compared. In Experiment I, the
algae were grown in 1-liter beakers; in Experiment II, in the usual
500-ml Erlenmeyer flasks. In both experiments, average yields were
considerably greater in the etched culture containers. Of more im-
portance, is the decreased variability in yields from the etched flask
cultures. In each experiment, both the range in yield and the standard
deviation were less in the etched culture vessels. Before specific
recommendations can be made, additional similar tests should be carried
out.
59
-------
SECTION IX
CALIBRATION OF FITZGERALD TESTS FOR FIELD
EVALUATIONS OF NUTRIENT SUPPLIES
Two types of bioassays have been used on this project to determine
whether the growth of Cladophora sp. in specific aquatic environ-
ments was limited by supplies of certain nutrients. The plant
analysis bioassay was developed for use with aquatic plants by one
of the Principal Investigators (Gerloff and Krombholz, 1966; Gerloff,
1969). Application of plant analysis to assaying nutrient supplies
for C_. glomerata was discussed in Section III. The second bioassay
(Fitzgerald tests) which is based on POit-P extracted in boiling water
and rate of NHit-N uptake as measures of P and N sufficiency was de-
veloped by the other Principal Investigator (Fitzgerald and Nelson,
1966; Fitzgerald, 1969) . The laboratory efforts required to cali-
brate the Fitzgerald tests in evaluating nutrient supplies for
Cladophora sp. will be presented in this section.
RESULTS AND DISCUSSION
Extractable
Several experiments were carried out to determine the relationship
between algae yield, total P concentration, and extractable PO^-P
when Lake Michigan Cladophora sp. was grown in different solution
concentrations of POi+-P. The results of representative experiments
are presented in Figures 5 and 6.
In both experiments, algae yields increased with increasing con-
centrations. of P in the medium up to 0.2-0.3 mg P/l. The total P
concentration in '-the Cladophora sp. which corresponded with slightly
less than maximunv-yi eld was approximately 0.10%. This is an addi-
tional evaluation of the critical P concentration. It corresponds
reasonably well with the 0.06% in earlier experiments of Section VI.
The minimum concentration of hot-water extractable PO^-P associated
with maximum yield was approximately 0.07 mg/100 mg algae (%P) . This
correlates well with results with other algae in which less than
0.08% extractable PO^-P was considered to indicate the algae were
60
-------
YIELD
0.4 0.8
mg P04-P/I
- .4
o
Figure 5. Comparison of yield and total P concentration of Lake
Michigan Cladophora sp. after a 17-day culture period in solu-
tions varying in P concentration.
P-limited (Fitzgerald and Nelson, 1966).
To demonstrate that the Cladophora sp. isolated from all five of the
Great Lakes had similar P requirements, yields obtained after culture
at several POit-P concentrations are summarized in Table 35. In general,
responses of the five isolates were similar. Comparative yields at
0.05 ppm P probably are of most interest because that approximates a
61
-------
125
100
o>
1 75
Q
UJ
CD
<
50
25
YIELD
I
1
I
0.2 0.4
mg P04-P/I
.25
.20
15
10
.05
0.6
O
UJ
x
Ul
Figure 6. Comparison of yield and extracted PO^-P of Lake Michigan
Cladophora sp. after a 17-day culture period in solutions varying
in P concentration.
growth-limiting P supply. Yields of the Lake Huron isolate were the
lowest or nearly the lowest at each P concentration.
P Availability from Various Sources
The availability to algae of an essential nutrient in a specific
source usually is determined by comparing growth of the algae in
62
-------
Table 35. THE RESPONSE OF Cladophora sp. ISOLATED FROM EACH
OF THE GREAT LAKES TO DIFFERENT P CONCENTRATIONS21
Source of
Cladophora
Lake Michigan
Lake Superior
Lake Huron
Lake Erie
Lake Ontario
Ave. algae dry-wt yield, mg/1
0 rag P/l
18
30
11
18
20
0.05 mg P/l
113
97
75
92
105
0.15 mg P/l
168
130
115
112
142
aThe nutrient medium contained Gorham's Medium major elements (except
1/2 N and 2x Ca) vitamins BI and 612 and 10% Lake Mendota water.
Average of triplicate cultures in each treatment; 25-day culture
period.
different concentrations of that source and known available sources
of the nutrient. The time required for these tests usually exceeds
two weeks for Cladophora sp. During this interval non-available
sources of a nutrient may break down to usable forms. Therefore,
alternative tests that can be carried out rapidly with a minimum
requirement for laboratory space are desirable. These relatively
simple procedures are not meant to replace growth tests or short-
term-exposure-and-growth tests (Fitzgerald, 1970), but can be used
for comparative studies and surveys of a large number of potential
sources of available nutrients.
It has been demonstrated that Cladophora sp. in which growth is
limited by P, N, or Fe are capable of absorbing these nutrients,
if they are presented in available forms. Therefore, nutrient-
limited field collections of Cladophora sp. were used to evaluate
the availability of different nutrient sources.
Approximately 5 mg (dry weight) of P-limited Cladophora sp. from
Lake Wingra were added to 500 ml of Gorham's (-P) Medium containing
-------
different concentrations of ortho-PC^, pyro-PC%, tripoly-POi*, and
phosphite-P. These P sources contained 100, 2.4, 1.6 and 0.4% ortho
POit-P, respectively. After incubation for 20 hours at 25°C, the
amounts of PO^-P extractable from the algae were determined (Table 36),
The data show that pyro-POi* and tripoly-P(K are readily available to
Lake Wingra Cladophora sp.; the P of phosphite is unavailable. These
data agree with the results from comparable studies with other algae
(Fitzgerald, 1970). The value of the type of information obtained
from this test was demonstrated in the failure of algae to utilize
some of the P in Yahara River sewer discharge with chemical tests
indicated to be soluble PO^-P (Fitzgerald and Uttormark, 1974).
The availability of different sources of P for the growth of Lake
Michigan Cladophora sp. also was tested (Table 37). There was an
increase in yield with each increase in P concentration as ortho-,
pyro-, meta-, and tripoly-POit. All of these P sources were available
to the Cladophora sp. The relatively low amount of growth (43 mg/1)
in the presence of 0.4 mg phosphite-P/1 was probably due to the ortho-
P present (12%). Additional tests demonstrated the low yield was
due to P deficiency and not to phosphite toxicity.
An experiment was carried out to test the availability to Cladophora
sp. of P from the littoral muds of several lakes. Previous tests had
indicated that algae could utilize only a small part of the P present
in bottom muds (Fitzgerald, 1970). There was no detectable POit-P in
the water above the sample area in one of the lakes tested (Wingra);
P was barely detectable (0.02 mg POit-P/1) in the waters of Monona Bay;
the overlying waters of Lake Waubesa (east shore) and Lake Kegonsa
(east shore) had 0.07 and 0.10 mg P04-P/1, respectively. The yields
°f Cladophora sp. from cultures containing lake muds and several
concentrations of added P are summarized in Table 38. Comparisons
of yields of Cladophora sp. from the lake mud cultures with yields
from cultures with known concentrations of POi^-P indicated the
concentrations of available P and the percentage of the total P of
the lake muds available for algae growth.
64
-------
Table 36. THE RELATIVE AVAILABILITY OF DIFFERENT SOURCES OF P TO
P-LIMITED LAKE WINGRA Cladophora sp.
Sample
A
B
C
D
E
F
G
H
I
J
K
L
Phosphorous
source
None
Ortho-POit
Ortho-POit
Ortho-POi,
Ortho-POi,
Pyro-POi*
Pyro-POij
Tripoly-POit
Tripoly-POit
Phosphite-POit
Phosphite-PO^
None
Phosphorous
cone., mg/1
0.00
0.04
0.08
0.12
0.16
0.08
0.12
0.08
0.12
0.16
0.80
0.00
Algal extractable P0i+-P,a
mg P/100 mg algae
0.046
0.11
0.16
0.19
0.24
0.18
0.23
0.17
0.19
0.047
0.052
0.049
aAverage of duplicate cultures in each treatment; 5 mg Cladophora sp./
500 ml Gorham's Medium (-P) after a 20 hr incubation at 25°C.
