ATP measurements in laboratory cultures and field populations of lake plankton

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ATP measurements in laboratory cultures and field populations of lake plankton
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xi, 130 leaves. : ill. ; 28 cm.
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Browne, Francis Xavier, 1943-
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Water -- Pollution -- Measurement   ( lcsh )
Adenosine triphosphate   ( lcsh )
Plankton   ( lcsh )
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theses   ( marcgt )
non-fiction   ( marcgt )

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Thesis:
Thesis--University of Florida, 1971.
Bibliography:
Bibliography: leaves 118-129.
Statement of Responsibility:
by Francis Xavier Browne.
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Manuscript copy.
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Vita.

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University of Florida
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Full Text











ATP Measurements in Laboratory Cultures and
Field Populations of Lake Plankton

















By

FRANCIS XAVIER BROWNE


A IS-SERTATION -PPESFNTLP! TC; rTIIE CIRAD-U.A,-,TE COUNCIL OF
TH~E ENITRSYIY 'F FLOCI R TD IN PTIAL
PLL TLL1:EA:T CF -I' PFl UIRE'LXTP EC TS, F -7 EH
07 D'lOCTTOR, OF PHILOSOIPHlY







ULiYF.1>TY CF FLORIDA
1971














ACKNOWLEDGMENTS


The research effort reported in this dissertation

has been accomplished with the support of many people.

Particular thanks are expressed to Mr. Roger Yorton, Miss

Virginia Morgan, and Mrs. Zena Hodor for their helpful

assistance.

I would like to thank Dr. W. H. Morgan for his

friendship and support during these years. Special thanks

go to Jenny Hunt for her assistance in many little things

over the years and for one big thing typing this

dissertation.

I wish to thank my Supervisory Committee for their

advice and encouragement, but especially for their

friendship during this period. Particular gratitude is

extended to my principal advisors, Drs. Patrick L.

Brezonik and Jackson L. Fox. I also thank the rest of

Imy committee, Drs. Hugh Putnam and Frank Nordlie, for

their ideas and insights.

I also wish to thank my parents for all the help

and support they gave me throughout the years. For

without them, truly. none of this would have been possible.









Finally, I thank my wife, June, for everything:

the frustrations, the sacrifices, the added responsibili-

ties she endured. Her encouragement and understanding

have made all this possible.

This investigation was made possible through the

financial support of the Department of Health, Education,

and Welfare, PHS Traineeship Grant No. 5-T01-EC-00035-10.


iii















TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ........................................ ii

LIST OF TABLES ..................... ................. vi

LIST OF FIGURES ..................................... viii

ABST A CT .............................................. x

CHAPTER I. INTRODUCTION ................... ... ....... 1

CHAPTER II. MECHANISM OF ATP METABOLISM IN ALGAE ... 4

CHAPTER III. LITERATURE REVIEW ..................... 9
Introduction ................................... 9
Eiomass Parameters .................. ......... 10
Pigment Concentration ..................... 10
Particulate Organic Carbon ................ 14
Cell Number ............................... 15
Packed Cell Volume and Dry Weight ......... 16
DNA ......................................... 17
Limiting Nutrient Bioassay Methods ........... 18
Carbon-14 Uptake .... ...................... 19
Enzymatic and Extractive Techniques ....... 21
ATP .......................................... 24

CHAPTER IV. EXPERIMENTAL METHODS AND MATERIALS ..... 36
Analytical Techniques .......................... 36
Experimental Materials ................. ...... 38

CHAPTER V. METHODS OF ATP ANALYSIS ................. 41

CHAPTER VI. EXPERIMENTAL RESULTS ................... 55
ATP Bionass Results .................. ...... 55
ATP vs. Chlorophyll a ................... 56
ATP vs. Dry Weight ......................... 68
ATP vs. Cell Number ........................ 76
ATP vs. Absorbance ......................... 76
Zooplankton Study ......................... 80














ATP Response Results
Light-Dark Study ...
pH Studies .........
Toxicity Studies ...
Nutrient Studies ...

CHAPTER VII. DISCUSSION .....

CHAPTER VIII. CONCLUSIONS AND

Conclusions ...........

Recommendations .......

BIOBLIOCGRAPHY ................

BIOGRAPHICAL SKETCH ..........


........
........
l.......
........





COMMIENDA

........

....o...

........

........


* *









TIONS

* *

* *

* *


Page

80
81
83
88
93


110

115

115

117

118

130















LIST OF TABLES


Page


TABLE 1. STANDARD FREE ENERGY OF HYDROLYSIS OF
COMMON INTRACELLULAR PHOSPHATE
COMPOUNDS ..................................

TABLE 2. COMPARISON OF BIOMASS ESTIMATED BY ATP,
CHLOROPHYLL, DNA AND ORGANIC CARBON ........

TABLE 3. ATP AS PERCENT DRY WEIGHT ..................

TABLE 4. ATP TO CHLOROPHYLL RATIO FOR VARIOUS
ALGAE UNDER DIFFERENT ENVIRONMENTAL
CONDITIONS .................................

TABLE 5. EFFECT OF INCUBATION ON FIREFLY EXTRACT
AND ATP LIGHT EMIISSION (6-SEC COUNT) .......

TABLE 6. ATP VS. CHLOROPHYLL A CORRELATION
COEFFICIENTS FOR UNIALGAL CULTURES .........

TABLE 7. CHEMICAL AND BIOLOGICAL CHARACTERISTICS
OF EXPERIMENTAL LAKES ......................

TABLE 3. ATP VS. CHLOROPHYLL A CORRELATION
COEFFICIENTS FOR LAKE PHYTOPLANKTON ........

TABLE 9. ATP TO CHLOROPHYLL A RATIOS FOR ALGAL
CULTURES AND LAKE PHYTOPLANKTON ............


TABLE 10.


TABLE 11.


TABLE 12.


TABLE 13.


ATP VS. DRY WEIGHT CORRELATION
COEFFICIENTS FOR UNIALGAL CULTURES ..........

ATP TO DRY WEIGHT CORRELATION
COEFFICIENTS FOR LAKE PHYTOPLANKTON ........

ATP TO DRY WEIGHT RATIOS FOR ALGAL
CULTURES AND LAKE PHYTOPLANKTON ............

COMPARISON OF ATP/DRY WEIGHT RATIO WITH
AVERAGE TURBIDITY OF EXPERIMENTAL LAKES ....


vi












TABLE 14.


TABLE 15.


TABLE 16.


TABLE 17.


TABLE 18.


TABLE 19.


ATP TO ABSORBANCE CORRELATION
COEFFICIENTS FOR UNIALGAL CULTURES .......

PERCENT REDUCTION AFTER ONE-HOUR
INCUBATION WITH MERCURIC CHLORIDE ........

RESPONSE OF CELLULAR ATP 'O ADDITION
OF PHOSPHATE .............................

RESPONSE OF CELLULAR ATP TO NITRATE
ADDITION .................................

ATP TO DRY WEIGHT RATIOS OF VARIOUS
MARINE ALGAE .............................

