ODOROUS METABOLITE AND OTHER SELECTED
STUDIES OF CYANOPHYTA
APPROVED!
Graduate Committee,!
Major Professor
MinoriProfess
Committee Membe
Committee Member .
Ut
Director of^the Departmen^ of Biology
Dean or the Graduate School
ODOROUS METABOLITE AND OTHER SELECTED
STUDIES OF CYANOFHYTA
DISSERTATION
Presented to the Graduate Council of the
North Texas State University in Partial
Fulfillmant of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
By
Don E. Henley, B. A., M. A.
Denton, Texas
August, 1970
TABLE OF CONTENTS
Page LIST OF ILLUSTRATIONS . . . . . . . . . . . iv
Chapter
ODOROUS METABOLITE STUDIES OF ANABAENA CIRCINALIS . . . ,
Introduction Materials and Methods Results Discussion Summary
II. A COMPARATIVE STUDY OF SELECTED ODOR-PRODUCING CY&NOPHYTES 28
Introduction Materials and Methods Results Discussion Summary
III. THE EFFECT OF GERANIOL AND GSOSMIN ON THE GROWTH AND DEVELOPMENT OF ANABAENA CIRCINALIS . . . . . 35
Introduction Materials and Methods Results Discussion Summary
LITERATURE CITED . . . . . 49
LIST OF ILLUSTRATIONS
Figure Page
1. Standard Curve for Dry Weight Quantitation of A. circinalis . . . . . . . . . .. 8
2. Fractionation Scheme for Preparation of Volatile Odor Fraction . . . . . . . . . . . 11
3. Gas Chromatogram of Volatile Odor Fraction from
A. circinalis 13
4. Series Mass Culture of A. circinalis . . . . . . . 16
5. Growth Curves for Each Vessel of the Mass Culture 17
6. Biomass Curve for Each Vessel of the Mass Culture . . . . . . . . . . . . . . . . . . . 19
7. Infrared Spectrum of the Earthy-Musty Odor Compound from A, circinalis . . . . . . . . . 20
8. Nuclear Magnetic Resonance Spectrum of the Earthy-Musty Odor Compound from A, circinalis . . . . . . . . . . . . . . . . 22
9. Mass Spectrum of the Earthy-Musty Odor Compound from A. circinalis . . . . . . . . . . . . . . 23
10. Typical Gas Chromatogram of Volatile Odor Fraction from Four Species of Cyanophyta Showing Common Odor Constituent . . . . . . . 32
11. Gas Chromatogram of Pure Geosmin . . . . . . . . . 33
12. The Effect of Geraniol on Heterocyst Frequency in A. circinalis 40
13. The Effect of Geosmin on Heterocyst Frequency in A. circinalis . . . . . . . . . . . . . . . 41
Figure Page
14. The Effect of Geraniol on the Growth of A. circinalis . . . . . . . . . . . 42
15. The Effect of Geosmin on the Growth of A. circinalis 43
CHAPTER I
ODOROUS METABOLITE STUDIES OF
ANABAENA CIRCINALIS
Introduction
The cyanophyta, or blue-green algae, are a distinctive
group of organisms sharply differentiated from other algae
in respect to both morphology and physiology. These
organisms do not possess a typically organized nucleus
and, in addition, contain assessory pigments phycocyanin
and phycoerythrin. An additional characteristic of certain
members (Nostocales) is their ability to fix atmospheric
nitrogen. Another peculiarity, although not unique to this
group, is their ability to develop rapidly into "water-
blooms" or "blooms". The term water-bloom or blooms has
been used by several workers (Trelease, 1889; Olive, 1918;
Lackey, 1945; Welch, 1952; Ruttner, 1953; Allee et al., 1955;
Palmer, 1959; Klein, 1962; Kackenthun et al., 1964; Veatch
and Humphrey, 1964; Pennak, 1946) to denote or describe
unusually large concentrations of algae in the natural
habitat. In most instances, the term has been used in a
rather subjective and qualitative manner with the exception
2
of Lackey (1945), who defined it as "an arbitrary value of
500 organisms/ml," A water-bloom, regardless of the
definition applied, appears to be associated with the
increasing cultural activities of man. An example of such
cultural activities would be the construction of sewers in
Madison, Wisconsin during 1880. These sewers pumped raw
sewage into the Madison Lakes. Trelease (1889) cited the
year 1882 as one in which heavy scums developed in both
Madison area lakes, Mendota and Monona. Singh (1953) cited
the relationship between sewage discharge into Lake Kakki
and blooms of the blue-green alga, Microcystis aeruginosa.
Fogg (1969) discussed the occurrence of blue-green algae in
organically polluted aquatic environments. Vinyard (1967)
stated that organic pollution almost always insures an
abundance of blue-green algae. In 1965, Vance suggested
a direct relationship between high organic content of aquatic
habitats and dense cyanophycean blooms.
There is little doubt that the change from oligotrophic
to mesotrophic to eutrophic, is dependent upon and enhanced
by the supply of plant nutrients entering a given body of
water. Hasler (1947) defined eutrophication as "enrichment
of water, be it intentional or unintentional." The overt
evidence of the process of eutrophication, however, is the
increasing rate of primary production and, in many extreme
cases, extensive algal blooms develop either periodically
3
or continually. The result of such manifestation is the
decreased suitability of the water for municipal supply and
recreational purposes.
Cyanphycean blooms have produced unpleasant odors,
depleted the oxygen supply of freshwater habitats resulting
in extensive fish kills, and, on occasions, have produced
toxic substances which, in turn, have resulted in mortality
of waterfowl and livestock (Gorham, 1964) and intestinal
disorders in man (Schwimmer and Schwimmer, 1964).
The association of tastes and odors and algae in water
supplies has been recognized since the early works of George
C. Whipple in the 1890's. In 1924# John Bayliss, then
principal sanitary chemist for the Baltimore Water Department,
declared!
There are objectionable tastes in many of the waters of our country that will always remain objectionable, unless removed. It is removal or prevention of these that should receive more consideration from our water works officials than heretofore. Changes in our modes of living and this vast amount expended for things that add comfort to our lives justify the assumption that we are now ready for more rapid progress in improving the palatableness of our drinking water.
In a nationwide survey regarding tastes and odors in
water supplies which was conducted by the United States Public
Health Service in 1955 (Sigworth, 1957), 82 per cent of the
responding facilities reported tastes and odors resulting from
algae. In the same survey, algal types responsible for
various odors were also evaluated. The diatoms ranked first,
4
cyanophytes arid protozoans were second# chlorophytes third,
and the fungi were ranked fourth in production of odors.