Table 37. THE AVAILABILITY TO LAKE MICHIGAN Cladophora sp. OF
P FROM DIFFERENT SOURCES WHEN MEASURED IN GROWTH TESTS
Source
Ave. algae dry-wt yield, mg/1
of Pa
Ortho-P
Pyro-P
Meta-P
Tripoly-P
Phosphite-P
0.05 mg P/l
18
--
20
42
--
0.10 mg P/l
110
137
121
135
0.20 mg P/l
165
195
177
175
0.40 mg P/l
--
--
--
--
40
The nutrient medium contained Gorham's Medium major elements (except
1/2 N and 2x Ca), vitamins BI and Bja, and 5% Lake Mendota water.
Yield with no P added was 5 mg/l; average of triplicate cultures
in each treatment; 27-day culture period.
65
-------
Table 38. THE AVAILABILITY OF THE P OF LITTORAL MUDS TO LAKE
MICHIGAN Cladophora sp.
Phosphorous
source
POi,-P
PO^-P
PO^-P
POif-P
PCK-P
Wingra a
Wingra a
Wingra b
Wingra b
Monona Bay a
Monona Bay a
Monona Bay b
Monona Bay b
Waubesa a (W)
Waubesa a
Waubesa b (E)
Waubesa b (E)
Kegonsa a (E)
Kegonsa a
Kegonsa b (SE)
Kegonsa b
Cone. ,
mg P/l
0
0.025
0.050
0.10
0.15
0.5
1.0
0.5
1.0
0.5
1.0
0.5
1.0
0.5
1.0
0.5
1.0
0.5
1.0
0.5
1.0
Ave. algae dry-wt
yield, mg/1
5
26
86
134
150
41
74
34
91
37
106
123
133
15
24
139
152
241
243
136
228
Calculated
available
P, rag/1
0.04
0.05
0.03
0.05
0.03
0.06
0.08
0.10
0.01
0.02
0.10
0.15
0.15
0.15
0.10
0.15
Available
P, %
--
--
--
--
--
8
5
6
5
6
6
16
10
2
2
20
15
30
30
20
30
aThe nutrient medium contained Gorham's Medium major elements (-P)
and vitamins Bj and 612; 19-day culture period.
It is evident that very little of the P (less than 10%) was available
to Cladophora sp. from Lake Wingra muds, from one Monona Bay sample,
and from mud from the west shore of Lake Waubesa. The greater availa-
bility of the P (20-30%) in lake muds collected from Lake Kegonsa at
66
-------
a time when the overlying water contained 0.10 mg PO^-P/1 is not
surprising. These muds probably were saturated with P. In contrast,
muds from Lake Wingra had already lost any P that could have been
exchanged with the overlying water. These results point out that
studies of the capacities of lake muds to provide available P for
algae growth should be tested at different seasons, so that results
when the overlying waters contain PCK-P can be contrasted with re-
sults when the overlying waters have been stripped of available P.
Nitrogen Availability
To utilize rate of NHit-N uptake as a bioassay of N availability to
Cladophora sp. it was necessary to establish the specific values
that indicated adequate or deficient N supply for Cladophora sp.
To accomplish this, the algae were grown in different concentrations
of NOs-N in the culture solution, and after a 24-day culture period,
yields, total N concentrations, and rates of NH^-N uptake were
determined (Figure 7) .
Growth of Cladophora sp. increased as the NOs-N concentration in the
medium increased up to approximately 8 ppm. There was a slight fur-
ther yield increase at higher N concentrations, but not in proportion
to the amount of N added. At solution NOs-N concentrations of less
than 8 ppm, total N concentrations in Cladophora sp. decreased and
NH^-N absorption rates increased in proportion to the decrease in N
in the medium. Therefore, a total N content of 1.8% and an NHi^-N
absorption rate in excess of 15 yg N/10 mg algae/hour would define
N-limited Cladophora sp. The 1.8% critical N concentration is
slightly higher than established in experiments in Section VI; the
NHij-N uptake critical value is equal to the value established for
other algae (Fitzgerald, 1968).
To evaluate the availability of different sources of N, algae can be
grown at various concentrations of the N sources and rates of NHit-N
absorption or algae yields can be compared. Ammonium-N uptake tests
were carried out to compare the effectiveness of NHit-N, nitrite (NOs-N)
glutamate-N, and urea-N as N sources for Cladophora sp. Twenty to
67
-------
-i 250
Z
UJ
§
ct
10 20 30 40
NITROGEN IN MEDIUM (mg/l)
Figure 7. Comparison of yield, total N in the algae, and rate of
NHij-N uptake in the dark when Cladophora. sp. was grown at different
solution concentrations of NOs-N.
25 mg samples of Cladophora sp. from Lake Mendota known to be nitrogen
limited (47 yg NH^-N absorbed/10 mg algae/hour) were incubated in 1,
2, and 6 mg N/l provided from each N source for 22 hours with shaking
at 20°C. All three concentrations of NHit-N and N03-N caused reductions
in the rates of NHit-N absorption when tested after the incubation
period (1-8 yg NH^-N/10 mg algae/hour). In contrast, absorption rates
with the added glutamate-N were only reduced to 20-32 yg/10 rag/hour
68
-------
and with the added urea to 34-43 Ug/10 mg/hour. Thus, all of the nitrite
nitrogen seemed to be available to the nitrogen-limited Cladophora sp.,
but only part of the glutamate-N and none of the urea-N could be uti-
lized in such short exposure times.
The results of a more definitive test of N availability in which the
capacity of C_. glomerata to utilize sources of N for actual growth are
presented in Table 39. The yield of Lake Michigan Cladophora sp. was
obtained with different concentrations of NOs-N, NOg-N, NHit-N, and
dried abysmal Lake Waubesa muds (1.3% N).
Yield increased with increasing concentrations of all the sources of
N. All four could be used for Cladophora sp. growth. Yield with the
highest concentration of NHii-N, 5 mg N/l, was considerably less than
with 2 mg N/l. This is probably due to a lower pH in the media with
higher amounts of NHit-N. The NHij-N absorbed by algae is replaced with
H which results in lowering of the pH. The yield per mg of N was
fairly uniform for NOa , NOa , and NHi^-N, but the yield per mg of N
from the lake muds was very low, only 1 to 2% of the N of the lake
muds being available under the conditions tested. Similar experiments
have corroborated the poor availability of the N of lake muds to
Cladophora sp. and to other algae under aerobic conditions (Fitzgerald,
1970) and to the aquatic weed Lemna minor (duckweed) (Fitzgerald §
Uttormark, 1974).
Iron Availability
The availability to Cladophora sp. of Fe from several sources was
measured in tests in which the algae were grown for 24 days at diff-
erent concentrations of each Fe source. The data obtained (Table 40)
show that Fe-EDTA and Fe-citrate gave increasing yields at the con-
centration of Fe increased, but there was almost no growth when FeCls
was the Fe source.
In another growth experiment (Table 41), Lake Michigan Cladophora sp.
was grown with different concentrations of iron derived from Fe-EDTA
or littoral lake muds. By using the yields of the cultures with Fe-
EDTA to construct a growth versus concentration of iron relationship,
69
-------
Table 39. THE AVAILABILITY OF DIFFERENT SOURCES OF NITROGEN TO
LAKE MICHIGAN Cladophora sp. WHEN MEASURED IN GROWTH
EXPERIMENTS
Nitrogen
source
None
N03-N
N03-N
N03-N
N03-N
N02-N
N02-N
N02-N
N02-N
NH^-N
NHit-N
NH^-N
NHirN
L. Waubesa dried
abysmal muds
(1.3% total N)
Concentration
of N, mg/1
0
0.5
1.0
2.0
5.0
0.5
1.0
2.0
5.0
0.5
1.0
2.0
5.0
13.0
26.0
52.0
Ave. algae dry-wt
yield, b mg/1
6
62
106
132
131
57
97
111
110
53
87
116
63
25
64
102
aThe nutrient medium contained Gorham's Medium major elements(-N)and
vitamins BI and Bi2.
Average of triplicate cultures; 22-day culture period.
the concentration of available iron in the lake mud cultures could
be calculated from algae yields. From these data, the percentage of
the iron in the different lake muds which was available for the
growth of Cladophora sp. also was calculated. Although the Cladophora
sp. could use the Fe from Fe-EDTA, the iron of the different lake muds
tested was no more than 10% available.