ATP TO DRY WEIGHT PATIOS OF SELEHASTRUM .


vii


Page


79


90


99


108


111

112















LIST OF FIGURES


Page

FIGURE 1. CHEMICAL STRUCTURE OF ATP ................ 5

FIGURE 2. CELLULAR CONTENTS OF ATP IN EUGLiNA
GRACILIS DURING ALTERNATING PERIODS OF
LIGHT AND DARK .......................... 27

FIGURE 3. ATP CONCENTRATION VS. CELLULAR ORGANIC
CARBON ................................... 29

FIGURE 4. LUMINESCENCE DECAY OF ATP FIREFLY
LANTERN EXTRACT REACTION WITH TIME ....... 48

FIGURE 5. DEPENDENCE OF QUANTITATIVE ACTIVATED
SLUDGE ATP EXTRACTION ON TEMPERATURE OF
EXTRACTION SOLUTION ...................... 51

FIGURE 6. STANDARD ATP CURVE ........... ............ 53

FIGURE 7. CORRELATION OF ATP WITH SELEI.ASTPUM
CONCENTRATION ............................ 54

FIGURE 8. ATP VS. CHLOROPHYLL A OF SELENASTRUM AND
CHLORELLA .................................. 57

FIGURE 9. ATP VS. CHLOROPHYLL A FOR A7PABAENA ...... 58

FIGURE 10. ATP VS. CHLOROPHYLL A FOR MICROCYSTIS .... 59

FIGURE 11. ATP VS. CHLOROPHYLL A OF DILUTED
MICROCYSTIS .............................. 62

FIGURE 12. ATP VS. CHLOROPHYLL A FOR LAKE
PHYTOPLANKTON ............................ 64

FIGURE 13. VARIATION OF ATP/CHLOROPHYLL A RATIO WITH
CELL AGE ................................ 69

FIGURE 14. ATP VS. DRY WEIGHT OF UNIALGAL CULTURES .. 70

FIGURE 15. ATP VS. DRY WEIGHT FOR LAKE
PHYTOPLANKTON .............................. 73


viii









Page

FIGURE 16. ATP VS. CELL COUNT FOR IICROCYSTIS ....... 77

FIGURE 17. ATP VS. ABSORBANCE OF UNIALGAL CULTURES .. 78

FIGURE 18. RESPONSE OF ATP TO PERIODS OF LIGHT AND
DARK ...................................... 82

FIGURE 19. EFFECT OF pH ON ATP CONTENT OF SELENASTRUM
CELLS ..................................... 84

FIGURE 20. EFFECT OF pH ON ATP CONTENT OF ANDERSON-
CUE LAKE WATER ...; ....................... 86

FIGURE 21. EFFECT OF pH ON ATP CONTENT OF BIVENS
ARM LAKE WATER ........................... 87

FIGURE 22. ATP POOL RESPONSE TO INCUBATION WITH
VARIOUS CONCENTRATIONS OF MERCURY ........ 89

FIGURE 23. RESPONSE OF MERCURY-POISONED CHLORELLA
AFTER TRANSFER TO FRESH MEDIUM ........... 92

FIGURE 24. RESPONSE OF BIOMASS PARAMETERS IN BIVENS
ARM TO ADDITION OF COPPER ................ 94

FIGURE 25. ATP RESPONSE IN ANAJBAEA TO ADDITION OF
CARBON DIOXIDE ........................... 96

FIGURE 26. ATP RESPONSE OF BIVENS ARM TO ADDITION
OF CARBON DIOXIDE ........................ 98

FIGURE 27. RESPONSE OF ATP IN ANABAENA TO ADDITION
OF PHOSPHORUS ............................ 101

FIGURE 28. RESPONSE OF ATP IN MICROCYSTIS TO
ADDITION OF PHOSPHORUS ................... 102

FIGURE 29. RESPONSE OF ATP IN SELENAS2RUM TO
ADDITION OF PHOSPHORUS ................... 103

FIGURE 30. RESPONSE OF ATP IN CHLORELLA TO
ADDITION OF PHOSPHORUS ................... 104

FIGURE 31. RESPONSE OF ATP IN AJA3BAEEA TO
ADDITION OF NITROGEN ..................... 106

FIGURE 32. RESPONSE OF ATP IN SELEIASTRUM TO
ADDITION OF NITROGEN ..................... 107









Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ATP MEASUREMENTS IN LABORATORY CULTURES AND
FIELD POPULATIONS OF LAKE PLANKTON

By

Francis Xavier Browne

December, 1971

Chairman: Patrick L. Brezonik, Ph.D.
Co-Chairman: Jackson L. Fox, Ph.D.
Major Department: Environmental Engineering

ATP measurements in laboratory unialgal cultures and

lake plankton were made to investigate the use of ATP as a

biomass and activity parameter. No account was taken of

the bacterial population in the unialgal cultures or the

lake plankton. Although the algal cultures were not axenic,

efforts were made to minimize bacterial growth. ATP was

analysed using the luciferin-luciferase firefly reaction.

The ATP analysis proved to be sensitive and reliable for

quantitative determination of cellular ATP concentrations.

Good correlation was observed between ATP and

chlorophyll a, dry weight, and cell number. ATP to chloro-

phyll a ratios were relatively constant, ranging from 0.09

to 0.35. These values agree with those reported by other

investigators. ATP to dry ,weight ratios were also rela-

tively constant. The presence of detrital material in

laboratory and field samples reduced the significance of

any particular ATP to dry weight ratio.









The ATP concentration was observed to be relatively

constant under both light and dark conditions. Maximum

cellular ATP concentrations occurred at a oH level at or

in the range of the normal pH of the organisms tested.

Rapid decreases in ATP concentration occurred

immediately after addition of toxic substances. Results of

one experiment indicate that toxic substances, at least

in the first few hours, cause a reduction in ATP per cell

rather than a reduction in viable biomass.

Additions of nutrients to nutrient-deficient cultures

resulted in rapid increases in ATP concentration.

The advantages of ATP over existing biomass para-

meters are manifold. The results of this research indicate

that ATP analysis is applicable as a biomass parameter for

aquatic systems. It is also a sensitive activity parameter

which can be applied to toxicity bioassays. These results

also indicate that ATP could be used as a qualitative

activity parameter in limiting nutrient bioassays.














CHAPTER I. INTRODUCTION


Pollution of surface waters is well recognized as a

serious problem in the United States and other highly

developed countries. The effects of pollution range from

the death of aquatic organisms as a result of oxygen deple-

tion from organic discharges or poisoning from toxic

industrial waters to over population with nuisance organ-

isms as a result of enrichment from nutrient discharges.

To properly measure and evaluate the effects of various

pollutants on a particular water, reliable measures of

biomass and activity are needed. The biomass parameters

most often used in lake and stream studies are chlorophyll

concentration, suspended solids, or plankton count. The

application, theory and problems associated with these

parameters are discussed in Chapter III, but it suffices to

say here that better measures of phytoplankton biomass and

activity are needed.

The deficiencies of present methodologies are

evidenced by the many conflicting reports in the literature

concerning limiting nutrients and the effects of municipal

and industrial discharges on a body of water. In 1969 a

Provisional Algal Assay Procedure (Joint Industry-Government

Task Force on Eutrophication 1969) was developed to









standardize existing methods of investigating phytoplankton

responses in natural waters. However, this procedure has

not been wholly successful probably because it utilizes

inadequate biomass parameters.

