EPA-600/3-76-103
September 1976
ASPECTS OF PHOSPHATE UTILIZATION
BY BLUE-GREEN ALGAE
by
Thomas E. Jensen and Linda Sicko-Goad
Herbert H. Lehman College of CUNY
Bronx, New York 10468
Project R-800431-03-0
Project Officer
William Miller
Special Studies Branch
Corvallis Environmental Research Laboratory
Corvallis, Oregon 97330
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
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DISCLAIMER
This report has been reviewed by the Corvallis Environmental
Research Laboratory, U. S. Environmental Protection Agency, and
approved for publication. Approval does not signify that the con-
tents necessarily reflect the views and policies of the U. S. En-
vironmental Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommendation for
use.
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FOREWORD
Effective regulatory and enforcement actions by the Environmental
Protection Agency would be virtually impossible without sound scien-
tific data on pollutants and their impact on environmental stability
and human health.. Responsibility for building this data base has
been assigned to EPA's Office of Research and Development and its
15 major field installations, one of which is the Corvallis Environ-
mental Research Laboratory (CERL).
The primary mission of the Corvallis Laboratory is research on the
effects of environmental pollutants on terrestrial, freshwater, and
marine ecosystems; the behavior, effects and control of pollutants
in lake systems; and the development of predictive models on the
movement of pollutants in the biosphere.
This report is an attempt to define the basic physiological phosphate
kinetics in blue-green algae. As such it is an important beginning
towards the understanding of phosphorus dynamics in the aquatic
environment.
A. F. Bartsch
Director, CERL
111
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CONTENTS
List of Figures vii
List of Tables x
Acknowledgements xi
I Introduction 1
II Summary 2
III Conclusions 3
IV Recommendations 5
V Literature Review 6
Biological Occurrence of Polyphosphate 6
Parameters Affecting Active Uptake of Phosphate 7
Microscopy of Polyphosphate Bodies 9
Aspects of Phosphate Metabolism 11
Phosphagen Hypothesis vs. Phosphate Storage 14
Ecological Aspects of Phosphorus in Natural
Waters 16
Nutrient Sources in Aquatic Ecosystems 16
Hydrolysis of Condensed Phosphates 20
VI Material and Methods 23
Growth Curves 23
Dry Weight Determinations 23
Correlation of Dry Weight to Cell Counts 23
Phosphate Assay and Calibration 24
Starvation Conditions 25
"Overplus" or Rapid Uptake 25
Inhibitors and Temperature Studies 25
Total Phosphate Determinations 25
Extraction Scheme for Polyphosphates 26
Electron Microscopy 28
Light Microscopy 28
X-ray Energy Dispersive Analysis 28
VII Results 30
Growth Curves 30
Total Phosphate Present as a Function of
Growth 38
Determination of Optimum Phosphorus Starvation
Time 38
Determination of Uptake Parameters 40
Physical Parameters Affecting Phosphate Uptake
Rates 40
v
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CONTENTS (Continued)
Normal Levels of Phosphorus Containing
Compounds 51
Phosphorus Distributions During Starvation
and Rapid Uptake 69
Hydrolysis of Condensed Phosphates 77
Rapid Uptake of Condensed Phosphates 77
Cell Ultrastructure Under Normal Culture
Conditions 80
Changes in Ultrastructure Associated with
Several Phosphate Concentrations 80
Phosphate Starved Cells 85
Rapid Uptake 89
Energy Dispersive X-Ray Analysis 93
VIII Discussion 97
Growth Curves, Phosphate Depletion and Minimum
Cellular Phosphate Levels 97
Physical Parameters Affecting Uptake Rates 98
Ion Effects 99
Phosphorus Distributions in Cellular Extracts 99
Hydrolysis and Uptake of Condensed Phosphates 102
Electron Microscopy 103
X-Ray Energy Dispersive Analysis 104
IX Bibliography 106
vi
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LIST OF FIGURES
Figure P_age
1 Extraction Scheme for Polyphosphates 27
2-5 Growth Curves of ^. boryanum and Phosphorus
Depletion of Culture Medium 31
6-8 Growth Curves of ^. boryanum and Phosphorus
Depletion of Culture Medium 32
9-12 Growth Curves of _0. tenuis and Phosphate in
the Culture Medium 33
13-15 Growth Curves of _0. tenuis and Phosphate in
the Culture Medium 34
16 Growth Curve of _P. boryanum and Phosphate
Depletion of the Culture Medium 35
17 Total Phosphorus of j>. boryanum as a Function
of Culture Age 36
18 Total Phosphorus of j?. boryanum as a Function
of Culture Age 37
19 Semi-logarithmic Plot of Increase in Total Cell
Phosphorus as a Function of Increasing the
Length of time in Phosphate-free Medium 39
20-21 Increase in Total Cell Phosphorus and Cold TCA
Extractable Phosphorus during Rapid Uptake 42
22 Semi-logarithmic Plot of the Decrease in Total
Cell Phosphorus vs. Uptake time as a Function
of Temperature 43
23 Increase in Total Cell Phosphorus during Rapid
Uptake as a Function of Light Intensity 44
24-26 Change in Total Cell Phosphorus and Phosphate
Depletion of the Medium 45
27 Effect of Metabolic Inhibitors on the Rapid
Uptake Process 46
28 Semi-logarithmic Plot of Total Phosphorus in
Cells after Rapid Uptake 47
29 Phosphorus Left in the Medium during Rapid Uptake 48
30 Total Phosphorus in Cells after Rapid Uptake at
pH 7 and PH 9 49
31 Phosphorus Left in the Medium after 4 hrs Rapid
Uptake at pH 7 and pH 9 50
32-33 Total Phosphorus in Cells during Rapid Uptake -
Ion Effects 52
34 Total Phosphorus in Cells during Rapid Uptake at
pH 9 - Ion Effects 53
vii
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LIST OF FIGURES (Continued)
Figure
35 Total Phosphorus in Cells during Rapid Uptake at
pH 7 - Ion Effects 53
36 Phosphorus Left in the Medium during Rapid Up-
take - Ion Effects 54
37 Phosphorus Left in Medium during Rapid Uptake -
Ion Effects 55
38 Phosphorus Left in Medium during Rapid Uptake
at pH 9 - Ion Effects 56
39 Phosphorus Left in Medium during Rapid Uptake
at pH 9 - Ion Effects 57
40 Total Cell Phosphorus in Cells during Rapid
Uptake without PO^ and K4" 58
41 Total Cell Phosphorus in Cells during Rapid Up-
take without PO, and Ca++ 58
42 Comparison of Total Cell Phosphorus during Rapid
Uptake without PO^ and MgSC>4 59
43 Total Phosphorus in Cells during Rapid Uptake
without PO^, K+ and Ca++ 59
44 Total Cell Phosphorus in Cells during Rapid
Uptake without PC^, Ca4"1" and Mg4"4" 60
45 Comparison of Total Cell Phosphorus during Rapid
Uptake without MgS04 60
46 Total Phosphorus in Cells during Rapid Uptake
without PC^, Mg*4", Ca++ and K+ 61
47 Comparison of Rapid Uptake of Phosphorus without
K+ 61
48 Comparison of Rapid Uptake of Phosphorus without
CaCl, 62
49 Comparison of Rapid Uptake of Phosphorus without
Mg++ 63
50 Comparison of Rapid Uptake of Phosphorus without
CaCl2 64
51 Comparison of Rapid Uptake of Phosphorus without
CaCl2 and MgSO^ 64
52 Phosphorus Left in the Medium during Rapid Uptake
without Mg++ and K4" 65
53 Phosphorus Left in the Medium during Rapid Uptake
without K+, Ca++ and Mg++ 65
54 Comparison of Phosphorus Left in the Medium
during Rapid Uptake without K and Ca4"4" 67
55 Total Phosphorus during Rapid Uptake with Excess
Mg++ 67
56 Total Phosphorus during Rapid Uptake with Excess
¥+ 68
57 Total Phosphorus during Rapid Uptake with Excess
Ca+ 68
58 Semi-logarithmic Graphic Presentation of the
Change in Total Phosphorus 72
viii
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LIST OF FIGURES (Continued)
Figure
59 Semi-logarithmic Graphic Presentation of the
Change in Cold TCA Extractable Phosphorus 73
60 Semi-logarithmic Graphic Presentation of the
Change in Phosphorus Content of the Lipid
Fraction 74
61 Semi-logarithmic Presentation of the Change
in Hot TCA Extractable Phosphorus 75
62 Semi-logarithmic Graphic Presentation of the
Change in Phosphorus Content of the Residue 76
63-65 Hydrolysis of Sodium Tripolyphosphate 78
66 Increase in Phosphorus Containing Fractions
using Sodium Tripolyphosphate 79
67-68 Cell Ultrastructure in Normal Medium 81
69 Cell Ultrastructure in Presence of 100 mg P04/1 82
70-72 Cell Ultrastructure in Presence of 1,000 mg PC>4/1 83
73-76 Cell Ultrastructure in Presence of 1 mg PC>4/1 84
77-79 Cell Ultrastructure of Phosphate Starved Cells 86
80-83 Cell Ultrastructure of Phosphate Starved Cells 87
84-88 Cell Ultrastructure of Phosphate Starved Cells 88
89-93 Cell Ultrastructure during Rapid Uptake 90
94-97 Cell Ultrastructure during Rapid Uptake 91
98-101 Cell Ultrastructure during Rapid Uptake 92
102-103 Scanning Transmission Electron Micrographs 94
104-109 Energy Dispersive X-Ray Analysis of Polyphosphate
Bodies 95
ix
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LIST OF TABLES
Table Page
1 Sources of Nutrients in Natural Waters 17
2 Changes in Cell Size and Weight during
Starvation and Rapid Uptake 40
3 Summary of Phosphate Depletion in the
Medium and Decrease in Total Cell
Phosphorus 66
4 Composition of Fitzgerald's Modified Medium 66
5 Phosphorus Content of Cell Fractions as a
Function of a Phosphate Concentration in
the Culture Medium 70
6 Phosphorus Content of Cell Fractions during
5 Days of Phosphate Starvation and 4 Hours
of Phosphate Uptake 71
7 Peak to Background Ratios of X-Ray Analysis
Data 96
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ACKNOWLEDGEMENTS
The authors thank the following for technical assistance,
Mr. Robert Ayala, Ms. Marian Zucker, Ms. Enid Levine and Mr.
Nelson Lawry.
The authors also thank Mrs. Hortense Levine and Mrs. Helen
Schwartz for typing the draft manuscript. Special thanks are
expressed to Ms. Dorothy Divack for typing the final manuscript.
xi
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SECTION I
INTRODUCTION
A paradox exists in the apparent success of blue-green algae in eutrophic
waters. Blooms of these species often occur when the supply of nutrients is at
its lowest point (Pearsall, 1932). Hutchinson (1973) suggests that a mecha-
nism exists in these organisms which makes them extremely efficient at taking
up phosphorus at very low concentrations. This suggestion is supported by
evidence from Mackereth (1953), Rigler (1956), Stewart and Alexander (1971),
and Lean (1973). The mechanism suggested by Hutchinson is most likely a physi-
ological condition referred to by other workers as "Phosphat-Uberkompensation"
(Liss and Langen, 1962), "Polyphosphate Overplus Phenomenon" (Harold, 1964,
1965; Harold and Harold, 1963; Voelz _et al, 1966), or "rapid uptake" (Shapiro,
1967) and provides for a restoration of phosphorus supply following phosphate
starvation or phosphate limitation. The specific mechanism involved results
in the formation of polyphosphates, long chain, osmotically inert polymers of
inorganic phosphate.
The biochemical nature of polyphosphates, their periodicity and role in
phosphorus metabolism in the cell, and the significance of their presence in
algae common in eutrophic waters require further examination. Difficulties
arise when one attempts to assess the extensive literature available on phos-
phate metabolism. Both environmental and physiological parameters of the up-
take phenomenon described in the literature vary. In addition, no exhaustive
study has been conducted on one test organism.
Consideration of these factors led to studies which were designed to
determine if the polyphosphate overplus phenomenon exists in a blue-green
alga, Plectonema boryanum Gomont. The experiments are an attempt to eluci-
date the effect of the environmental parameters on the physiological and
cytological responses of the alga to conditions of both phosphate limitation
and excess available phosphate.
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SECTION II
SUMMARY
The effects of various external phosphate concentrations on physiological
and cytological aspects of Plectonema boryanum have been studied. _P. boryanum
was found to tolerate a wide range of phosphate concentrations, from 1 to
1000 mg of phosphate per liter. Growth of the alga in these concentrations
was characterized by changes in the subcellular distribution of phosphorus-
containing compounds and in ultrastructural changes which were monitored by
transmission electron microscopy.
Culturing the alga in phosphate-free or phosphate deficient medium led
to general reductions of phosphate in all cell fractions examined, with the
most dramatic decrease in both short and long chain polyphosphates. Cyto-
logically, the phosphate starvation period was characterized by the develop-
ment of areas of medium electron density, and vacuolization resulting from
expansion of intrathylakoidal spaces.
Inoculation of the phosphate-starved algae into a medium containing a
known amount of phosphate led to increases in all phosphorus-containing frac-
tions, particularly the polyphosphates. Increases in both short and long
chain polyphosphates were greater than an order of magnitude. The satisfac-
tion of the "phosphorus debt" was met essentially within an hour. Examination
of the cells revealed that the cells develop polyphosphate bodies in the char-
acteristic areas of medium electron density that develop during phosphate
starvation or phosphate limitation. X-ray energy dispersive analysis of the
polyphosphate bodies confirmed that they are deposits consisting of two major
elements, phosphorus and calcium.
The alga was not able to utilize the condensed phosphate, sodium tri-
polyphosphate, without prior hydrolysis of this compound to orthophosphate.
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SECTION III
CONCLUSIONS
On the basis of experiments described in the main text of this report,
the following conclusions have been derived.
Physiological and cytological aspects of phosphate utilization by the
blue-green alga Plectonema boryanum were studied. It was found that the
external phosphate concentration influenced the distribution of phosphorus-
containing compounds as well as cell architecture.
Culturing the alga in concentrations of 10, 100, and 1000 mg P04 per
liter resulted in increases in the levels of acid-insoluble and acid soluble
polyphosphates. The values reported for 100 and 1000 mg PO//liter were the
same, indicating that the algae were able to assimilate and utilize only
fixed amounts of phosphates. This value was calculated to be 6.5 ug P per
10 cells. Increased external phosphate concentration led to increases in
size and in the frequency of encountering polyphosphate bodies in the cells.
However, cell death and lysis also increased at these higher concentrations.
When the algae were grown in a concentration of 1 mg P0//liter, the
phosphate present in all cell fractions decreased. Cytologically, the cell
was also altered. Large areas of medium electron density developed, and the
area of intrathylakoidal spaces increased.
The changes that were encountered in the cells grown in 1 mg PO^/liter
were similar but reduced, when compared to algae grown in phosphate-free
medium for 5 days. It was determined that cells grown in the absence of ,-
phosphate for five days had total cell phosphorus levels of 0.76 ug per 10
cells. Cells in culture for two months or longer were found to have total
cell phosphorus levels of 0.73 ug P/10 cells. This was determined to be the
medium cell phosphorus level, limiting growth. Transfer of cells from either
of the two culture conditions previously described to a medium containing
phosphate led to an "overplus" phenomenon.
The phosphate overcompensation reaction was characterized by increases
in all cell phosphorus fractions. The most dramatic increase was that of
both soluble and insoluble polyphosphates. These fractions often increased
by more than an order of magnitude. The greatest phosphate uptake occurs
within one hour of transfer of phosphate starved cells into a medium contain-
ing a known amount of phosphate, and is essentially complete at four hours.
Phosphate uptake is light dependent and is inhibited by 2,4-dinitrophenol,
mercuric chloride, and sodium fluoride. It is suggested that the energy
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required for the uptake process is the result of the metabolism of photo-
synthetic products. The total cell phosphorus levels for uptake never in-
creased beyond 18.9 jug P/106 cells.
The phosphate overplus phenonemon was also quite distinct cytologically.
Large polyphosphate bodies developed in the areas of medium electron density.
These areas developed during phosphate-free or phosphate-limited growth. The
areas of medium electron density, and consequently, polyphosphate bodies
developed in five different locations in the cell: 1. In ribosomal areas,
2. intrathylakoidally, 3. in nucleoplasmic areas, 4. in polyhedral bodies,
and 5. in apparent association with strands of DNA. All developmental stages
of polyphosphate bodies can be found during this uptake process.
It was also determined that the alga could not utilize sodium tripoly-
phosphate, a condensed linear phosphate, for the uptake phenomenon. It
appeared that hydrolysis of the condensed phosphate was essential for sub-
sequent utilization by the alga.
X-ray energy dispersive analysis of polyphosphate bodies revealed that
there are two major components of a polyphosphate body--phosphorus and cal-
cium. Magnesium or other elements are not present in amounts that are
greater than background emission. The amounts are below the limit of detecta-
bility as a major component. Traces of an element would probably be lost in
background emission.
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SECTION IV
RECOMMENDATIONS
This investigation was limited to a laboratory study of certain aspects
of phosphate metabolism in selected blue-green algae. It was not possible
during the tenure of this proposal to evaluate the response of a wide variety
of blue-green algae to varying amounts of phosphorus. This aspect should be
examined and should include the organisms responsible for "blooms". Further
work should be carried out to determine if the overplus phenomenon exists in
a wide variety of blue-greens. Work should also be directed toward a determi-
nation of what other factors will induce the cells to exhibit the polyphos-
phate overplus phenomenon.
Now that some basic groundwork has been done it should be imperative to
determine if blue-green algae in nature will exhibit the rapid uptake phe-
nomenon and how this relates to "bloom" production. Investigations should
also be carried out to determine if blue-greens in nature under "bloom" and
"non-bloom" conditions have a store of polyphosphate available. Work should
also be carried out on factors affecting phosphorus release from polyphos-
phate bodies.
From a more basic point of view work should be done to ascertain the
exact nature of the polyphosphate and whether the calcium associated with it
is essential for its formation and subsequent use. It would also be of basic
interest to determine if other ions can replace calcium in the polyphosphate
bodies.
We suggest that lines of research, such as outlined above, be pursued
in future studies to help us understand the complicated relationship between
phosphorus and other factors in the environment on the undesirable growth of
blue-green algae.
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SECTION V
LITERATURE REVIEW
BIOLOGICAL OCCURRENCE OF POLYPHOSPHATE
Polyphosphate has been a subject of controversy for at least twenty-five
years. In 1944, Jeener and Brachet noted a massive accumulation of a baso-
philic substance within yeast cells after the addition of phosphate to a
previously phosphate-starved suspension. Wiame (1947a,b, 1949) and Schmidt
et al (1946) isolated this substance and identified it as polyphosphate.
Thus, granules earlier referred to as "metachromatic" or "volutin" granules
were actually discovered to be deposits of inorganic polyphosphates.
Polyphosphates have been reported to occur in a variety of organisms
from bacteria and blue-green algae to higher plants and animals (Harold,
1966). The occurrence of polyphosphates seems to be related to two distinct
nutritional conditions (Smith_et _al, 1954). The first of these is nutrient
imbalance. Exhaustion of an essential nutrient can result in the formation
of many types of reserve materials in the cell such as poly B-hydroxybutyric
acid, glycogen, polyphosphates, or lipids, which are all osmotically inert
(Voelz et al, 1966). Pesch (1924) showed that Corynebacterium diptheriae
volutin production increased when the amount of growth was limited on glucose-
blood agar medium due to insufficient blood content. Duguid _e_t al (1954) found
in Klebsiella aerogenes that volutin production was increased on inadequately
buffered sugar-containing agar medium. Smith et al (1954) obtained evidence
that volutin formation in Aerobacter aerogjen.es occurred when cultures were
limited by nitrogen or sulfur deficiencies, but not carbon, potassium, or
phosphorus. In a similar manner, Spitznagel and Sharp (1959) reported that
magnesium deficiency interfered with volutin formation, whereas sulfate defi-
ciency promoted volutin formation in Mycobacterium bovis.
The second nutritional condition that can stimulate the formation of
polyphosphate is the restoration of a phosphorus supply following phosphate
starvation. This phenomenon has been referred to as Phosphate-Uberkompen-
sation" (Liss and Langen, 1962) or "polyphosphate overplus" (Harold, 1964,
1965; Harold and Harold, 1963; Voelz .et .al, 1966). The ability of cells
synthetically to accumulate phosphorus was first investigated by Ketchum
(1939) in the marine diatom Nitzschia closterium. He referred to the defi-
ciency as the phosphorus or phosphate debt, and measured this debt by the
amount of phosphate absorbed from the medium by the diatom, or by direct
analysis of the cells. The magnitude of the phosphorus debt was directly
related to the length of time the cells grew in the light in phosphorus-free
medium.
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Jeener and Brachet (1944) found that in yeast cells, basophilia as-
sociated with volutin granules decreased when the cells were grown on a phos-
phorus-deficient medium, and increased when the cells were transferred to a
medium containing phosphorus. Wiame (1947b) demonstrated abundant volutin
synthesis when phosphate-starved Saccharomyces cerevisiae was transferred to
a phosphate-rich medium. This phenomenon has also been studied in such bac-
teria as Caulobacter (Grula £t al, 1954), Klebsiella aerogenes (Duguid, 1948),
Aerobacter aerogenes (Smith et _al, 1954), and Myxococcus xanthus (Voelz et al,
1966).