70
-------
Table 40. COMPARISON OF THE EFFECTIVENESS OF SEVERAL SOURCES OF
Fe FOR Cladophora sp. GROWTH IN NUTRIENT CULTURES
3.
Concentration,
rag Fe/1
0.075
0.15
0.30
0.60
Ave. algae dry-wt yield, mg/1
Fe-EDTA
33
72
87
108
Fe citrate
13
41
70
100
Fe C13
4
3
6
9
aThe nutrient medium contained Gornam's Medium major elements and
vitamins Bj and 612.
Average of triplicate cultures in each treatment; 24-day culture
period.
Although laboratory tests may indicate that some sources of Fe are
not readily available for Cladophora sp. growth, these sources may
be available in natural environments when certain chelates are added
or other algae decay in the immediate environment. However, the
relative availability of the iron in different natural environments
can be evaluated by analyses of the iron concentrations of ill situ
algae.
Confirmation of Fitzgerald Tests
To confirm the validity of the Fitzgerald bioassays when applied to
algae collected from field environments, tests were carried out to
demonstrate that algae which are alive but limited by available P,
N, or Fe are able to accumulate the nutrients with corresponding
measurable enzymatic or nutrient changes when placed in an environ-
ment with adequate supplies of N, P, or Fe.
Cladophora sp. from Lake Michigan which were indicated as P-limited
by low extractable POij-P tests were incubated 14 hours in Lake
Michigan water to which different amounts of POi,-P had been added.
The results of extractable PO^-P analyses of these algae are summarized
71
-------
Table 41. THE AVAILABILITY OF THE IRON OF LITTORAL MUDS FOR THE
GROWTH OF Cladophora sp.
Iron
source
Fp-FDTA
Fp-FDTA
FP FDTA
Fp-FDTA
Fp-FDTA
Wingra a
Wingra a
Wingra b
Wingra b
Monona Bay a
Monona Bay a
Monona Bay b
Monona Bay b
Waubesa a
Waubesa b
Waubesa b
Kegonsa a
Kegonsa a
Kegonsa b
Kegonsa b
Iron added,
mg Fe/1
n
0 nyq
n 1 c;
n 3D
n 4^
o fin
0.3
0.6
0.3
0.6
0.3
0.6
0.3
0.6
0.3
n ft
0.3
n f\
0.3
0.6
0.3
0.6
Yield/
mg/1
c
OQ
1 ^8
1 ftQ
26
64
6
50
22
27
7
21
63
23
28
48
6
24
Calculated
available Fe, mg/1
0.02
0.05
0
0.045
0.015
0.02
0
0.015
0.05
0.015
0.03
0.04
0
0.015
Available
Fe, %
7
8
0
8
5
3
0
2
2
5
10
7
0
2
aAve. algae dry-wt yield of duplicate cultures; 20-day culture period
in Gorham's Medium major elements (-Fe) and vitamins BX and Bia.
72
-------
Table 42. THE ABSORPTION OF PHOSPHORUS BY PHOSPHORUS-LIMITED
Cladophora sp. FROM LAKE MICHIGAN
0
Sample
A (Original)
B
C
D
E
Concentration
of added PO^-P,
mg P/l
0
0.16
0.24
0.32
Extractable
POit-P of algae,
mg P/100 mg algae
0.057
0.043
0.12
0.20
0.18
in Table 42. The extractable POn-P increased from 0.057 mg POit-P/100
mg for the original algae and from 0.043 for algae incubated without
added POi^-P to values as high as 0.20 with increasing amounts of
added POij-P. Thus these algae were alive and capable of increasing
their phosphorus content if more phosphorus had been available in the
lake environment sampled.
Cladophora sp. collected from Lake Mendota (Madison, Wisconsin) during
mid-August, 1973, were found to have high rates of NHif-N absorption
in the dark and were considered to be nitrogen-limited. This was
substantiated by the observation that the algae were relatively free
of epiphytes and had a pale yellow-green color (Fitzgerald, 1969).
After samples of these algae were incubated overnight in Lake Mendota
water fortified with different amounts of NOs-N and NHa-N, the rates
of NHit-N absorption were measured. The results are summarized in
Table 43.
The NHij-N absorption rates of the algae incubated with 0.8 and 3.2 mg
NOa-N/1 were only slightly reduced in comparison to algae incubated
with no added nitrogen. When 13 mg NOs-N/l was present, the NHi^-N
absorption rate was reduced to one-half; the same concentration of
73
-------
Table 43. THE EFFECT OF AMMONIUM AND NITRATE NITROGEN ON THE
NH^-N ABSORPTION RATES OF NITROGEN-LIMITED Cladophora
sp. FROM LAKE MENDOTA
Sample
A
B
C
D
E
Nitrogen
source
None
N03-N
N03-N
N03-N
NH^-N
Concentration of
added nitrogen,
mg N/l
0
0.8
3.2
13.0
13.0
Rate of NH^-N
absorption,
Ug N/10 mg algae/hour
84
64
60
32
0
a20-40 mg Cladophora sp./250 ml Lake Mendota water; incubated with
shaking for 22 hours at 25°C.
Table 44. THE EFFECT OF INCUBATION WITH ADDED IRON ON THE IRON
CONTENT OF Cladophora sp. FROM LAKE MENDOTA
0
Sample
A
B
C
D
E
F
G
H
Concentration
of iron,
mg Fe/1
0
0.12
0.25
0.38
0.50
0.75
1.25
0
Iron content
of algae,
% Fe
0.036
0.10
0.12
0.15
0.18
0.22
0.25
0.033
a!5-20 mg Cladophora sp./400 ml Lake Mendota water; incubated with
aeration at 25 C.
Fe-EDTA source of iron.
74
-------
NHifN resulted in no absorption of NH^-N by the algae. Therefore, if
the Cladophora sp. from Lake Mendota had grown with adequate supplies
of available nitrogen, it would have had relatively low NHi^-N absorp-
tion rates. Subsequent NHij-N uptake tests after a rainfall in this
area resulted in lower NH^-N absorption rates and a change in color
from pale yellow-green to dark green, presumably as a result of N in
the rainfall.
Cladophora sp. with relatively low iron concentrations were collected
from Lake Mendota during August and September, 1973. Samples of these
algae were incubated in lake water and synthetic media to which a
soluble form of iron had been added to determine if algae with low
iron concentrations were capable of absorbing additional iron. The
source of iron was Fe-EDTA, so there would be less chance of high
iron concentrations in the algae due surface adsorption of insoluble
iron precipitates. The results of one such experiment are summarized
in Table 44. The iron concentration in the Cladophora sp. increased
from 0.036 to 0.25% as the amount of iron added to the lake water
increased, indicating the algae were capable of absorbing more
iron if available.
75
-------
SECTION X
EVALUATION OF NUTRIENT SUPPLIES IN THE GREAT LAKES
FOR Cladophora sp. GROWTH
The results of evaluations of nutrient supplies and primary growth-
limiting nutrients that might be critical in nuisance growths of
Cladophora sp. in the Great Lakes are presented in this section.
These evaluations were accomplished primarily by two bioassays, plant
analysis and Fitzgerald tests.
EXPERIMENTAL PROCEDURES
The procedures used in culturing and analyzing C_. glomerata to estab-
lish the base values for plant analysis and the Fitzgerald tests were
described in Section VI and IX.
Samples of Cladophora sp. collected from lakes for analyses were
thoroughly rinsed in water from the sampling area, squeezed between
dry filter papers, and dried with forced warm air in a manner that
permitted storage until chemical analyses were made.
GENERAL NUTRIENT EVALUATIONS BY PLANT ANALYSIS
Nutrient assay by plant analysis involves comparison of the critical
concentration for each required inorganic nutrient in the aquatic
plant under study with the concentrations of the same elements in
field samples of the alga or macrophyte. A concentration of an
element above the critical concentration indicates the element was
in adequate supply at the time of sampling in the environment from
which the sample was obtained. A concentration below the critical
concentration indicates that available supplies of an element were
inadequate for optimum growth. The element had become growth-limiting.
Routine sampling of a body of water throughout the summer months will
indicate whether a specific nutrient became growth-limiting at any
time during the growing season.
A number of Cladophora sp. samples were collected from Lake Michigan.