An adequate biomass parameter must have a relatively

constant cellular concentration under most environmental

conditions, and it must not be associated with non-living

material. Cellular biochemistry offers a number of possibil-

ities for measurement of activity and biomass. One bio-

chemical parameter that seems to offer an appropriate measure

of biomass and metabolic activity is adenosine triphosphate

(ATP). The overall objective of this study was to investi-

gate the use of adenosine triphosphate (ATP) as a measure

of phytoplankton biomass and metabolic activity. Biomass

parameter evaluation was divided into two phases. First, the

ATP content of batch unialgal cultures was measured and

correlated with traditional biomass parameters. In the second

phase, the ATP content of natural lake phytoplankton was

measured and correlated with biomass parameters. ATP as a

measure of metabolic activity was evaluated by observing the

ATP changes in laboratory algal cultures and lake phytoplankton

which were subjected to varying environmental conditions.

Correlation of cellular ATP concentration with current

biomass parameters would indicate the validity of using ATP

to measure phytoplankton biomass. The lack of response of





3



cellular ATP to various environmental conditions would

indicate the stability of cellular ATP to minor environ-

mental changes. The response of cellular ATP to additions

of nutrients and toxic substances would indicate its useful-

ness as a rapid bioassay parameter.














CHAPTER II. MECHANISM OF ATP METABOLISM IN ALGAE


ATP hIs been called the "energy currency" of living

cells. It occurs in all living cells. ATP is a nucleotide

containing adenine, a 6-amino derivative of purine, D-ribose,

a 5-carbon sugar, and three phosphate groups as shown in

Figure 1. The ATP molecule within the cell is highly

charged with negative charges concentrated around the

polyphosphate structure. Most of the ATP in cells is

present as a M.g'2 complex.

It is coirmon practice to refer to ATP as a "high

energy" phosphate compound. However, as shown in Table 1,

ATP has an intermediate energy value when compared with

other phosphate compounds. It is this intermediate position

which makes ATP so important.


TABLE 1

STANDARD FREE ENERGY OF HYDROLYSIS OF
CC',:ON INTRACELLULAR PHOSPHATE COMPOUNDS

AG
Kcal/Mole
Phosphoenolpyruvate -14.80
1,3-Diphoyphaglycerate -11.80
Phosphocreative -10.30
Acelyi phosphate -10.10
Phosphoarginine 7.70
ATP 7.30
Glucose 1-phosphate 5.00
Fructose 6-phoaphate 3.80
Glucose 6-phosphate 3.50
Glycerol l-phosphate 2.20
























Z
UU
20


u u




-1


I I











C C-C
0 1 --- -








0
C
I u

0 ^-1
) 5









The function of the ATP-ADP (adenosine diphosphate)

system is to act as an intermediate between high-energy

phosphate compounds and low-energy phosphate compounds. One

set of enzymes helps transfer phosphate from high-energy

compounds to ADP, forming ATP; while another set of enzymes

help transfer the terminal phosphate group of ATP to low-

energy phosphate acceptors. No enzymes exist that can

transfer phosphate groups directly from high-energy donors

to low-energy acceptors. Thus, all high-energy phosphate

transfers must use the ATP-ADP system.

Inside living cells there is usually very little ATP

since it is continually being used up and remade. The energy

in the energy-rich bonds of ATP is used to do cellular work.

It is used to synthesize large molecules from simple subunits,

to transport substances in and out of the cell, and to

perform mechanical work.

In algae, ATP is formed from two basic processes:

photosynthesis and respiration. Eucaryotic algal cells

contain chloroplasts, where photosynthesis occurs, and

mitochondria, where respiration occurs. Procaryotic cells,

like the blue-green algae, do not have chloroplasts and

mitochondria. Although eucaryotic and procaryotic cells

differ in structure, the chemical reactions occurring in

photosynthesis and respiration are similar.

Photosynthesis consists of both light and dark

reactions; the light reactions drive the entire process.

In the light reactions, water is split, ATP is formed, and









oxygen is liberated. The dark reactions use carbon dioxide

to form siLmple sugars, some of which are converted into

glucose as the product of photosynthesis. In photosynthesis,

chlorophyll molecules absorb light energy and give off high-

energy electrons. It is believed that molecules of ferredoxin,

a protein which contains iron, pick up the high-energy

electrons. The energy gained by ferredoxin is released in a

series of electron transfers. This released energy is used

to form ATP. The electrons, after losing their energy, return

to the chlorophyll molecule again. This process is called

cyclic photophosphorylation. Another process called non-

cyclic photophosphorylation also occurs and is similar to

the cyclic process except the original electrons do not return

to the chlorophyll molecule. Instead, the electrons are

transferred to NADP (nicotinamide adenine dinucleotide

phosphate) which subsequently is reduced. Electrons from

the hydroxyl groups of water are transferred to the chlorophyll

molecules, filling the electron gaps. Thus the primary

purpose of photosynthesis is to form ATP and reduced NADP,

with oxygen evolved as a by product. Under normal condi-

tions one would expect non-cyclic photophosphorylation to

occur since this process forms ATP and reducing power (in

the form of reduced NADP). Both ATP and the reducing power

are used in the dark reactions to form glucose from CO2.

The role of cyclic photophosphorylation is not completely

understood. Some believe it is a shunt mechanism used to









form ATP without formation of reduced NADP. It appears that

light-induced cyclic electron flow is used to generate ATP

at whatever rate is required by the cell, without the

necessity of generating reduced NADP or of evolving molecular

oxygen.

ATP production from respiration is basically the

same for algae and other microorganisms. Catabolic processes

which terminate in the Krebs Cycle product ATP. For example,

one molecule of glucose is transformed into two molecules

of pyruvic acid, forming two molecules of ATP. These two

molecules of pyruvic acid are broken down into six molecules

of carbon dioxide and water, liberating 36 molecules of ATP.

Thus, a net ATP production of 38 molecules of ATP results

from the oxidative breakdown of one glucose molecule.














CHAPTER III. LITERATURE REVIEW


Introduction


Vollenweider (1970) defines eutrophication as any-

thing that accelerates the nutrient loading, increases the

nutrient level and directly increases water productivity.

lH explains that a mere increase in the nutrient level is

not important if it does not produce unpleasant or undesira-

ble effects on the metabolism of the water. Vollenweider's

definition emphasizes the two primary constituents of

eutrophication the addition of nutrients to a water and

the concomitant response of the phytoplankton. Measuring

the standing phytoplankton biomass and its response to

nutrient additions is becoming increasingly important; yet,

there is little consensus as to which parameter or method

is best to use. A variety of methods is presently used to

measure biomass and its response to nutrient additions,

including pigment concentration, particulate organic carbon

concentration, cell number or areal units, packed cell

volume and dry weight, and DNA concentration. While commonly

used each suffers from certain deficiencies. The following

sections will briefly describe the merits and drawbacks of

each.









Biomass Parameters


Pigment Concentration

The algal pigments consist primarily of the chloro-

phylls, the carotenoids, and the phycolilins. Chlorophylls

are lipo-soluble molecules characterized by strong absorp-

tion of red (650 680 my) and blue light (400 450 mu)

and by their red fluorescence in organic solvents. Chloro-

phylls are divided into three groups: chlorophyll a which

is present in all photosynthesizing cells; chlorophyll b,

an oxidized derivative of chlorophyll a, which is found in

green algae; and chlorophyll c which is found in brown

algae and diatoms. Carotenoids are C40 hydrocarbons with

numerous conjugated double bonds. There are two major

groups of carotenoids: the carotenes, which are pure

hydrocarbons, and the xanthophylls, which contain from 1

to 8 hydroxyl groups making them more polar. The carote-

noids serve as accessory pigments, extending the range of

visible light useful in photosynthesis. They are usually

yellow or orange and absorb light in the blue-violet region

of the spectrum. All photosynthesizing cells contain

carotenoids. Odum and Nixon (1970) have studied the role

of carotenoids in photorespiration. Odum et al (In Press)

note that the carotenoid-chlorophyll ratio may help mTeasure

the relative use of solar energy in photosynthesis and

photorespiration. The third major class of pigments are

the phycobilins which are related structurally to the









chlorophylls. They consist of an open conjugated system

of four pyrrol rings, the fundamental structure of the bilin

pigments, so called because they were discovered in bile.