Blum (1966) studied several parameters of phosphate uptake by phosphate-
starved Euglena gracilis. He reported that many features of this uptake sys-
tem were similar to those of an active transport system. That is, in Euglena,
phosphate uptake requires an energy source, can be limited by dinitrophenol,
is saturable by substrate, and can be competitively inhibited by a phosphate
analog, arsenate. Similar evidence for an active uptake process is presented
by Kylin (1966) in Scenedesmus, by Simonis and Urbach (1963) in Ankistrodesmus
braunii, and by Borst-Pauwels and Jager (1969) in Saccharomyces cerevisiae.
Two exceptions have been reported regarding "luxury consumption" of phos-
phate and involvement of an active uptake process. Butt and Lees (1960) have
reported that phosphorus deficient cells of Nitrobacter assimilate orthophos-
phate from the medium at a slow rate during nitrite oxidation. Whitton (1967),
studying phosphate accumulation by Nostoe colonies, found that softer Nostoc
colonies could accumulate phosphate from the environment by non-active means.
This accumulation of phosphate was reduced by pre-treatment with chelating
agents.
PARAMETERS AFFECTING ACTIVE UPTAKE OF PHOSPHATE
As has been demonstrated many times, the uptake of phosphorus in "luxury
consumption" is a process requiring energy. This uptake can be limited by
physical and chemical parameters such as light, pH, temperature, concentration
gradient, source of available phosphorus, ion effects (Fogg, 1973), and other
factors collectively referred to as a "phosphate-sparing" factor (Shapiro,
1968).
Light stimulation of absorption of 32p labelled phosphate has been re-
ported by Talpassayi (1962) for the blue-green alga Anabaena cylindrica.
Simonis and Urbach (1963) found that pre-illumination of cultures of the
green alga Ankistrodesmus braunii stimulated subsequent assimilation of phos-
phate in the dark. Other reports of dark assimilation of phosphate have been
made in Chlorella by Kanai, Miyachi, and Miyachi (1963), in Anacystis nidulans
by Batterton and Van Baalen (1968), in several algae by Stewart and Alexander
(1971), and Overbeck (1962) who demonstrated a dark "overplus" phenomenon in
Scenedesmus. Light and/or dark uptake of phosphate has been found to exist in
Selenastrum capricornutum (Fitzgerald, 1970) and in Ankistrodesmus braunii
(Kanai and Simonis, 1968). Harris and Riley (1956) suggested that a dark
uptake of phosphate may allow replenishment of phosphorus to phytoplankton
which become phosphorus deficient during the day. Fitzgerald (1970) inter-
prets these findings as a means of nutrient absorption by algae in areas of
low light intensity. The algae could then rise to the photic zone of a lake
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where growth could later take place.
Phosphate uptake rates can be affected markedly by such physical para-
meters as stream current, concentration of other nutrients, and the aquatic
environment itself (lake water vs. laboratory conditions). Fogg (1973) dis-
cusses the influence of motion on uptake of nutrients by planktonic species.
Those organisms which are non-motile achieve changes in concentration gradient
by sinking or by motion of currents. Motile organisms are able to change
their position and can seek out optimal growth conditions; however, they still
may be affected by currents. Schumacher and Whitford (1965) found that in a
variety of alga, stream currents as low as 1-4 cm/sec increased the rate of
phosphate uptake. The concentration of phosphorus in natural waters is usual-
ly low, ranging from about 1-20 ug of phosphorus per liter. These low con-
centrations do not always repress growth. Atkins (1923, 1925) found that the
diatom Nitzshia closterium grew well in cultures until it completely utilized
all the available phosphate. The growth of the diatom in natural waters ap-
peared to be seasonal; the phosphate concentrations become higher in the
winter, accounting for the summer development of phytoplankton. Kuenzler and
Ketchum (1962) showed that Phaeodactylum tricornutum was able to take up phos-
phorus from solutions containing less than 11 nm phosphate, and suggested that
concentration levels of phosphate this low do not depress growth rate. Pilson
and Betzer (1973), studying the phosphate flux across a coral reef, found that
the phytoplankton take up phosphorus at a nearly constant rate, independent of
light quantity, or the magnitude of photosynthetic activity.
The normal low value of phosphate in water has led several authors to
discuss the possibility of a "phosphate-sparing" factor (Shapiro, 1968) that
is, a substance, quite possibly organic, which allows lake water organisms to
respond quite differently to varying phosphate concentration. In 1948, Rodhe
demonstrated that the diatom Asterionella formosa would grow well in lake
water with concentrations of phosphate as low as 0.002 mg/1. This same diatom,
under laboratory growth conditions and in a medium consisting of inorganic
salts in distilled water, would only grow when the phosphate concentration was
0.20 mg/1. Mackereth (1953) investigated the active uptake of phosphate by
this diatom, and found that luxury consumption of phosphate occurred in lake
water supplemented with phosphate. No uptake occurred in phosphate solutions
(30 ul) made up in distilled water. Mackereth drew no conclusions as to why
the diatoms behaved so differently in lake water and in artificial medium.
Shapiro (1968), using a similar system, postulated that in his test organism,
Microcystic aeruginosa, the concentration of inorganic anions was probably the
most important parameter affecting uptake.
Ion effects and pH can also markedly affect uptake of orthophosphate and
distribution into the phosphorus-containing compounds of the cell. Both types
of experiments indicate that fixed charges on either the plasmalemma or cell
wall can prevent an active ion uptake. Ullrich (1972) found that in synchro-
nized cultures of Ankistrodesmus braunii, 32p - labelling is strongly depen-
dent upon the pH of the culture medium. In alkaline ranges and in the absence
of C02, organic phosphates and ATP are labelled most strongly, whereas poly-
phosphate labelling is highest in the acidic range. Using the same organism,
Ullrich-Eberius (1973) reported that maximal rates of phosphate uptake occur-
red between pH 5.5 and 6.5, in agreement with Ullrich. Ullrich-Eberius also
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found that Na+ enhanced phosphate uptake 8 to 9 times in the light and in the
dark. Belsky _et al (1970) also reported a specific Na requirement for phos-
phate uptake in the marine fungus Dermocystidium sp. Ullrich-Eberius and
Simonis (1970) investigated the effect of both sodium and potassium ions on
phosphate uptake, by A. braunii. Again, 0.002M sodium chloride increased
phosphate uptake, whereas uptake was constant over a long period of time in
the presence of 0.002M KC1. The authors, considering these ion effects
coupled with pH dependency, suggested that Ankistrodesmus braunii meta-
bolically transports t^PO^-, but not HPO^ , and that these effects are exert-
ed at the plasmalemma.
Other ion effects have also been reported in the literature. Polyphos-
phate formation in Saccharomyces mellis is almost completely inhibited by
0.5 M KC1 (Weimberg, 1970). Any phosphate assimilated remains as orthophos-
phate. However, the potassium effect is reversible, and Weimberg suggests
that uptake of orthophosphate and subsequent release of polyphosphate are
related to changes in the conformation of the cell membrane, the probable
site of action of the high concentration of K*". Baker's yeast, on the other
hand, seems to have a specific requirement for potassium ions in the process
of phosphate uptake (Schmidt .et .al, 1946). In fact, 0.01M potassium ions
have an enhancing effect on the assimilation of orthophosphate.
MICROSCOPY OF POLYPHOSPHATE BODIES
Many organisms possess cellular inclusions which stain metachromatically
with certain basic dyes (Rosenberg, 1966). These inclusions have collectively
been referred to as metachromatic, volutin, or Babes-Ernst granules (Harold,
1966). The term "volutin" was first used by Meyer (1904) who noticed an ac-
cumulation of distinctive granules in Spirillum volutans. Wiame (1947a,b;
1949) and Schmidt .et al (1946) were the first to identify volutin granules as
deposits of inorganic polyphosphates.
Polyphosphate bodies or granules have long been confused with other cyto-
plasmic inclusions. Two controversies developed based on microscopic studies
and histochemical staining. Early electron microscope studies showed that
many bacteria contained granules which were highly electron scattering, and
had smooth, sharply defined margins, as in Mycobacteria (Lembke and Ruska,
1940; Knaysi et .al, 1951; Mudd et al, 1956), Corynebacterium diphteriae
(Morton and Anderson, 1941; Konig and Winkler, 1948; Bringmann, 1950), Staphy-
lococcus flavocyaneus and Neisseria meningitidis (Knaysi and Mudd, 1943).
Konig and Winkler (1948) first correlated the identity of these electron-
scattering granules with the metachromatically staining volutin granules. This
was accomplished by examining the same stained films by light microscopy and
electron microscopy. Before Konig and Winkler's correlation was made, the
electron-scattering granules were frequently believed to be prokaryotic nu-
clear bodies. This confusion arose out of light microscopy and histochemical
staining, and electron microscopy.
Knaysi and Mudd (1943) and Bringmann (1950) found evidence that the
electron-scattering granules contained deoxyribonucleic acid and believed they
corresponded to nuclear bodies. Similar cytological evidence was presented by
Lindegren (1948) who demonstrated that volutin appeared in previously phos-
-------
phate-starved yeast cells on the "chromosomes" within three minutes.
From a histochemical point of view, the confusion between volutin gran-
ules and "nuclear bodies" is quite understandable. The linear array of
negatively charged phosphate groups in polyphosphate bodies, and the phosphate
backbone of nucleic acids are similar enough to give confusing results in
histochemical staining (Fuhs, 1969). Both polyphosphates and nucleic acids
are basophilic and stain metachromatically with basic dyes. Polyphosphates
are more strongly basophilic, however, and retain a basic dye at a pH as low
as 1.0 whereas nucleic acids destain at pH 3.5 (Fuhs, 1969). Ebel et al
(1958a) have described a technique for staining polyphosphates based on their
ability to form an insoluble lead salt either at pH 3.5, or pH 1.0, depending
upon the chain length of the linear polymer. Polyphosphates of chain length
eight or greater are stained at either pH 3.5 or 1.0, while those with a chain
length of fewer than eight retain a stain only at pH 3.5.
It had also been suggested that the electron-scattering granules in bac-
teria were mitochondrial equivalents. This was based on evidence by Mudd
(1953) and Mudd _et al (1951a,b; 1956) that the electron-scattering granules
stain intravitally with tetrazolium salts and Janus green B. They suggested
that these granules are organized centers of oxidative-reductive activity
like mitochondria of animal cells and, under certain circumstances, are capa-
ble of accumulating volutin. Smith et al (1954) suggested that volutin may
accumulate in more than one kind of cellular structure or inclusion.
In addition to the references already cited, polyphosphate accumulations
as distinct granules have been reported by Rosenberg (1966) in Tetrahymena
pyriformis, by Widra and Wilburn (1959) in Aerobacter aerogenes by Talpasayi
(1963) in several blue-green algae, by Jensen (1968, 1969) in Nostoc pruni-
forme and Plectonema boryanum, by Stewart and Alexander (1971) in several
blue-green algae, by Ebel _et al (1958b) and Keck and Stitch (1957) in a va-
riety of organisms, and by Voelz et al (1966) in Myxococcus xanthus.
Association of polyphosphates with other cellular inclusions has been
reported by several authors. Weimberg and Orton (1965) concluded that the
ortho and polyphosphates of Saccharomyces mellis are located in the protoplast
and occupy a position which is different from that of phosphomonoesterase.
Indge (1968) reported that in another yeast, Saccharomyces carlsbergensis,
polyphosphate was located in the cell vacuole. Polyphosphates deposition in
Myxococcus xanthus was found to vary in relation to the growth of the organism
by Voelz et al (1966). They found three areas of deposition in the cells:
1. Dense granules around polysaccharide inclusions in the cytoplasm when
phosphate in a final concentration of 5x10 M was added to cells grown to log
phase in 5xlO~^M phosphate; 2. Depositions in the cytoplasm which began as
dense strands, subsequently forming tightly wound bodies partially or totally
surrounded by nuclear fibers, in cells which were grown in phosphate-free
medium and replenished with 5xlO~3 or 5xlO"^M phosphate; and 3. Dense strands
scattered throughout the cytoplasm in cells grown for 20 hours without buffer,
and replenished with 5xlO~2M phosphate.
In describing a developmental sequence of polyphosphate bodies in _P.
boryanum, formed under conditions of excess phosphate and continuous light,
10
-------
Jensen (1969) observed that all cytoplasmic inclusions are excluded from the
area of polyphosphate body formation. This evidence is supported by Fuhs
(1958) who demonstrated cytochemically that the polyphosphate bodies of
another blue-green alga, Oscillatoria amoena, contained only polyphosphate.
The most frequently observed images of polyphosphate bodies are those
of round granules, quite electron dense, and not limited by a membrane. The
most thorough descriptions of polyphosphate bodies and their developmental
sequences have been made by Jensen (1968, 1969). These descriptions include
such features as sublimation under high electron beam intensity, also re-
ported by Drews and Niklowitz (1957), Stewart and Alexander (1971), Konig and
Winkler (1948), and Drews (1960). Polyphosphate bodies, due to their dense
nature, often fall out or chip out in sectioning, may compress during section-
ing, shrink under the electron beam, or "smear" in the direction of sectioning
(Jensen, 1968). The general size range of polyphosphate bodies has been re-
ported to be between 0.1 to 2.0 urn (Stewart and Alexander, 1971; Sicko, 1972).
Formation of polyphosphate bodies in Plectonema boryanum, under conditions of
excess phosphate and continuous light, appears to be in the following sequence
(Jensen, 1969): 1. Development of electron-lucent areas in the cytoplasm or
at the cross walls; 2. Increase in size of this area to approximately the
size of a mature, dense polyphosphate body; 3. Development of a porous area of
medium electron density in the electron-lucent area and simultaneous depo-
sition of polyphosphate in the cytoplasm; 4. Penetration of polyphosphate into
the porous structure, resulting in the usual image of a dense granule. This
work has subsequently been confirmed by Stewart and Alexander (1971).
In addition to the descriptions previously cited, several reports have
occurred in which polyphosphate is said to be deposited in morphologically
different structures. Munk and Rosenberg (1969) reported that Tetrahymena
pyriformis deposited polyphosphates in spherical granules which appeared to be
surrounded by membranes. "Electron-scattering alveolar bodies" were reported
by Fisher (1971) in a lichen phycobiont, Trebouxia erici. The appearance of
these vaculolar granules varied with fixation. The polyphosphate-containing
particles of Micrococcus lysodeikticus differed markedly in shape and organi-
zation from the volutin granules described in other microorganisms. Fried-
berg and Avigad (1968) described the electron-dense granules ranging from
40-80 nm in diameter as part of a more complex structure. The polyphosphate
bodies appeared to be organized around a granulated center in a rosettelike
pattern.
Thus it can be seen from the previous discussion that the morphology and
location within the cell of polyphosphate bodies seem to vary in different
organisms, and under different growth conditions.
ASPECTS OF PHOSPHATE METABOLISM
There are two distinct classes of polyphosphates found in cells: "acid-
soluble", polymers which are readily extracted in cold trichloroacetic acid
(TCA), and "acid-insoluble", polymers which are not extracted by cold TCA,
but may be extracted by a short exposure to hot TCA (Wiame, 1949; Krishnan
et al, 1957). Yoshida (1955) first reported the preparation of soluble and
insoluble polyphosphates from yeast, and found that polymerization grades of
11
-------
acid-soluble and acid-insoluble were 10 and 50, respectively. Thus, the
references in the literature are to acid-soluble forms as being short chain
polyphosphates, and acid-insoluble forms as being long chain polymers (Harold,
1966; Terry and Hooper, 1970). Kanai and his associates (1963, 1965) and
Miyachi and Tamiya (1961), were able further to subdivide these classes of
polyphosphates into four fractions, obtained by a modified Schmidt-Thannhauser
method. These fractions, obtained by successive extractions, were classified
as follows: 1. poly-Pi "A" - cold 8% TCA; 2. poly-Pi "B" - cold KOH at pH
9.0; 3. poly-Pi "C" - 2NKOH, Reprecipitable by neutralizing the extract; and,
4. poly-Pi "D" - 2NKOH, soluble after neutralizing the extract.
The physiological and metabolic functions of the acid-soluble and acid-
insoluble polyphosphates also seem to be distinct, and vary as a function of
growth. Wiame (1949) first reported that the acid-insoluble form is meta-
bolically more active in the cell, and that it is rapidly and reversibly
transformed to orthophosphate. Katchman and Petty (1955) substantiated this
evidence in Saccharomyces cerevisiae by demonstrating that the soluble in-
organic polyphosphate fraction maintained a steady-state concentration from
generation to generation, while the insoluble fraction appeared only in the
later stages of logarithmic growth in a nonsteady-state concentration indepen-
dent of the concentration of the soluble polyphosphates. Katchman and Van
Wazer (1954) postulated that the protein-complexing ability of polyphosphates
differing in chain length might determine the different metabolic activities
of the polyphosphates in yeast.
The differences in the amounts of the two classes of polyphosphates
appear to be a function of growth. Early studies by Smith et al (1954) and
Wilkinson and Duguid (1960) demonstrated that Aerobacter aerogenes contains
little or no polyphosphate during exponential growth. A similar system
appears to operate in Corynebacterium xerosis. Hughes and Muhammed (1962)
observed an accumulation of polyphosphate in these bacteria during lag phase
after transfer to fresh medium, a decrease during the exponential phase, and
an accumulation in the stationary phase. Similar results were also reported
by Drews (1968), Mudd et al (1958), and Winder and Denney (1957). As a con-
trast to the above systems, Terry and Hooper (1970) found that short chain
polyphosphates were present in constant amounts in Nitrosomenas europaea
throughout growth and had a negligible turnover rate. Acid-insoluble long
chain polyphosphates decreased upon transfer to fresh medium, then increased
as growth proceeded and remained fairly constant. Terry and Hooper (1970)
suggested that rapid hydrolysis of polyphosphate after transfer to a fresh
medium was triggered primarily by the higher pH of the fresh growth medium.
It seems evident that the accumulation or degradation of polyphosphate
is a function of cell metabolism. Conditions of polyphosphate accumulation
under conditions of nutrient imbalance fall into two distinct patterns -
(a) the "polyphosphate overplus" phenomenon as previously described, and (b)
cessation of nucleic acid synthesis due to exhaustion of an essential meta-
bolite.
Lindergren (1948) first suggested that volutin, which Wiame (1949) had
later identified as metaphosphate, was essential for cell division. Sail
_e_t _al (1956, 1958) reported that in Corynebacterium diphtheriae accumulation
and disappearance of polyphosphate were physiological events related to cell
12
-------
division, with minimal amounts of polyphosphate present after recurrent
periods of cell division. Mudd et al (1958) demonstrated that there was a
competitive relationship between nucleic acid synthesis and the accumulation
of polyphosphate in mycobacterial cells. This relationship was studied using
P and tracing the exchange from labelled polyphosphate to RNA.-P. Earlier
work by Schmidt et al (1956) had demonstrated that in Baker's yeast, acid-
insoluble intracellular polyphosphates were utilized as efficiently as ortho-
phosphate present in nutrient solutions for nucleic acid synthesis. Large
accumulations of inorganic polyphosphates did occur in these yeast cells under
conditions of complete RNA inhibition. Baker and Schmidt (1964) found that in
synchronized cells of Chlorella pyrenoidosa, there was a recurrent decrease in
the polyphosphate level immediately prior to and during nuclear division.
This study also demonstrated that accumulations of total nucleic acid phos-
phorus and acid-insoluble polyphosphate phosphorus exhibited an inverse re-
lationship to each other. Sauer et al (1969) obtained similar results with
the slime mold, Physarum polvcephalum. Inhibition of RNA synthesis in the
••^•^••wmi^—w^v— ii ' • M.P^^(JiM^^wm— •* Q O
plasmodium by actinomycin D resulted in a marked stimulation of J P incor-
poration into polyphosphate. No such correlation was found after inhibition
of either DMA synthesis by 5-fluoredeoxyuridine or protein synthesis by cyclo-
heximide.
Evidence which does not support the hypothesis that polyphosphate-phos-
phorus may serve as a source of RNA-phosphorus comes from the work of Miyachi
and Tamiya (1961). They demonstrated that in Chlorella ellipsoidea the phos-
phorus used in the synthesis of DNA and protein was taken primarily from poly-
phosphates, while that used in the synthesis of RNA., phospholipid, and other
polyphosphates was generally taken from an extracellular phosphorus source.
This work is supported by evidence from Kanai et aJL (1965) who found that
poly-Pi "A" and "C" function as intermediates transferring phosphate from
orthophosphate to DNA and phosphoprotein.
Extensive studies on phosphate metabolism have been made by Harold and
his coworkers both on Aerobacter aerogenes and Neurospora crassa. As has been
discussed before, exhaustion of an essential metabolite results in a cessation
of nucleic acid synthesis. This same situation was found in A. aerogenes.