The locations of the sample sites, the dates of sampling, and the
analyses for most of the essential elements are presented in Table 45.
76
-------
The critical concentrations with which to compare the analytical data
from field samples were presented in Table 23 in Section VI. Based
on the critical concentrations presented in Table 23, there were no
indications that N or P supplies were growth limiting at the sample
points listed in Table 45. The critical N concentration was estab-
lished as 1.1% in Section VI; the range in the field samples was
from 1.71 to 4.30% with every sample except the one from Manastique
on July 23, 1973, at least 100% in excess of the critical concentration.
The critical P concentration established in Section VI was 0.06%;
the range in P concentration was 0.17% to 0.86% critical concentration.
The low P concentration of 0.17% also was well above the 0.10% critical
P concentration established in another phase of the project (Section IX).
Concentrations of the essential major element cations Ca, Mg, and K also
were far in excess of the critical concentrations of 0.30% Ca, 1.30% K,
and 0.15% Mg. Again the lowest values were at least 2x the critical
concentrations.
The high concentrations of Ca and Mg in the algae probably reflect
the hard water and high concentrations of these two elements in Lake
Michigan. The very high K concentrations in the algae are somewhat
surprising. In three samples, K concentrations were in excess of 5.0
per cent, values comparable to concentrations in heavily fertilized
agricultural crops.
The concentrations of Fe, Mn, and Zn in Lake Michigan Cladophora sp.
were far above the critical concentrations of 45 ppm Fe, 16 ppm Mn,
and <_ 5 ppm Zn. Very high Fe concentrations, in excess of 1000
ppm, may have been due to sample contamination with silt and sand
particles or surface precipitation. There was no indication of a
growth-limiting role for Fe, Mn, or Zn.
Cladophora sp. concentrations of two of the trace elements, Cu and B,
were low enough to suggest they might have become or could become
growth-limiting. The critical Cu concentration is <^ 6.0 ppm. The Cu
concentration in three algae samples was less than 6.0 ppm and in 10
of 20 samples the Cu concentration was 10 ppm or less. The critical
77
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"lotal N analyses were by Micro-Kjeldahl procedure; all other analyses were with the Jarall-Ash
Multichannel Spectrometer at the Wisconsin Alumni Research Foundation Laboratories, Madison,
Wisconsin.
79
-------
B concentration was established as 110 ppm, a value far in excess
of the critical concentration in other plants, both agricultural and
aquatic. In 9 of the 20 samples, the B concentration was below the
critical concentration. In two samples from North Point Park in
Milwaukee, B concentrations were only 43 and 34 ppm.
Analyses of Cladophora. sp. from three other Great Lakes are presented
in Table 46. For the most part the results are similar to the data
from Lake Michigan. No N value was below the 1.1% critical concen-
tration, although in two samples from Eastern Lake Erie N concentrations
were 1.69 and 1.48%, only slightly above the critical concentration.
Phosphorus concentrations were extremely high, averaging 0.49% in
the 12 samples. Independence in the N and P concentrations is
apparent. For example, Cladophora sp. samples from Eastern Lake
Erie with the lowest N values were among the highest in total P
concentration. One sample from Eastern Lake Erie (Geneva, Ohio)
was very low in both Ca and Mg. Algae concentrations of K, Fe,
and Mn were far above the critical concentrations.
No copper concentration was below the <_ 6.0 ppm critical concentration,
although four were below 10.0 ppm. In contrast to the Lake Michigan
data, the Zn concentration in two samples from Lake Erie, Pt. Pelee
Park and Geneva, Ohio, were below the critical concentration of 16 ppm.
In common with the Lake Michigan data, the B concentration in two of
the samples was below the critical concentration of 110 ppm. One sample
was from Western Lake Erie; the other was from Lake Ontario.
Data on the concentrations in Cladophora sp. of several elements
currently not considered essential for the growth of most plants are
presented in Tables 47 and 48. The Jarrell-Ash Multichannel Emission
Spectrometer routinely determines these elements. The elements are
Na, Al, Ba, Sr, and Cr. At this time, the critical concentrations
which would permit evaluation of these analyses have not been estab-
lished. The data are made available for possible future use. It
can be stated that except for Cr the Cladophora sp. data are compar-
able to values reported for economic crops (Chapman, 1966). In general,
80
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the lowest Cr concentrations in Cladophora sp. were comparable to high
values in economic plants. Very high Al concentrations correlate with
high Fe concentrations, probably because both reflect soil contamination
of samples.
ADDITIONAL EVALUATIONS OF P SUPPLIES
Because of the key role assigned P in promoting nuisance aquatic plant
growths and the emphasis placed on P removal to reduce these growths,
additional bioassays and evaluations were carried out on P availability
in Great Lakes samples. The P data in the previous section obtained
with a Jarrell-Ash Emission Spectrometer are not as reliable as data
obtained by the best quantitative procedures. For that reason P
analyses in this section were by standard colorimetric procedures.
Some data on Fitzgerald tests for NH4-N uptake as a measure of N
availability to Cladophora sp. and on Fe concentrations as a measure
of Fe availability are included.
Lake Superior and Northern Lakes Michigan and Huron
Algae samples were collected at several points on the north shore of
Lake Superior on two trips during the summer of 1974. Most of the
north shore is free of readily collectable algae. Cladophora sp.
were only found in the harbor area at Duluth. This could be due to
the relatively warm water temperature in this enclosed area compared
to the open lake. The most common alga found in different sampling
areas was a species of Ulothrix, perhaps because of relatively low
water temperatures in Lake Superior even in midsummer. The growth
of Ulothrix sp. usually precedes the growth of Cladophora sp. as
lake temperatures increase. A summary of the concentrations of the
total P and Fe in algae from Lake Superior is presented in Table 49.
The relatively high concentrations of P in several species of fila-
mentous green algae from the harbor area of Duluth (0.38-0.54%) in-
dicates there are readily available sources of P in this area. In
contrast, samples of Ulothrix sp. from the northern outskirts of
Duluth (Lester River outlet) contained less than half as much P
(0.14-0.17%). Relatively low concentrations of P also were found
85
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*n Ulothrix sp. from the harbor at Grand Marais, Michigan, (0.07 -
0.10%) and in diatoms from the harbor at Thunder Bay, Ontario
(0.10%). Ulothrix sp. from near Tofte, Michigan, contained the
highest concentrations of P (0.68-0.70%) found in any of the Lake
Superior samples, perhaps due to input of sewage from a small nearby
community. These results of P analyses of in_ situ algae agree with
the data and conclusions of Plumb and Lee (1974) using other methods
for measuring nutrient availability in this area of Lake Superior.
Duluth Harbor does not appear to be a relatively important source
of available Fe for Lake Superior, since Fe concentrations in
Ulothrix sp. from the harbor area and the open lake areas (Table
49) were approximately the same (0.10-0.18% Fe). Iron concentrations
in Ulothrix sp. from near Tofte, Michigan, were exceptionally high
(0.37-0.38% Fe). It would seem worthwhile to study this north shore
area of Lake Superior to determine if Fe entering the lake from
different mining areas is available to algae without the addition of
chelation chemicals that can be contributed by municipal sewage
effluents.
Phosphorus concentrations in Cladophora sp. from northern Lakes
Michigan and Huron were relatively low (0.088-0.19%). In contrast,
P concentrations in algae from Petosky and St. Ignace, Michigan, were
relatively high (0.24-0.40%) probably due to local pollution.
Iron concentrations in algae collected from the Manistique, Michigan,
area of Lake Michigan were high (0.21-0.66% Fe) in comparison to Fe
concentrations in other samples from northern Lakes Michigan and
Huron (0.034-0.075% Fe). The high concentrations of Fe in algae
from Manistique were expected since the reddish color of rivers in
this area is associated with dissolved and suspended iron compounds
derived from iron ore in the area.