Within the division Cyanophyta, the genus Anabaena ranked
first as an offender. So important an offender are certain
species of this genus that Anabaena circinalis has been
listed as the representative species in regard to taste and
odor production (Palmer, 1962). Palmer stated that blue-
green algae are well known for developing very foul "pigpen"
odors in water and that several, including Anabaena, are
capable of collection into larger masses sufficient to form
water-blooms. The foul pigpen odor was thought to develop
as a result of decomposition products as the algae began to
die off in large numbers. Anabaena, together with
Gomphosphaeria, Cylindrospermum, and Rivularia, have a
natural odor which is commonly described as "grassy".
Palmer stated further that the odor changes to that of
nasturtium, probably as a result of oxidation.
History of the Problem
Jackson and Ellrns (1897) characterized the various odors
produced by certain noxious algae in Massachusetts reservoirs.
These workers reasoned that, in general, tastes and odors
originate as a result of decomposition and natural growth.
In regard to Cyanophyta (Anabaena), the "pigpen" odor was
placed in the first category while the moldy—grassy odor was
5
placed in the latter category. Qualitative work indicated
to these workers that the moldy-grassy odors of Anabaena were
essential oils.
Laboratory studies by Gerber and Lechevalier (1965) led
to the isolation of an earthy-smelling compound from several
actinomycetes. The compound was named geosmin. Safferman
et al.,(1967) presented evidence which suggested that an
earthy-smelling compound produced by the cyanophyte, Symploca
muscorum, was identical to geosmin. Medsker et al., (1968)
described work wherein geosmin was isolated from two substrate
cyanophytes, Symploca muscorum and Oscillatoria tenuis,
Geosmin was later shown to be trans-1, 10-»dlmethyl-trans-9~
decalol (Gerber, 1968).
During the spring of 1969, a bloom of Anabaena clrclnalis
occurred in Garza-Little Elm Reservoir, which serves as the
water supply for Denton, Texas. Coincident with this bloom
occurrence, a taste and odor problem of the earthy to musty
variety developed in the Denton municipal water supply.
Samples of the bloom-laden water were secured and returned
to the Water Research Laboratory at North Texas State
University. Immediately, cultivation procedures were
initiated which led to the isolation of Anabaena circinalis
into unialgal culture. The cultured organism, when sensed
by olfaction, was observed to produce odors of the same
variety as that occuring during the spring bloom.
Purpose
Anabaena clrcinalls possessed several characteristics
which made it an organism of choice for odorous metabolite
studiesJ (a) The organism grew well under laboratory
conditions, (b) the organism could be cultured in defined
inorganic media, (c) odor production of the earthy to musty
variety was intense, (d) the organism was a euplanktonic
species with a long history of bloom and associated odor
production, (e) and due to its growth and morphological
characteristics, the organism could be easily quantitated,
Therefore, the purpose of this study was as followss
1. Elucidate the laboratory conditions under which
Anabaena circinalis produces the noxious odor
component or components in maximum concentration,
2. Isolate the major noxious odorous metabolite(s).
3. Structurally define the odorous metabolite(s).
4. Quantitate the amount of metabolite(s) that a
given amount of the organism produced.
Materials and Methods
Algae and Stock Cultures
The unialgal stock cultures of Anabaena circinalis
(North Texas State University Culture Collection No. NT69-2,
maintained in ASM-1 culture medium (Gorham et al., 1964),
served as the experimental organism in this study. Stock
cultures were routinely grown for purposes of subsequent
inoculation into mass culture, The stock cultures were
grown by inoculating a 1.0 ml. suspension of the organism
(O.D. of 0.02 at 660 mp) into 250 ml. Erlenmeyer flasks
containing 75 mis, of the ASM-1 culture medium. The
inoculated cultures were placed on a rotary shaker making
147 rpm. The temperature was maintained at 24.5 ± 1.0° C.
while illumination was held at 60 ft-c. by cool-white
overhead fluorescent lamps. After IS days, the cultures
displayed an approximate O.D, of 0.41. Pooled aliquots of
these cultures were used for the preparation of mass
cultures«
Growth Measurement
Stock and mass culture growth was measured photometrically
at 660 myi on a Klett-Summerson, Kodel 900-3, photoelectric
colorimeter. Growth was expressed as O.D, versus time in
days. Dry weight determinations were made by filtering
aliquots of cultures during exponential-growth on preweighed
Gelrnan, Type E. glass filters. The filter and adhering
organism were dried overnight at 75° C. in a force-draft
hot-air oven. For purposes of biomass quantitation, a curve
was prepared by plotting O.D. against dry weight as shown
in Fig. 1.
8
8.0-
g 60-
x O o «o
§4.0-
2.0
2.0 4.0 6.0 8.0 DRY WT. (MG./3D ML.)
10.0
Fig. I--Standard curve for dry weight quantitation of A* r"{ rva 1 •{ I
Mass Cultures
The culture vessels utilized were 5 gallon capacity-
glass carboys. Seventeen and one-half liters of ASM-1
culture medium were prepared in each culture vessel. A
500 ml. stock-culture inoculum at an O.D. of 0.41 was added
to each, yielding a final volume of 18 liters. The culture
vessels were closed with #5 rubber stoppers, into which
had been fitted two lengths of glass tubing (3.5 mm. I.D. x
5.0 mm. O.D.). One was 51 cm. in length and served as an
air inlet and for agitation of the culture. The other tube
was 15 cm. in length and served as an air exhaust port. The
culture vessels were set up in series and agitated with
filtered laboratory air. Agitation was adjusted to 1000 ml.
of air per minute. Routinely, three vessels were placed
in series. The static pressure above the medium was adjusted
so that vessel #1 (closest to the air supply) was 1,5 inches
of Hg above atmospheric pressure, vessel #2 was 1,0 inches
of Hg above atmospheric pressure, and vessel #3 was 0,5
inches of Hg above atmospheric pressure. The mass cultures
were maintained at ambient room temperature and illuminated
from above with 150 ft-c. by cool-white fluorescent lamps.
After approximately one month, the biomass of organism in
each vessel was determined as described previously (Fig, 1),
The organism was harvested by means of a continuous-flow
Sharpel's Super Centrifuge. The harvest was then stored at
—20° C. to await subsequent volatile fraction preparation.