When growth and nucleic acid synthesis were blocked by depriving the organisms
of sulfate, assimilation of inorganic phosphate from the growth medium result-
ed in a slow accumulation of polyphosphate (Harold and Sylvan, 1963). This
accumulation could be reversed if growth were allowed to resume. That is,
polyphosphate was rapidly degraded, and the phosphate was transferred to the
nucleic acid fraction. Again, evidence was accumulated for an inverse
relationship between nucleic acid synthesis and polyphosphate accumulation
(Harold, 1963, 1965; Harold and Harold, 1965). Using a series of mutants of
A. aerogenes deficient in some aspect of phosphate metabolism, Harold (1966)
has arrived at the following scheme for regulation of polyphosphate synthesis:
1. There is little or no deposition of polyphosphate in normally growing
cells. Synthesis of nucleic acids inhibits polyphosphate synthesis and stimu-
lates polyphosphate degradation; 2. If growth and nucleic acid synthesis are
inhibited by exhaustion of an essential nutrient, polyphosphate degradation is
inhibited. Possibly, the competition for ATP is relieved, and the levels of
polyphosphate kinase determine the amount of polyphosphate accumulated; and,
3. The basis of the overplus phenomenon is elevated levels of kinase. Cells
13
-------
subjected to phosphate starvation are derepressed in the snythesis of kinase.
Thus, exposure to inorganic phosphate results in rapid polyphosphate synthe-
sis.
The second organism investigated by Harold was Neurospora crassa. In
1948, Houlahan and Mitchell observed an accumulation of polyphosphate in vari-
ous mutants of Neurospora crassa (Harold, 1966). Harold (1960) found that
Neurospora contained high levels of polyphosphate, even during logarithmic
growth. Exhaustion of a nutrient in the growth medium resulted in an accumu-
lation of polyphosphate at the expense of RNA. During subsequent starvation,
polyphosphate was degraded for RNA synthesis. Harold (1962) also found that
ATP was a precursor for the overplus phenomenon, but not an intermediate in
the degradation of polyphosphate.
The relationship between nucleic acids and polyphosphates is further
complicated by studies which indicate that ribonucleic acid and polyphosphate
may exist as a complex in several organisms. This complex was first postu-
lated by MacFarlane (1936). RNA-polyphosphate has been demonstrated by Kulaev
and Belozuskii (1958) in Aspergillus niger by Chayen et al (1955) in Torulop-
sis utilis, by Winder and Denneny (1957) in Mycobacteria, by Ebel et al (1958b,
1962) in yeast by Correll and Tolbert (1962, 1964) and by Correll (1965) in
Anabaena and Chlorella, and by Wang and Mancini (1966) in Russel wheat.
Correll and Tolbert (1962, 1964) found that in Anabaena, the complex accounted
for 25-357o of the total phosphorus, and a major portion of the alga's RNA.
However, there was an additional 40-50% of the total phosphorus present as
uncomplexed polyphosphate. The polyphosphate-RNA complex in Chlorella was
more variable; the relative amounts of the complex varied with respect to the
synchronized growth cycle. Wang and Mancini (1966) isolated a RNA-polyphos-
phate fraction from wheat leaves. Leaves fed with 32p phosphate were found
to contain this complex with nearly all of the label in the polyphosphate of
the complex.
It can be concluded from the previous discussion that there appears to
be a correlation between levels of polyphosphate in cells, and the amount of
nucleic acids synthesized. In fact, there might be a direct transfer of phos-
phate between them, coupled with an energy prerequisite. Evidence such as
this has led to the following theories of polyphosphate function.
PHOSPHAGEN HYPOTHESIS VS. PHOSPHATE STORAGE
Phosphagen Hypothesis
Much attention has been paid to the role of polyphosphate as a phospha-
gen. Ennor and Morrison (1958) have defined phosphagens as "those naturally-
occurring phosphorylated compounds which function as stores of phosphate-bond
energy from which phosphoryl groups may be transferred to ADP to form ATP as
a result of enzymatic catalysis." The essential point is the direct formation
of ATP at the expense of the phosphagen. Evidence exists in the literature
both for and against this hypothesis.
Kornberg (1956) and Kornberg et al (1956) described in detail an enzyme
purified from Escherichia coli which reversibly catalyzed the formation of
14
-------
highly polymerized inorganic polyphosphate from the terminal phosphate of ATP,
according to the following equations.
1. xATP -f j (P03)n; ->xADP -f- POs (n+1)
I primerj
2. (P03)n + nADP ^nATP
Polyphosphate synthesized chemically or enzymically is utilized quantita-
tively in the phosphorylation of ADP to ATP. Kornberg (1957) discusses the
significance of this reaction and concludes that when cellular levels of ADP
are high due to metabolic activity, the ADP could be phosphorylated by poly-
phosphate. Yoshida (1955) measured the heat of enzymic hydrolysis of insolu-
ble polyphosphate and found it to be 10 Kcal per P-O-P linkage. Winder and
Denneny (1955), using cell-free extracts of Mycobacterium smegmatis, found
that this extract was capable of utilizing metaphosphate for the phosphoryl-
ation of glycerol in the presence of ATP. Since their experiments were con-
ducted under anaerobic conditions, they concluded that the metaphosphate must
have provided the energy for this phosphorylation. The phototrophic Chloro-
bium thiosulphatophilum appears to synthesize polyphosphate when ATP is formed
in excess of other requirements by photophosphorylation (Cole and Hughes,
1965). The authors also postulate that under conditions where ATP utilization
is high, net breakdown of polyphosphate could occur. Purification and a study
of the properties of the enzyme polyphosphate kinase from Mycobacterium smegmatis
(Suzuki et al, 1972) support these earlier results.
Evidence contradicting the role of polyphosphate as a phosphagen is dis-
cussed in detail by Harold (1966). Two lines of research are responsible for
this interpretation: 1. Harold (1962) has demonstrated that in intact cells
of Neurospora crassa, polyphosphate does not break down if energy generation
is limited or blocked. Similar evidence is provided by Kaltwasser (1962) for
Hydrogenomonas. Polyphosphate breakdown can occur in yeast treated with iodo-
acetate, but the pathway is not through conversion to ATP, (Langen and Liss,
1958; Langen, 1965); and 2. In Aerobacter aerogenes, there is direct evidence
polyphosphate degradation is hydrolytic, resulting in dissipation of the energy-
rich bond (Harold and Harold, 1965). From this evidence, Harold (1966) con-
cludes that the hypothesis of polyphosphate as a phosphagen is not tenable.
Polyphosphate as a Phosphorus Reserve
Much evidence has been presented to support the theory that polyphosphate
is a storage form of phosphorus. As has already been discussed, insoluble
polyphosphate can serve as a source of phosphorus in nucleic acid synthesis,
most often RNA snythesis. Polyphosphate, due to its structure, minimizes
disturbance of osmotic equilibrium in the cell, and thus makes it an attrac-
tive compound for storage in the cell (Voelz _et _al, 1966; Harold, 1966). As
will be discussed in a later section, the concentration of phosphorus in the
environment is usually low. If the scheme proposed by Harold as discussed
previously were operative, cells subjected to low concentrations of phosphorus
would have elevated levels of polyphosphate kinase, the enzyme responsible for
polymerization of orthophosphate. Thus, when the organisms come into contact
with a phosphate source, the organism would be able to accumulate a phosphorus
15
-------
reserve for later synthesis of cellular compounds (Harold, 1966). This theory
is also substantiated by Kaltwasser (1962) who found similar results in Hydro-
genomonas.
ECOLOGICAL ASPECTS OF PHOSPHORUS IN NATURAL WATERS
The role of phosphorus in the eutrophication processes has long been a
controversial subject. Nutrients such as carbon, nitrogen, and phosphorus
are often referred to as "limiting" or regulating nutrients in an aquatic
ecosystem, limiting in the sense that they control photosynthetic producti-
vity. In fact, nutrient concentrations of these and other elements are often
used to classify lakes into either oligotrophic or eutrophic categories
(Rodhe, 1969).
Eutrophication, in the most popular definition, is usually mancentered,
and refers to an enrichment process which is vastly accelerated by man (Hasler,
1947; Likens, 1972). From an ecosystem point of view, eutrophication may be
broadly defined as follows (Likens, 1972): "Eutrophication ... nutrient or
organic matter enrichment, or both, that results in high biological producti-
vity, and a decreased volume within an ecosystem." Likens (1972) also points
out that eutrophication is often considered as a form of pollution, but the
two terms are actually not synonymous.
Phosphorus, especially in the form supplied by effluents containing
synthetic detergents, is usually implicated as the main source of nutrient
enrichment responsible for accelerating eutrophication. This topic, as will
be reviewed, is quite extensive, and yet the role of phosphorus, as it in-
volves organisms in an aquatic ecosystem, is not clearly defined.
NUTRIENT SOURCES IN AQUATIC ECOSYSTEMS
Phosphorus can be found in a variety of forms in water. The sources of
phosphorus-containing compounds are equally diverse (Table 1). It has been
estimated that millions of pounds of phosphates per year are contributed from
these sources (Ferguson, 1968). The phosphate source most frequently cited
as the principle factor causing blooms of algae is synthetic detergent build-
ers, consisting for the most part, of sodium tripolyphosphate.
Evidence exists again, for both sides of this question. An important
factor lies in the definition of a "limiting" nutrient, and changes in the
concentration of this nutrient. Gibson (1971) reviews the concept of nutrient
limitation from the following three definitions: 1. An organism is limited
when it is not growing as fast as it is theoretically able to; 2. A factor
is said to be limiting when it is in such short supply that no growth is pos-
sible; and 3. A factor is not limiting, if when it is increased, no effect
on growth is observed. The third definition is most commonly encountered, and
evidence both for and against phosphate being a limiting nutrient is approached
from this viewpoint.
Phosphorus has long been found to be an important minimum factor for
plant growth in natural waters (Sawyer, 1947, 1952; Ohle, 1953; Fuhs et al,
1972). Thomas (1953) observed that phosphorus and nitrogen were minimum
factors for algal growth. The high phosphorus content of waste waters was a
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CO 4J -H CU 4J
CU 4J > ^4 CO
> O -H CO 0)
i-l pQ ,J nJ PM
CU
CO
-------
key factor in the acceleration of eutrophication above its natural rate. If
the phosphorus demand of the algae had been met, nitrogen replaced phosphorus
as the minimum factor. Fuhs et al (1972) also observed multiple nutrient
limitation in Lake George, New York. They found that nitrogen and phosphorus
could act as limiting nutrients, either simultaneously or alternating with
time and space. With nitrogen limitation in effect, low concentrations of
phosphorus could not be interpreted as indicating phosphorus limitation, be-
cause nitrogen limitation favored luxury uptake of phosphorus. Schelske and
Stoermer (1972) found that in natural phytoplankton assemblages enclosed in
plastic bags in Lake Michigan, phosphorus was the limiting nutrient. This
resulted in lower concentrations of silica in the lake, and replacement of
diatoms with nonsiliceous forms, such as blue-green and green algae. Edmunson
(1961, 1969, 1970, 1972) demonstrated that the phytoplankton population of
Lake Washington (Seattle) was dependent upon the increasing volumes of efflu-
ent from secondary sewage treatment plants. There was a strong correlation
between the abundance of phytoplankton in the summer, and high levels of phos-
phate, but not nitrate or carbon dioxide, in the water during the winter
months. Diversion of the effluents resulted in a decreased phosphate concen-
tration, and a reversal of cultural eutrophication in Lake Washington. In
fact, by the time half the effluent had been diverted, the phosphate concen-
tration started to decrease. Edmunson (1972) then made predictions about the
possible changes that could occur. Since about one-half of the phosphate in
the sewage came from detergents, elimination of all detergents from sewage
should lead to the same results as following diversion of one-half of the ef-
fluent. The lake could then tolerate a much larger human population without
their accompanying detergents. Similar results in determining in situ effects
of added phosphates in natural waters were obtained by Powers et_ _al (1972).
Lakes of varying productivity in both Minnesota and Oregon were studied, and
it was found that phosphorus appeared to be the primary controlling nutrient
in enrichment experiments. They postulated that several lakes could be re-
stored by the removal of phosphorus from municipal waste by advanced treatment
methods. Sonzogni and Lee (1972) also demonstrated that effluent diverted
from a number of Wisconsin lakes resulted in a reduction of phosphorus content
of the lakes, as well as a decrease in the frequency and severity of blue-
green algal blooms. Pitcairn and Hawkes (1973) demonstrated that there was a
general positive correlation between the standing crop of Cladophora and the
phosphorus concentration of several river waters. They confirmed the impor-
tance of phosphorus by showing that the growth of Cladophora in waters upstream
of sewage discharges could be increased to downstream levels by the addition of
phosphorus.
Taylor (1967) reviewed the phosphorus concentrations that limit algal
growth. Sawyer (1952) found that phosphorus concentrations below 0.01 parts
per million severely limit algal growth, while concentrations of 0.05 parts
per million or higher permit profuse growth. Most uncontaminated lakes ,con-
tain between 0.01 and 0.03 parts per million of phosphorus; thus, adding a
relatively small amount of phosphorus to the lakes, to increase the level to
0.05 ppm, is likely dramatically to increase productivity (Sawyer, 1952).
This view is not supported by a number of workers. Kuentzel (1969) re-
viewed the carbon dioxide-phosphate controversy and concluded that CC>2 and
organic matter supporting growth of bacteria were responsible for massive
19
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algal blooms rather than phosphorus. He suggested that a symbiotic relation-
ship between bacteria and algae could open up another possibility for reduc-
tions in algal growth via control of bacteria. The line of succession sug-
gested was: organic matter -> bacteria >C02 >algae. This review
was attacked heavily by Shapiro (1970) who pointed out the fact that C02 is
not limiting, and in spite of high concentrations of C02, algae are not able
to grow unless there is a sufficient supply of phosphorus.
Mitchell (1971) found that the eutrophication potentials of a phosphate-
containing detergent and two phosphate-free detergents were not significantly
different. He pointed out that elimination of phosphates in domestic sewage
due to detergents would still result in a concentration of 3-4mg P/l, using
conventional primary water treatment. He did not consider the use of activated
sludges. His estimates of phosphate concentrations in domestic sewage are in
agreement with those of Hudson and Marson (1970), and Marson (1971). However,
Marson reported up to 90% phosphorus removal by any of the precipitation pro-
cesses currently available, and 8070 by luxury consumption by activated sludge.
Levin and Shapiro (1965) demonstrated that luxur^ uptake of orthophosphate
by sludge organisms occurred in the absence of growth. Uptake was dependent
upon the dissolved oxygen and pH of the mixed liquor, with maximum uptake occur-
ring in the pH range 7.0-8.0. Shapiro (1967) later determined that settling of
activated sludge in settling ponds of water treatment plants led to anoxia of
the organisms, and subsequent release of the phosphate. Yall et al (1970) sup-
ported the concept of biological luxury uptake by activated sludges using both
radioactive tracers and inhibitor studies.
Jenkins et al (1973) studied the environmental impact of detergent builders
in California waters, and found that detergents accounted for about 35% of the
total phosphorus released to surface waters. They suggested that the algae in
the California waters did not appear to be phosphorus-limited, so that control
of phosphorus input would do little to control eutrophication. If phosphate
control were necessary, they suggested point-source elimination for a particu-
lar area and not solely detergent phosphate elimination.
The controversy of phosphate-removal still exists. The problem is further
complicated by the fact that snythetic detergent builders are condensed phos-
phates, usually sodium tripolyphosphate (Davis and Wilcomb, 1967, 1968). The
studies discussed so far were concerned with utilization of orthophosphate in
natural waters by phytoplankton. Thus, two more problems are involved: 1. The
natural hydrolysis of condensed phosphates in natural waters, and 2. Uptake
and metabolism of condensed phosphates by phytoplankton.
HYDROLYSIS OF CONDENSED PHOSPHATES
Synthetic detergent builders, condensed phosphates, appear in sewage efflu-
ents and receiving waters. These compounds, under various conditions, have a
tendency to react with water and ultimately form orthophosphate. Engelbrecht
and Morgan (1959) found that sodium tripolyphosphate, and tetrasodium poly-
phosphate were subject to degradation in natural waters, the hydrolysis rates
varying with the water sample. They also indicated that biological life exert-
ed an effect on the rate of degradation, since filtration of raw water decreased
20
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the rate of degradation.
Clesceri and Lee (1965a,b) studied the rates of hydrolysis of condensed
phosphates in non-sterile and sterile environments. They found that pyro-
phosphate and tripolyphosphate were apparently not as available a phosphorus
source as orthophosphate. Using both unialgal and axenic cultures of Chlo-
rella, the condensed phosphate compounds were completely hydrolyzed in a short
time by the unialgal cultures, but not by the axenic cultures. They attributed
the increased hydrolysis rates to the presence of non-algal microorganisms in
the unialgal culture. Clesceri and Lee (1965b) also showed that the rate of
hydrolysis of condensed phosphates was higher by several orders of magnitude
in sterile lake water and algal culture media than in distilled water at a
similar pH and temperature. Hydrolysis rates of both pyrophosphate and tripoly-
phosphate were highest in those solutions containing the highest concentrations
of calcium ion. When comparing these rates to those determined in the presence
of microorganisms under similar conditions (Clesceri and Lee, 1965a) it was
found that enzymatic processes controlled the aqueous environmental chemistry
of the condensed phosphates.
Clesceri and Lee (1965a) summarized the studies on the factors that influ-
ence the rate of condensed phosphate hydrolysis as follows:
Factor
A. Temperature
B. pH
C. Enzymes
D. Colloidal gels
E. Complexing cations
F. Concentration
G. Independence of
hydrolysis in a
mixture of phos-
phates
Effect on rate
5 6
10 -10 faster from freezing to boiling.
Rates of hydrolysis of all condensed phosphates
are higher in acidic media; tripolyphosphate hydro-
lysis can be base-catalyzed, but it is most stable
in the pH 9-10 range.
Divalent cations in combination with enzymes ac-
celerate hydrolysis, especially magnesium; adaptive
enzymes, usually phosphatases, are produced by many
organisms.
Hydrated oxides of iron, cobalt, nickel, aluminum,
and rare earths accelerate hydrolysis.
pH effect is absent when there are no cations;
calcium increases hydrolysis rates more than sodium.
Hydrolysis of condensed phosphates are first order
processes; the rates are proportional to the con-
centration.
Hydrolysis of different species in the same dilute
solution proceeds independently.
The data presented by Clesceri and Lee (1965a,b) were later substantiated
21
-------
by Davis and Wilcomb (1967, 1968). Cultures of several green algae, grown
in nutrient medium with phosphate concentrations close to those expected in
sewage, were capable of degrading polyphosphate to orthophosphate. The rate
of orthophosphate assimilation was dependent upon the environment. Axenic
cultures of the algae demonstrated hydrolytic ability, a greater hydrolytic
capacity existing in moving systems than in static ones. Davis and Wilcomb
(1968) also demonstrated that several genera and species of blue-green algae
were able to utilize condensed phosphates, and, in fact, return them to the
aqueous environment during certain growth phases. Again, uptake of the con-
densed phosphates by the blue-greens was greater in moving columns than in
static cultures.
It has already been suggested (Clesceri and Lee, 1965a) that microbial
activity is responsible for the degradation of condensed phosphates to a much
greater extent than other environmental parameters. This microbial activity
appears to be the result of phosphatases, enzymes which catalyze the release
of bound phosphates. Hydrolysis of tripolyphosphate results in a unit of
pyrophosphate and a unit of orthophosphate, whereas hydrolysis of pyrophos-
phate yields two units of orthophosphate (Clesceri and Lee, 1965a) . Kornberg
(1956) isolated tripolyphosphatase, pyrophosphatase, and trimetaphosphatase
from yeast cells. Eppley (1962) found that orthophosphate was released when
living pieces of Porphyra were incubated with several condensed phosphates and
ATP. Eppley (1962) indicated enzymatic catalysis of these compounds, and also
concluded that both calcium and magnesium were essential for hydrolysis.
The presence of adaptive phosphatases has been observed in a variety of
organisms. Galloway and Krauss (1963) found an adaptive pyrophosphatase
associated with the cell wall of Chlorella. Overbeck (1961a,b) found inter-
cellular phosphatase activity in Scendesmus quadricauda when the substrates
were condensed or organically bound phosphates. A number of correlations have
been made numerous times and in a variety of organisms between the levels of
alkaline phosphatase and the amount of phosphate in the growth medium. Alka-
line phosphatase appears to be induced when the external phosphate source is
limiting in E. coli (Torriani, 1960), B. subtilis (Cashel and Freese, 1964),
Vibrio parahaemolyticus (Sakaguchi et al, 1972), Anacystis nidulans (Reichardt,
1971), and Anabaena flos-aquae (Bone, 1971), and a variety of algae, (Fitz-
gerald and Nelson, 1966). Torriani (1960) suggested that it is a means of
obtaining phosphate from organic phosphate when the supply or concentration
of orthophosphate becomes limiting in the medium.
22
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SECTION VI
MATERIALS AND METHODS
GROWTH CURVES
The test organism was chosen after series of growth curves in various cul-
ture media were determined. The two blue-greens initially selected were Plecto-
nema boryanum Gomont (Indiana Culture Collection No. 581) and Oscillatoria te-
nuis Ag. (Indiana Culture Collection No. 428), obtained from the Starr Culture
Collection (Starr, 1964). This selection was based on their ability to grow
well in a defined medium, Modified Fitzgerald (Fitzgerald et al, 1952; Zehnder
and Gorham, 1960). Growth curves for the algae were determined by inoculating
a known dry weight of the algae into sterile culture tubes containing 20 ml of
Modified Fitzgerald's medium. The tubes were then placed in a Sherer-Gillette
growth chamber adjusted to: 1. 500 ft-candles of illumination (5330 lux) from
incandescent and fluorescent sources; 2. 25°C; and, 3. an alternating 12 hour
day/night cycle. Growth was monitored for a period of 28 days as a function of
increase in dry weight per unit volume. Stock cultures were also maintained
under these conditions for one month, and subsequently placed under 200 ft-
candles of illumination. Transfers to fresh medium were made from cultures
which were between two and four months old.