Milwaukee Area of Lake Michigan
The total P and Fe concentrations in Cladophora sp. collected from
the Milwaukee area during the summer of 1974 are presented in Table
50. The data in general indicate that P concentrations in algae
-------
TABLE 50. TOTAL P AND Fe ANALYSES OF Cladophora sp. COLLECTED
FROM THE MILWAUKEE AREA OF LAKE MICHIGAN IN 1974
Sampling date
6-17-74
6-17-74
7-08-74
7-08-74
7-15-74
7-15-74
10-21-74
10-21-74
6-17-74
7-16-74
10-21-74
6-17-74
6-17-74
7-08-74
7-08-74
7-16-74
7-16-74
10-21-74
10-21-74
Sampling area
North Point Park, North
North Point Park, South
North Point Park, North
North Point Park, South
North Point Park, North
North Point Park, South
North Point Park, North
North Point Park, South
Bradford Beach
Bradford Beach
Bradford Beach
Breakwater, outer side
Breakwater, inner side
Breakwater, outer side
Breakwater, inner side
Breakwater, outer side
Breakwater, inner side
Breakwater, outer side
Breakwater, inner side
Total P,a %
0.29
.29
.12
.11
.15
.14
.12
.16
.25
.16
.14
.26
.39
.22
.38
.23
.38
.14
.20
Total Fe,a %
0.098
.080
.24
.18
.18
.40
.12
.10
.09
.31
.11
.080
.16
.24
.56
.084
.31
.11
.093
aData reported are averages of duplicate analysis.
collected from outside the Milwaukee Harbor (North Point Park,
Bradford Beach, and Outer side of breakwater) were considerably
lower than in algae collected within the harbor (Inner side of
breakwater). Average total P values were 0.19% and 0.34%, res-
pectively, for the two areas. The low concentrations of P in
algae from areas outside the harbor and to the north (North Point
Park) indicate that, whereas the Milwaukee Harbor can be a source
89
-------
of P for the algae within the harbor, it does not provide surplus
P for algae outside the harbor area.
The concentration of Fe in the Cladophora sp. from the harbor area
varied considerably between the different sampling dates and sites,
but in general samples from within the harbor area had higher Fe
concentrations than samples from outside the harbor.
Fitzgerald tests on NH^-N uptake, extractable POit-P, and total Fe
concentration in several algae samples collected outside the Milwaukee
harbor in 1973 are presented in Table 51. Two of the three samples
were indicated to be N-deficient by NHit-N uptake values in excess of
15 Ug N/10 mg algae/hr. All three samples were from algae indicated
to be deficient or close to deficient in P by comparisons with the
critical extractable POit-P concentration of 0.08%. There was no
indication that the algae were deficient in Fe.
Lake Erie Collections
Total P and Fe analyses of Cladophora sp. from Lake Erie (Tables
52 and 53) show that P concentrations in algae from western Lake
Erie (average, 0.34%) are considerably higher than in algae from the
eastern part of the lake (average, 0.089%). An exception is the two
samples from the northwestern side of Lake Erie (Ontario, Canada)
which had comparatively low P concentrations of 0.13 and 0.20%. Algae
with high P concentrations (0.22-0.56%) were collected from the Put-in-
Bay area to Cleveland, Ohio, which would indicate that the entire area
of the southwestern shore of Lake Erie has excessive amounts of avail-
able P for algae growth. These data agree with the report by Glooschenko
and Moore (1972) which stated that primary productivity in the western
basin of Lake Erie was much higher than in the central or eastern basins.
The fact that P concentrations in Cladophora sp. from the eastern area
of Lake Erie during August, 1974, indicated the algae were P-limited or
nearly P-limited has considerable ecological significance. These algae
were P-limited at a time when Cladophora sp. from the western basin
of the lake contained surplus P. Therefore, the lake muds of the
eastern part of Lake Erie do not provide adequate available P during
90
-------
TABLE 51. FITZGERALD TESTS FOR N, P, AND Fe SUPPLIES TO Cladophora
sp. COLLECTED FROM THE MILWAUKEE HARBOR AREA OF LAKE
MICHIGAN, 1973
Sampling
date
8-17-73
8-17-73
9-26-73
Sampling
area
North Point Park
End of breakwater
North Point Park
NHi»-N absorption,
N/10 mg/hr
30
32
4
Extract
P0^-P,a %
0.084
0.10
0.057
Total Fe,a
%
0.046
0.033
0.075
aData reported are the averages of triplicate analyses.
late summer to meet algae demands and cannot be considered an in-
exhaustible P source. As a result, reduction in local additions of
P, such as effluents from municipal and industrial sewage plants
should aid in the further control of Cladophora sp. in this part of
Lake Erie. In developing measures to control the algae, more detailed
algae sampling of eastern Lake Erie should be carried out to determine
the seasonal cycle of nutrient levels in the algae and to locate signi-
ficant nutrient sources.
Lake Ontario
The few analyses of the nutrient content of Cladophora sp. from the
Olcott, N.Y., area of Lake Ontario (Table 54) indicate these algae
have high concentrations of P (0.58-0.66%), but relatively low
concentrations of Fe (0.061-0.10% Fe). More detailed studies should
be carried out to determine the seasonal cycle of nutrients in the
algae of this and other areas of Lake Ontario, which would permit
evaluation of the significant sources of nutrients for the algae.
Studies of other deep fertile lakes have shown that despite signi-
ficant contributions of nutrients by spring and fall lake turnover,
some of these lakes become depleted of N, P, and Fe during midsummer.
Thus, midsummer pollution can have a significant influence on mid-
summer algae growth (Fitzgerald and Uttormark, 1974).
91
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TABLE 54. TOTAL P AND Fe CONCENTRATIONS IN Cladophora sp. COLLECTED
FROM LAKE ONTARIO
Sampling date
8-30-73
5-23-74
5-23-74
6-27-74
6-27-74
Sampling area
Olcott, N.Y., breakwater
Olcott, N.Y., breakwater
Olcott, N.Y., beach rocks
Olcott, N.Y., breakwater
Olcott, N.Y., beach rocks
Total P, %
--
0.63
0.66
0.58
0.58
Total Fe, %
0.066
0.10
0.061
0.068
0.086
Green Bay, Lake Michigan
Concern over pollution of Green Bay, Lake Michigan, from heavy industrial
and municipal waste loadings along the Fox River led to a study of ef-
fects of these pollutants on the nutrition of Cladophora sp. from the
area. The sources of pollution in Green Bay and the degree of pollution
in various parts were correlated with nutrient concentrations in algae
from different parts of the study area.
Sources of Nutrients
The major tributary to Green Bay, the Fox River, flows into the extreme
southern end of the bay (Figure 8). It is severely polluted because
of the heavy waste loadings contributed by industry and municipalities
(approx. 275,000 population). Because of its large input volume, the
river has a significant influence on the water quality and biota of
the bay.
Sources of P and N to the Lower Fox River, the only section of the
river subjected to severe deterioration, are enumerated in Table 55.
(Epstein, et_ al., 1974; Sager and Wiersma, 1972; Schraufnagel et a 1.,
1968; Shridharan, 1972). On an annual basis, Lake Winnebago provides
about 85% of the total N added to the Lower Fox River; municipal
sewage treatment effluents and effluents from the pulp and paper
industry add another 24,000 Ibs N/day, or 13% of the total. Dairy
94
-------
MANISTIQUE
N
PENSAUKEE
LAKE
WINNEBAGO
Figure 8. Green Bay and Lake Michigan area.
and other industries contribute the remaining 2%. In contrast to
N, the input of P from Lake Winnebago to the Lower Fox nearly equals
the phosphorus added by the municipal treatment plants which line the
river. Pulp and paper plants contribute about 1,100 Ibs P/day, or
half the amount contributed either by Lake Winnebago or by the
municipal plants. Other industrial sources of P are negligible.
South of Long Tail Point (Figure 8), Fox River water constitutes
95
-------
TABLE 55. NITROGEN AND PHOSPHORUS INPUTS TO THE LOWER FOX RIVER
Source
Lake Winnebago
Municiple treatment plants
Pulp and paper mills
Dairy
Other industry
Total
Nutrients entering Green Bay
at the Fox River (annual ave.)
Nitrogen,
Ibs N/day
153,000
10,400
13,400
4,300
16
181,116
232,000
Phosphorus,
Ibs P/day
2,160
2,200
1,100
--
16
5,476
4,310
50-80% of the total water volume present in Green Bay. Under southerly
winds, a tongue of Fox River water was traced northward 9.3-12.4 mi
along the east side of the bay during the summer of 1969. Modlin and
Beeton (1970) confirmed the presence of the Fox River along the eastern
shore of the bay in the summers of 1968 and 1969.