The phycobilins (algal bilins) are water-soluble pigments

consisting of the phycoerythrins and tne physocyanins. Both

pigments are found in the red marine algae (Rhodophyceae)

and the primitive blue-green algae (Cyanophyceae).

Phvcoerythrin, present primarily in red marine algae, absorbs

light in the -riddle of the visible spectrum. Phycocyanin,

present primarily in blue-green algae, absorbs red light at

about 630 mp.

Chlorophyll is the major pigment of algae and occurs

in all species. Because of its universal distribution,

chlorophyll a is often used to estimate phytoplankton

biomass. Weber and McFarland (1969) and Keup and Stewart

(1966) used chlorophyll a as an estimate of the phytoplankton

standing crop and found it to correlate well with carbon,

nitrogen, and phosphorous content.

One problem with using chlorophyll as a measure of

photoplankton biomass is that the chlorophyll concentration

varies with environmental conditions. Spoehr and Milner

(1949) measured extremes of 0.1 percent and 6 percent for

chlorophyll content as percent of dry weight. Usually the

chlorophyll content is about 0.5 to 1.5 percent of the dry

weight (Round 1967). Studies on algae cultured under

controlled laboratory conditions demonstrate that mineral

nutrition, light intensity, and cell age affect the cellular









chlorophyll concentration. Emerson (1929) found a direct

correlation between photosynthetic activity and chlorophyll

content in ChlorelZa pyrenoidca and concluded that a

reduced chlorophyll content was the only factor responsible

for the reduced photosynthesis of iron-deficient cells.

Sargent (1940) noted that chlorophyll constituted 6.6 per-

cent of the dry weight of "shade-grown" Chlore Zla cells

but only 3.3 percent of "sun-grown" cells. Myers (1946)

found that chlorophyll per milliliter of packed cells was

inversely related to light intensity, but he also found a

direct relationship between light intensity and cell size.

Fogg (1965) observed that the cellular chlorophyll concen-

tration decreased with cell age and nutrient deficiency.

Ryther and Yentsch (1957) found that marine phytoplankton

at light saturation has a reasonably constant assimilation

ratio of 3.7 grams of carbon assimilated per hour per gram

of chlorophyll. Calculated production rates based on this

ratio and on chlorophyll-light measurements were similar

to those obtained by simultaneous use of the light-and-dark

bottle oxygen method. Bain (1968) used a similar method

to predict primary production in San Francisco Bay. Some

controversy exists as to whether chlorophyll a can best be

used to measure biomass or primary production (Odum et al

1958).

The analytical technique for extracting and calcu-

lating chlorophyll concentration suffers from many weak-

nesses. Strickland and Parsons (1968) note that the only









rapid chemical method known fcr estimating living plant

matter in the particulate organic matter of sea water is

to determine the characteristic pigments. However, they

also note that the amount of organic material associated

with a given plant pigment is variable, depending on the

class of algae and the nutritional state. A factor ranging

from 25 to 100 is used to convert chlorophyll a concentra-

tion to total plant carbon. According to Strickland and

Parsons (1968) extraction with 90 percent acetone gives

results undoubtedly low in many instances because of the

presence of plant cells that are not fully extracted.

These authors state that some species may retain 50 percent

or more of the pigments in their cells. Bogarad (1962)

notes that it is generally more difficult to extract

chlorophylls from algae than from higher plants; he adds

that extraction with hot or cold methanol is usually more

effective than acetone. Another weakness of the extraction

process is that inactive chlorophyll and degradation products

may be determined along with the active chlorophyll of the

living phytoplanktons. Glooschenko and Moore (1971)

analyzed Lake Ontario water samples for chlorophyll a and

its degradation products, pheophorbide a and pheophytin a.

These authors found the degradation products made up less

than 20 percent of total chlorophyll a until the decline of

the spring growth, after which degradation products accounted

for up to 100 percent measured chlorophyll a in some regions

of the lake. These studies indicate the necessity of

correcting chlorophyll a data for degradation products.









Particulate Organic Carbon

Particulate organic carbon is often used as a

measure of phytoplankton biomass. Menzel and Ryther (1964)

measured the composition of particulate matter in the

western North Atlantic to determine the relationship

between carbon, nitrogen, phosphorus, and chlorophyll.

Regression of phosphorus or chlorophyll vs. nitrogen or

carbon, when extrapolated back toward the origin, indicated

appreciable amounts of nitrogen and carbon in the absence

of phosphorus and chlorophyll. In contrast, the regression

of phosphorus vs. chlorophyll had its intercept at the

origin. The authors concluded that chlorophyll and phospho-

rus are decomposed or mineralized at about the same rate,

while carbon and nitrogen are more refractory. Menzel and

Goering (1966) studied the decomposition of particular

matter from surface and deep Atlantic waters. Changes in

carbon were related to the initial biomass of phytoplankton.

They found that the living carbon represents a variable

fraction of the total organic particulate matter present

in surface waters. Superimposed on this is detrital carbon

which is refractory to decomposition. Parsons and Strickland

(1962), Holm-Hansen (1969) and others have also found

variable amounts of refractory particulate organic carbon

in phytoplankton samples. Because of this refractory

nature of carbon its use as an estimate of bionass is

questionable.









Cell Number

Early work on algal physiology utilized the cell

number, determined by counting cells with a hemocytometer,

whipple disc, or a Segwick-Rafter Cell (Myers 1962). With

unialgal cultures the cell count can be relatively meaning-

ful because of the approximate uniformity of the cell size

and volume. Cell counts on a heterogeneous culture of algae

are tedious and open to misinterpretations. Oswald and

Gaonkar (1969) in a review of the Provisional Algae Assay

Procedure (Joint Industry-Government Task Force on Eutro-

phication 1969) note the difficulty in counting cells in

mixed populations: taking the size of ChllorelZa pyrenoidosa

as unity, the size of Scenedesmus obliguus is 1.2,

ChlZarydoou.cas 6.9, Euglena gracilis 54, Euolcna viridis 84,

and a Phacus sp. 71. Besides this variability among species,

cell size differs with both age and nutritional state of

each species. EugZena cells tend to become larger with age

while ChlorcZla and Scendesmzus cells become smaller.

Nutrient starved EugZena usually decrease in size while

nutrient starved Chloretla increase in size. There is also

a difficulty in counting filamentous and agglomerated cells

like Anabaena and Microcystis. Oswald and Gaonkar also

note that many algae are photo regulated. Chlorella, for

example, accumulates nutrients during the day and divides

during the night; thus, unless cells are counted at the same

time each day, the number of cells could vary significantly.