DRY WEIGHT DETERMINATIONS
Dry weight of the samples was found to be the most consistent and easiest
method for determining the growth of the algae. Both blue-greens tested are
filamentous and have sheaths which make dispersal and rupture of filaments into
individual cells difficult for counting or spectrophotometric analysis. Milli-
pore filters, type HA with a pore diameter of 0.45 p.m predried by heating to
100°C for 24 hours, and weighed after cooling to determine the dry weight.
Known volumes of algal suspensions were then syringed sequentially ten times
each through 14, 18 and 22 gauge sterile disposable needles, and then passed
through the predried Millipore filters. After drying the filter under an
incandescent lamp, the filters were then dried again under the previously de-
scribed conditions, and weighed when cool. The difference in the weight of
the filters was taken to be the dry weight of the algae per unit volume fil-
tered. Normally a 10 to 20 ml aliquot containing between 200 and 1000 mg algae
per liter was filtered.
CORRELATION OF DRY WEIGHT TO CELL COUNTS
Although dry weight analysis was chosen to be the method for quantita-
ting the algal suspensions, correlations were made between actual cell number
23
-------
and dry weight. This was accomplished by extensive syringing of the cultures
through 22 gauge needles and recording the following information using a hemo-
cytometer and reticule: 1. number of algal filaments per unit volume of cul-
ture; 2. length of individual filaments; and, 3. number of cells per filament.
In this manner, the number of cells per unit volume was determined, and cor-
related to a dry weight analysis of that culture. Analysis of cell number,
dry weight, and average cell size were made for logarithmic phase cultures,
phosphate starved cultures, and cultures at the end of the four hour rapid up-
take period.
PHOSPHATE ASSAY AND CALIBRATION
Phosphate depletion of the medium was also monitored during all growth
determinations. This was accomplished by testing the growth medium without
the algae. In all cases, the algae were removed by Millipore filtration as
previously described. A known volume of the filtrate was diluted volumetrical-
ly so that the spectrophotometric absorbance values were in the range 0.3 to
0.7 whenever possible. The two tests used for the determination of orthophos-
phate were the stannous chloride method (American Publ. Health Assoc. 1965)
and the single solution method of Murphy and Riley (1962).
The method selected as the most reliable was that of Murphy and Riley
(1962). The orthophosphate determinations are colorimetric tests; the absorb-
ance of the phosphomolybdate complex formed is a function of orthophosphate
concentration in solution. The colored complex formed during the Murphy-Riley
determination is stable for a period of 24 hours, and there is less inter-
ference due to arsenic or salts. The stability of the colored complex formed
in the stannous-chloride method is considerably less. Forty ml of the solution
to be tested were placed in a 50 ml graduated cylinder. Eight ml of the mixed
reagent, and two ml of glass distilled water were added so that the final vol-
ume of the solution was fifty ml. The solution was then mixed thoroughly, and
color development was allowed to proceed from at least 10 minuffis up to a peri-
od of 1 hour. Percent transmittance was read directly and converted to an
absorbance value. All readings were taken at 880 nm on a Spectronic 20 spectro-
photometer equipped with infrared sensitive phototubes and filters. Calibra-
tion curves were used to determine the sensitivity of the test. The absorbance
value divided by the slope of the calibration curve gave a direct reading of
phosphorus concentration as phosphate. All phosphate determinations were car-
ried out in glassware which was acid washed and used only for these tests.
Calibration curves were determined in the following manner: A standard
phosphate solution containing 0.1757 grams of potassium dihydrogen phosphate
per liter was prepared. This solution contains 40 mg P (as phosphate) per
liter. Dilutions of the stock solution were made to the following concentra-
tions:
80 pg P/l (3.2 jig P/40 ml)
160 jug P/l (6.4 ug P/40 ml)
320 jug P/l (12.8 ug P/40 ml)
400 ug P/l (16.0 ug P/40 ml)
640 jag P/l (25.6 ug P/40 ml)
The percent transmittance values were determined, and converted to absorbance.
24
-------
The values were measured at 880 run in 2.56 cm cells. Beer's law is obeyed in
this concentration range, and the method gives an excellent reproducibility.
STARVATION CONDITIONS
Cells of Plectonema boryanum were grown in Modified Fitzgerald's medium
containing 8 - 10 mg PO^ per liter for 14-17 days at 25dC, 500 ft-candles of
illumination, and an alternating 12 hour day/night cycle. To induce phosphorus
starvation, cultures were harvested aseptically by centrifugation in a Sorvall
refrigerated centrifuge at 21°C and 12,100 xg for 10 minutes. The medium con-
taining phosphate was decanted, and the cells were washed 3 times in sterile
medium free of phosphate. After the final centrifugation, the cells were re-
suspended in phosphate free medium and then placed under the original environ-
mental conditions. Optimal starvation conditions were determined by prolonging
starvation up to a length of 12 days.
"OVERPLUS" OR RAPID UPTAKE
Luxury storage of phosphate was induced by starving the algae of phosphate
for 5 days, and then inoculating into medium containing a known amount of phos-
phate. This phosphate concentration was varied in the range 0.1 to 112 mg PO^
per liter. The rapid uptake was monitored in one or more of several ways:
1. increase in dry weight of the algae; 2. depletion of phosphate from the medi-
um; 3. increase in total phosphate in the cells; 4. increase in various phos-
phate-containing fractions; or, 5. light or electron microscopy of the samples.
INHIBITORS AND TEMPERATURE STUDIES
For uptake studies involving inhibitors or temperatures other than 25sC,
the algae were treated in the following manner. At the end of the starvation
period, the algae were pelleted by centrifugation and resuspended in medium
containing no phosphate and containing the appropriate concentration of inhibi-
tor. The algae were then placed under the appropriate growth conditions and
incubated with the inhibitor for a period of 1 hour prior to the addition of
phosphate in the normal uptake manner. The three inhibitors tested were 2,4-
dinitrophenol (5 x 10"%), mercuric chloride (1 x 10"%), and sodium fluoride
(Ix 10"^M). Uptake studies involving two different temperatures were treated
somewhat differently, depending on the temperature. For uptake at 37*C, the
algae were first acclimated to that temperature for a period of one month,
then logarithmic phase cells were starved in the normal manner. This was pos-
sible since the algae grow well at this temperature. Uptake at low tempera-
tures involved the growth of the algae at 25°C, and preincubation of the algae
at 4 C for 1 hour prior to uptake. This procedure was followed because there
is virtually no growth at this low temperature.
TOTAL PHOSPHATE DETERMINATIONS
Batterton and Van Baalen's (1968) modification of the Menzil-Corwin (1965)
potassium persulfate (K2S20g) digestion was used for the assay of total phos-
phorus levels in the cells. A known aliquot of washed cell suspension contain-
ing no more than 1000 mg algae per liter was placed in screw cap culture tubes
containing 0.5 grams of potassium persulfate. The tubes were then sealed
25
-------
loosely and autoclaved at 121 C for thirty minutes. The clear contents of
the tube were transferred quantitatively and assayed for orthophosphate by the
Murphy-Riley technique. Total phosphorus was reported as ug P per ug dry
weight algae.
EXTRACTION SCHEME FOR POLYPHOSPHATES
The extraction scheme chosen was Harold's (1960; 1963) modification of
the Schmidt-Thannhauser extraction scheme for nucleic acids (1945). Both tri-
chloroacetic acid (TCA) and perchloric acid were tested initially. TCA was
found to be more reproducible, and was chosen subsequently for all extractions.
A flow chart of this scheme is presented in Figure 1.
Cold TCA Extraction
Washed algal suspensions were extracted twice with 5 ml of cold (4°C) 5%
TCA. The supernates were pooled along with the distilled water wash of the
residue. This supernate was assayed both for orthophosphate and total phos-
phate. In all cases, the residue was separated from the supernate by a centri-
fugation in a VWR MSE GT 2 centrifuge with a swinging bucket rotor at 3280 rpm
(1745 xg) for 10 minutes.
Lipid Extraction
The residue of the cold TCA extraction was then extracted with 5 ml of
absolute ethanol at room temperature for 30 minutes, followed by extraction in
5 ml of ethanol-ethyl ether (3:1). The mixture was boiled for 1 minute, and
left to stand at room temperature for 20 minutes. The two supernates were
pooled along with a distilled water rinse of the residue and assayed both for
orthophosphate and total phosphate.
Hot T CA Extra ct ion
The lipid-free residue was then dried by heating the test tubes in a 70°C
water bath, and then extracted twice with 5 ml portions of 5% TCA warmed to
70 C at 15 minute intervals. The supernates were pooled along with a distilled
water rinse of the residue and assayed both for orthophosphate and total phos-
phate. The residue remaining after this extraction was then digested with
potassium persulfate as previously described, and then analyzed for orthophos-
phate content.
Separation of Nucleic Acids
200 mg of sulfuric acid washed Norit A was added per 5 ml of extract.
Nucleic acids were then eluted with alcoholic ammonia from the charcoal, and
either analyzed for total phosphorus concentration, or the extinction at 260 nm
was determined.
For all total extractions, the total phosphate content of the algal suspen-
sion was determined before equivalent aliquots were fractionated. In this man-
ner, percent recovery could be calculated. Secondary precautions were also
taken by total phosphate determinations on residues after each extraction, and
26
-------
WASHED CELL
SUSPENSION
COLD TCA
EXTRACTION
|5ml.of5XTCAat4°C.
J2 changes, 30min./change
I Super note ] | Residue 1
orthoP total P total P
LIPID
EXTRACTION
7\
K»XEtOH.30min..210C.
EtOH-EthylEmer(3:l)boil
1 min., room temp. 20 min.
Supernote | [ Residue |
/\ ^™~^T
orthoP total P total F
HOT TCA
EXTRACTION
A
5n
2<
5ml. of 5XTCAat70°C,
2 changes, ISmin./change
Supernote | I Residue j
/\
orthoP total P
total P
Figure 1. Flow chart of the extraction scheme for polyphosphates.
27
-------
by processing triplicate samples in all cases.
ELECTRON MICROSCOPY
Algae were harvested from cultures grown under the various culture con-
ditions previously described, and prepared for electron microscopy in the fol-
lowing manner. Algae were pelleted from the original medium by centrifugation
at 1745 xg. The supernatant medium was discarded and the algae resuspended in
Modified Kellenberger according to the method of Pankratz and Bowen (1963).
The fixative employed was 1% OsO^ in Michaelis buffer of pH 6.2 for 3 hours at
room temperature. The algae were then dehydrated in a graded ethanol series
and embedded in Epon 812 according to the method of Luft (1961).
Sections approximately 500 A thick were cut with a DuPont diamond knife on
a LKB Ultrotome III, and collected on clean 300 mesh copper grids. The sections
were post stained with saturated uranyl acetate in methanol (Stempak and Ward,
1964) for 10 minutes or with lead salts (Reynolds, 1963), separately or in com-
bination with uranyl salts, and examined in an Hitachi HU HE electron micro-
scope operating at 75 kv. Pictures were taken on Kodak Contrast Projection
Plates and developed with D-19.
LIGHT MICROSCOPY
Algal suspensions were stained for polyphosphates by the method of Ebel
et. _al (1958a) and Jensen (1968). This staining procedure employs the ability
of lead salts to complex with polyphosphate and remain stable at low pH values.
The algae were initially fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer
adjusted to pH 6.2 for a period of 1 hour at 4"C. The algae were then rinsed
five times in 0.1 M cacodylate buffer, pH 6.2, and incubated in 20% lead ni-
trate, adjusted to pH 3.4 with acetic acid, for 4 hours at room temperature.
After the incubation in lead nitrate, the algae were washed thoroughly five
times with distilled water, then placed in 1% ammonium sulfide at room tempera-
ture for \ hour. They were again washed with distilled water, and then examin-
ed with a Zeiss light microscope (Jensen, 1968). Pictures were taken either
with Polaroid 55 PN film, or Tri-X film and developed in diluted Microdol X.
X-RAY ENERGY DISPERSIVE ANALYSIS
Sections, approximately 0.5 jum in thickness, were cut on glass knives and
mounted on 200 mesh copper grids. The Sections were then examined and analyzed
in a JEM-100B analytical electron microscope fitted with a scanning attachment,
a * 60^ side entry goniometer stage, and either EDAX or KEVEX energy disper-
sive X-ray analysis systems having 180 eV resolution. For orientation purposes,
the sections were examined at either 80 or 100 KV in the transmission or scan-
ning modes. The areas to be analyzed were then selected, and the accelerating
voltage reduced to 40 KV for elemental analysis. The beam spot can be reduced
to less than 50A in diameter, so the beam was in all cases reduced to the size
of the polyphosphate body, or a comparable area if no polyphosphate body was
present.
All analyses were carried out in one of 2 ways: either total counts of
emitted X-rays were read for a fixed time period, or total counts, an arbitrary
28
-------
number, and regardless of time, were read. The fixed time was usually 100
seconds, and the fixed count was usually 20,000.
Samples examined were treated in three ways: 1. Glutaraldehyde - fixa-
tion and Epon embedding; 2. Osmium - fixation and Epon embedding; and, 3.
Osmium - fixation and Durcupan embedding. Durcupan is a water-soluble embed-
ding medium. All samples examined were starved of phosphate for five days,
and fixed after four hours of rapid uptake.
29
-------
SECTION VII
RESULTS
GROWTH CURVES
The test organism, Plectonema boryanum, was chosen after a series growth
curves were determined both for _P. boryanum and Oscillatoria tenuis. P.
boryanum is able to grow well with phosphate concentrations ranging from 0.8
mg PO^ to 1100 mg PO^ per liter (Figures 2-8). The growth of the alga was
also monitored simultaneously for phosphate depletion of the medium by the
alga. In all phosphate concentrations monitored, the phosphate remaining in
the medium was reduced substantially the first day after transfer, rose slight-
ly on the third or fourth day, and then decreased gradually through the re-
mainder of the 28 day period (refer to figures 2-8).
The growth curves varied somewhat with the different concentrations. All
growth curves were characterized by an increase in dry weight, often more than
four-fold, on the first day after transfer. This initial peak then diminished
during the lag period. The growth of Plectonema boryanum is sigmoidal; lag,
log, stationary, and decline phases are recognizable. The length of the lag
and log periods seem to be a function of the phosphate concentration in the
medium. Longer log periods of growth are encountered at higher phosphate con-
centrations (Figures 6 and 7). Lag period appears to be longer at lower phos-
phate concentrations.
This data led to routine culture of P. boryanum at either 8 or 10 mg of
phosphate per liter. At these concentrations, log phase occurs approximately
between 13 and 17 days of culture (Figures 5 and 6). These are also normal
phosphate levels for synthetic media.
Oscillatoria tenuis was also cultured at the same phosphate concentrations
as _P. boryanum, but with quite different results, (Figures 9-15) _0. tenuis did
not grow well in phosphate concentrations above 10 mg PO^ liter (Figures 14 and
15). At 1000 mg PO^7!, _0. tenuis increased in dry weight 2 days after transfer.
However, the culture was not able to survive at this high phosphate concentra-
tion for more than 4 days (Figure 15). The phosphate depletion from the medi-
um was similar to that of J?, boryanum (Figures 9-15). Plectonema boryanum was
then selected for all subsequent experiments due to its ability to tolerate a
wide range of phosphate concentrations.
Figure 16 demonstrates the growth of the alga at 37~C. This rate was
determined so that the alga could be acclimated to that temperature for uptake
studies. By raising the temperature 12'C above room temperature, the growth
rate was approximately doubled so that log phase occurred at 7 days. The 10 mg
30
-------
4 8 12 16 20
Days in Culture
28
Figures 2-5.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Growth curves of Plectonema boryanum and phosphate depletion of
culture medium. All growth curves were determined at 25"C, 500
ft-candles of illumination, and a 12 hour alternating day/night
cycle. Growth is represented by circles and phosphate depletion
by squares.
1 mg PO^/ liter culture medium
2 mg PO^/ liter culture medium
5 mg PO^/ liter culture medium
8 mg PC>4/ liter culture medium
31
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1150 _g
noo a.
1050 ™
1000 «
950
900
8 12 16 20 24
Days in Culture
28
Figures 6-8.
Figure 6.
Figure 7.
Figure 8.
Growth curves of Plectonema boryanum and phosphate depletion of
medium. All growth curves were determined at 500 ft-candles of
illumination, 25"C, and 12 hour alternating day/night cycle.
Growth is represented by circles and phosphate depletion of the
medium by squares.
10 mg PO,/ liter
100 mg PO^/ liter
1000 mg P04/ liter
32
-------
no
90
70
50
I 30
•o
g 130
E 100
Q> 70
± 40
N 10
V
§> '7°
"5 130
^ 90
£ 50
X 10
"O
0) 90
S 70
50
30
10
• /
\A x-7
fv \ v^»
rv* >c •
» \ Xs*. • • • _ • • — •—
\' •
^ 1O
/
1
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•^^*
/
• /**
9 •'
I
12
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J\..^ *\
I _• • B *~-i i ,^_
" •-•-•—•!._
0.6
0.5
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0?
0.2
1.6
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50
40
30
20
10
7.5
6.0
4.5
3.0
1.5
E
Tj
E
k
±
s
•5
^
a
M
JZ
01
%
8 12 16 20 24
Days in Culture
28
Figures 9-12. Growth curves of Oscillatoria tenuis and phosphate of the culture
medium. All growth curves were determined at 25°C, 500 ft-candles
of illumination, and a 12 hour day/night cycle. Growth is repre-
sented by circles and phosphate depletion by squares.
Figure 9. 1 mg PO// liter culture medium
Figure 10. 2 mg PO^/ liter culture medium
Figure 11. 5 mg PO^/ liter culture medium
Figure 12. 8 mg PO^/ liter culture medium
33
-------
200
150
E 100
TJ
0) 50
E
^m
*• 100
^
S 75
0)
O
JJ> 50
"5
•
*• 25
* 100
•o
•
yft ~f c
w» 75
50
25
13
1 / ^^"^^v
1 /* •
1 y
•1 •
/ T\ /
h • • • H • •••
r 14
\
\
"\ -
• \"^ •_•-•_
\ /
7x« •
/ \
./* — •• *x»^ 0-
15
1
|\
\
10.5
9.0
7.5
6.0
4.5
3.0
1.5
110
100
90
80
70
60
50
1150
lf\Cf\
1050
950
•o
0)
E
».
-------
E
3
TJ 200
0)
E 180
0>
IZ 160
S
0>
0 140
. 120
A 80
16
10
8 E
TJ
0)
S
0)
Q.
0
Q.
24 6 8 10
Days in Culture
12
14
Figure 16. Growth curve of Plectonema boryanum and phosphate depletion of
the culture medium. Growth conditions were 10 mg PO, / liter
culture medium, 37°C, 500 ft-candles of illumination, and an
alternating 12 hour day/night cycle. Growth is represented by
circles and phosphate depletion of the culture medium by squares,
35
-------
52
48
44
40
36
28
N 24
a.
0)
* 20
16
12
8
17
\
17 25 33 41
Weeks in Culture
49
Figure 17. Total phosphorus Content of Plectotiema boryanum as a function of
culture age. The distribution is for cultures maintained at 25"C,
and under the conditions as listed for Figures 3-9.
36
-------
14
13
12
11
0) 10
•^
0)
* 9
X
"O 8
7
6
5
4
3
E
X
Q.
V)
O
Q.
6)
18
6 10 14 18 22
Days in Culture
26 30 34
Figure 18. Total Phosphorus of _P. boryanum as a function of culture age.
Cultures were maintained at 37°C.
37
-------
of phosphate initially present in the medium was depleted after 1 day of cul-
ture, and remained so for the 14 day period.
TOTAL PHOSPHATE PRESENT AS A FUNCTION OF GROWTH
The initial increase in dry weight of the alga upon transfer to fresh medi-
um was of particular interest. It was initially thought to be an artifact, but
it was also accompanied by a tremendous loss of phosphate from the medium and
was present during all growth determinations. The total cell phosphorus was
then measured for cultures of the alga between 1 and 51 weeks old. These re-
sults are presented in Figure 17. P. boryanum, inoculated into fresh Modified
Fitzgerald medium containing between 7 and 10 mg PO^ per liter, reaches a peak
in total cell phosphorus at approximately 1 month of culture. This value is
53 ug P per mg dry weight algae (Figure 17). The cell phosphorus then declines
and begins to attain a constant value at about 2 months of culture, this lower
limit of phosphorus concentration being approximately 4 ug P per mg dry weight
algae (Figure 17). Thus, transfers to fresh medium were always made from cul-
tures which had reduced total cell phosphorus levels.
Similar values for total cell phosphorus levels were also determined for
the 37°C cultures (Figure 18). It was found that the highest levels occurred
on the seventh day after transfer of the cultures. This value was 14 ug P mg
dry weight algae. The total cell phosphorus then declines to approximately 3.5
ug P per mg dry weight algae at the end of 34 days in culture. The lower values
for the total cell phosphorus are similar for both the 25 and 37 cultures, while
the maximum values are considerably higher in the 25°C cultures (Figures 17 and
18).