10
Volatile Fraction Preparation
The method utilized for preparation of the volatile
fraction from Anabaena circinalis is diagrammed in Fig. 2.
A suspension consisting of 1 per cent organism and 99 per
cent distilled water (dry wtsvol.) was prepared. Approximately
5.0 grams of organism in a total volume of 500 mis, was steam-
distilled at any given time. After 50 per cent of the total
volume had been distilled, the residue was discarded since
only trace amounts of the volatile components remained. The
distillate was extracted two times with 30 per cent (vsv)
nannograde petroleum ether. Prior to the final extraction,
the distillate was saturated with reagent KaCl. The petroleum
ether extracts were pooled and dried with 5 per cent (wsv)
reagent NaCl. The pooled extracts were concentrated by
distilling at 60° C. The residual volume, containing the
volatile odorous metabolites, was concentrated by a factor
of 120. The residual volume was then stored at -20° C. to
await subsequent instrumental analyses.
Gas Chromatographic Analysis and Collection
An Aerograph Autoprep, Model 705 gas chromatograph,
equipped with a hydrogen flame detector and linear splitter
was utilized in this study.
The column packing employed was SE-30, 10 per cent on
60/80 mesh chromasorb G. acid washed. Three types of columns
11
1 g. organism + distilled H2O to 100 ml. volume
steam distill 50%
50 ml. of odorous distillate
extract with 30% _ (viv) petroleum ether
Distillate saturated with NaCl
extract with 30% (viv) petroleum ether
Discard aqueous phase
Pooled extracts
dried with 5% (wrv) NaCl
Raw concentrate
concentrated 120 X by distillation at 60° C.
Volatile Odor fraction
GC Analysis
Fig. 2—Fractionation scheme for preparation of volatile odor fraction.
12
were used: an analytical column (10 feet by one-eighth inch)
for detailed analysis and resolution, a semi-analytical column
(five feet by one-quarter inch) for general analysis, and a
preparative column (10 feet by three-eighths inch) for
collection purposes«
Microliter samples of the odorous volatile fraction
were injected into the gas chrornatograph utilizing the
analytical column for separation of the volatile components,
A chromatogram was produced as shown in Fig. 3. Upon sensing
each peak during elution at the exit port, a subjective
olfactory description could be assigned. The olfactory
description of each peak is also shown in Fig. 3. The most
noxious and odoriferous component, peak #5, was also the
component present in the greatest concentration as evidenced
by peak area analysis. When sensed by olfaction, it was of
an intense earthy-musty nature. The earthy-rausty component
was chosen for collection and further instrumental analysis
since it was obviously responsible for the over-all earthy-
musty odor of the organism in culture and, no doubt, responsible
for the odorous nature of the water supply during the
described bloom incident.
Gas chromatographic collection of the earthy-musty
component was achieved by inserting a 2.0 mm. glass capillary
tube into the exit port of the gas chrornatograph at the time
of peak elution as evidenced by the peak response on the
13 CS Det. N " H
2 min./in. flame
Air DA "
150 cc/ m i n. 24 cc/min.
210 cc/min. &s s hown
Date 3-21-70 Samp. volatile odor faction of A. circlnalis" C°1«—SE-30, 10/o 10» X3/8" Col. Temp. 205<> C In. Temp. 165° C " " Det. Temp". 240° c ~
100-J
90-
on eu-
70-
4" V.
60-a •L u 50
H-
0 40
NP 30-
20
10-
solvent
#1 spoiled hay X2
#5 earthy-musty X4 #2 grassy
X4
#4 no odor X2
#3 cucumber X2
Msn.
Fig. 3—Gas chromatograra of volatile odor f A. circinalis. fraction from
14
recorder. An ice-pack placed against the wall of the glass
capillary facilitated condensation of the odorous component.
After approximately 10 microliters of the earthy-musty
component had been collected, the preparative column was
removed from the gas chromatograph and replaced with the
analytical column. Microliter samples of the collected
metabolite were then reinjected into the gas chromatograph
utilizing widely differing temperatures and flow conditions.
If more than one component had been present, theoretically,
resolution into a number of peaks would have occurred. Only
one peak was observed. This technique afforded a semi-
quantitative purity analysis prior to further instrumental
analyses.
- -Other Analytical Instrumentation
Mass spectra of the odorous metabolite were recorded
with a Hitachi-Perkin-Elmer RMU-6E double focusing spectro-
meter. Liquid samples were injected directly into the
heated inlet system with a 10 microliter syringe.
Infrared spectra were recorded by means of a Perkin-
Elmer Model 621 spectrophotometer. The samples were diluted
to 10 per cent with carbon tetrachloride and placed into a
1 microliter NaCl cell and the spectrum recorded.
Nuclear magnetic resonance spectra were recorded on a
Jeolco KH-60 60 MHg spectrometer. Due to the small sample
15
size, a microcell supplied by Nuclear Magnetic Resonance
Specialities, Incorporated was utilized. The solvent was
carbon tetrachloride with one per cent tetramethylsilane
as internal reference.
Results
Mass Culture and Associated Studies
The "series" technique for mass culturing Anabaena
circinalls is shown in Fig. 4. The growth curve for each
vessel is shown in Fig. 5. Once peak exponential growth had
been reached in vessel #1, the air supply was disconnected
from each vessel. The vessels were then allowed to sit for
a period of time prior to harvest. The organism in vessel
#1 was more dense than the medium as evidenced by the organism
settling to the bottom of the vessel. The organism in vessel
#2 was dispersed homogeneously throughout the volume of the
medium. In vessel #3, the organism formed a dense mat or
layer on the surface of the medium. In reference to the
overt buoyancy, vessel #1 was referred to as the "submergent"
culture, vessel #2 as the "homogeneous" culture, and vessel
#3 as the "emergent" culture. Microscopic observation of
the organism revealed that vessel #1 contained filaments
whose cells possessed a large number of very small gas-vacuoles.
The organism in vessel #2 possessed a fewer number of gas-
vacuoles per cell but each vacuole was considerably larger
16
"I 'a <L)
Fig. 4—Series mass culture of A. circinalis
17
'culture vessel #1 culture vessel #2
culture vessel #3
WEEKS
Fig. 5—Growth curves for each vessel of the mass culture
18
than a vacuole in the cells of vessel #1. Vessel #3
contained organism having gas-vacuoles even larger, yet
fewer in number per cell than those of vessels #1 and #2.