Aside from the problem of size, there are difficul-

ties with counting technique. Ilee (1954) studied varia-

bility in cell counts using the hemocytometer, a popular

and simple cell counting device. He found that for Euglena

the variation in cell counts exceeds 30 percent when the

number of cells counted in the hicmocytometcr grid is less

than 30, but that clumping occurs when the number of cells

exceed 100. For Scenedesmus he found that a minimum of

about 15 percent variation occurs when the number of cells

counted in the five central squares exceeds 80. The

variation is a minimum for Chlorella when the cells in the

five squares exceed 100.

Finally, a problem exists with respect to count

variations among technicians (Oswald and Gaonkar 1969).

Although this problem occurs with all laboratory techniques,

it is particularly acute for cell counting since personal

judgement and knowledge of algae identification play a

major role.


Packed Cell Volume and Dry Weight

Oswald and Gaonkar (1969) recommended the volumetric

technique, commonly known as the packed-cell volume, be

added to the Provisional Algal Assay Procedure as a method

for evaluating algal growth. As Oswald (1967) noted, the

volurmetric (packed-cell volume) technique is a non-

destructive procedure in which the sample need not be dis-

carded after analysis and can be used for further analysis.









According to Oswald, the packed volume usually contains

about 14 percent dry weight of algae and the approximate

algal content in dry weight is 1400 times the packed cell

volume plus or minus 400. Myers (1962) observed that

sufficient centrifugal force and time must be used to

obtain a constant and minimum packed cell volume. A major

objection to the packed cell volume as a measure of biomass

is that it measures detrital as well as living matter. Lee

et a7. (1971) found a significant amount of detrital material

in unialgal cultures of SeZenastrum capricornutum after only

four days of growth in batch systems. Thus, quantitative

interpretation of packed cell volume results can be mis-

leading.


DNA

Deoxyribonucleic acid (DNA) consists of two poly-

nucleotide chains twisted upon each other to form a double

helix; it is the genetic substance which contains the

hereditary information of the cell. The use of DNA as a

measure of phytoplankton biomass has been investigated by

Holm-Hansen and colleagues (1968). After measuring concen-

trations in the Atlantic and Pacific Oceans and comparing

them with measurements of chlorophyll and organic carbon,

the authors concluded that there is either a considerable

quantity of living material that is high in DNA or that DNA

is associated with particulate, non-living material. Holm-

Hansen (1969) measured ATP, chlorophyll, DNA, and organic









carbon off the coast of California; he converted, ATP,

chlorophyll, and DNA concentrations to organic ca-rbon using

conversion factors of 250, 50, and 100 respectively as

shown in Table 2.


TABLE 2

COMPARISON OF BIOMASS ESTIMATED BY ATP, CHLOR(OPHYLL, DNA
AND ORGANIC CARBON


Depth (m)
Biomass (pg c/liter) 0 50 100 200

Direct Examination 13 25 3.8 1.1
ATP 24 22 5.6 1.9
Chlorophyll 14 22 5.0 0.2
DNA 250 200 95 50


The biomass estimates as determined from DNA measurements

are, according to Holm-Hansen, "impossibly high." He

concluded that DNA is an excellent biomass parameter for

laboratory cultures but not for work in the natural environ-

ment.

None of the methods discussed in this section are

very good estimates of biomass. With biomass, nothing is

constant or absolute; all parameters vary and this variation

makes it difficult to say which is the best measure of

biomass.


Limiting Nutrient Bioassay Methods


Many methods are used to determine which physical or

chemical conditions limit phytoplankton growth. One general

approach is to monitor the phytoplankton response over a









period of time after enriching the sample with some nutrient.

Although popular, this method depends on the response para-

meter measured and may give misleading results if another

nutrient or condition becomes limiting. Response parameters

include the various biomass measures and "C assimilation

rates. The former are usually insensitive to short term

changes while insufficient time for response to enrichment

may give inaccurate results with the latter. Other nutrient

bioassay procedures are based on analysis of planktonic

constituents (e.g.,adaptive enzymes, percent nitrogen or

phosphorus) which vary in response to nutritional conditions.


Carbon-14 Uptake

Carbon assimilation rates, as measured by the fixation

of carbon-14, are often used as a measure of algal growth

in bioassays (Goldman 1960, 1961, 1962, 1964, 1965, 1967;

Goldman and Carter 1965; McAllister et aZ 1964; Menzel and

Ryther 1960, 1961; Putnam 1966). Primary production

measured by the carbon-14 method is a function of the environ-

mental conditions prevailing at the time of incubation.

Light intensity, for instance, is an important factor in

the amount of carbon fixed. After incubation formaldehyde

is usually added to kill the organisms, but Strickland

and Parsons (1968) note that even small concentrations of

formaldehyde may affect the excretion or loss of organic

material from delicate algae. In situ measurements of

productivity require two to three hours of incubation, thus









limiting the number of sampling stations that can be tested

in one day. On the other hand, laboratory incubation using

a constant light intensity gives more precise but inaccurate

results. Oswald and Gaonkar (1969) state that the technique

of radiocarbon measurements as set foith in PAAP seems

n;:edlessly complex, delicate, and subject to error in the

hands of inexperienced personnel.

Goldman (1960, 1962, 1963) used carbon-14 uptake for

bioassays of limiting nutrients. Carbon-14 was added to a

phytoplankton culture to which nutrients were added.

Periodically, subsamples were removed and the uptake of

carbon-14 measured. This method measured the total carbon

taken up from the beginning of the experiment. An alternate

method consisted of adding nutrients to an unlabeled phyto-

plankton culture and periodically removing a subsample,

adding carbon-14 to it, incubating for three to six hours, and

then measuring the amount of carbon taken up during the

incubation period. Interpretation of the results of these

methods can be misleading due to the inherent variation in

measuring carbon-14 uptake. Since net productivity is not

being measured by this method, it is questionable what

exactly the results mean; is an increase in biomass also

occurring?

Many basic questions concerning the carbon-14 method

must be answered. Is there any luxury uptake of carbon;

how much fixed 4C is excreted, how much is internally

recycled? What is the difference between C and "C









uptake? Strickland and Parsons (1968) use the factor of

1.05 to account for this difference, buc note that this

value is uncertain.


Enzymatic and Extractive Techniques

Enzymatic and extractive methods of analysis have

been used to study limiting nutrient concentrations in algal

cultures (Fitzgerald 1966, 1969; Shapiro and Ribeiro 1965).

Surplus phosphorus, the internal concentration in excess of

the amount needed for maximum growth, can be extracted from

algae by boiling the algal sample in water for 60 minutes

and measuring the orthophosphate in the extract. Fitzgerald

(1969) used this procedure and demonstrated that algae limited

by phosphorus contain little or no extractable phosphate

while algae grown with surplus phosphorus released more than

0.08 mg P04-P/100 mg algae. Gerloff and Skoog (1954, 1957)

used a tissue analysis method to evaluate nutrient availa-

bility in Wisconsin lakes for the growth of .'icrocystis

aer-:igincsa. Tissue analysis was also used to measure the

nutrient availability for the growth of angiosperm aquatic

plants (Gerloff and Kromholz 1966). The tissue analysis

method requires the establishment of a critical level for

each element. The critical level is the minimum tissue

content in a particular species that is required for maximum

growth. Tissue contents below the critical concentration

are associated with deficiencies of that element resulting

in less than maximum yields. This method requires the









determination of the critical level for each species

encountered; the maximum growth rate for each species is

a function of many conditions besides nutrient levels (light,

temperature, and pH for instance), and may be difficult to

evaluate. In mixed phytoplankton populations the complexity

of critical levels tend to make the method impractical.