DETERMINATION OF OPTIMUM STARVATION LENGTH FOR RAPID UPTAKE FOR ALL THE
FOLLOWING EXPERIMENTS
Logarithmic phase cells were used for all following experiments. Cells in
this metabolic state were chosen because the amount of polyphosphate present
should be at its lowest level (refer to Aspects of Phosphate Metabolism Section
of Literature Review). Since the length of time necessary to deplete the cell
of any phosphorus reserves was not known, a series of experiments were set up
to determine the effects of phosphate starvation on rates of phosphate uptake.
Cells taken from logarithmic phase were inoculated into phosphate-free medium
for 3, 5, 11, and 12 days. The cells were then reinoculated into medium con-
taining a known amount of phosphate, and the cells were then analyzed for total
phosphorus content. These results are presented in Figure 19.
Phosphate uptake is represented by increase in total cell phosphorus for
these experiments. Virtually no uptake occurred after 3 days of starvation
(see legend to Figure 19 for environmental parameters). After 5 days of starva-
tion, total cell phosphorus at the end of the uptake period increased from ap-
proximately 5 to 50 ug phosphorus per mg dry weight algae. Prolonging the star-
vation period to 12 days had no significant effect on the uptake rate. Star-
vation of the algae for 11 days and subsequent inoculation into a lower phos-
phate concentration (10 mg P04 per liter as opposed to 92.50 mg PO, per liter
as above) resulted in uptake, but with reduced magnitude. Thus, 5 days of in-
cubation in phosphate-free medium at 25°C, 500 ft-candles of illumination, and
38
-------
,6'
4>
(A
3
Q.
O
Q.
d)
io3
19
o
1 2 3
Uptake Time (hours)
Figure 19. Semi-logarithmic plot of increase in total cell phosphorus as a
function of increasing the length of time in phosphate-free
medium (starvation). All uptakes were at 25°C, and 500 ft-candles
of illumination. Closed squares represent 12 days of starvation
and subsequent inoculation into medium containing 92.51 mg PO^/
liter.
Closed Circles represent 5 days of starvation and uptake from
92.51 mg P04/ liter.
Open Squares represent 11 days of starvation and uptake from 10 mg
PO^/ liter.
Closed Triangles represent 3 days of starvation and uptake from
10 mg PO^/ liter.
39
-------
a 12 hour alternating day/night cycle was chosen to be the minimum time length
necessary to induce phosphate starvation.
DETERMINATION OF UPTAKE PARAMETERS
After the determination of starvation conditions, it was necessary to
select parameters to monitor for the uptake of phosphate. The first two physi-
cal parameters considered were the dry weight of the algae and loss of phosphate
from the medium (refer to Figure 21). Orthophosphate in the medium was essen-
tially depleted at the end of the 4 hours of uptake. During this same period
of time, the dry weight of the algae increased. Studies correlating cell num-
ber and dry weight analysis of normal, starved, and uptake cells indicate that
the dry weight of the cells decreases during starvation and subsequently in-
creases during rapid uptake, but not to the levels of normally grown cells
(Table 2).
TABLE 2. CHANGES IN CELL SIZE AND WEIGHT DURING STARVATION AND RAPID UPTAKE
Cell Size (um) Dry Weight(mg) Average Wt. Cell Cells/Mg Dry Wt.
Normal 4.01 + 1.93
Starved 2.87 -t 0.73
Uptake 2.80 ± 9.69
330
60
200
1.98 x 10"7 rag/cell
2.20 x 108
7.48 x 10"8
5.5 x 106
The average cell size decreased during starvation and uptake, indicating
unusual and/or rapid cell division. During the starvation period, the average
weight of the cell decreased by an order of magnitude. This value increased
during uptake, but not to the value of a normal cell. These values were ob-
tained by counting 300 filaments averaging 8 cells per filament, and taking dry
weight measurements of the culture. For most cultures, 1 mg of dry weight cor-
responds to 5.5 x 10" cells.
Figure 20 demonstrates that during the four hour uptake period, the larg-
est increase in total cell phosphorus and cold TCA extractable phosphorus oc-
curs at 1 hour. The total cell phosphorus increases from about 8 to 13 ug P/mg
dry weight and the cold TCA extractable phosphorus increases from 1 to 7 ug P/mg
dry weight algae. This uptake then begins to level off, and is virtually com-
plete at four hours. It was for these reasons that uptake was usually monitored
for a four hour period.
PHYSICAL PARAMETERS AFFECTING PHOSPHATE UPTAKE RATES
Temperature
Rapid uptake of orthophosphate by the algae was monitored at three dif-
ferent temperatures, 4, 25, and 37°C. The 25 and 37°C uptakes were carried
out on algae acclimated to those respective temperatures as previously described.
Since the algae did not grow at 4CC, they were grown at 25 , and chilled for 1
hour prior to inoculation into a phosphate-containing medium. These results are
presented in Figure 22. This graph demonstrates that there was little uptake at
4 C, and a definite reduction in total cell phosphorus after 2 hours of incuba-
40
-------
bation at this temperature. The uptake response at 37 is essentially the
same as that of 25°C. This is unlike the growth rate response at 37°C, which
was a doubling.
For all rapid uptakes at different light intensities, the algae were in-
cubated during the starvation period at 300 ft-candles of illumination and then
incubated at different light intensities for the uptake. Variations in light
intensity were accomplished by placing the culture flasks at different levels
from the light source in the growth chamber, or in the case of total darkness,
by covering the flasks entirely with aluminum foil. Figure 23 shows the results
of these experiments. In total darkness or at 100 ft-candles, there was little
uptake of orthophosphate, as expressed by increase in total cell phosphorus.
The total cell phosphorus increased when the algae were exposed to orthophos-
phate at 500, 1000, and 2000 ft-candles of illumination. This increase was
directly related to the light intensity. The greatest increase in total cell
phosphorus for all light intensities occurred within the first hour of uptake
(Figure 23).
Effect of Phosphate Concentration on Uptake
Monitoring the phosphate depletion from the medium led to studies which
were designed to test the maximum amount of phosphate that could be utilized
by the algae. It was found that the algae could essentially deplete the medium
of phosphorus if given limited amounts (Figures 21, 24, and 25). These uptakes
were carried out in medium containing 10 mg PC>4 (3.26 mg P) per liter or less.
By increasing the amount of orthophosphate available in the medium to 92.51 mg
per liter, the algae removed more phosphate from the medium, and the total cell
phosphorus of the algae also increased (Figure 26). These results are summarized
in Table 3.
Effects of Inhibitors on Uptake
For all studies of inhibitors, the algae were pre-incubated with the in-
hibitor for one hour prior to the addition of phosphate. Figure 27 illustrates
the results of these experiments. There was virtually no uptake when 1 x 10~3
sodium fluoride, 1 x 10"^ mercuric chloride, or 5 x 10" 3 2,4-dinitrophenol were
used as inhibitors. Of the three tested, sodium fluoride was the least effec-
tive. The upper curve in Figure 27, an uptake with no inhibitors present, under
otherwise identical conditions, is plotted as a reference curve.
Phosphate Uptake from Starvation Period with .01 m% PO^/l
Figures 28 and 29 show that even when cells are starved for 5 days in cul-
ture medium with .01 mg PO^/l a rapid uptake is induced in the cells. The rate
and amount of uptake is similar to that of cells starved in PO^ free medium.
pH Effect on Uptake
Figures 30 and 31 show that uptake is very pH dependent. Very little phos-
phate is taken up at pH 7 but at pH 9 there is considerable uptake. When starved
cells at pH 9 are placed in medium containing 100 mg P04/1 they can reduce the
41
-------
21
19
17
I'5
«*
1,13
O>
•S 11
r
oi> 9
s
£ 3
1
3.5
~ 30
i
UJ
= 1 5
05
0
20
O—
Cold TCA Extract
21
Medium P
1000
800
600
400
200
1234
TIME (HOURS)
Figure 20. Increase in total cell phosphorus and cold TCA extractable phos-
phorus during rapid uptake at 25"C, 500 ft-candles of illumination
and 10 rag PC>4/liter.
Figure 21. Increase in dry weight of the algae and depletion of medium phos-
phorus during rapid uptake as described for Figure 20.
42
-------
101
.2*
"Z
-o
•
O)
10'
0
O.
0
a.
0)
,63
22
0
Uptake Time (hours)
Figure 22. Semi-logarithmic plot of the increase in total cell phosphorus
vs. uptake time as a function of temperature.
Triangles represent uptake at 25^0.
Squares represent uptake at 37JC.
Circles represent uptake at 43C.
43
-------
102
1 2 3
Uptake Time (Hours
Figure 23. Increase in total cell phosphorus during rapid uptake as a function
of light intensity. The graphs from top to bottom, represent 2000,
1000, 500, 100, and 0 ft-candles of illumination.
44
-------
100
80
60
40
20
12
10
8
6
4
X28
tft
3
JJ 12
0
°". 8
2
4
25
26
A-
2.0
1.5
1.0
0.5
0
1 2 3
Uptake Time (hours)
3 .2
•o
0)
2 E
0)
3
3
w
30
i.
0)
29 =
283
27-
25
24
a
(A
0
Figures 24-26.
Figure 24.
Figure 25.
Figure 26.
Change in total cell phosphorus and phosphate depletion of
the medium as a function of orthophosphate concentration in
the medium. Total cell phosphorus is represented by open
symbols, and phosphate depletion of the medium by closed
symbols.
6.58 mg PO^/ liter culture medium
10.13 mg P04/ liter culture medium
92.51 mg P04/ liter culture medium
45
-------
.5'
27
JJ>
"Z
(A
D
i.
O
t
0
Q.
O)
,o3
0
Uptake Time (hours)
Figure 27. Effect of metabolic inhibitors on the rapid uptake process. Semi-
logarithmic plot of increase in total cell phosphorus vs. time.
Variation of phosphate levels at time 0 can be explained by the
standard deviations encountered during sampling.
Open Circles represent a normal uptake.
_ o
Closed Circles represent 1 x 10 M sodium fluoride
_ r\
Closed Triangles represent 1 x 10" M mercuric chloride
_ O
Closed Squares represent 5 x 10 M 2,4-dinitrophenol
46
-------
o>
X
k.
X
o.
0)
40
30
20
10
28
o
1234
Uptake Time (hours)
Figure 28. Semi-logarithmic plot of total phosphorus in cells after rapid
uptake in 10 mg TO./I. Cells were starved at 500 ft-candles,
75°F for 5 days in medium containing 0.01 mg P04/l. Medium
replenished daily. Rapid uptake done at 500 ft-candles at 75 F.
Open and closed circles represent separate experiments.
47
-------
10
8
-
0)
E
a!
o
a.
29
o
1234
Uptake Time (hours)
Figure 29. Phosphorus left in the medium during rapid uptake by cells which
were starved of phosphorus for 5 days at 500 ft-candles, 75°F in
medium containing 0.01 mg PO^/1. Medium replenished daily.
and closed circles represent separate experiments.
Open
48
-------
30
•; 50
£ 40
x
a.
"O
0)
E
Q.
0)
30
20
10
0
1234
Uptake Time (hours)
Figure 30. Total phosphorus in cells after rapid uptake at pH 7 in 10 mg
PO./l (•) and 100 mg P0,/l (O). Rapid uptake done at pH 9 in
10 mg PC>4/1 (•) and 100 mg P04/l (D). Cells grown and starved
of phosphorus under normal culture conditions. Semi-log plot.
49
-------
no
90
31
-o-
•o-
70
I30
•5
o
£
i.
-------
level more than 10 mg PO^/l.
Uptake From Distilled Water—Ion Effects
Figures 32-39 show the effect of ions in distilled water on rapid uptake.
When the phosphorus left in the medium is monitored it appears in several cases
that considerable uptake has occurred. However, when total cell phosphorus is
determined little uptake is revealed. This is probably due to some sort of
differential trapping of the phosphate in the sheath under these conditions.
Thus it would not be present in the culture medium alone and could be washed
out during preparation of the samples for a total phosphorus determination.
As can be seen from this data at a pH of 7 virtually no uptake occurs with
any of the ions tested (Figures 32 and 33). At pH 9 potassium, calcium, sodi-
um and magnesium either alone or in combination with one other ion seems to be
necessary for uptake (Figure 34). However, in no case does the uptake equal
that of the complete medium. Figure 35 shows that with sodium, calcium and
magnesium present the greatest uptake is found with various combinations of
these ions and the greatest uptake with all present.
Effect of Starvation of Phosphate and Ions
Figures 40-54 show the effect of phosphate and ion starvation on the up-
take of phosphorus in the absence of certain ions. These results indicate that
as long as potassium, calcium or magnesium are present either alone or in combi-
nation the rapid uptake will occur. Some of the data however indicate that
calcium may be of special importance (Figures 41, 50 and 53). Magnesium does
not seem to be critical for uptake (Figure 42). Table 4 shows the concentra-
tion of the various ingredients in Fitzgerald's modified medium.
Effect of High Ionic Concentration During Starvation and Uptake
Figures 55-57 show the effect of a high ionic concentration during star-
vation and during rapid uptake. Figures 55 and 56 demonstrate that a high
ionic concentration of mg and K have no effect on total amount of phosphate
assimilated. However, calcium in high concentrations enhance the uptake of
phosphorus (Figure 57).
NORMAL LEVELS OF PHOSPHORUS-CONTAINING COMPOUNDS IN PLECTONEMA BOR"¥ANUM
After preliminary data were obtained about the "overplus" phenomenon in
P. boryanum, studies were conducted to determine the distribution of phosphorus
in various extracts. Nucleic acids as determined in each of these fractions,
and determined for whole cell digests was no greater than 10 mg P per mg dry
weight in any case, which is considerably less than any fraction reported. It
is for this reason that they are not included in the data.
_P. boryanum was grown for 14 days to logarithmic phase under normal cul-
ture conditions in Modified Fitzgerald containing various concentrations of
phosphate. The algae were then extracted as previously described, and the ex-
tracts were measured for total phosphorus concentration. These results are
presented in Table 5. Average values for triplicate samples, varying no
51
-------
5 *
x
•6 20
10
32
23
TIME [hours]
«. 40
f 30
*
•o 20
a
E
<- 15
10
33
1 2 3
TIME [hours]
Figure 32. Total phosphorus in cells during rapid uptake by cells starved
for 5 days in PO^ free modified Fitzgerald's medium at a pH of 7.
Uptake done at 500 ft-candles, 75°F, pH 7 (adjusted with HC1) in
300 ml of distilled water containing IT ( • ), K+ and Ca44" ( A ),
K+ and Mg44" ( • ) and K4", Ca44" and Mg44" ( V ). Ions added at
same concentration as in Fitzgerald's modified medium. Semi-log
plot.
Figure 33. Total phosphorus in cells during rapid uptake by cells starved
for 5 days in P04 free modified Fitzgerald's medium at a pH of 7.
Uptake done at 500 ft-candles, 75SF, pH 7 (adjusted with HC1) in
300 ml of distilled water containing Na4" ( • ), Na+ and Ca*+ ( A ),
Na4" and Kg44" ( • ), and Na4", Ga-H- and Mg44" ( y ). Ions added in
same concentration as in Fitzgerald's modified medium except Na
was kept at the same level as K4". Semi-log plot.
52
-------
4. 4O
• 30
X
r*°
«* 15
a.
o>
a
10
34
2 3
TIME [hours]
°
* 30
•o
.
15
10
35
2 3
TIME [hours]
Figure 34. Total phosphorus in cells during rapid uptake by cells starved
for 5 days in PO^ free modified Fitzgerald's medium at a pH of 9.
Uptake done at 500 ft-candles, 75°F, pH 9 (adjusted with NaOH) in
300 ml of distilled water containing K+ ( O ), K+ and Ca44" ( A ),
K + and Kg44" ( D ) and K4", Ca44- and Mg++ ( V ). Ions added in
same concentration as in Fitzgerald's modified medium. Semi-log
plot.
Figure 35. Total phosphorus in cells during rapid uptake by cells starved
for 5 days in P04 free modified Fitzgerald's medium at a pH of 9.
Uptake done at 500 ft-candles, 75°F, pH 9 (adjusted with NaOH) in
300 ml of distilled water containing 10 mg PO/./1 and Na+ ( O )
Na+ and Ca4* ( A ) Na+ and Mg44 ( D ), and Na*, Ca44 and Mg44 ( V )
Ions added in same concentration as in Fitzgerald's modified medi-
um except Na4" was kept at the same level as IC^. Semi-log plot.
53
-------
12
1O
8
E
a
•*m
•o
0)
E
0)
3
0)
-------
12
10
I
Hi
o>
0)
9
8
D>
37
23
TIME [hours]
Figure 37. Phosphorus left in the medium during rapid uptake by cells starved
for 5 days in P(>4 free modified Fitzgerald's medium at a pH of 7.
Uptake done at 500 ft-candles, 75aF and in 300 ml of distilled
water containing 10 mg PCWl and Na+ (
-------
110
'•5
4)
oj 3
5
?4
38
TIME [hours]
Figure 38. Phosphorus left in the medium during rapid uptake by cells starved
for 5 days in PO^ free modified Fitzgerald's medium at a pH of 9.
Uptake done at 500 ft-candles, 75'*F, pH 9 (adjusted with NaOH) in
300 ml of distilled water containing 10 mg PO^/1 and K ( O ),
K+ and Ca^ ( A ), K+ and Mg44- ( D ), and ^, Ca^ and MS** ( v )•
Ions added in same concentration as in Fitzgerald's modified
medium.
56
-------
12
1O
0)
8
0)
3
39
Figure 39.
01234
TIME [hours]
Phosphorus left in medium during rapid uptake by cells starved
for 5 days in PO^ free modified Fitzgerald's medium at a pH of 9.
Uptake done at 500 ft-candles, 75°F, pH 9 (adjusted with NaOH) in
300 ml of distilled water containing 10 mg PO^/1 and Na+ ( Q ),
Na+ and Ca^ ( A ), Na+ and Mg"14 ( D ), and NJI , Ca++ and Mg^ (v).
Ions added in same concentration as in Fitzgerald's modified medi-
um except Na"*" was kept at the same level as K^".
57
-------
0)
*»
X
L.
•o
0)
E
s
0)
a.
40
30
20
15
10
40
1 2 3
TIME [hours]
0)
k
•o
O)
E
s
o.
0)
a.
40
30
20
15
10
41
1 2 3
TIME [hours]
Figure 40. Total cell phosphorus in cells during rapid uptake by cells
starved for 5 days in modified Fitzgerald's medium without P04
and K*. Total cell phosphorus during rapid uptake from complete
medium ( A ) and total cell phosphorus during rapid uptake from
medium lacking id" ( • ) . Nal^PO/^.I^O was used as the phosphorus
source. Semi-log plot.
Figure 41. Total cell phosphorus in cells during rapid uptake by cells
starved for 5 days in modified Fitzgerald's medium without PO^
and Ca"^. Total cell phosphorus during rapid uptake from com-
plete medium ( A ) and total cell phosphorus during rapid uptake
from medium lacking Ca** ( • ). Semi-log plot.
58
-------
40
X
•o
0)
E
s
a
o>
a.
30
20
15
10
42
123
TIME [hours]
«
x
0)
g 20
S
o>
43
01234
TIME [hours]
Figure 42. Comparison of total cell phosphorus during rapid uptake of phos-
phorus after 5 days of starvation in medium without phosphorus and
MgSO^. Total cell phosphorus during rapid uptake from complete
medium ( A ). Total cell phosphorus during rapid uptake from medi-
um without MgSO^ ( • ). Semi-log plot.
Figure 43. Total phosphorus in cells during rapid uptake by cells starved for
5 days in modified Fitzgerald's medium without PO^, K*" and Ca"^".
Total cell phosphorus during rapid uptake from complete medium (A)
and total cell phosphorus during rapid uptake from medium lacking
id" and Ca++ ( • ). Nal^PO^.l^O was used as the phosphorus source.
Semi-log plot.
59
-------
40
£ 30
g>
x 20
en
E
\
oi
a.
15
44
2 3
TIME (hours)
O)
Ol
E
a.
O)
40
30
20
10
45
o
2 3
TIME (hours)
Figure 44. Total cell phosphorus in cells during rapid uptake by cells
starved for 5 days in modified Fitzgerald's medium without PO.,
Ca"^ and Mg . Total cell phosphorus during rapid uptake from
complete medium ( A ) and total celljphosphorus during rapid up-
take from medium lacking Ca"1^" and Mg ( • ). Semi-log plot.
Figure 45, Comparison of total cell phosphorus during rapid uptake of phos-
phorus after 5 days of starvation in medium without phosphorus
and MgSO^. Total cell phosphorus during rapid uptake from com-
plete medium ( A ). Total cell phosphorus during rapid uptake
from medium without MgS04 and KH2P04 ( • ) . Nat^PO/^ was used
as the phosphorus source. Semi-log plot.
60
-------
> 40
X
•g 30
E 20
** 15
&
2 'o
46
23
TIME [hours]
10
E
3
1 •
I 6
3
O
I '
47
TIME (hours)
Figure 46. Total phosphorus in cells during rapid uptake by cells starved
for 5 days in modified Fitzgerald's medium without PC>4, Mg"^,
Ca"H" and KT1". Total cell phosphorus during rapid uptake from
complete medium ( A ) and total cell phosphorus from medium
lacking Mg-H-, Ca-H- and K+ ( • ). NaH2P04.H20 was used as the
phosphorus source. Semi-log plot.