North of Long Tail Point, the proportion of Fox River water in Green Bay
decreases rapidly. More than 15.5 miles north of the river mouth,
values exceeding 25% of the total volume attributable to the Fox are
seldom observed (Ahrnsbrak and Ragotzkie, 1970). Still farther north,
the effects of the Fox River are minimal with respect to water volume
and chemistry.
Sager and Wiersma (1972) recorded high concentrations of orthophosphate
south of the Grassy Islands during the summer of 1971. The concentration
gradient in the first few miles from the mouth of the Fox River was
considered steep (from 33 to less than 8 yg P/l in 5 mi). Generally,
concentrations along the east half of the bay were higher than in the
west. These lower values illustrate the dispersal of Fox River water
following entry into Green Bay.
96
-------
Rousar and Beeton (1973) measured total P in transects across lower
Green Bay in the summer of 1971. Phosphorus concentrations decreased
with increasing distance from the mouth of the Fox River. Inshore
concentrations exceeded offshore concentrations. Within a transect,
the highest values for total P were generally found along the eastern
shore of the bay.
Results
Seventy-one samples of Cladophora sp. were collected on three sampling
dates during September, 1974, from shoreline stations in Green Bay and
nearby Lake Michigan. For comparative purposes, some Cladophora sp.
samples also were collected from other areas of the Great Lakes during
July and August, 1974.
The results of triplicate analyses of the Cladophora sp. samples from
each station for P, N, and Fe are summarized in Table 56. The plot
(Figure 9) of P concentrations in the Cladophora sp. samples compares
the analyses from the different areas sampled.
Phosphorus concentrations in the Cladophora sp. from along the east
shore of the lower bay were relatively high, decreasing from 0.35%
P for the bright green algae in the dense stands near the University
of Wisconsin-Green Bay campus to 0.20% P at sampling sites 10 to 20
miles farther north. The total N and Fe concentrations also decreased
with increasing distance from the Fox River outlet. However, only N
decreased to values considered close to or below the critical nutrient
concentration.
All samples collected 30 miles or more north of the Fox River outlet
on the shores of the Door County peninsula (areas B and C) were rela-
tively low in P, were usually from sparse growths, and were of a
yellowish-green color. Algae in these areas also had relatively low
Fe content, especially those samples collected from area C on the
northern part of the peninsula. The samples from area D on Lake
Michigan illustrate the effects small rivers and cities (Algoma)
can have on the P concentration of local stands of Cladophora.
At the river outlet in Algoma, Cladophora sp. growing on piers and
97
-------
10 MILES
Figure 9. Green Bay and Lake Michigan Sampling Area and Total P (%)
of Cladophora sp.
rocks was especially thick and dark green, and the P concentrations
were very high at 0.52%. The Fe content, however, was not much
different from that of Cladophora sp. growing along the northern
part of the peninsula.
The P concentrations in algae collected along the western shore of
Green Bay were low compared with concentrations in algae from the
eastern part of the bay, as would be expected if the flow of the
98
-------
TABLE 56. SUMMARY OF ANALYSES OF Cladophora sp. COLLECTED DURING
SEPTEMBER, 1974, FROM GREEN BAY AND LAKE MICHIGAN
Average of triplicate analyses, %
Sampling area
Area A
Campus, U. of Wi.
Bay Shore Park
Sugar Creek
Area B
Claflin Park
Potawatomi State Park
Sturgeon Bay Outlet
Area C
Egg Harbor, Wi.
Gill's Point
Baileys Harbor, Wi.
Area D
Clay Banks
Algoma, Wi.
Area E
Little Suamico, Wi.
Brookside, Wi.
Area F
Menominee, Mi.
J. W. Wells State Park
Area G
Manistique, Mi.
Total P
0.35
0.21
0.25
0.10
0.11
0.11
0.069
0.062
0.057
0.18
0.52
0.15
0.16
0.063
0.058
0.11
Total N
3.8
2.9
1.4
1.9
-
2.4
1.8
-
2.1
-
-
-
2.9
2.2
-
2.6
Total Fe
0.18
0.14
0.11
0.085
0.11
0.11
0.051
0.053
0.038
0.060
0.056
0.20
0.15
0.14
0.20
0.48
Fox River is mainly along the eastern shore.
The general conclusion from this study was that the growth of algae seems
generally to be limited by available P during the algal growing season,
99
-------
except in polluted areas. This corroborates the work of Schelske et al.
(1974) which also indicated that P limits phytoplankton growth in Lake
Michigan.
Despite an annual average contribution of more than two tons of P per
day to Green Bay by the Fox River, the Cladophora sp. within 30 miles
of the river outlet are P-limited during late summer. Therefore, in
September and November, Green Bay does not supply Lake Michigan with
major quantities of algal nutrients that can enhance algal growth. If
Green Bay can effectively purify itself, then other areas of the Great
Lakes may do so also. A combination of tertiary treatment of wastes
and the ability of Green Bay to purify itself should result in a
marked quantitative improvement in the algal crop of the lower Green
Bay area.
DISCUSSION
The studies described in this section were concerned primarily with
evaluating nutrient supplies and identifying growth-limiting nutrients
for Cladophora sp. in the Great Lakes. These are the principal practical
applications of the project. Information on growth-limiting nutrients
and nutrient supplies should be valuable in developing plans and measures
for reducing nuisance algae growths.
Several aspects of the results justify additional comment. First, the
results suggest that the development and use of plant analysis bioassays
for evaluating nutrient supplies is worthwhile and to be recommended.
This is supported by close correlations between plant bioassay evaluations
of N and P supplies in several areas of the Great Lakes and the recognized
N and P pollution of those areas.
The best example of such a correlation was the degree to which the total
P in Cladophora sp. from Green Bay decreased as the distance of the points
of sample collection from the point of discharge of effluent from the Fox
River increased. The plant analysis bioassay also offers an advantage
in making possible evaluations of supplies of inorganic nutrients other
than N and P. This includes the essential trace elements. Because the
base data on water concentrations which represent adequate and potentially
100
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deficient supplies of elements other than N and P are not available
at this time, chemical analyses of water samples for elements other
than N and P are difficult to interpret in terms of potential for
plant growth.
Of the plant bioassay data, the results on the N and P status of
Cladophora sp. probably are of most interest, because of the general
recognition of the critical role of these elements in nuisance algae
growths and the possibilities for control measures oriented toward
these elements, particularly P. There was little indication that
N supplies were reduced to levels that limited Cladophora -sp. Nitrogen
concentrations in the algae usually were well in excess of the 1.1%
critical concentration. However, P concentrations in the Cladophora sp.
not only correlated well with the general pollution of areas, but also
in samples from relatively unpolluted areas approached the critical P
concentrations, both total P and extractable POit-P. Low P concentrations
were observed in some Cladophora sp. samples from northern Lakes Michigan
and Huron, from Lake Michigan near the Milwaukee Harbor, from the upper
parts of Green Bay, and from Eastern Lake Erie. In contrast, most
samples from Western Lake Erie, lower Green Bay, and Lake Ontario were
very high in P. Although positive recommendations probably should be
based on more data, the results suggest that reduction in P supplies
would be a practical means of reducing nuisance growths of Cladophora
sp. in the Great Lakes.
The assays by plant analysis for supplies of elements other than N
and P are interesting because they suggest that elements other than
N and P actually become limiting for Cladophora sp. growth in the
Great Lakes. This was true of Zn and particularly of B. The practical
value of these observations cannot be assessed until further samples
are taken and analyzed. A potential growth-limiting role for elements
other than N and P also was indicated in a Lake Michigan water enrich-
ment experiment reported in Section VII. In addition, relatively
little Cladophora sp. growth developed in Lake Michigan water unless
vitamin BI was added. Vitamin BI, therefore, also is a potentially
101
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growth-limiting nutrient in the Great Lakes. Again, further studies
are needed to establish the significance of this observation.