Gerloff and Skoog (1954, 1957), using tissue analysis on

Microystis aervgiuosa in Wisconsin lakes, concluded that

nitrogen was more likely to become growth limiting than was

phosphorus. However, Gerloff and Krombholz (1966), using

tissue analysis on angiosperm aquatic plants in the same

Wisconsin lakes, concluded that phosphorus was more likely

to limit higher aquatic plant growth than nitrogen. These

results demonstrate the species-dependency of the tissue

analysis technique, and highlight the qualitative and

relative nature of the method.

Alkaline phosphatase activity is another measure of

algal phosphorus nutrition. When algae are phosphorus

limited, the alkaline phosphatase activity per unit weight

is as much as 25 times that of algae grown with surplus

phosphate (Fitzgerald 1966, 1969). Fitzgerald cautions that

the effect of the local environment such as recent rains

or unusual circulation patterns in lakes cause changes in

the distribution of algae and must be considered when

interpreting nutritional data. He also notes that results

vary according to the species of algae under consideration.









Iitzgerald (1968) found that the rate of NH4-N

absorption by algae in the dark is 4-5 times greater for

algae which are nitrogen limited compared to plants with

available nitrogen. The comparative rate of ammonium

nitrogen absorption in the dark of algae containing surplus

nitrogen versus algae limited by available nitrogen was

measured. The test consisted of placing 5-20 mg (dry weight)

of algae, washed in nitrogen-free medium, into 10-30 ml of

Gorham's medium (minus N) and adding 0.1 mg ammonium nitro-

gen. After one hour incubation at 250 C in the dark the

ammonium nitrogen content of the supernatant was compared

to controls not containing algae. The procedure is based

on the fact that nitrogen-starved cells can assimilate

ammonium-nitrogen in the dark while normal cells require

light and carbon dioxide (Wetherell 195S). It is believed

that nitrogen-starved cells have a carbohydrate reserve

which is lacking in normal cells and nitrogen-deficient

cells assimilate ammonium-nitrogen until their carbohydrate

reserves are exhausted (Syrett 1962).

All of the above techniques whether they be extrac-

tive or enzymatic are best interpreted when applied to

unialgal test cultures rather than natural waters. These

methods give results which are relative and qualitative,

and standardization is difficult, that is, a given number

by itself means little and interpreting what it means is no

easy task.









ATP


In the past, most studies on the ATP pool in micro-

organisms were confined to cultures of bacteria grown on

synthetic substrates. By using pure cultures of bacteria

in a defined stage of growth, biochemists have been able

ro study the role of ATP in the bioenergetics of the cell.

Recently, however, some researchers have proposed that ATP

could be used to measure microbial biomass. Determination

of microbial biomass by measurement of ATP depends upon

the assumptions that ATP is not associated with non-living

particulate material and that the ratio of ATP to cell

carbon is fairly constant (Holm-Hansen and Booth 1966).

It is also important that the cellular ATP pool does not

vary substantially under different environmental conditions.

Forrest and Walker (1965) observed that the ATP pool

in starved cultures of Streptococcus faecalis remained

constant for almost three hours under endogenous conditions.

They concluded that an energy balance kept the ATP pool

constant until all the stored substrate was utilized. They

also found that the length of time the ATP pool remained

constant during endogenous conditions was proportional to

the initial substrate concentration.

Bauchop and Elsden (1960), working with three species

of bacteria, showed that under anaerobic conditions the

amount of ATP synthesized was proportional to new cell

yield. Elsden (1963) reported that the "ATP growth









coefficient," that is, the grams of new. cells produced per

mole of ATP synthesized, was approximately constant for all

the organisms studied. D'Eustachio et at (1968) reported

that cell counts based upon ATP concentration were linearly

correlated to standard plate count. D'Eustachio and Levin

(1367), studying the ATP pool in three aerobic bacterial

species during lag, exponential and stationary growth phases,

found that E coli had a relatively constant level of ATP

throughout all growth phases. Pseudomoncs fluorescens and

Bacilzus subrtillis were also fairly constant except for a

small increase in ATP pool during exponential growth.

Holm-Hansen and his co-workers have used ATP to

measure phytoplankton in the ocean (Holm-Hansen and Booth

1966; Hamilton and Holm-Hansen 1967; Holm-Hansen, Sutcliffe

and Sharp 1968; Holm-Hansen 1969, 1970). Experiments have

shown that ATP is not associated with non-living material

(Holm-Hansen and Booth 1966). These experiments included

killing of various algae and bacteria with heat, repeated

freezing, or cyanide. The measured residual ATP was

negligible. The ATP content in three cultures of bacteria

studied averaged between 0.1 and 0.2 percent of the dry

weight while the content in eight species of algae ranged

from 0.003 to 0.016 percent of the dry weight. (Holm-Hansen

and Booth 1966.) These were maximum variations in ATP over

a wide variety of growth conditions and stages in batch

cultures.









HT-n-iltcn and Holm-HInsen (1967) determined the ATP

content of seven marine bacterial isolates cultured in both

batch and chemostat conditions. The range of ATP in the

chemostat grown cells was 0.5 to 6.5 x 10-9 jg ATP/cell,

or 0.3 to 1.1 percent of the cell carbon. Senescent cells

in batch cultures and starved cells in general had an ATP

content about one-fifth that of exponentially growing cells.

Averaging a representative number of observations from the

chemostat and batch grown cells, the authors calculated the

average ATP of these bacteria to be 1.5 x 10-9 vg ATP/cell.

On a per unit cellular carbon basis the ATP was calculated

to be 0.4 percent of the cell carbon.

To determine whether the cellular ATP of algae

changed with light and dark periods, Holm-Ilansen (1970)

studied the response of algal cells to periods of light

and dark. As shown in Figure 2, an initial decrease in

ATP pool size occurred in the dark followed by a gradual

increase to approximately the initial ATP pool level. His

test, however, only covered a time scale of 22 minutes and

doesn't conclusively demonstrate the constant nature of

cellular ATP under light and dark conditions.

Holm-Hansen (1969) measured the total particulate

carbon, nitrogen, ATP, DNA, and chlorophyll in profiles to

600 meters and 1000 meters off the coast of southern

California. As shown in Table 2, the biomass estimates

based on ATP measurements are in good agreement with those

based on chlorophyll data and direct microscopic measurements.














C


0

















--------------- j












2I

0 0
s( w

















C r-C
ZO
O1-


















/T 'd
o ^ c- c o oU
(< 3i S; *
. -- -%----------------- =-. C &

1 I-l -rL1 Y









Holm-Hansen concluded that there is a considerable uniformity

in algal A1P concentration over the size range from 1 pg

c/cell to 215,000 pg c/cell. Further the average algal

ATP value (0.35 percent of the organic carbon) is close to

those concentrations reported for bacteria.

Holm-Hansen (1970) investigated the cellular ATP

content in 30 different algal cultures under different

environmental conditions. As shown in Figure 3, the average

concentration of ATP as a percent of the cellular organic

carbon is 0.35 percent during exponential growth. Extreme

nitrogen deficiency in cultures dropped the ATP level to

35 percent of that found during exponential growth for

SkZeltonermna costatum, to 46 percent for T!onochnysis lutheri,

and to 14 percent for DunaZiella tertiolecta. Phosphate

deficient cultures of M. lutheri showed that ATP dropped

to about 0.03 percent of the carbon content, but that it

increased to 0.15 percent one day after phosphate was added.