An increasing degree of cellular fragility was also noted
when samples of organisms from vessel #1 through vessel #3
were observed under the microscope.
The diameter of the filament spiral of Anabaena circinalis
decreased from cultural vessel #1 through cultural vessel #3
such that the organism in vessel #3 assumed the morphology
of Anabaena flos-aquae i. e,, lacking spirals altogether. A
coloration gradient was also apparent when organisms from
each cultural vessel were compared. Organisms from vessel
#1 possessed a deep blue-green coloration, from vessel #2
a lighter blue-green, and from vessel #3, a yellowish pale
blue-green.
As can be seen in Fig. 6, the biomass increase per unit
time decreased from vessel #1 through vessel #3. The biomass
at harvest, beginning with vessel #1, decreased in the ratio
1.7si.4:1.0. The biomass yield ratio varied directly as the
static pressure ratio of the cultures (1.5:1.0:0.5).
Instrumental Analyses
The infrared spectral (IR) results of the earthy-musty
compound are shown in Fig. 7. Sharp absorption at 3630 cm"1
is suggestive of a hydroxyl group. Absorption in the region
19
10.0
2 O
- 6.0 o 2
>-
u 0
4.0
2.0
<<r
culture vessel #1
culture vessel #2
culture vessel #3
3 4 WE E KS
Fig. 6—Biomass curve for each vessel of the mass culture
3ooo
Frequency (cm'1) Zooo (boo 1400 1200
20
1000 9oo 600
V
Wavelength (p )
Fig. 7—-Infrared spectrum of the earthy-musty odor compound from A. clrcinalls.
21
from 2800 to 3000 cm""1 is indicative of aliphatic C-H stretch.
The possibility of double bonds and aromaticity are precluded
by the infrared spectrum. Therefore, the two degrees of
unsaturation required by the empirical formula can only be
satisfied by a ring system.
The nuclear magnetic resonance spectral (NMR) results
of the isolated odor compound are shown in Fig, 8. The
spectrum is complicated but consistent with the spectrum to
be expected with a ring system wherein a great deal of
shielding would occur. The hydroxyl group is located at
0.9 5 and at an extremely high field position, and due, no
doubt, to steric factors which result in extreme shielding
(Medsker et al., 1968).
High resolution mass spectral (MS) results of the
collected odor component, represented by peak #5 of Fig. 3,
are shown in Fig. 9. The apparent parent-ion peak occurs
at M/e 164 (p-18), representing loss of H2O. The base peak
of the MS occurs at M/e 57. The remaining fragments of
the spectrum are indicative of an aliphatic structure.
The instrumental analyses, when considered collectively,
allowed deduction of the empirical formula ci2H22°* T h e I R
and NMR spectra.7 data are consistent with those of Medsker
et al. (1968) and Marshall (personal communication) for
geosmin (trans-1, 10-dlmethyl-trans-9-decalol; Gerber, 1968),
although the mass spectrum does differ from that reported
by Medsker et al. (1968).
22
, v maanetic resonance spectrum of the earthy-Pia, 8—Nuclear magnetic circinalis . F i g musty odor compound A. c y ^ M L — -
23
80
SO 1 0 0 110 120 130 140 150 160 170
m/e
Fig. 9—Mass spectrum of the earthy-musty odor compound from A- HrrlnaHo
24
Quantitation of the Odor Metabolite
Since the amount of organism which had been steam
distilled in the isolation procedure was known and the final
volume of the concentrate was also known, the amount of odor
metabolite could be easily quantitated, Quantitation was
achieved by collection of known amounts of the odor metabolite
utilizing the capillary technique described. Known volumes of
the odor metabolite were then reinjected into the gas chromat-
ograph and peak area analysis performed. The amount of geosmin
produced by Anabaena circinalis in culture vessel #1 was 0,027
microliters per milligram dry weight of the organism. In
culture vessel #2, the yield of geosmin was 0,013 microliters
per milligram dry weight, and in culture vessel #3, the yield
was 0,0018 microliters per milligram dry weight.
Discussion
The laboratory conditions under which Anabaena circinalis
produced the earthy-musty odor metabolite in maximum concen-
tration were found to be in culture vessel #1 of the series
mass culture. The concentration of geosmin (trans-1,
10-dimethyl-trans-9-decalol) produced in culture vessel #1,
culture vessel #2, and culture vessel #3 was 0,027 microliters
per milligram of dry weight, 0,013 microliters per milligram
of dry weight, and 0,0018 microliters per milligram of dry
weight of organism, respectively.
25
A study of the organism at peak exponential growth
from culture vessel #1 through culture vessel #3 revealed
several characteristic gradients. A decreased growth
gradient, an increasing cellular fragility when observed
under the microscope, an increasing buoyancy due to
increased gas—vacuole volume, a decreased production of
geosmin, a coloration gradient from deep blue-green to a
yellowish pale blue-green,and a gradual loss of the spiral
nature were all symptomatic gradients and tended to confirm
an increasing aberration in physiology.
The series culture technique has much to be said for
it as a potential laboratory method for studying bloom
formation, since the hypothesis that natural blooms develop
in deep water under anaerobic conditions has been advanced
by Fritsch (1945). The series culture technique could be
considered as simulatory of the transitions which occur
during a natural bloom. The only differences being that in
the series culture, all stages (submergent, homogeneous, and
emergent) were present simultaneously at peak exponential
growth.
Geosmin was originally isolated from several species of
actinomycetes (Geber and Lechevalier, 1965}. Rosen et al.
(1967) presented evidence that the substrate cyanophyte,
Symploca muscorum, also produced geosmin. In addition,
26
Medsker et al. (1968) demonstrated that another substrate
form, Oscillatoria tenuis, also produced geosmin, However,
no data have been presented on the nature of volatile
odorous metabolites of frequent bloom-forming planktonic
blue-green algae. Isolation and identification of geosmin
in this group presents stronger evidence that the metabolite
may be primarily responsible for the noxious tastes and
odors which develop in water supplies during blue-green
algal bloom periods.
Summary
1. The laboratory conditions under which Anabaena
circlnalis produced the maximum concentration of geosmin
were found to be in culture vessel #1 of the series mass
culture. Geosmin production was found to be directly
related to biomass yield at harvest. The series mass
culture technique devised for this study offers potential
as a means of studying the transition phase~physiology of
a bloom in the laboratory.