Figure 47. Comparison of rapid uptake of phosphorus after 5 days of starva-
tion in medium without phosphorus and K*~. Uptake of phosphorus
from complete medium ( A ). Uptake of phosphorus from medium
without K+ ( m ).
61
-------
48
2
10
E
3
• IB
"O
-------
12
£
3
• M
•o
o
0)
k.
Q>
10
8
O)
49
o
1 2 3
TIME (hours)
Figure 49. Comparison of rapid uptake of phosphorus after 5 days of starva-
tion in medium without phosphorus and Mg . Uptake of phosphorus
from complete medium (
without MgS04 ( • ).
). Uptake of phosphorus from medium
63
-------
10
s fl
I
"a
w
«
N
0)
E 2
50
2 3
TIME (hours)
I 10
"8
S 4
a.
o>
51
2 3
TIME (hours)
Figure 50. Comparison of rapid uptake of phosphorus after 5 days of starva-
tion in medium without CaCl2 and KH2P04. Uptake of phosphorus
from complete medium ( A ). Uptake of phosphorus from medium with-
out CaCl2 and K*" ( • ). Nal^PC^.H^O was used as the phosphorus
source.
Figure 51. Comparison of rapid uptake of phosphorus after 5 days of starva-
tion in medium without phosphorus, CaCl2 and MgSC^. Uptake of
phosphorus from complete medium ( A ). Uptake of phosphorus from
medium without CaCl2 and MgSO^ ( • ).
64
-------
10
i
•o
I 8
e
I «
V
f
52
2 3
TIME (hours)
10
E
N 4
8*
r *
53
2 3
TIME (hours)
Figure 52.
Figure 53.
Phosphorus left in the medium during rapid uptake by cells starved
for 5 days in modified Fitzgerald's medium without P04, Mg"^*" and
K . Uptake by cells in complete medium ( A ) and uptake by cells
in medium without Mg"^" a°d K+ (•). NaH^PO^.I^O was used as the
phosphorus source.
Phosphorus left in the medium during rapid uptake by cells starved
for 5 days in modified Fitzgerald's medium without P04, K*", Ca"*"*"
and Mg"*""". Uptake by cells in complete medium (A) and uptake by
cells in medium without fC*", Ca^ and Mg"^ (•).
used as the phosphorus source.
NaH2P04.H20
was
65
-------
TABLE 3.
Medium
Phosphate
(mg./l)
6.58
10.13
92.51
SUMMARY OF PHOSPHATE DEPLETION IN THE MEDIUM AND INCREASE IN TOTAL
CELL PHOSPHORUS AS A FUNCTION OF EXTERNAL ORTHOPHOSPHATE DEPLETION
Medium
Phosphorus
(mg./l)
cone, time
2.15 0
0.09 4
3.30 0
0.33 4
30.16 0
24.65
Dry
Weight
(mg./l)
20
80
75
Medium
Phosphorus
Dry Weight
(mg.P/mg.dw)
1.03 x 10'1
3.7 x 10"2
7.4 x 10"2
Largest
increase in
Total Cell
Phosphorus
9.36 x 10"2
1.19 x 10"2
2.73 x 10'2
Phosphorus
Medium after
4 hours of
uptake
8.5 x 10"2
3.3 x 10'1
24.65
TABLE 4. COMPOSITION OF CULTURE MEDIUM. MODIFIED FITZGERALD'S MEDIUM
CONCENTRATIONS OF COMPONENTS EXPRESSED IN MG/LITER
Component s
Concentrations
(mg/1)
NaN03
Na2Si03.9H20
Ferric Citrate
Citric Acid
Gaffron's minor element
solution - 0.04 ml
124
13
25
36
20
58
3
3
Gaffron's Solution
H3B03
MnS04.4H20
ZnS04.7H20
(NH)46M07C24.4H20
CuS04.5H20
Co(N03)2.6H20
. 24H20
Cd(N03)2.4H20
Cr(N03)3.7H20
V204(S04)3.16H20
Na2W04.2H20
KBr
KI
(g/D
3.10
2.23
0.287
0.088
0.125
0.146
0.474
0.198
0.154
0.037
0.035
0.033
0.119
0.083
66
-------
•
£
•
N
t '
9
E
54
1234
TIME [hours]
60
50
30
20
a
3
10
55
1 2 3
TIME [hours]
Figure 54.
Figure 55.
Comparison of phosphorus left in the medium during rapid uptake
by cells after 5 days starvation of phosphorus and K ( • ) phos-
phorus and Ca"*~^ ( A ), and phosphorus and Mg""" ( T ). Uptake was
from complete medium.
Total P in cell digest during a four hour rapid uptake of 10 mg
PO, /I from MFN ( A ) and MFN with 5 x Mg*2 concentration ( A
Cells were starved of P in medium lacking PO^, but with 5 x
concentration.
) .
67
-------
X
*5
60
50
o>
30L
10
56
2 3
TIME [hours]
X
O)
a
a
60
50
40
30
20
10
57
1 2 3
TIME [hours]
Figure 56. Total P in cell digest during a four hour rapid uptake of 10 mg
PO./l from MFN ( A ) and MFN with 5 x K concentration ( A ).
Cells were starved of P in medium lacking P04 but with 5 x K*
concentration.
Figure 57. Total P in cell digest during a four hour rapid uptake of 10 mg
PO,/I from MFN ( A ) and MFN with 5 x Ca+2 concentration ( A j.
Cells were starved of P in medium lacking PO^ but with 5 x Ca"^
concentration.
68
-------
greater than 1 percent transmittance, in three different extractions are sum-
marized in this table. Residues from each successive extraction were also
tested as a check on the extraction procedure.
It can be seen that the phosphate levels for the total cell digest, cold
TCA extraction, and hot TCA extraction increase with increasing external ortho-
phosphate concentration. This effect is not seen with the lipid extractions or
residue. The phosphorus levels in these fractions were highest when the algae
were grown in 10 mg PO^ per liter. Since the algae were normally cultured in
medium containing between 8 and 10 mg PO^ per liter, the values listed for these
concentrations in Table 5 were used as the normal phosphorus levels in these
fractions.
PHOSPHORUS DISTRIBUTIONS DURING STARVATION AND RAPID UPTAKE
Table 6 summarizes the changes in phosphorus content of the various ex-
tracts during five days of starvation and four hours of rapid uptake. These
results are also presented in Figures 58-62. It was not possible chemically to
extract and analyze hourly all fractions during rapid uptake from one sample due
to the length of time required for one extraction. The time required for one
total extraction is approximately four hours. Consequently, hours of uptake
were monitored in all phosphorus containing fractions whereas 1 and 3 hours were
monitored for total cell phosphorus.
Total Cell Digest
Figure 58 demonstrates the changes in phosphorus content of the algae dur-
ing starvation and uptake. The values of these fractions are also presented in
numerical form in Table 6. During the 5 day starvation period, the total phos-
phorus content varied along with the dry weight of the algae. Five days of star-
vation led to a reduction of the total phosphorus content of the algae by ap-
proximately one-half. Upon the addition of phosphate, this value increased by
an order of magnitude of one hour and shows an additional small increase at four
hours.
Cold TCA Extractable Phosphorus
The values for this fraction follow a similar pattern for the starvation
period (Figure 59 and Table 6). Again, the phosphorus content of the cold TCA
extract was reduced by about one-half at the end of the 5 day starvation period.
However, the most substantial increase was found at four hours of uptake, when
the value was one order of magnitude higher than that of the value for 5 days of
variation.
Lipid Extraction
The phosphorus content of the lipid fraction was reduced at the end of the
five day starvation period by 81 percent (Figure 60 and Table 6). At the end of
the four hour uptake period, this value did not return to the levels found in
normal cells, indicating that the phosphate lost during starvation was not re-
covered in this fraction within four hours.
69
-------
TABLE 5. PHOSPHORUS CONTENT OF CELL FRACTIONS AS A FUNCTION OF A PHOSPHATE
CONCENTRATION IN THE CULTURE MEDIUM. VALUES REPORTED ARE mg P/mg
DRY WEIGHT
Phosphate
in
Medium
Img.
lOmg.
100 mg.
1000 mg.
Phosphorus Content of Cell Fractions
TOTAL
CELL
DIGEST
3.01 x 10"3
i0.38xlO~3
8.25xlO~3
i3.0x!0'3
3.28xlO"2
il.OxlO"2
3.58xlO"2
il.46xlO~2
COLD
TCA
EXTRACTION
1.72xlO'3
±0.36x1 0"3
5.51 xlO'3
±2.33xlO~3
2.79xlO"2
±2.31x10'2
2.00x10~2
±1.85x10'2
RESIDUE
FROM
COLD TCA
1.97xlO'3
±0.32X10'3
5.85xlO'3
±1.40xlO"3
7.58xlO"3
±1.31xlO"3
4.80xlO~3
i3.08xlO"3
LIPID
EXTRACTION
1.20xl04
±0.33xl04
7.54xl04
±1.67xl04
3.37xl04
2.30 xlO4
LIPID
FREE
RESIDUE
1.44xlO~3
±0.25xlO~3
6.71xlO"3
i2.16xlO~3
6.54xlO~3
±2.06xlO~3
2.53xlO~3
HOT
TCA
EXTRACTIOh
1.56 xlO~3
±0.18xlO~3
4.49 xlO~3
±2.08xlO~3
5.67xlO~3
±0.72 xlO3
1.95xlO~3
RESIDUE
1.13xlO~3
3.23 xlO~3
il. 07x10
1.89* 10~3
±1.34xio3
1.27xlO~3
70
-------
TABLE 6. PHOSPHORUS CONTENT OF CELL FRACTIONS DURING 5 DAYS OF PHOSPHATE
STARVATION AND 4 HOURS OF PHOSPHATE UPTAKE. SAMPLE DRY WEIGHTS FOR
STARVED CELLS ARE PRESENTED TO DEMONSTRATE THE FLUCTUATIONS IN DRY
WEIGHT THAT OCCUR DURING STARVATION. THE INCREASE IN DRY WEIGHT IN
A REPRESENTATIVE UPTAKE CAN BE SEEN IN FIGURE 21. VALUES REPORTED
ARE me P/me DRY WEIGHT
growth
condition.;
Normal
Cells
8- lOmg.
1 Day
Starved
2 Day
Starved
3 Day
Starved
4 Day
Starved
5 Day
Starved
1 Hour
Uptake
2 Hour
Uptake
3 Hour
Uptake
4 Hour
Uptake
TOTAL PHOSPHORUS IN CELL FRACTIONS
Dry
Wt.
1200
2400
1400
2000
Total Cell
Digest
8.25 x 10'3
±3.00x!0"3
5.35x10"3
2.48xlO"3
5.38xlO"3
2.98xlO"3
4.19 xlO""3
±0.92xlO~3
4.43xlO"2
±1.86xlO~2
4.78xlO-2
±1.96xlO~2
4.69xlO"2
+1.75X10"2
7. 83 x 10"2
*1.52xl02
Cold TCA
5.51 xlO"3
±2. 33 xlO3
2.52xlO'3
1 . 1 7 x 10"3
1.97xlO"3
1.27x|0"3
2.35xKT3
±1.08x)o"3
7.02xlO"3
7. 70 x 10'3
7. 20 x 103
2. 35 x 1CT|
±0.77x10
Cold TCA
Residue
5. 85 xlO"3
+ 1.40xlO"3
5.73xlO"3
4.40xlO"3
Lipid
Extract
7.54xlO"4
±1.18x10
1.09 x 10"4
7.0 x 10"5
9.3 xlO"4
1.4 x id4
1.03 xlO4
3.5X10'3
5.6xlO~3
Lipid Free
Residue
6.71 XlO"3
±2.16xlO*3
5.1X10"3
Hot TCA
Extract
4.49x10~3
±2. 08 KlO3
2.52KIO"3
l.29x)0"3
1 .91 x I0"3
1.21X10"3
2.68xlO"3
±1.56xlO~3
3. 88 KlO2
Residue
3.23KlO~3
+ 1.07KIO
I.75KI03
8.7K104
3- 12 KlO3
1.72 KlO3
3-3 x 10"3
+2.12XIO3
8.6 * id3
9-0 xlO3
71
-------
101
0)
"5
X
U)
10"
3
i.
O
Q.
M
O
58
0
1234
— Starvation
2 3
Uptake -
Figure 58. Semi-logarithmic graphic presentation of the change in Total Cell
Phosphorus during starvation and rapid uptake. The standard error
of the samples is presented in this figure and in all subsequent
figures as open circles connected by lines. Standard errors are
plotted where four or more samples were analyzed. All other data
represents the mean of three extractions.
72
-------
51234
1 2 3
•Starvation
Figure 59. Semi-logarithmic graphic presentation of the change in cold TCA
extractable phosphorus during starvation and rapid uptake.
73
-------
60
103
.5P
"3
x
i.
"O
•
O) -4
Eio4
o
a
o
a
d>
2 4
Starvation —
2
Uptake
Figure 60. Semi-logarithmic graphic presentation of the change in phosphorus
content of lipid fraction during starvation and rapid uptake.
74
-------
1 2 3
Starvation—
-•-Uptake-
Figure 61.
Semi-logarithmic presentation of the change in hot TCA. extractable
phosphorus during starvation and rapid uptake. Hourly sampling of
the hot TCA extract was difficult, and the results obtained were
questionable. Therefore, only the sample taken on the fourth hour
was plotted.
75
-------
•Starvation-
Uptake
Figure 62. Semi-logarithmic graphic presentation of the change in phos-
phorus content of the residue from the threefold extraction
during starvation and rapid uptake.
76
-------
Hot TCA Extractable Phosphorus
The greatest increase in phosphorus content of any cell fraction during
the uptake period was found in the hot TCA extract (Figure 61 and Table 6).
Five days of starvation led to a reduction in this fraction by approximately
40 percent. At the end of the four hour uptake period, the increase in phos-
phorus content was greater than an order of magnitude, the value increasing
from 2.68 to 38.8 ug P per mg dry weight algae.
It is also interesting to note that although in normal cells the amount
of phosphorus present in the hot TCA extract was less than that in the cold
extract, the values at four hours of uptake were reversed. There was more
phosphorus present in the hot TCA extract. During the five day starvation
period, these values were approximately the same.
Residue From The Three-Fold Extraction
The residue from the three-fold extraction as previously described showed
the greatest variation in phosphorus content during the starvation period
(Figure 62 and Table 6). However, the phosphorus content of this fraction was
essentially the same for a 5 day starved culture and a normal culture. The
increase after four hours of uptake was also not as large as the other extrac-
tions. This fraction probably represents all phosphorus compounds in the cell
other than some nucleic acids, short and long chain polyphosphates, orthophos-
phate, and phospholipids.
HYDROLYSIS OF CONDENSED PHOSPHATES
Hydrolysis of the condensed phosphate, sodium tripolyphosphate, was tested
both with and without the experimental organism. Modified Fitzgerald's medium
was made with three different concentrations of the condensed phosphates, 4.59,
153.7 and 1204 mg PO, per liter culture medium. The tubes were incubated under
normal culture conditions as previously described. These results are presented
in Figures 63-65.
Figure 63 demonstrates that at a concentration of 4.59 mg PO- per liter,
the condensed phosphate apparently hydrolyzes at a greater rate with the alga
present in the medium. At a concentration of 153.7 mg PO//1 (Figure 64), the
condensed phosphate hydrolyzes more rapidly in the presence of the alga. How-
ever, this value begins to decrease on the tenth day of culture, and does not
rise again. Hydrolysis in all cases is determined by an increase in orthophos-
phate concentration in the medium. Figure 65 illustrates the rapid hydrolysis
of a considerable amount of condensed phosphate on the first day of culture,
and a subsequent steady increase in the amount of orthophosphate present in
medium. At the higher concentrations of condensed phosphate (Figures 64 and 65),
hydrolysis in the absence of algae proceeded at a constant rate.
RAPID UPTAKE OF CONDENSED PHOSPHATES
Sodium tripolyphosphate was substituted for orthophosphate in a normal
starvation and rapid uptake experiment. Total cell fractionations of phosphorus-
containing compounds were assayed. These results are presented in Figure 66.
77
-------
Figures 63-65.
Figure 63.
Figure 64.
Figure 65.
26 10 14 18 22 26 30
Days
Hydrolysis of sodium tripolyphosphate in Modified Fitzgerald's
medium both in the presence and absence of _p. boryanum. Circles
represent hydrolysis without cells, and squares represent hy-
drolysis with cells present. Hydrolysis is indicated by an
increase in orthophosphate present in the medium.
4.59 mg PO^/liter culture medium
153.7 mg PO^/liter culture medium
1204 mg PO^/liter culture medium
78
-------
Cold
Hot
Residue
Total
Figure 66. Increase in phosphorus-containing fractions during starvation
and rapid uptake from 10 mg PO,(as sodium tripolyphosphate) per
liter. The left bar of each pair represents starved cells, and
the right bar represents 4 hours of uptake. The uptake with
condensed phosphate was only repeated twice. Consequently the
values reported represent the mean.
79
-------
There was no substantial increase in any cell fraction after four hours of up-
take. This is in contrast to an uptake with orthophosphate as previously dis-
cussed. Refer again to Table 6 and Figures 58-62.
CELL ULTRASTRUCTURE UNDER NORMAL CULTURE CONDITIONS
Cells of _P. boryanum grown under normal culture conditions have an appear-
ance which is similar to that of other blue-green algae. Cellular inclusions
such as polyhedral bodies, areas of DNA, ribosomes, thylakoids, lipid droplets,
cyanophycin granules, and a few small polyphosphate bodies are found in the cell
with regularity (Figures 67 and 68). A four-layered cell wall, as described in
most blue-green algae is also present (Figure 68). Some intrathylakoidal vacu-
olization is present, with the thylakoids usually arranged at the periphery of
the cell (Figure 67). Cell division occurs by transverse binary fission. A
newly formed septum can be seen in Figure 67.
CHANGES IN ULTRASTRUCTURE ASSOCIATED WITH SEVERAL PHOSPHATE CONCENTRATIONS
100 mg PO per liter
H-
_P. boryanum grown in culture medium containing 100 mg PO/ per liter had a
number of alterations in cell structure. Figure 69 demonstrates these changes.
Generally, there is a marked increase in cell death and cell lysis, as evidenced
by the debris in the sectioned pellet. The cells which appear to be dead have
cytoplasm which is devoid of ribosomes. The thylakoids are also fewer in number
and are rather prominent in the cell. Occasionally, large polyphosphate bodies,
or areas where polyphosphate bodies have fallen out, can be seen. Cells which
are normal in appearance, as previously described, can also be found.
1000 mg P04 per liter
Changes that occured in culture at 100 mg per liter were even more pro-
nounced when the algae were grown in medium containing 1000 mg P04 per liter
(Figures 70-72). Figure 73 demonstrates some expansion of thylakoids (IT), and
a large hole in the center of the cell, presumably an area where a polyphosphate
body has fallen out during sectioning. In the more normal appearing cells, the
thylakoids are still located at the periphery, and the DNA is located in the
center of the cell. Figure 71 shows in greater detail a cell which appears to
be dead, and is devoid of ribosomes. The frequency of encountering such cell
types increases with greater phosphate concentrations. Figure 72 shows a por-
tion of a filament containing several polyphosphate bodies which have sublimated
under the electron beam or have been partially lost during the section prepara-
tion procedures. The ribosomes also have an unusual appearance, being much
larger than in cells grown in normal culture medium.
1 mg PO^ per liter
Cells grown in culture medium containing 1 mg P04 per liter have approxi-
mately one-tenth of the normal phosphate concentration available to them. This
particular culture condition also leads to changes in cell architecture, but
these changes are unlike those of higher phosphate concentrations (Figure 73-76),
The most pronounced changes that occur are the expansion of intrathylakoidal
80
-------
67
68
Figure 67. Cells of _P. horyanum grown for 14 days in 10 mg PO,/liter at 500
ft-candles of illumination with an alternating 12 hour day/night
cycle. Note the relatively small intrathylakoidal spaces, and
inclusions normally present in blue-green algae such as lipid drop-
lets (L), polyhedral bodies (Pb), and areas of DNA (D). Cell divi-
sion which will result in two equal daughter cells can also be
seen (S).
Figure 68. _P. boryanum grown under normal conditions described above and shown
at a higher magnification. Note the arrangement of the cell wall
outside the plasma membrane (PM). Layer 2 is the mucopolymer-
containing layer.
81
-------
\'f, , -•. */»
** - < 'VjJS*
.r>5?
.»
/;
rx^rvi
Figure 69. Low power micrograph of a sectioned pellet of J?. boryanum grown in
Modified Fitzgerald's Medium for 14 days containing 100 mg PO,/,./
liter at 500 ft-candles of illumination. Note the cellular debris
in the pellet, and the appearnace of several types of cells. The
filament in the upper left corner of the picture can be considered
as "normal", resembling cells grown under the conditions listed
for Figure 38. Other cell types are also evident.
82
-------
Figures 70-72.
Figure 70.
Figure 71.
Figure 72.
Cells of J?. boryanum grown for 14 days in medium containing
1000 mg PO^/liter at 500 ft-candles of illumination. Note the
cell types present, as in the culture containing 100 mg PO,/
liter.
Portions of cells showing some expansion of intrathylakoidal
spaces (IT), thylakoids (T) at the periphery of the cells, and
DNA. (D) and polyhedral bodies (Pb) surrounding a space where a
polyphosphate body has fallen out.