Holm-IHansen maintains that although ATP pool size varies

significantly under extreme nutrient deficient conditions,

these conditions would rarely be found in nature. He notes

that the apparent change in ATP concentration could result

from a significant amount of detrital carbon in his cultures,

giving the appearance of a drop in cellular ATP.

The ATP content of microorganisms calculated as

percent of dry weight seems relatively constant. Table 3

shows some of the values reported for bacteria and algae.

For natural lake and ocean samples of ATP per dry weight





















z
S00
o

















U
;s










O








Z






00
OI U





S 0 0






O d<
0


*< f-i


0 r-
r--q















L.0


U oo ri .: if-
fi Lt \C ) \-




0 I W+* r-




C 0 0 (















H*





t
,-- z


H l 0















4 tC
E o













c. Qr E



*,-U U E




n r- n P- *
Fb- -- a II If II


Ofl & 3 0 0 0

co .. 0 X O O O


0 0 *0 C C) t i
Mi: "3 t r
r-i rt o4>&oE~









value may be mneaningless because of the presence of detrital

material. Holm-IHansen (private communication) does not

believe it is feasible to report ATP/dry weight ratios and

reports most of his values as percent of cellular carbon

or as ATP per cell.

For algal samples the ratio of ATP to chlorophyll

seems to have some meaning. As shown in Table 4, the ATP

to chlorophyll ratio is relatively constant even under

different nutritional and physical conditions. Using the

ratios of ATP to chlorophyll seems more reliable than using

the ATP to dry weight ratio, especially for natural popula-

tions. Even in unialgal cultures the ATP to dry weight

ratio may not be reliable. Lee et aZ (1971), working with

batch cultures of Selenastrum capricornutum, only calculated

the ATP to dry weight ratio up to four days of batch growth.

After four days they observed cell debris and dead cells,

making the dry weight meaningless.

Lehninger (1971) estimated that the turnover time

of the E. coli ATP pool is only a fraction of a second.

Forrest and Walker (1965) have found the turnover time in

Streptococcus faesalis to be about 5 seconds. In light of

the rapid turnover rate of cellular ATP, many researchers

have studied the response of cellular ATP to different

environmental conditions.

Forrest (1965) showed that the ATP pool in Strep

faecalis increased after addition of glucose and later

returned to its endogenous level when the substrate was








































0000 -Lf) C -C
(I --C C) r-i C

C CD D *) (


U)U)U)U)U)n

-e -X e -


- 4-
!I II
4-J 4-


ii II II II


co




o o


in
a
Io




0
O


C)


Ia-

0r


II) o1

II II II
I4- 4-. -


Oi-i

















c 4rt rt"




11 II II
+I 4-' 4-J


C) _
C1) ~C
0Na
~ r. -H
3-- ,-
=T cd
4ci)


II ii II II


&F-


V) U) Ln
, -, I-,





II II II
+ ICI


C)



Q m


e N
N
II II




















4-J

t,.C
,,il




1-3










i--




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rU


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t)














4.

y *9-
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r-i.
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4- 4-
000
m o


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oV







a00


C i-n Un 00 \o o C ( Z)
i- CJ i CN1 trc) Ln CiD CIA r r-l

0000 000 0000:0









used up. I orking with E. ccli, Winpenny (1967) also noted

an increase in ATP pool size when glucose or pyruvate was

added.

Patterson et at (1969) and Patterson (1970), working

with bench scale activated sludge units, studied the

response of the ATP pool to changes in the incubation tempera-

ture and pH, to extended anaerobiosis, to starvation and

enrichment, and to inhibition by nickel, chromium, and

chromate. He also studied the ATP response of activated

sludge to additions of toxic materials including mercury,

copper, and cyanide. Maximum ATP pool occurred in the pH

range of 7.5 to 8.0, the normal operating range of the

units used in his research. Within 15 minutes after addition

of a lethal dose of mercuric chloride to activated sludge,

no ATP was detected.

Brezonik and Patterson (1971), reporting on the

effects of environmental stress on ATP in activated sludge,

observed increased ATP pool levels following addition of

substrate. They noted, however, that previous studies on

endogenous ATP pool indicated that only a small fraction

of activated sludge is viable. Patterson et at (1970)

reported 15 to 20 percent viability in a full scale activated

sludge unit and 35 to 40 percent in a laboratory unit.

Prezonik and Patterson questioned whether the increased ATP

pool levels following substrate addition reflect an increase

in cell population or an increase in ATP/cell. Recalculating

the ATP response on a viable fraction basis, Brezonik and









Fatterson, showed that substrate addition can effect an

increase in ATP per cell.

Coomb et aZ (1967a) subjected cultures of the marine

diatom CyZindrotheca fusiforms to 24 hours of darkness

followed by re-illumination at a high light intensity and

addition of silicon. During the time of silicon uptake,

the ATP pool size decreased with a subsequent increase when

the silicon uptake ceased. Although this indicated that

ATP participates in silicon metabolism of cell wall forma-

tion, the presence of other cellular activities such as

cell division could also account for the decrease in ATP

pool size. Coomb et al (1967b) also studied the same

phenomena using iavicuia peiiiculosa, a diatom which does

not divide when subjected to a period of silicon starvation.

Addition of silicon induces synchronous uptake of silicon

and wall formation. When silicon was added, a rapid

temporary increase in the ATP pool occurred followed by a

sharp decrease. The ATP pool increased slowly throughout

the remainder of the silicon uptake period. Similar changes

occurred in a synchronous culture kept in the dark.

Santarius and Heber (1965) studied the changes in

ATP, ADP, AMP and inorganic phosphate in leaf cells.

Leaves were exposed to light and dark, killed and

fractionated into a chloroplastic and a residual fraction.

When the chloroplast was exposed to light, the ATP

increased rapidly and it decreased rapidly when the.

chloroplast was placed in the dark. The ATP pool responded









in a similar manner in the cytoplasmic fraction. The ADP

change was opposite to the ATP change and the AMP change

followed that of ADP. From these results, Santarius and

Heber concluded that the controlling factor for inhibition

of glycolysis and respiration by light is the increased

ratio of ATP to ADP rather than a drop in the orthophosphate

concentration.

Because of the energy balance occurring within cells,

ATP seems to be relatively constant under normal environ-

mental conditions. This constant ATP pool may allow the

use of ATP as a measure of microbial biomass. On the other

hand, the rapid turnover time and sensitivity of ATP to

environmental stress may permit the use of ATP analysis

as a rapid bioassay method.














CHAPTER IV. EXPERIMENTAL METHODS AND MATERIALS


Analytical Techniques


The procedures used in this study were basically

those commonly used to analyze the biological and chemical

constituents of aquatic systems. Analysis of ATP is

discussed separately in Chapter V.

Chemical oxygen demand (COD) was measured using the

dichromate reflux method described in Standard Methods

(APHA 1971). Distilled water blanks were analyzed simul-

taneously. Samples were not filtered prior to analysis

thus the reported values represent combined soluble and

suspended particulate chemical oxygen demand.