2, The major odorous metabolite (geosmin) was isolated
by steam distilling gram quantities of the organism,
extracting the distillate with petroleum ether, concentrating
the extract, and followed by gas chromatographic analysis
and collection.
27
3, Nuclear magnetic resonance and infrared spectral
results of the odorous metabolite were identical to those
obtained by Medsker et al. (1968) and Marshall (personal
communication) for geosmin (trans-1, 10-dimethyl-trans-9-
decalol). Mass spectral analysis showed the odorous
metabolite to have an apparent parent-ion peak at K/e 164.
(p-18), indicating a loss of 1^0. Infrared spectral analysis
confirmed the presence of the hydroxy1 group (absorption
at 3630 cm"-'-), thus substantiating that the M/e 164 of the
mass spectrum resulted from a loss of from the parent-ion
(182). Methylene C-H stretch occurred in the region from
2800 to 3000 cm"1.
4. The amount of geosmin produced by Anabaena
circlnalis was found to be dependent upon the cultural
state of the organism. Geosmin production, as quantitated
by gas chromatographic peak area analysis, was calculated
to be 0.027 microliters per milligram dry weight of the
organism in cultural vessel #1, 0.013 microliters per
milligram dry weight of the organism in cultural vessel #2,
and 0,0018 microliters per milligram dry weight of the
organism in cultural vessel #3.
CHAPTER II •
A COMPARATIVE STUDY OF SELECTED
ODOR-PRODUCING CYANOPHYTES
Introduction
Two species of the Oscillatoriaceae (Symploca muscoruro
and Oscillatoria tenuis) have been found by Safferman et al.
(1967) and Medsker et al. (1968), respectively, to produce
the earthy-smelling compound, geosmin. This compound was
originally isolated from several actinomycetes by Gerber
and Lechevalier (1965). Later, Gerber (1968) showed geosmin
t o k® trans-It 9-dimethyl-trans-lO-decalol.
During routine isolation of several genera of Cyanophyta
from the natural habitat, several species were observed to
produce the earthy-musty smelling odor of geosmin. In one
instance (September, 1968), a surface bloom of Oscillatoria
sp. developed in Garza-Little Elm Reservoir which serves as
the water supply for Denton, Texas. Coincident with the
bloom, an earthy-musty odor developed in the Reservoir and
in the city tap water.
Purpose
The purpose of this study was to perform a comparative
qualitative analysis on the volatile odorous metabolites
28
29
from four species of earthy-odor producing Cyanophyta in
order to determine whether the odor was due primarily to
one or more constituents.
Materials and Methods
Algae
The algal sample material used in this study was either
cultured in the laboratory or harvested during the natural
bloom incident. The organisms cultured in the laboratory
included #NT68-8 (Oscillatoria sp.), #RT68-20 (Oscillatoria
sp.), and #NT69-2 (Anabaena circinalis). The cultures were
all unialgal and, in addition, #NT68~20 was bacteria-free.
The organism harvested directly from the natural habitat
was designated #NT69-1 and since collection, has been
obtained in unialgal cultute condition.
Culture
Samples of the organism to be cultured were routinely
inoculated into 250 ml. Erlenmeyer flasks containing 125 ml.
of ASM-1 medium (Gorham et al., 1964). In culturing #NT68-20,
aseptic techniques were followed throughout. The flask
cultures were rotated by a rotary shaker at 147 rpm. Light
was continuous and was maintained at 150 ft-c. by cool
white overhead fluorescent lamps. The temperature was
maintained at 24.5 i 1° C. After 12 to 15 days of growth,
the organisms were harvested.
30
Harvest
The sample material of each laboratory cultured organism
was harvested by filtration. Excess moisture was removed by
pressing the "harvest" firmly between two additional filter
pads. The sample was stored at -20° C, until approximately
5.0 grams (wet weight) had been accumulated.
In the instance of #NT69~1, approximately one kilogram
(wet weight) was harvested during the bloom incident by
means of a bottom fauna seive (#40 mesh). The material was
returned to the laboratory and odor fraction preparation
initiated immediately.
Volatile Fraction Preparation
The volatile odorous fractions from the sample materials
were prepared as shown diagrammatically in Fig. 2. The
resulting odor concentrates were analyzed gas chromat©graph-
ically.
Gas Chromatography
An Aerograph Autoprep, Model 705 gas chromatograph,
equipped with a hydrogen flame detector was utilized for
performing the comparative qualitative analyses. The column
packing employed was SE-30, 10 per cent on chromasorb G,
acid washed. The columns utilized were ten feet by three-
eighths inch and ten feet by one-eighth inch. Microliter
31
samples of the odor concentrate from each organism were
injected into the gas chromatograph by means of a 10 micro-
liter syringe.
Results
Gas chromatographic analysis of the odor concentrate
from each organism revealed a common constituent in each.
Under the physical parameters employed (Fig. 10), the common
constituent had a retention time of 18.0 minutes. In each
instance, the peak produced an earthy-musty sensation when
sensed by olfaction during elution. The common peak in each
case matched the retention time and odor of pure geosmin
(Fig. 11). Finally, the common peak also matched the retention
time and odor of the primary odor component of Oscillatorla
tenuis (IUC #428) which had been shown previously (Medsker,
et al., 1968) to be geosmin.
Discussion
The results demonstrate that geosmin is of frequent
occurrence in Cyanophyta. However, what is perhaps more
important is the fact that, in one instance, the compound was
isolated from a bloom-organism in the natural habitat wherein
a taste and odor problem of the earthy to musty variety was
manifest. Isolating the compound from a purified blue-qreen
culture (#NT68-20) dispells any notions that the compound may
result from a symbiotic relation between blue-green algae and
bacteria.
32
CS 2 mm«/in * flame Det.
N "150 cc/min H 1 2 4 cc/min Air, DA "
"270 ̂ /roln XI
Date 12-15-69 Samp. see Fig. 10 below Col. SE-30, 10% 10' X 3/8" Col. Temp. 205^ C In• Temp. 165° C Det. Temp. 240° C
solvent 100-
90-
80-
70-
+ • 60-u 0
50-X 50-u 50-
H-0 40-
-vO 0s- 30-
20-
10 J
earthy-musty XI
1 I 6 8 i r~
10 12 Min.
~r 14
~l r~ 16 18
Fig. 10—Typical gas chromatogram of volatile odor fraction from four species of cyanophyta showing common odor constituent.