A cell, apparently dying, which is devoid of ribosomes. Note
the small lipid droplets (L) throughout the cell, and the
prominent thylakoids (T).
Portion of a filament showing numerous polyphosphate bodies
(P) and enlarged ribosomes (R).
83
-------
75
Figures 73-76.
Figure 73.
Figure 74.
Figure 75.
Figure 76.
Cells of J?. boryanum grown in culture medium for 14 days con-
taining 1 mg POA/liter at 500 ft-candles of illumination and
a 12 hour alternating day/night cycle.
Cell showing expansion of intrathylakoidal spaces (IT) and the
presence of an area of medium electron density (arrow). DNA
(D) and polyhedral bodies (Pb) are located in the center of
the cell.
Cell showing a greater degree of expansion of intrathylakoidal
spaces (IT). An immature polyphosphate body (arrow) can also
be seen.
A second cell type found in phosphate-limited cultures. These
cells are characterized by unusually large ribosomes (R).
Portion of an elongate cell of j?. boryanum. The cell contains
many small lipid droplets (L) and ribosomes (R) .
84
-------
spaces (IT) and the development of areas of medium electron density (arrows in
Figures 73 and 74). In these figures, the appearance of the cytoplasm and some
of the ribosomes is normal in all other respects. Some ribosomes (Figure 76)
are unusually large. Polyhedral bodies and areas of DNA are also present.
One also encounters, with great frequency, elongate cells (Figure 76). These
cells often attain lengths of 6-9 urn, whereas a normal cell is about 3 urn.
They frequently have abnormally large ribosomes.
PHOSPHATE-STARVED CELLS
In some cells the cell division process is abnormal. Some cells are ab-
normally long (Figure 77). In normal cultures the cells are about 1.5 by 3.5
um. The longest cells observed under phosphate-starved conditions are about
1.5 urn by 10 um. Generally an entire filament is composed of oblong cells.
In other filaments the forming septum, rather than being located in the middle
of the cell, is displaced toward one end (Figure 78) resulting in a very short
cell (Figure 79). Some of the short cells have a length of only about 10 run and
contain only JJ granules, ribosomes, thylakoids, and some DMA. (Figure 79). In
other cells the septa are displaced at points around the filament. If the dis-
placement is quite significant, i.e., the septa are not opposite (unpublished
results), a bend occurs in the filament when the septa join. All of these con-
ditions are observed with greater frequency as the length of time in the phos-
phate-free medium increases.
In certain cells numerous polyphosphate bodies can be seen in association
with the DNA fibrils (Figures 80, 81). They vary in size from about 20 nm to
200 nm. Three morphological varieties of polyphosphate bodies can be observed.
These are (a) bodies which are porous in appearance, (b) bodies having a dense
exterior with an electron-transparent central area, and (c) bodies which are
completely electron-dense (Figures 80, 81). A few polyphosphate bodies are
found in the ribosomal areas away from nucleoplasmic areas. Several of these
polyphosphate bodies have a crystal-like substructure (Figure 83). In other
cells an area of medium electron density develops in the nucleoplasmic area
(Figure 82). Within this area, which seldom exceeds 0.5 um in diameter, elec-
tron-transparent areas develop (Figure 82). Within the electron-transparent
areas polyphosphate bodies are formed. The sequential development appears to
be nearly the same as previously reported for the development of polyphosphate
bodies in ribosomal areas of _P. boryanum (Jensen, 1969). Some polyphosphate
bodies also form intrathylakoidally (Figure 77). The frequency of observation
of these morphological changes seems to increase with increased time the phos-
phate-free medium.
In some cells the thylakoids expand greatly to form a large electron-
transparent area with a density that resembles the areas found in the nucleo-
plasmic area (Figure 87).
Electron-dense inclusions, sometimes with a mottled structure, appear in
many of the cells as phosphate starvation proceeds (Figures 80, 84, 86). These
spherical inclusions are between 0.2 um and 0.4 um in diameter and sometimes
appear to be limited by an osmiophilic layer about 30 A in thickness (Figure
86).
85
-------
Figure 77.
Figure 78.
Figure 79.
Elongate cell from phosphate- starved culture. Note the expanded
thylakoids (T) and small polyphosphate bodies (P) . OsO^ fixation.
Section of portion of a filament from phosphate-starved culture.
Note the forming septa (S) which would cut off a small cell.
fixation.
Portion of a filament from a phosphate-starved culture. Note the
small cell (arrow) which has been formed. Septa (S) are also
forming which are not opposite. OsO/ fixation.
86
-------
80
Figure 80.
Figure 81.
Figure 82.
Figure 83.
Phosphate- starved cell. Note the large lipid-like inclusions (L)
and the numerous polyphosphate bodies (P) associated with DNA
fibrils (D) . OsO, fixation.
Phosphate-starved cell. Note the fibrils (arrow) associated with
the polyphosphate body. OsO^ fixation.
Phosphate- starved cell showing an area of medium electron density
(M) with electron-transparent areas. OsO^ fixation.
Phosphate-starved cell showing a polyphosphate body which has
developed in an RNA area. Note the crystal-like organization of
part of the body.
87
-------
8ft
Figure 84. Phosphate-starved cells showing a cyanophycin granule (C) and lipid-
like inclusion (L). Os04 fixation.
Figure 85. Phosphate-starved cells showing an elongate polyhedral body (Pb).
These bodies are always associated with the nucleoplasmic areas (N).
OsO, fixation.
Figure 86. Phosphate-starved cell with a lipid-like inclusion (L) with a
"mottled" appearance which appears to be limited by a 30-A "mem-
brane" (arrow). OsO, fixation.
Figure 87. Phosphate-starved cell showing a very large intrathylakoidal space
(I) which formed from an expanded thylakoid. OsO^ fixation.
Figure 88. Micrograph showing the cell debris which is common in phosphate-
starved cultures. OsO, fixation.
88
-------
In a number of cells the polyhedral bodies elongage (Figure 85). General-
ly the polyhedral bodies are about 0.2 um in diameter. In many starved cells
they increase to a length of about 0.6 um.
Small cyanophycin granules develop during phosphate starvation and general-
ly do not exceed 0.2 um (Figure 84).
Two types of cells appear in the culture based on the appearance of their
ribosomes. Type I has ribosomes which are the usual 20 nm size, and type II
has ribosomes which appear abnormally large and measure about 40 nm. Numerous
cells fragments are observed in pelleted material (Figure 88), with the 40 nm
ribosomes appearing most frequently and suggesting lysis of the type II cells.
RAPID UPTAKE
In cells exposed to 10 tng PO^/liter after 5 days of starvation nearly every
cell, after 2 h, contained at least one large polyphosphate body (Figure 92).
These bodies are visible at the light-microscope level after staining by the
Ebel procedure (Ebel et al. 1958). Some cells contain several polyphosphate
bodies (Figure 92). Some of these polyphosphate bodies are dense around the
periphery while others are completely dense (Figure 92). They vary in diameter
from 1.5 pm to the limit of visibility with the light microscope.
As rapid uptake proceeds many more elongate polyhedral bodies are observed
than in starved cells (Figure 89). In many of these polyhedral bodies areas of
medium electron density develop in which a smaller electron-transparent area is
located (Figures 90, 93). These areas of medium electron density are about
0,05 um in diameter. Within the electron-transparent areas polyphosphate bodies
develop (Figure 91).
The medium electron-transparent areas in the nucleoplasmic area seen oc-
casionally in normal cultures and with increasing frequency in the phosphate-
starved cells become very pronounced and most cells contain such areas (Figures
91, 93, 94, 101). Within these areas polyphosphate bodies appear to develop in
the electron-transparent areas (Figures 93, 94, 97, 98-101). In some cells only
one polyphosphate body is visible in a section through this area (Figure 94)
while in other cells several polyphosphate bodies are seen (Figure 97). The di-
ameter of these polyphosphate bodies varies from about 100 nm to 1.5 um. As-
sociated with most of these polyphosphate bodies are apparent DNA fibrils (Fig-
ures 94, 97). These polyphosphate bodies are quite variable in morphology. In
addition to the three morphological varieties already described for the starved
cells, one can also observe bodies which are irregular in shape (Figures 97, 99,
101) and those which have a compound structure (Figures 98, 99, 100).
In some cells the polyphosphate bodies are small (10-15 nm in diameter) and
are associated with the DNA (Figure 95). Their morphology varies as previously
described.
In other cells the polyphosphate bodies develop intrathylakoidally (Figure
96). These polyphosphate bodies vary in diameter from about 10 to 150 nm.
Numerous polyphosphate bodies often develop within one expanded thylakoid as
three may be seen in one thin section (Figure 96).
89
-------
Figure 89.
Figure 90.
Figure 91.
Figure 92.
Figure 93.
Rapid uptake cell showing an elongate polyhedral body (Pb}
Glut-OsO, fixation.
4
Portion of a rapid uptake cell showing an area of medium electron
density in a polyhedral body (Pb) . OsO fixation
4
/
polyhedral body (Pb) .
Sh°Wing a
body in a
Filament from rapid uptake culture which has been stained to show
f%S r^°SP °dieS' N°te the vario"s sizes and morphologies
of the bodies. Bar marker equal 5 urn.
Rapid uptake cell showing a large medium electron-dense area which
has developed in a nucleoplasmic area. Note the electron-trans-
parent areas (arrows) in which polyphosphate bodies will develop.
OsO fixation. v
90
-------
Figure 94. Rapid uptake cell showing several polyphosphate bodies, one (arrow)
has many apparent DNA fibrils associated with it. OsO/ fixation.
Figure 95. Rapid uptake cell showing many polyphosphate bodies associated with
the DMA. fibrils. Note the variation in their morphology. OsO/
fixation.
Figure 96. Rapid uptake cell showing polyphosphate bodies which have developed
intrathylakoidally. Glut-OsO^ fixation.
Figure 97. Rapid uptake cell showing two large polyphosphate bodies (P) which
have developed in an area of medium electron density OsO/ fixation.
91
-------
101
Figures 98-101.
Rapid uptake cells showing morphological variation of poly-
phosphate bodies which have developed in areas of medium
electron density. OsO^ fixation.
92
-------
In a very few cells polyphosphate bodies are seen in the ribosomal areas
as previously described in _P. boryanum (Jensen, 1969).
Few lipids and cyanophycin granules are seen in the cells during rapid
uptake.
ENERGY DISPERSIVE X-RAY ANALYSIS
Analysis was performed in all cases after morphological identification of
the polyphosphate bodies in the transmission mode of the scanning electron
microscope (STEM). Typical polyphosphate bodies are shown in Figures 102 and
103. The results obtained with the various fixation and embedding procedures
are as follows:
Osmium Fixation
Glutaraldehyde fixation does not yield the best preservation of morpho-
logical integrity in prokaryotes, hence they are usually fixed with osmium
tetroxide. The presence of osmium, however, poses a problem in X-ray analysis
because the osmium M spectral line (1.978 keV) cannot be resolved from the K
spectral lines (Ka, 2.015 and KB, 2.016 KeV). The limitation to resolution of
these spectral lines, which are 28 ev apart, is the detector, which has a reso-
lution of 152 ev. In all cases, this osmium contribution to the phosphorus
peak results in a shoulder making analysis or semi-quantitative estimates dif-
ficult (Figures 104 and 105). This interference was somewhat reduced by de-
creasing the range of the X-ray analysis to 10 ev/channel. Other components
normally encountered in the analysis of polyphosphate bodies were chlorine
(Ka at 3.691 keV), Figures 104 and 105. Chlorine is a common component of
epoxy resins and was found in the analysis of the embedding medium with no cells
present (Table 7).
Glutaraldehyde Fixation
Glutaraldehyde fixation without post-fixation in OsO^ was chosen as an
alternate method of preserving the algae in an attempt to avoid osmium con-
tribution to tne phosphorus peak during X-ray energy dispersive analysis. Al-
though this method does not provide the best image for routine electron micro-
scope studies, it does provide sufficient preservation of the cells to allow
identification of the constituents and X-ray analysis of the inclusions. Poly-
phosphate bodies are somewhat less dense and more difficult to identify with
this fixation (Figure 103). However, analysis with this method of fixation
results in a more distinct phosphorus peak without osmium interference (Figures
106 and 107). The other components present are calcium, and traces of chlorine
(Figures 106 and 107). The P/B ratio of chlorine in polyphosphate bodies is
significantly lower than in the cytoplasm due to the lack of permeability of
epoxy resins into the bodies as well as the greater X-ray emission character-
istics of phosphorus and calcium compared to organic constituents of the cyto-
plasm. Analyses of material fixed with either glutaraldehyde or OsO^ are quali-
tatively similar (Table 7). Polyphosphate bodies consistently reveal a phos-
phorus and a calcium component. The cytoplasm of the cells (Figures 107 and
108) or the embedding medium (Figure 109) does not contain detectable amounts
of these elements.
93
-------
f*':
102
103
Figure 102. Scanning Transmission Electron Micrograph (STEM) of a filament
of Plectonema boryanum fixed in 0864. Note the large, dense
deposits of polyphosphate.
Figure 103. STEM of cells in cross section which have been fixed only in
glutaraldehyde. Note the polyphosphate bodies which are not as
dense as those in Figure 102.
94
-------
104105
1JIJIC 2I47SIMT 1O6I107 • J55SEC 149C1INT
1ft* HS 2IEV/CH I VS Itlt HS 2IEV/CM
Figure 104. Analysis of a polyphosphate body fixed in osmium and embedded in
Epon. The major identifiable peaks are phosphorus (2.015 and
2.016 keV), chlorine (2.622 keV) and calcium (3.691 keV). The
figure shows a marker at 1010 ev to demonstrate the location of
the osmium emission on the spectrum. Note also the K emission
of calcium at 4.012 keV.
Figure 105. Analysis of a polyphosphate body fixed in osmium and embedded in
Durcupan. Note the reduced separation at 50 ev/channel and the
absence of chlorine in the Durcupan. Other elements present are
osmium and copper.
Figure 106. Analysis of a polyphosphate body fixed in glutaraldehyde and em-
bedded in Epon. Note the absence of a shoulder on the phosphorus
peak.
Figure 107. Comparison spectra of a polyphosphate body and the adjacent cyto-
plasm. Dots correspond to the polyphosphate body whereas lines
correspond to the cytoplasm analysis.
Figure 108. Analysis of cytoplasm fixed in glutaraldehyde.
Figure 109. Comparison spectra of a polyphosphate body and the epoxy embed-
ding medium.
95
-------
TABLE 7. P/B EATIOS. PEAK TO BACKGROUND RATIOS WERE CALCULATED BY DIVIDING
THE COUNTS FROM A SINGLE CHANNEL FROM THE BACKGROUND INTO THE HIGH-
EST CHANNEL OF A GIVEN EMISSION PEAK
Fixation and Embedding Area Analyzed
Procedures
Osmium-Epon Poly P body
Osmium-Epon Cytoplasm
Glutaraldehyde- Poly P body
Durcupan
Glutaraldehyde- Cytoplasm
Durcupan
Durcupan Plastic without
cells
Element
Os
P
Cl
Ca
Os
P
Cl
Ca
Os
P
Cl
Ca
Os
P
Cl
Ca
Os
P
Cl
Ca
P/B
2.2
5.6
2.1
4.0
1.9
2.2
21.9
1.1
2.9
7.6
1.0
2.3
1.1
2.6
1.4
1.2
1.1
1.6
1.6
.9
96
-------
SECTION VIII
DISCUSSION
GROWTH CURVES, PHOSPHATE DEPLETION, AND MINIMUM CELLULAR PHOSPHATE LEVELS
The growth curves of Plectonema boryanum and Oscillatoria tenuis all demon-
strate an increase in dry weight on the first day after transfer into fresh me-
dium, regardless of phosphate concentration in the medium. The increase in dry
weight is also accompanied by a corresponding decrease in phosphate concentra-
tion of the external medium (Figures 2-15). This increase in dry weight and
depletion of phosphate from the external medium is probably due to an uptake phe-
nomenon. Transfers of cultures were routinely made from cultures which were at
least 2 months old. Analysis of the total phosphate content of cells as a func-
tion of culture age revealed that at this age, cultures had already been reduced
in phosphorus content to 4 ug P per mg dry weight algae (Figure 17). Studies
correlating dry weight analysis to cell number revealed that for all cultures,
1 mg of dry weight algae corresponded to 5.5 x 10 cells. Thus, the total phos-
phorus content of a 2 month old culture would equal 0.73 ug P per 10^ cells.
Similar analysis of normal, 5 day starved, and 4 hour rapid uptake cultures
yield the following information: Normal cultures - 1.5 Mg P/10^ cells; Starved
cultures - 0.76 ug P/106 cells; and, Uptake cultures - 14.2 ug P/106 cells. It
can be seen that the total phosphorus content of a two month old culture is
slightly lower than that of a culture starved for 5 days. Normal culturing of
the algae also leads to depletion of phosphorus reserves, but over a much longer
period of time. Thus, transfer of these phosphate depleted cells in reality,
sets the stage for a rapid uptake of orthophosphate.
These results for internal phosphorus concentration are similar to those
reported for other organisms. Mackereth (1953) reported that the limiting phos-
phorus concentration of Asterionella formosa was about 0.06 jug P/10 cells.
This diatom could also store phosphorus in concentrations as high as 7 ug P/10
cells. The marine diatom Phaeodactylum tricornutum was found by Kuenzler and
Ketchum (1962) to contain phosphorus concentrations of 2.1 ug P/10 cells at its
highest levels, and 0.06 Mg P/10° cells at its lowest levels. Kholy (1956) re-
ported that the minimum value below which growth did not occur in Chlorella was
0.1 jug P/10" cells. The maximum level of phosphorus in the cells was 1.5 jug P/
10° cells. This value did not increase further when the starved algae were
reinoculated into higher phosphate concentrations.
The values reported in the literature are similar to those values obtained
for Plectonema boryanum in this study. However, it would appear that _P.
boryanum contains more total phosphorus even at its lowest levels, especially
when one considers that this alga is much smaller in size than the diatoms or
green alga utilized in these previous studies.
97
-------
Table 2 indicates that there is an appreciable increase in dry weight of
the algae during the uptake process. This increase in dry weight is also com-
mon to routine culture transfer (Figures 2-15). These results also lend sup-
port to the occurrence of the uptake phenomenon associated with transfer of
algae to fresh medium during routine culture.
PHYSICAL PARAMETERS AFFECTING UPTAKE RATES
Light has been demonstrated to stimulate uptake of phosphate in many organ-
isms (Talpasayi, 1962; Kanai and Simonis, 1968; Fitzgerald, 1970). Dark as-
similation of phosphate has also been reported (Kanai, Miyachi, and Miyachi,
1963; Batterton and Van Baalen, 1968; and Overbeck, 1962). j?. boryanum appears
to be an organism which requires light for maximal uptake of phosphate (Figure
23). The increase in total cell phosphorus is directly related to the light
intensity available. Higher light intensities during the uptake period result
in higher cell phosphorus levels, up to 18.9 ug P/10" cells at 2000 ft-candles.
Preillumination of cultures of JP. boryanum during the starvation period and sub-
sequent inoculation into a phosphate-containing medium in the dark resulted in
uptake, but at greatly reduced levels. These results are unlike those reported
by Simonis and Urbach (1963) who reported dark uptake in preilluminated cultures
of Ankistrodesmus braunii and Anacystis nidulans (Simonis et al, 1974).
The effect of light intensity on the uptake process suggests several possi-
bilities. Energy is required for phosphate uptake to occur. This energy can be
supplied by photosynthesis, respiration, or a combination of both processes. If
photosynthesis alone were involved, one would expect results such as those pre-
sented in Figure 23. ATP produced during photophosphorylation would supply the
energy required for uptake. Another possible source of energy would be the
metabolism of products of photosynthesis. If this were true, then inhibition
of metabolic pathways involved in utilization of photosynthetic products would
result in either complete or partial inhibition of the uptake process. A third
alternative is the metabolism of storage materials in the cell through respira-
tion. In this case, one would expect increased uptake with increasing light
intensity, and also phosphate uptake in the dark with the metabolism of storage
materials.
Figure 23 demonstrates that increasing light intensity does accelerate
phosphate uptake, but there is virtually no uptake in the dark. Figure 27 illu-
strates the effects of several metabolic inhibitors on the phosphate uptake pro-
cess. These results indicate that phosphate uptake requires a supply of meta-
bolic energy. If metabolism of the products of photosynthesis were not involved
as an energy source, one would expect no effect exerted by these inhibitors.
This does not appear to be the case. Increasing light intensity most likely in-
creases the supply of sugars to be metabolized by the alga. Some dark uptake
probably indicates that sugars resulting from photosynthesis during the star-
vation period were probably metabolized in the dark, but no further production
was possible. It is also likely that there are no other storage forms of sugar
present in the alga which could readily be available as an energy source.
It is a well known fact that the optimal temperature for photosynthesis is
25 C. Temperature effects on uptake (Figure 22) and growth rates (Figure 16)
indicate that although elevated temperatures (25-37 C) increased in the growth
98
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rate, they do not increase the rate of uptake.