Definite and reliable critical concentrations are essential in
successful applications of the plant analysis bioassay. The con-
siderable difference in the critical concentration for P established
in independent efforts by the two Principal Investigators in different
aspects of this project (Sections VI and IX) indicates there can be
difficulties in obtaining and interpreting the data from which critical
concentrations are derived. Additional studies are necessary to estab-
lish the critical concentration of P and other key elements more pre-
cisely. It is possible that the critical concentration of P, and other
elements as well, varies somewhat with the age of the Cladophora
glomerata, as a result of changes in non-protoplasmic constituents
and food reserves. The 0.08% critical value for hot water extractable
P in the Fitzgerald test seems too high. It should not exceed the
critical concentration based on total P in the algae cells. Again
additional calibration tests are required. The data do seem to justify
the view that, compared with other algae, the critical concentrations
of N and P are relatively low for Cladophora glomerata, 0.06-0.1% for
total P and 1.1-1.5% for total N.
102
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SECTION XI
RELIABILITY OF SAMPLING AND ANALYTICAL METHODS
To attach ecological significance to differences in the inorganic
composition of algae from various environments, the variability
associated with the sampling procedure and with the analytical
procedures should be known. Tests were carried out to evaluate
both aspects of experimental variability.
RESULTS AND DISCUSSION
Replicate Analyses of Cladophora sp. Samples
A sample of Cladophora sp. was collected from Lake Monona and from
Lake Wingra, Madison, Wisconsin, and total P and extractable PO^-P
analyses were made on ten replicate aliquots from each sample. The
range in variation and the standard deviation for the analyses of
each sample are presented in Table 57. The mean P concentration in
the Lake Monona sample was considerably higher than in the Lake
Wingra algae. In fact, the data would be interpreted to indicate
the supply of P in Lake Monona was adequate for Cladophora sp.
growth but P was limiting and inadequate for the Lake Wingra algae.
The agreement in the replicate analyses in general is equal to the
agreement obtained in routine use of the best quantitative proce-
dures in plant analyses. Also, the data indicate that the analytical
procedures employed are suitable for distinguishing by plant bio-
assay algae adequately supplied and deficient in P.
Two separate laboratories have been involved in this project. To
further evaluate the reliability of the analytical procedures used,
replicated analyses for P, Fe, N, and Ca were made on aliquots from
samples collected from Lakes Monona and Wingra on July 11, 1974.
The analyses included a comparison of results obtained by the sepa-
rate laboratories involved in this project. The P method used in the
Algae Laboratory is an acid-persulfate digestion (autoclave) followed
by stannous chloride color development (Standard Methods, 13th
Edition); the procedure used in the Botany Department Laboratory
involved dry-ashing at 600°C followed by development of a
103
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Table 57. AGREEMENT IN REPLICATE ANALYSES FOR TOTAL AND EXTRACTABLE
P IN FIELD SAMPLES OF Cladophora sp.
Replicates, %
Ave., %
Range, %
S.D.
L. Monona
Extract POi^-P, %
0.16
0.16
0.17
0.17
0.18
0.16
0.16
0.18
0.18
0.19
0.17
0.16-.19
0.01
Total P, %
0.33
0.36
0.38
0.36
0.39
0.44
0.40
0.41
0.35
0.33
0.38
0.33-.44
0.04
L . Wingra
Extract PO^-P, %
0.018
0.016
0.017
0.018
0.015
0.013
0.015
0.017
0.030
0.018
0.018
0.013-.030
0.005
Total P, %
0.056
0.066
0.056
0.058
0.062
0.061
0.061
0.064
0.065
0.065
0.061
0.056-.066
0.004
vanadomolybdate complex which is yellow in color (Jackson, 1958). The
results of the analyses are presented in Table 58. Again the data
indicate that the agreement among replicates equals that expected
from procedures in general use for plant analyses. This included
total P analyses by the slightly different procedures used in the
two participating laboratories.
Replicated Sampling of Cladophora sp.
Tests were carried out to measure the variability in the concentra-
tions of several nutrients in Cladophora sp. collected from within
a small area in a lake. This information is important in determining
whether differences in nutrient concentrations in algae truly reflect
variations in environmental nutrient supplies or merely normal
104
-------
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biological variation in the algae.
Ten samples of Cladophora sp. were collected from within an area 5 feet
in radius within the breakwater of the Milwaukee Harbor; 10 samples
were obtained at North Point Park, about one-half mile north of the
harbor; and six samples were collected at Put-in-Bay, Lake Erie. The
results of analyses of these samples for total P and Fe are presented
in Table 59. The results indicate a high degree of reproducibility in
the samples, particularly in the total P analyses. The 0.33-0.44%
range in P concentration in the Milwaukee Harbor samples was the same
as the range in the replicate analyses of the algae collected from
Lake Monona and reported in Table 57. In other words, the analytical
procedure could have been solely responsible for the differences ob-
served. There was considerable variation in the total Fe concentra-
tion in the North Point Park samples, perhaps because the concentra-
tions were relatively low. In general, the high degree of reproduci-
bility in analyses of Cladophora sp. samples from within small areas
supports the concept that variations in the concentration of an
element in the algae do reflect variations in environmental supplies.
106
-------
Table 59. AGREEMENT IN THE TOTAL P AND TOTAL Fe CONCENTRATIONS
IN SAMPLES OF Cladophora sp. COLLECTED FROM WITHIN
A FIVE-FOOT RADIUS
Ave., %
Range, %
S.D.
L. Mich.
Milw., Wis.
breakwater
Total P,
%
0.44
0.39
0.40
0.37
0.44
0.39
0.33
0.41
0.39
0.39
0.40
0.33-.44
0.03
Total Fe,
%
0.28
0.35
0.30
0.36
0.33
0.37
0.40
0.30
0.33
0.40
0.34
0.28-.40
0.04
L. Mich.
Milw., Wis.
N. Pt. Park
Total P,
%
0.16
0.18
0.20
0.19
0.18
0.21
0.15
0.17
0.15
0.20
0.18
0.15-.21
0.02
Total Fe,
%
0.04
0.12
0.06
0.06
0.10
0.08
0.03
0.13
0.06
0.07
0.08
0.03-.13
0.03
L. Erie
Put- in- Bay }
Ohio
Total P,
%
0.39
0.44
0.42
0.45
0.41
0.44
0.46
0.39
0.48
0.43
0.43
0.48
0.43
0.39-.48
0.03
Total Fe,
%
0.064
0.063
0.066
0.065
0.066
0.071
0.077
0.079
0.065
0.069
0.068
0.063
0.068
0.063-.079
0.005
107
-------
SECTION XII
REFERENCES
Ahrnsbrak, W. F. and R. A. Ragotzkie. 1970. Processes in Green
Bay. In: Proc. 13th Conf. Great Lakes Res. International
Association for Great Lakes Research, Ann Arbor, p. 880-890.
Arnon, D. I. 1938. Microelements in Culture Solution Experiments
with Higher Plants. Amer. Jour. Bot. 25: 322-325.
Blanchar, R. W., G. Rehm and A. C. Caldwell. 1965. Sulfur in Plant
Materials by Digestion with Nitric and Perchloric Acid. Soil Sci.
Soc. Am. Proc. 29: 71-72.
K
Chapman, H. D. 1966. Diagnostic Criteria for Crops and Soils. Uni-
versity of California Div. of Agricultural Sciences, Riverside,
California. 793 pp.
Epstein, E., M. Bryans, D. Mezei, and D. Patterson. 1974. Lower
Green Bay: An Evaluation of Existing and Historical Conditions.
EPA Great Lakes Initiative Contract Program. U.S. Environmental
Protection Agency, Madison, Wisconsin. EPA-905/9-74-006. 281 pp.
Fitzgerald, G. P. 1968. Detection of Limiting or Surplus Nitrogen
in Algae and Aquatic Weeds. J. Phycol. £: 121-126.
Fitzgerald, G. P. 1969. Field and Laboratory Evaluations of Bio-
assays for Nitrogen and Phosphorus with Algae and Aquatic Weeds.
Limnol. Oceanogr. j.4: 206-212.
Fitzgerald, G. P. 1970. Evaluations of the Availability of Sources
of Nitrogen and Phosphorus for Algae. J. Phycol. 6_: 239-247.
Fitzgerald, G. P., and S. L. Faust. 1967. Effect of Water Sample
Methods on the Release of Phosphorus from Algae. Limnol. Oceanog.
12_: 332-334.
Fitzgerald, G. P., and T. C. Nelson. 1966. Extraction and Enzymatic
Analyses for Limiting or Surplus Phosphorus in Algae. J. Phycol.