33
cs 2 rain./in. Det. flame f N 140 cc/min. H 24 cc/rnin. Air 270 cc/min. DA XI Date . 11-17-69 Samp. qeosmin Col. SS-30, 10% 10' X 3/8"
In, Temp. 165° C Det. Temp. 215° C
100-1
so-
80-
70-
*-60-
0 50-JZ 50-u 50-
»•-0 40-
vO 3fr
20-
geosmin peak
1 2 3 5 6 Min.
1 8
Fig. 11—Gas chromatogram of pure geosmin
34
Summary
1. Geosmin was found to be the primary odor component
in four species of Cyanophyta.
2. In one case, the compound was isolated from algal
material collected during a bloom in the natural habitat
wherein an earthy-musty odor problem was manifest. This
finding demonstrates that geosmin can and does occur in the
natural habitat.
3. Isolation of geosmin from a pure blue-green culture
dispells any notion that the compound may originate as a
result of symbiotic relations between blue-green algae and
bacteria.
CHAPTER III
THE EFFECTS OF GERANIOL AND GEOSMIN ON THE GROWTH
AND DEVELOPMENT OF ANABABNA CIRCINALIS
Introduction
It is noteworthy that several investigations have
demonstrated the occurrence of ethereal oils of isoprenoid
origin in red, brown, and green algae (Katayama, 1962), but
only recently have such metabolites been shown to occur in
blue-green algae (Sirenko and Sakevich, 1967; Medsker et al.,
1968). The cyanopnytes are of particular theoretical and
practical interest in this regard since isoprenoid secondary
by-products (terpenes) have been shown to be stable taxonomic
characters (Harley and Bell, 1967) and since certain
cyanophytes (including Anabaena circinalis) frequently
develop into noxious and odorous water blooms.
Purpose
Since geraniol (a terpene alcohol) and geosmin
(possibly a sesquiterpene which has lost an isopropyl
groupj Gerber, 1968) have been isolated from blue-green
algae (Sirenko and Sakevich, 1967; Medsker et al., 1968)
and since that suggestion has been made that isoprenoids may
36
exert an effect at the genetic level by controlling protein
biosynthesis (Nicholas, 1968), a study was initiated to
determine if the two compounds had any observable effect
on the growth and developmental cycle of Anabaena circinalis.
Materials and Methods
Algae and Culturing-
Unialgal cultures of Anabaena circinalis. maintained in
a nitrate-free modification of ASM-1 medium (Wyatt, 1969),
were the experimental orgamisms. Three days prior to
treatment, a one ml. suspension of the stock culture was
inoculated into each of twenty-seven 225 ml. Erlenmeyer
flasks containing 125 ml. of medium. The cultures were
rotated on a rotary shaker at 147 rpm. The temperature was
maintained at 24.5 t 1° c. while illumination was held at
60 ft-c. by cool white fluorescent lamps. On the third day
of post-inoculation, the cultures had an opitacl density
(O.D.) of 0.03 as measured photometrically by a Klett-
Summerson, Model 900-3, photoelectric colorimeter at 660 mp.
The twenty-seven cultures were then separated for chemical •
treatment into nine groups with three cultures per group.
Chemicals and Treatment
The chemicals utilized in this study were geraniol and
geosmin. Geraniol was obtained from Pflatz-Bauer, Inc.,
37
Flushing, New York. Geosmin was isolated and purified by
preparative gas chromatography from gram quantities of
Anabaena circinalis (Chapter I).
Group I cultures served as controls and, therefore,
received no treatment. Groups II-IV were treated with
geraniol such as to yield a final concentration of
5.4 X 10"~̂ molar, 10.8 X 10""̂ molar, 16.2 X 10*"̂ molar,
~5
and 21.6 X 10 molar, respectively. Groups VI-IX were
treated with geosmin to yield final concentrations of
1.9 X 10"*® molar, 3.8 X 10~® molar, 7.6 X 10""̂ molar, and
15.2 X 10 molar, respectively.
Observations and Measurements
Microscopic observations were made daily. Morphological
variations such as gas-vacuole presence or absence in
vegetative cells, the production of spore chains, and the
production of heterocyst chains were noted,
Heterocyst frequency counts were made on alternate
days beginning day two post-treatment. The method employed
was that of Wolk (1965) except, to qualify for counting, a
filament had to possess at least two heterocysts. The
intervening vegetative cells were then counted and this
expressed as a ratio of one heterocyst to the number of
intervening vegetative cells, A total of forty filaments
were counted from each culture in each group and the count
expressed as an average.
38
Results
In a period of twelve hours after treatment of the
cultures, a dramatic decrease in gas-vacuoles was clearly
evident within each vegetative cell. The degree of gas-
vacuole loss was treatment related and in those cultures
receiving the highest concentration of geraniol and
geosmin, no gas-vacuoles were observed at the twelve-hour
post-treatment observation period. Numerous vegetative
cells were lysed by the treatment as evidenced by the
amount of cellular debris and cellular fragility upon
microscopic examination. The degree of fragility of the
vegetative cells was dependent upon treatment level and
upon its location in the filament in reference to the
heterocyst. The higher the treatment level and the more
remote the vegetative cell was from the heterocyst, the
greater the degree of fragility. The point of rupture
always occurred at the cell wall junction adjacent to the
next vegetative cell wall,
Heterocyst chains (two or more adjacent heterocysts
in a filament) were observed microscopically in Group III
and Group IV cultures. The appearance of heterocyst chains
seemed to be growth-related since it occurred in both groups
at approximately the same O.D. (0,14). Heterocyst chains
were never observed in the geosmin-treated cultures.
39
Heterocyst Frequency Counts
The results of the heterocyst frequency counts in the
geraniol-treated cultures and in the geosmin-treated cultures
are shown in Figures 12 and 13, respectively. Each point on
the graph represents the mean count of three cultures,
Sporulation
Within twenty-four hours after treatment, those
filaments which possessed heterocysts were observed to be
sporulating. In those filaments where heterocysts were
not present, incipient spore chains had developed. These
chains were similar to vegetative cell filaments with the
exception of increased cellular size and nucleoplasm!c
inclusions in the former. Spore germination into hormogones
occurred within one to two days after sporulation in the
geraniol-treated cultures. However, in those cultures
treated with geosmin, spore germination into hormogones
was not observed until day ten in Group VI, day eighteen in
Group VII, and day twenty-five in Group VIII. The resulting
hormogones developed into filaments whose vegetative cells
and heterocysts were smaller in diameter than those of the
"parent filament." This developmental or morphogenetic
cycle occurred several times in each geraniol and geosmin-
treated group, and is reflected as "humps" in the growth
curves (Figs. 14 and 15).