It has been demonstrated many times that an energy source is required for
phosphate uptake. Blum (1966) demonstrated that phosphate uptake by Euglena
gracilis was inhibited by 2,4-dinitrophenol. Borst-Pauwels and Jager (1969)
found that phosphate uptake in Saccharomyces cerevisiae was inhibited by both
0.1 mM 2,4-dinitrophenol, and 20 iriM fluoride. Jungnickel (1970) found that
2,4-dinitrophenol reduced, but did not completely inhibit phosphate uptake in
Candida utilis. Ullrich (1972) found that rates of polyphosphate formation
were rather low when compared with the probable rates of ATP formation under
various conditions of photophosphorylation in Ankistrodesmus braunii. The up-
take rate of phosphate after a starvation period was higher than the rate of
polyphosphate synthesis.
ION EFFECTS
Previous studies have indicated that the pH of the medium and ions effects
are related to phosphorus uptake. In Ankistrodesmus braunii (Ullrich, 1972),
Saccharomyces mellis (Weimberg, 1970), Dermocystidium sp. (Belsky_et _al., 1970)
and Baker's yeast (Schmidt et al., 1946) uptake was greatest in the acidic range.
However, these organisms, unlike J?. boryanum, grow best in an acidic environ-
ment. In _P. boryanum uptake was the greatest at pH 9. In culture media of this
pH organism grew at the fastest rate. It is also interesting to note that cal-
cium exerted the greatest effect on phosphorus uptake while in the above named
organisms potassium and sodium exerted the greatest influence on uptake. This
may be related to the fact that in _P. boryanum the polyphosphate is associated
with calcium as determined by X-ray analysis. It is also evident that ions in
combination lead to greater uptake than any single ion. Calcium also seems to
be necessary for rapid uptake but the uptake is enhanced by the presence of
other ions either singly or in combination.
PHOSPHORUS DISTRIBUTIONS IN CELLULAR EXTRACTS
Logarithmic Cultures in Different Phosphate Concentrations
Table 5 summarizes the influence of external phosphate concentration on
the distribution of phosphorus in various cell extracts. It can be seen that
increasing phosphate concentrations, in general, result in higher phosphorus
levels for all fractions. These results can also be correlated with the micros-
copy of the samples.
The ratios of acid-soluble (cold TCA extractable) phosphates to acid-in-
soluble (hot TCA extractable) phosphates seem to vary in many organisms.
Aitchison and Butt (1973) reported that acid-insoluble polyphosphates of Chlo-
rella vulgaris were slightly higher during logarithmic growth than acid-soluble.
Baker and Schmidt (1964) found that in Chlorella pyrenoidosa, the levels of acid-
insoluble polyphosphate decreased immediately prior to and during nuclear divi-
sion. Krishnan et al. (1957) found that both acid-soluble and acid-insoluble
polyphosphates were normally present in mycelia of Aspergillus niger. Soluble
polyphosphate was present in low concentrations in new mycelia, but accumulated
and reached a peak as the organism grew, after which it declined until the time
of general autolysis. On the other hand, they found that insoluble polyphos-
99
-------
phates were present in high concentrations in newly formed mycelia, and fell
after about one week at these concentrations.
Plectonema boryanum, during logarithmic growth, contains more acid-soluble
than acid-insoluble polyphosphates (Table 5). This is true for all phosphate
concentrations tested. The standard deviations expressed in this table are a
result of variability in different experiments, and not variability in the
triplicate samples of a single total extraction. The increasing total cell
phosphorus content is probably due to the uptake phenomenon occuring upon trans-
fer to fresh medium. It has been demonstrated earlier that the algae, under
optimal conditions, do not increase in total cell phosphorus to levels above
18.9 jug P per 10" cells. This maximum level occurred only at 2000 ft-candles
of illumination and was not used routinely for uptake studies because the alga
will not grow well at light intensities above 500 ft-candles.
In the case of growth in 1000 mg of phosphate per liter of culture medium,
the total phosphorus content of the cell digest represents 6.5 ug P per 10^
cells, a value approximately four times higher than that of the cultures grown
at 10 mg PO^ per liter. If the uptake phenomenon did occur upon transfer, one
would expect increased total cell phosphorus according to the available phos-
phorus in the medium, but probably reduced after 14 days of culture. The phos-
phorus content of the total cell digests of cultures grown in 100 and 1000 mg
P04 per liter culture medium again also indicates that the algae are able to
assimilate certain levels of phosphate, and beyond a certain value, no further
increase in phosphate concentration in the medium results in increased cellular
phosphate levels.
Electron microscopic examination of cultures grown at various phosphate
concentrations also reveals that there is increased cell death and lysis when
the algae are grown at high phosphate concentrations. This might indicate that
the measurements of phosphorus concentrations in the cell fractions of cells
grown in higher phosphate concentrations are more unreliable, since the number
of viable cells is reduced.
Starved and "Overplus" Cultures
Table 6 and Figures 58-62 demonstrate the changes in all phosphorus-con-
taining fractions that occur when the algae are cultured in phosphate-free
medium and subsequently inoculated into a medium containing phosphate. Cultures
starved of phosphate for a five day period generally decrease in phosphate
levels in all cell extracts examined. After the initial reduction on days 1
and 2, there was an increase, though small, on days 3 and 5. This pattern of
changes in phosphorus content can be related to the fluctuations in dry weight
which occur during the starvation period, and also the microscopic examination
of the cultures.
There is a loss in dry weight of the cultures on days 2 and 4 of starvation.
The increases in phosphorus occur when there is a reduction in dry weight.
Examination of the cultures during this period reveals that there is increased
cell lysis during the starvation period. Cells which do appear normal, however,
often have small polyphosphate bodies. Thus it seems likely that the increas-
ing cell lysis released phosphate back to the medium, and the surviving cells
100
-------
are able to utilize this phosphate for growth, even at these low phosphate
levels.
The two fractions of most interest both during starvation and rapid uptake
are the cold TCA extractable and hot TCA extractable phosphates. The cold TCA
extract (soluble polyphosphates) decreased by about one-half during five day
starvation period. The hot TCA extract (insoluble polyphosphates) initially
is present in lower concentrations than the acid-soluble fraction, but is
greater than the acid-soluble fraction at the end of the five day starvation
period. The increase in phosphate in the acid-insoluble fraction is also
greater after four hours of uptake than in the acid-soluble fraction.
These results are interesting in view of reports of starvation and uptake
in other organisms. Aitchison and Butt (1973) reported that phosphate star-
vation of Chlorella vulgaris resulted in a general reduction of all phosphate
fractions, and a 49-90 percent decrease in the acid-soluble fraction with 8
hours after starvation, and a less rapid but marked decrease in the acid-in-
soluble fraction. The uptake period was characterized by a rapid increase in
all phosphate fractions, with the greatest over-compensation occurring in the
acid-soluble fraction. The acid-insoluble fraction only recovered to the levels
of cells grown in normal culture. Wiame (1949) found that in yeast, phosphate
starvation resulted in a decrease in the insoluble polyphosphate fraction while
the soluble polyphosphate remained constant. Phosphate uptake after an induced
starvation period led to a large increase in soluble polyphosphate and no ap-
preciable change in the insoluble polyphosphate content. The results of Aitchi-
son and Butt (1973) using Chlorella vulgaris are comparable to those of Wiame
(1949).
It has been postulated that the soluble polyphosphates (short chain length)
are probably derived from insoluble polyphosphates (long chain length) by the
loss of phosphate residues (Krishnan et _al., 1957). If this postulate were true,
one would expect greater initial increases in soluble polyphosphates during the
uptake period, and a later increase in the insoluble polyphosphates due to the
addition of phosphate residues to already existing primers, a reversal of the
synthesis of short chain polyphosphates.
It is interesting to note that in _P. boryanum, the relative levels of acid-
soluble and acid-insoluble polyphosphates remain constant throughout the star-
vation period, even though the levels of acid-insoluble phosphate are lower in
normal cells. This would suggest that the acid-soluble fraction is maintained
at the expense of the acid-insoluble fraction. If synthesis occurs by addition
of phosphate residues to an already existing primer (acid-soluble fraction),
then the amount of acid-insoluble (long chain) phosphates is dependent upon the
amount of short chain phosphates and orthophosphate. The greater increase in
acid-insoluble polyphosphate at four hours of uptake is probably due to this
synthesis from increased short chain primers and available orthophosphate.
Evidence exists which suggests that an enzyme, polyphosphate kinase (ATP:
polyphosphate phosphotransferase, E. C. 2.7.4.1., Suzuki jit _al, 1972) is neces-
sary to polymerize orthophosphate into an osmotically inert form, polyphosphate.
This enzyme is synthesized during phosphate starvation (Harold and Harold, 1965;
Harold, 1966). Aitchison and Butt (1973) found that in Chlorella vulgaris,
101
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polyphosphate synthesis was a consequence of the stimulation of phosphate uptake
that was induced by phosphate starvation. These results, along with those of
Wiame (1949) lend support to the work of Harold (1966). The synthesis of poly-
phosphate during rapid uptake is most likely a consequence of an energy depen-
dent uptake of orthophosphate and subsequent polymerization by addition of phos-
phate residues to pre-existing primers in all organisms reported so far. The
polymerization is a result of increased levels of the inducible enzyme, poly-
phosphate kinase. The results of experiments with J?. boryanum are consistent
with this hypothesis.
HYDROLYSIS AND UPTAKE OF CONDENSED PHOSPHATES
The results of hydrolysis studies of sodium tripolyphosphate are presented
in Figures 63-65. From these graphs, it is apparent, especially at the higher
phosphate concentrations, that hydrolysis occurs more rapidly and to a greater
extent when the algae are present in the medium. At first glance, the graphs
are somewhat unclear. Rapid hydrolysis, expressed as orthophosphate present in
the medium, increased initially upon transfer, then decreased during the culture
period at concentrations of 4.59 and 153.7 mg of phosphate present as condensed
phosphate.
These results are consistent with the uptake associated with transfer to
fresh medium containing orthophosphate. The algae present in the culture tubes
are able to hydrolyze the condensed phosphate, but the resulting orthophosphate
is subsequently assimilated by the algae which are already low in total phos-
phorus levels. Thus there is a reduction in the orthophosphate resulting from
hydrolysis. This effect can be seen readily at the lower phosphate concentra-
tions (Figures 63 and 64). The reduction at 1204 mg PO^ per liter is not evident
due to the high phosphate concentration (Figure 65). Hydrolysis of condensed
phosphates in the absence of algae is essentially linear.
Figure 66 demonstrates that there is virtually no increase in phosphorus
levels in any cell fraction when phosphate-starved algae are inoculated into
medium containing condensed phosphates as the phosphorus source. This is pro-
bably due to the slow hydrolysis rate of the condensed phosphates. Maximum
hydrolysis of the condensed phosphates does not begin to occur until 2 days of
culture. Thus, inoculation of phosphorus deficient algae into medium containing
condensed phosphates does not result in uptake of phosphate. This is due to the
inability of the algae to assimilate condensed phosphates, and the low levels of
orthophosphate present as a consequence of hydrolysis at this early time.
Stewart and Alexander (1971) found that there was an uptake of condensed
phosphates present in detergent by the nitrogen-fixing, phosphate starved blue-
green alga, Anabaena flos-aquae. However, this uptake occurred over a period of
1 day, and uptake could actually be that of orthophosphate resulting from hydro-
lysis of the detergent during this time period. The available literature on the
utilization of condensed phosphates is scarce. However, it has been demonstrated
that hydrolysis of condensed phosphates and assimilation, probably in the form
of orthophosphate, occur in several green algae (Davis and Wilcomb, 1967), sever-
al blue-green algae (Davis and Wilcomb, 1968), and in the presence of various
microorganisms (Clesceri and Lee, 1965a). Hydrolysis is much slower in a ster-
ile environment (Clesceri and Lee, 1965b). Clesceri and Lee (1965b) also found
102
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a loss of phosphorus from the medium associated with the hydrolysis of condensed
phosphates, probably due to uptake by microorganisms. All these results are
consistent with the data presented for _P. boryanum.
ELECTRON MICROSCOPY
Cells of IJ. boryanum grown in different phosphate concentrations exhibit a
somewhat different morphology. Cells grown in either 100 or 1000 mg PO^ per
liter culture medium often possess polyphosphate bodies. They also appear to
undergo lysis at a much higher frequency. On the other hand, cells grown in 1
mg PO^ per liter are similar to cells which are grown in phosphate-free medium.
They possess large areas which are of medium electron density, and large intra-
thylakoidal spaces. Cells grown in 10 mg PO, per liter possess inclusions
normally found in blue-green algae, and the variations described for the other
phosphate concentrations, but to a much smaller degree.
Under conditions of phosphate starvation, the cells develop morphological
changes, namely, the areas of medium electron density, which later become the
areas of polyphosphate body development. These areas may develop at several
locations in the cell. The first of these is the nucleoplasmic area. This
results in the dispersion of DNA toward the periphery of the electron lucent
area and subsequent apparent association of DNA with polyphosphate bodies. The
electron lucent areas are also observed in the expanded intrathylakoidal spaces,
or in polyhedral bodies, with subsequent development of polyphosphate bodies in
these areas. The largest polyphosphate bodies are usually located in the nucleo-
plasmic areas. A few small bodies are usually encountered in phosphate starved
cultures. These might be correlated with the fluctuations in phosphorus content
of cellular extracts during starvation and the cellular debris, probably result-
ing from lysis, encountered in sectioned pellets. The polyphosphate bodies
could be formed from assimilation of phosphate released during the lysis. The
phosphate concentration never approaches that of the external medium, and con-
sequently the polyphosphate bodies formed are much smaller.
The mode of formation of the polyphosphate bodies in the areas of medium
electron density and in the polyhedral bodies appears to be essentially the same
as previously reported for the development of polyphosphate bodies in ribosomal
areas in Plectonema boryanum (Jensen, 1969). Polyphosphate bodies appear to
form in deposition loci, initially an area of medium electron density, and sub-
sequently a "porous body". Electron density increased from the periphery of the
body toward the center, until a completely electron dense body is formed (Jensen,
1969). This sequence was also found to be true in this study, regardless of the
location of the area of medium electron density.
Thus, in _P. boryanum grown under conditions of phosphate starvation and
rapid uptake, polyphosphate bodies can develop in 5 different areas of the cell:
(1) in ribosomal areas as previously described by Jensen (1969); (2) intra-
thylakoidally as previously described in Nostoc pruniforme (Jensen, 1968); (3)
in association with strands of DNA which is similar to a method previously
described by Voelz et al (1966) in Myxococcus xanthus; (4) in areas of medium
electron density; and, (5) in polyhedral bodies. Voelz et al (1966) described
three methods of formation of polyphosphate bodies under different environmental
conditions during the "overplus phenomenon" in Myxococcus xanthus. Only the
103
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third method described here is similar. Voelz et al (1966) also described
dense granules formed around polysaccharide inclusions and dense strands scat-
tered in the cytoplasm.
Another location of polyphosphate body formation has been reported by
Dierksheide and Pfister (1973). Under normal culture conditions, they found
that in the blue-green alga, Anacystis nidulans, polyphosphate body formation
occurred via the deposition of phosphate within the cyanophycin granule. This
site of deposition was never encountered in _P. boryanum.
Light microscopy of the cultures demonstrated that there was one apparent
polyphosphate body present. Numerous smaller bodies would not be distinct at
this limit of resolution. Thus it is difficult to state with any certainty
that one is viewing a single polyphosphate body.
The variation in cell length and the unusual division observed under con-
ditions of phosphate starvation is of interest. Phosphate has been suggested
to act as a control factor in cell division in Chlamydomonas reinhardti (Lien
and Knutsen, 1973). A similar type of variation in cell morphology has recently
been reported in phosphoglucomutase-deficient mutants of Bacillus licheniformis
grown under conditions of phosphate limitation (Forsberg jat al, 1973). These
mutants of J5. licheniformis often developed septa at angles to the normally
formed septa, and cell division did not always result in two equal daughter
cells.
X-RAY ENERGY DISPERSIVE ANALYSIS
X-ray energy dispersive analysis of inclusions identified as polyphosphate
bodies substantiates earlier physiological data. Polyphosphate bodies, examined
under a variety of conditions, contain as the major components phosphorus and
calcium. No magnesium is present as a major peak. This does not rule out its
presence, but it can be said that it is not a major constituent of polyphosphate
bodies in _P. boryanum.
At present, there is little available information on X-ray energy disper-
sive analysis of polyphosphate bodies in other organisms. There are several
reports of chemical identification, however, which substantiate the evidence
presented here. Rosenberg (1966) found that pyrophosphate granules isolated
from Tetrahymena pyriformis contained after ashing 48.5% phosphorus as pyrophos-
phate, 13.5% calcium, and 8.1% magnesium by weight. Munk and Rosenberg (1969)
investigating this same organism, found that equimolar concentrations of cal-
cium and magnesium ions were essential for the deposition of pyrophosphate
granules. Isolated granules were found to be deposits of calcium magnesium
pyrophosphate. Friedberg and Avigad (1968) reported that polyphosphate bodies
isolated from Micrococcus lysodeikticus contained 18.39 ug mg per mg dry weight
and 7.54 ug ca per mg dry weight. Cytoplasmic granules of Tetrahymena pyri-
formis analyzed in situ by electron probe analysis (Coleman et al, 1972) were
found to contain potassium, calcium, magnesium, phosphorus, and a lipid-like
material. The ratios of calcium to phosphorus and magnesium to phosphorus
tended to divide the granules into two distinct classes. These reports are in
some agreement with the data presented here.
104
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It can be seen that much work remains to be done in this area. At present,
no exhaustive study has been conducted previously on one organism, making a
comparison of these studies with those reported in the literature difficult to
interpret. The literature on phosphate metabolism is composed of a variety of
studies utilizing diverse organisms as well as a variety of environmental and
physiological parameters. Thus, selection of one organism to study seems es-
sential.
The relationship between the laboratory investigations reported here and
the field observations of other workers is also of interest. It has been sug-
gested that phosphorus is one of the key elements implicated in the limiting
nutrient controversy. The studies with J?. boryanum indicate that cultures
quite easily become phosphorus limited. When put into a variety of situations
where phosphorus is available as orthophosphate, the algae can easily assimi-
late large quantities of phosphates and store it as both soluble (short chain)
and insoluble (long chain) polyphosphates in the cellular inclusion known as a
polyphosphate body. This then probably serves as a phosphorus reserve. The
cells are still able to grow when phosphorus appears to be limiting in the
medium.
Studies with uptake of condensed phosphates do not rule out detergents as
sources of phosphate which are able to support the growth of JP. boryanum. They
merely indicate that condensed phosphates cannot be utilized directly, but must
first be hydrolyzed to orthophosphate. The orthophosphate can then be assimi-
lated and used for synthesis. It is most probable that algae, in their normal
environment, are phosphorus deficient. Possession of a mechanism whereby phos-
phate entering the environment could be assimilated rapidly and stored in an
osmotically inert form would enable organisms to survive in periods of low
available phosphorus. Phosphorus could enter the environment, for example,
through sewage, or land runoff after a rainfall. It is suggested that measure-
ment of phosphate concentrations in natural bodies of water should not be the
criterion for establishing phosphate levels which are conducive tu accelerating
eutrophication. A more meaningful estimation would be the total phosphorus
present in the cell, since phosphorus stored in the cell as polyphosphate is
capable of sustaining growth, even when there is no apparent available phosphate
in the environment.
105
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SECTION IX
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-76-103
2.
3. RECIPIENT'S ACCESSION>NO.
4. TITLE AND SUBTITLE
ASPECTS OF PHOSPHATE UTILIZATION
BY BLUE-GREEN ALGAE
5. REPORT DATE
September 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Thomas E. Jensen and Linda Sicko-Goad
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Herbert H. Lehman College of CUNY
Bronx, New York 10468
10. PROGRAM ELEMENT NO.
1BA031
11. CONTRACT/GRANT NO.
R-800431-03-0
12. SPONSORING AGENCY NAME AND ADDRESS
Ecological Effects Research Division
Corvallis Environmental Research Laboratory
Corvallis, Oregon 97330
13. TYPE OF REPORT AND PERIOD COVERED
final
14. SPONSORING AGENCY CODE
EPA/ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT : xhe eff-ects of various external phosphate concentrations on physiological
and cytological aspects of Plectonema boryanum have been studied. £. boryanum was
found to tolerate a wide range of phosphate concentrations, from 1 to 1000 mg of phos-
phate per liter. Growth of the alga in these concentrations was characterized by
changes in the subcellular distribution of phosphorus-containing compounds and in ultraj-
structural changes which were monitored by transmission electron microscopy.
Culturing the alga in phosphate-free or phosphate deficient medium led to general
reductions of phosphate in all cell fractions examined, with the most dramatic decrease
in both short and long chain polyphosphates. Cytologically, the phosphate starvation
period was characterized by the development of areas of medium electron density, and
vacuolization resulting from expansion of intrathylakoidal spaces.
Inoculation of the phosphate-starved algae into a medium containing a known amoun
of phosphate led to increases in all phosphorus-containing fractions, particularly the
polyphosphates. Increases in both short and long chain polyphosphates were greater
than an order of magnitude. The satisfaction of the "phosphorus debt" was met essen-
tially within an hour. Examination of the cells revealed that the cells develop poly-
phosphate bodies in the characteristic areas of medium electron density that develop
during phosphate starvation or phosphate limitation. X-ray energy dispersive analysis
of the polyphosphate bodies confirmed that they are deposits consisting of two major
elements, phosphorus and calcium.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COS AT I Field/Group
Phosphate
Polyphosphate
Physiology
Fine Structure
Blue-green Algae
08/H
13. DISTRIBUTION STATEMENT
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
121
RELEASE TO PUBLIC
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
122
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