2_: 32-37.
Fitzgerald, G. P., and P. D. Uttormark. 1974. Application of Growth
and Algal Assays. U.S. Environmental Protection Agency, Corvallis,
Oregon. EPA-660/3-73-023. 176 pp.
Gerloff, G. C. 1969. Evaluating Nutrient Supplies for the Growth of
Aquatic Plants in Natural Waters. In: Eutrophication: Causes,
Consequences, Correctives. National Academy of Sciences, Washington,
D.C. 661 pp.
108
-------
Gerloff, G. C. 1973. Plant Analysis for Nutrient Assay of Natural
Waters. U.S. Environmental Protection Agency, Washington, D.C.
EPA-R1-73-001. 66 pp.
Gerloff, G. C. 1975. Nutritional Ecology of Nuisance Aquatic Plants.
U.S. Environmental Protection Agency, Corvallis, Oregon. EPA-660/
3-75-027. 78 pp.
Gerloff, G. C. and K. A. Fishbeck. 1969. Quantitative Cation Require-
ments of Several Green and Blue-green Algae. J. Phycol. 5_: 109-
114.
Gerloff, G. C. and K. A. Fishbeck. 1973. Plant Content of Elements
as a Bioassay of Nutrient Availability in Lakes and Streams. In:
Bioassay Techniques and Environmental Chemistry. Ann Arbor Science
Publishers, Inc., Ann Arbor, Michigan, p. 159-176.
Gerloff, G. C., G. P. Fitzgerald, and F. Skoog. 1950. The Isolation,
Purification and Culture of Blue-green Algae. Amer. J. Bot. 37:
216-218.
Gerloff, G. C. and P. H. Krombholz. 1966. Tissue Analysis as a
Measure of Nutrient Availability for the Growth of Angiosperm
Aquatic Plants. Limnol. Oceanog. 11: 529-537.
Gerloff, G. C. and F. Skoog. 1954. Cell Contents of Nitrogen and
Phosphorus as a Measure of Their Availability for Growth of
Microcystis aeruginosa. Ecology. 35: 348-353.
Gerloff, G. C. and F. Skoog. 1957. Nitrogen as a Limiting Factor
for the Growth of Microcystis aeruginosa in Southern Wisconsin
Lakes. Ecology. 38: 556-561.
Glooschenko, W. A., and J. E. Moore. 1972. A Comparative Study of
Primary Production in Lakes Erie and Ontario. In: Abstracts of
15th Conf. on Great Lakes Res., International Association for
Great Lakes Research, Ann Arbor, p. 114.
Hewitt, E. J. 1966. Sand and Water Culture Methods Used in the
Study of Plant Nutrition, Second Edition, Commonwealth Bureau
of Horticulture and Plantation Crops, East Mailing, Maidstone,
Kent. Tech. Commun. No. 22.
Hughes, E. 0., P. R. Gorham, and V. A. Zehnder. 1958. Toxicity
of a Unialgal Culture of Microcystis aeruginosa. Canadian
Microbiol. £: 225.
Jackson, M. L. 1958. Soil Chemical Analysis. Prentice-Hall, Inc.
Englewood Cliffs, N. J. 498 pp.
Johnson, C. M. and T. H. Arkeley. 1954. Determination of Molybdenum
in Plant Tissue. Analytical Chem. 26: 572-573.
109
-------
Johnson, C. M., P. R. Stout, T. C. Broyer, and A. B. Carlton. 1957.
Comparative Chlorine Requirements of Different Plant Species. Plant
and Soil. _8: 337-353.
Johnson, C. M. and A. Ulrich. 1959. Analytical Methods for Use in
Plant Analysis. California Agricultural Experiment Station,
Berkeley, California. Exp. Bulletin 766. 78 pp.
Modlin, R. F., and A. M. Beeton. 1970. Dispersal of Fox River Water
in Green Bay, Lake Michigan. In: Proc. 13th Conf. Great Lakes
Res. International Association for Great Lakes Research, Ann Arbor.
p. 468-476.
Moore, L. F., and D. A. McLarty. 1975. The Influence of Soil Water
Extract and Thiamine on the Growth of Cladophora glomerata.
Canadian J. Bot. 53: 530-535.
Rousar, D. C., and A. M. Beeton. 1973. Distribution of Phosphorus,
Silica, Chlorophyll a and conductivity in Lake Michigan and Green
Bay. Wis. Acad. Sci., Arts, and Letters. LXI; 117-140.
Sager, P. E., and J. H. Wiersma. 1972. Nutrient Discharges to Green
Bay, Lake Michigan from the Lower Fox River. In: Proc. 15th Conf.
Great Lake Res. International Association for Great Lakes Research,
Ann Arbor, p. 132-148.
Schelske, C. L., E. D. Rothman, E. F. Stoermer, and M. A. Santiago.
1974. Responses of Phosphorus-Limited Lake Michigan Phytoplankton
to Factorial Enrichments with Nitrogen and Phosphorus. Limnol.
Oceanogr. 19: 409-419.
Schraufnagel, F. H., L. A. Lueschow, G. Karl, L. A. Montie. J. Tissack,
and J. R. McKersie. 1968. Report on the Investigation of the
Pollution in the Lower Fox River and Green Bay Made During 1966
and 1967. Dept. of Natural Resources, Madison, Wisconsin. 77 pp.
Sridharan, N. 1972. Aqueous Environmental Chemistry of Phosphorus
in Lower Green Bay, Wisconsin. Ph.D. Thesis, University of Wisconsin-
Madison.
Van Den Hock, C. 1963. Revision of the European Species of Cladophora.
E. J. Bull. Leiden, 248 pp.
Whitton, B. A. 1970. Biology of Cladophora in Freshwaters. Water Res.
£: 457-476.
Wojick, E. 1972. The Effects of Elevated Temperatures and Light Inten-
sity on Certain Aquatic Macrophytes and Algae. M.S. Thesis, University
of Wisconsin-Madison.
Zuraw, E. A. 1969. Culture and Physiological Requirements of a Bacterized
Cladophora glomerata (L.) Kiitz from Lake Michigan. J. Phycol. 5_: 83-85.
110
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-76-044
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
5. REPORT DATE
April 1976 (Issuing Date)
The Nutrition of Great Lakes Cladophora
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Gerald C. Gerloff and George P. Fitzgerald
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Botany and Water Resources Center
University of WisconsinMadison
Madison, Wisconsin 53706
10. PROGRAM ELEMENT NO.
1BA608
11. 88HXHHH5JVGRANT NO.
R-802464
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
Final - 6/15/73 to 12/31/75
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A synthetic culture medium was developed for the laboratory culture of Cladophora
glomerata from the Great Lakes. Recognition that Cladophora requires vitamins B^ and
#12 was a key feature in the successful culture of this alga. Systematic modifications
of the initial culture solution resulted in an optimum culture medium in which C^
glomerata routinely produced 500 mg oven-dry algae in a 21-day period.
The quantitative requirements of C. glomerata for essential inorganic nutrients were
expressed primarily as critical cell concentrations, that is minimum cell concentra-
tions which permit maximum yield. C^ glomerata has relatively low critical N and P
concentrations (1.1% and 0.06%, respectively) but very high B (110 ppm) and S (0.15%)
critical concentrations. Ortho-, pyro-, meta, and tripoly-P can be utilized in £._
glomerata growth as can Ntty and NO^-N. Phosphite-P is relatively unavailable.
Nutrient supplies and limiting nutrients for Cladophora sp. growth in the Great Lakes
were evaluated by two bioassays, primarily plant analysis and to some extent the Fitz-
gerald tests. Plant analysis assays for P showed that Cladophora P concentrations
correlated closely with the recognized pollution of areas sampled. Furthermore, P
was limiting or close to limiting in several relatively unpolluted areas. There were
indications that elements other than P at times limited Cladophora growth.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COS AT I Field/Group
Chlorophyta
Bioassay
Plant nutrition
Constraining
Cladophora
Green algae
Critical concentrations
Limiting nutrients
Algae culture medium
Great Lakes
6A
6F
3. DISTRIBUTION STATEMENT
RELEASE UNLIMITED
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
123
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
111
*USGPO: 1976 657-695/5406 Region 5-11
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