40
.10i
.08-
c D % .06
w X VI O | .04 o> X
.02
Gr. I (no treatment) Gr. II (5.4 X lCr5 M Geraniol) Gr. Ill (10.8 X 10-5 k Geraniol) Gr. IV (16.2 X 10~5 M Geraniol)
V // \>c // \ \ J
~r~
1Q 15 20 Days
Fig. 12—The effect of geraniol on hetemri"0
41
.1(h Gr. I (no treatment) Gr. VI (1.9 X 10""® geosrnin) Gr. VII (3.8 X 10~® geosrnin) Gr. VIII (7.6 X 10~6 geosmln)
.02-
Days
Fig. 13—The effect of geosrnin on heterocyst frequency in A - r*i •{ rss* 1 4 a
42
O
X
o o o
6 -
Gr. I (no geraniol treatment) Gr. II (5.4 X 10~5 M) Gr. Ill (10.8 X Gr. IV _ . . (16.2 XlO-SjO^*
/
Days
Fig. 14—The effect of geraniol on the growth of A. circinalis
43
8-
O
x
o o o
6-
Gr. I (no geosmin treatment) Gr. VI (1.9 X 10-6 m) Gr. VII (3.8X10"6M)
Gr. VIII (7.6 X 1CT6M),
/ /
/
Fig. 15—The effect of geosmin on the growth of A. circinalis
44
Growth
Growth curves for the geraniol-treated groups and for
the geosmin-treated groups are shown in Figs. 14 and 15,
respectively. Each point on the curves represents the mean
of measurements from three cultures.
Discussion
The increased loss of gas-vacuoles and increased
treatment levels of geraniol and geosmin, indicate an
initial surface active action. The site of lysis or rupture,
when observed microscopically, was always found to be at
the longitudinal-transverse cell wall junction. Similar
results, in reference to rupture sites, were reported by
Van 3aalen and Brown (1969) for the marine planktonic
blue-green alga, Trichodesmium erythraeum. Electron
micrographs showed the longitudinal and transverse cell
walls to be tapered at the point of junction.
The formation of heterocyst chains in the geraniol-
treated cultures is difficult to explain since heterocyst
formation and function is presently enigmatic. However,
since the appearance of heterocyst chains occurred at
approximately the same O.D. (0.14), it could be that
heterocyst chain formation is the manifest loss or removal
of growth inhibition.
45
The heterocyst frequency count curves (Figs. 12 and 13)
for the various treatment levels have four components each:
first, an initial decrease in heterocyst frequency! second,
an increasing frequency? third, a plateau frequency; and
fourth, another final decreasing frequency component. When
the control heterocyst frequency count curve is studied, only
three components are found. The missing component is the
initial decreasing component which was observed for the
various treatment levels. Further study reveals that the
lowest value of the initial decreasing component in each
treatment level is inversely related to the level of treatment.
The ultimate value of the increasing component, the plateau, is
also related to the treatment level, hut in a direct sense.
The data illustrate that geraniol and geosmin initially
inhibited heterocyst formation and, later, enhanced heterocyst
formation. The degree of initial inhibition and, subsequently,
the degree of enhancement was dose—dependent.
The developmental cycle of Anabaena circinalis was found
to bet vegetative filament with heterocysts, spore formation,
spore germination, and hormogone formation. The developmental
cycle occurred several times at each chemical treatment level
and was reflected as humps in the growth curves. The obser-
vation that sporulation was occurring within twenty-four hours
in all treated cultures indicates that both chemicals are
effective inducers of sporulation.
46
Growth
In all treatment levels of geraniol and geosmin, the
initial growth rate is seen to be inhibited when compared
to the control (Figs. 14 and 15). This finding was
substantiated by the observation that spore induction
occurred within twenty-four hours post-treatment* The
growth curves (Figs. 14 and 15) for Groups III, IV, VI,
and VII show a "stairstep" increase in growth. This type
of growth resembles that observed for C'hlorella pyrenoidosa
when grown under diurnal illumination (Davis et al., 1953),
However, each hump in this study represents a complete
developmental cycle. No growth curves are shown for Groups
V and IX since no growth had occurred after twenty-five days
and thirty days, respectively. Multiple developmental cycles
occuring in synchrony, have not, to this writer's knowledge,
been reported for a filamentous planktonic blue-green alga, -
although, the effects of various inorganic nitrogen sources
on the developmental morphology of Anabaena doliolum have
been described (Singh and Srivastava, 1968).
The physiological effects of geraniol and geosmin on
Anabaena circinalis appear to be hormonal in nature. In
cultures that have developed a maximum heterocyst frequency
(O.D. between 0,2 and 0.4), treatment to yield a final
concentration of 16.2 X 10"^ molar geraniol or 7,6 X 10"^ molar
geosmin resulted in an almost "pure spore" culture within
47
twenty-four hours in each instance. These results, however,
were not observed if the heterocyst frequency was low prior
to treatment. Geraniol and geosmin serve as effective
inducers of sporulation with geosmin being"effective at much
lower concentrations. The production of geosmin by Anabaena
circinalis (Chapter I), together with the resulting
physiological effects of the compound on the alga, would
indicate the possibility of the existence of an ecological
auto-control mechanism as a means of explaining bloom subsidence,
Additional studies are in progress to investigate the mode of
action of geraniol and geosmin in this alga.
Summary
The treatment of Anabaena circinalis with geraniol and
geosmin resulted in the following effects.
1. Gas-vacuole loss from vegetative cells.
2. Increased cellular fragility with cell lysis always
occurring at the longitudinal-transverse cell wall junction.
3. Induction of spores in those filaments possessing
heterocysts.
4. Initial heterocyst frequency depression, followed
by enhancement. The initial depression was inversely
related to the dose and enhancement was directly related to
the dose.
5. Repetition of the developmental cycle with the
various stages (vegetative filament, heterocyst formation,
48
sporulation, and germination into hormogones) occurring
in synchrony. The occurrence of this repetitive develop-
mental cycle was reflected as humps in the growth curves.
Demonstration of these physiological effects with such
compounds (naturally occuring in the instance of geosmin.
Chapter II) indicates the possibility of the existence of
an ecological auto-control mechanism as a means of explaining
bloom subsidence.
LITERATURE CITED
Allee, w. C., A. E. Emerson, O, Park, T. Park, K. P. Schmidt, 1955. Principles of Animal Ecology, W. B. Saunders Co., Philadelphia, p. 518-528.
Davis, E. A., J. Dedrick, C. S. French, H. W. Milner, J. Myers, J, H, C. Smith, and H. S, Spehr. 1953. Laboratory experiments on chlorella culture at the Carnegie Institution of Washington, Department of Plant Ecology, p, 105-153. In J. S. Burlew (Ed.) Algal Culture. Carnegie Inst. Washl. Publ. 600, Washington, D. C.
Fogg, G. E. 1969. The Leeuwenhoek Lecture, 1968sThe physiology of an algal nuisance. Proc. Roy. Soc. B. 173 *175—189.
Gerber, N. N. 1968. Geosmin from microorganisms is trans-1, 10-dimethyl-trans~9-decalol. Tetra. Lett. 25 *2971-2974.
Gerber, N. N. and H. A. Lechevalier. 1965. Geosmin, an earthy-smelling substance isolated from actinomycetes * App, Microbiol. 13i935-938.
Gorham, P. R. 1964. Toxic Algae, p. 307-336, In D. F. Jackson (Ed.), Algae and Man, Plenum Press, New.York,
Gorham, P. R., J. McLachlan, U. T. Hammer, and W. K. Kim. 1964. Isolation and culture of toxic strains of Anabaena flos-aquae (lyngb. de Breb.). Vehr. Internat. Verein. Limnol. 15s796-189.
Harley, R. M. and M. G. Bell, 1967, Taxonomic analysis of herbarium material by gas chromatography. Nature 213 *.1241-1242.
Kasler, A. D. 1947. Eutrophication of lakes by domestic drainage. Ecology 28s383-395.
50
Jackson, D. D, and J. W. Ellms, 1897, On odors and tastes of surface water, with special reference to Anabaena, a microscopical organism found in certain water supplies of Massachusetts. Tech. Quart. Proc. Soc. Arts 10J410-420,
Katayama, T. 1962, Volatile constituents, p. 467-473. In R. A. Lewin (Ed.), Physiology and Biochemistry of Algae. Acad. Press, New York.
Klein, L. 1962. River Pollution, II. Causes and Effects, Butterworth and Co., p, 125.
Lackey, J, B, 1945. Plankton Productivity of Certain southeastern Wisconsin lakes as related to fertiliza-tion. II. Productivity. Sewage Wks J. 17s795-802.
Mackentheen, K, M., W. M. Ingram, and R. Proges. 1964. Limnological Aspects of Recreational Lakes. Publ. Hlth. Serv. Pubis. Wash. No. 1167, p. 176.
Medsker, L. L., D. Jenkins, and J. F. Thomas, 1968, Odorous compounds in natural water - an earthy-smelling compound associated with blue-green algae and actinomycetes. Environ. Sci. Tech. 2s461~464.
Nicholas, H. J. 1968, The metabolism of isoprenoid compounds, p. 42-50, In Florkin and Statz (Ed,), Comprehensive Biochemistry. Vol. 20. Elsevier Publ. Co.
Olive, E. W. 1918, Blue-green Algae (Cyanophyceae), p. 100-114. In Ward and Whipple (Eds,), Fresh-Water Biology, John Wiley and Sons, Inc,, London,
Palmer, C. M. 1959. Algae in Water Supplies. An Illustrated Manual on the identification significance, and control of algae in water supplies. Publ. Hlth. Serv. Pubis. Wash. No. 657, 88 p.
Palmer, C. M. 1962. Taste and odor algae, p. 18-21, In Algae in Water Supplies, U.S.P.H.S. Div. Water Supp. and Poll. Cont,, Washington 25, D,C.
Pennak, R. W. 1946. The dynamics of fresh-water plankton populations. Ecol, Mcnogr. 16i339-356.
51
Ruttner, F. 1953. Fundamentals of Limnology, Univ. Toronto Press, p. 147 and 152.
Safferman, R. S., A. A. Rosen, C. I. Mashni, and M. E. Morris. 1967. Earthy-smelling substance from a blue-green alga. Eviron. Sci. Tech. 1:429-430.
Schwimmer, D. and M. Schwimmer. 1964. Algae and Medicine, p. 368-412, In D. F. Jackson (Ed.), Algae and Man. Plenum Press, New York.
Sigworth, E. A. 1957. Control of odor and taste in water supplies. J. Am. Water Works Assoc. 49»1507-1521.
Singh, R. N. 1953. Liiruiological relations of Indian inland waters with special reference to waterblooms. Verhr. Int. Verein. Theor. Angew. Limnol. 12s831-836.
Singh, H. N. and B. S. Srivastava. 1968. Studies on the morphogenesis in a blue-green alga. I. Effects of inorganic nitrogen sources on the developmental morphology of Anabaena doliolum. Can. J. Microbiol, 14s1341-1346.
Sirenko, L. A. and A. I. Sakevich. 1967. Terpene formation in blue-green algae. Doklady Akademii Nauk S. s. S. R. 177(4}s959~960.
Trelease, w. 1889. The working of the Madison lakes. Trans. Wis. Acad. Sci. Arts Lett. 7i121-129.
Van Baalen, C. and R. M. Brown, Jr. 1969. The ultra-structure of the marine blue-green alga, Trichodesmium erythraeum, with special reference to the cell wall, gas-vacuoles, and cylindrical bodies. Arch. Mikrobiol. 69:79-91
Vance, B. D. 1965. Composition and succession of Cyano-phycean water blooms. J. Phycol. 1*81-96
Veatch, J. 0., and C. R• Humphrys. 1964. Lake Terminology. Bull. Mich. Agric. Coll. Exp. Stn. Michigan state Univ., East Lansing, Michigan, P. 241.
Vinyard, W. C, 1967. Growth requirements of blue-green algae as deduced from their natural distribution, p. 81-85, In Environmental Requirements of Blue-green algae. U.S.D.I., Northwest Region, Corvalis, Oregon.
52
Welch, P. S. 1952. Limnology. McGraw-Hill Book Co., Inc., New York, p. 260.
Wolk, C. P. 1965. Control of sporulation in a blue-green alga. Devel. Biol, 12?15-35.
Wyatt, Jimmy T. 1969. Selected Physiological and Biochemical Studies on Blue-Green Algae. Unpublished doctoral dissertation. Department, of Biology, North Texas State University, Denton, Texas.