The dry weight of the algal cultures was determined

using the gravimetric procedure recommended in the Provi-

sional Algal Assay Procedure (Joint Industry-Government

Task Force on Eutrophication 1969). A measured portion

of algal suspension was filtered through a tared type AA

millipore filter with a 0.80 micron pore size. The filters

were dryed for several hours at 900 C in an oven, then

they were placed in a desiccator to cool. The filters

were weighed on a Mettler balance.









Turbidity was measured in Jackson Turbidity Units

(JTU) using a Hach Turbidimeter. Absorbance was measured

as described in the Provisional Algal Assay Procedure

(Joint Industry-Government Task Force on Eutrophication

1969). A Bausch and Lomb Spectronic 20 spectrophotometer

at a wavelength of 600 nm was used. Conductivity was

nmasured as described in Standard Methods (APHA 1971) using

a Beckman conductivity bridge.

Ortho-phosphate was measured by the single reagent

molybdenum blue method of Murphy and Riley (1962) adapted

to the Technicon Autoanalyzer. Total phosphorus was

measured by autoclaving samples at 15 psi for one hour in

the presence of potassium per sulfate and sulfuric acid.

The phosphorus concentration was measured using a Klett-

Sumerson photoelectric colorimeter.

The modified brucine method of Jenkins and Medsker

(1964) adapted to the Technicon Autoanalyzer was used to

measure nitrate. This procedure was essentially identical

to the automated method described by Kahn and Brezenski

(1967).

Alkalinity was measured by potentiometric titration

to pH 4.5 with 0.01 N H2S04 as described in Standard Methods

(APHA 1971).

Chlorophyll a was extracted and measured by the

procedure originally described by Richards and Thompson





38



(1952) and Crietz and Richards (1955). The equations of

Parsons and Strickland (1963) were employed to calculate

the chlorophyll a concentration.

A modification of the carbon-14 procedure developed

by Steeman-Nielsen (1951) was used to measure primary

production. Rather than inoculating 300 ml samples with

5 micro-curies of 1C, 30 ml samples were inoculated with

0.5 micro-curie of 4C. After incubation for three hours

in a constant temperature light box, aliquots were filtered,

dried, and counted in an internal proportional counter.

A method study was performed to determine the statistical

accuracy of this modified procedure. Replication of this

procedure yields a relative standard deviation of five

percent.

Alkaline phosphatase activity of the algal cultures

was measured using a modification of the method of

Fitzgerald and Nelson (1966). Instead of using Gorham's

medium as suggested, PAAP medium was used since all algal

cultures were grown on PAAP medium.

Cell numbers were determined using a hemocyto-

meter.


Experimental Materials


Four laboratory batch unialgal cultures were

operated for a period of seven months. The following









algal species were grown: Anabaena flos-aqae, 1i icro-

cystis aeruginosa, 1 Selenas trum capricornutum, and ChloreZa

sp. Initially, chemostat units were set up using Anabaena

flos-aquae, Selenastrum capricornutun, and NavicuZa minima,2

a diatom. A detention time of three days was maintained

by feeding three liters of PAAP medium to the nine liter

chemostats. A gradual wash-out of the algal cells occurred,

necessitating a change in detention time from three days

to nine days. After one month of operation all three

chemostats were contaminated with ChloreZla and new culture

material had to be ordered from the National Eutrophication

Research Center in Oregon and from the Starr Collection in

Indiana. At this time a decision was made to change to

batch cultures.

Batch unialgal cultures of Anabaena, Microcystis,

Selenastrum, and Navicula were grown. However, NavicuZa

continued to be contaminated with ChZorelta. Finally,

N7aviouZa cells declined in numbers and had to be discarded.

A unialgal culture of ChZorella was substituted for the

diatom.

The batch units contained four liters of culture

material which were kept well-mixed with magnetic stirrers.

Gro-Lux3 fluorescent lamps were used to illuminate the



Obtained from the National Eutrophication Research
Program, Pacific Northwest Water Laboratory, Corvallis,
Oregon 97330.
2Obtained from Dr.R.Starr, Dept. of Botany, Univ. of
Indiana Bloomington, Indiana.
Svylvania Electric Products Inc., Danvers, Mass.










cultures. Selenastrum and ChlorelZa received about 350

ft-c while Anabaena and Microcystis received about 200

ft-c. Each day 200 ml of culture material was removed

from each unit and 200 ml of fresh PAAP medium was added.

The PAAP medium was sterilized before it was fed to the

units. Because of the poor buffer capacity of PAAP medium,

the pH of the cultures often increased to a pH of 10 or

greater, indicating a carbon-limiting condition. Carbon

dioxide was occasionally bubbled into the units to lower

the pHl and add carbon. Frequent visible changes in the

algal pigments of the units occurred, especially when the

pH increased. At no time was a steady-state condition

obtained.













CHAPTER V. METHODS OF ATP ANALYSIS


McElroy (1947) found that luminescence in fireflies

has an absolute requirement for ATP. Light production by

firefly lantern extract (FLE) depends on the presence of

luciferin, the enzyme luciferase, oxygen, magnesium ions,

and ATP (McElroy 1947; McElroy and Strehler 1954). Hastings

(1968) has reviewed the biochemistry of luminescent reactions

in detail. Equations 1-3 illustrate the chemical reactions

involved in the firefly light production process.

Mg++
Lh2 + ATP + E E LH2 AMP + PP. (1)


E LH2 AMP + 02 Z E L O* + Product (2)


E L 0* E -L 0- + hv (3)


Luciforin and ATP, catalyzed by luciferase, react to form an

enzyme-luciferin-adenosine monophosphate complex (luciferyl-

adenylate) and inorganic pyrophosphate (PPi). Rapid oxida-

tion to oxyluciferyl-adenylate occurs (Equation 2). This

is an excited state and is immediately followed (Equation 3)

by the release of a quantum of light (Hopkins, Selinger

et ca 1968). One light quantum is emitted for each luciferin

molecule oxidized (Seliger and McElroy 1959, 1960).









The mixing of ATP with firefly lantern extract

results in a flash of light which rapidly declines to a

uniform level. Addition of arsenate or phosphate buffer

to the firefly reaction decreases the initial light flash

and produces an intermediate level of light which decays

exponentially with time. Arsenate buffer is now routinely

added to many commercially available firefly lantern

extracts used for ATP analysis.

ATP is the only nucleoside triphosphate that will

react with purified extracts of firefly lantern to produce

luminescence. The crude extracts of firefly lantern,

however, contain transphosphorylase enzymes, resulting in

light emission in the presence of high energy phosphate

molecules other than ATP (Balfour and Samson 1959). To

determine which high energy phosphate molecules might cause

light emission with firefly extract, Holm-Hansen and Booth

(1966) tested thirteen intracellular compounds including

adenylic acid, guanylic acid, cozymase, glucose-l-phosphate,

fructose-l-6-diphosphate, phosphocreative, adenosine, thia-

mine pyrophosphate, coenzyme-A, sodium glass, adenosine

diphosphate (ADP), cytidine-5-triphosphate (CTP) and inosine-

5-triphosphate (ITP). Of these compounds, only ADP, CTP,

and ITP affected light emissions. The amount of light

resulting from addition of ADP was less than 1 percent of

an equivalent amount of ATP. Both CTP and ITP stimulated

light production equivalent to that of ATP. Franzen and









Pinkley (1961) reported cellular ATP values 10 to 25 per-

cent higher using the firefly method than when determined

by chromatographic techniques. The fact that some light

emission may be due to nucleoside triphosphates other than

ATP is probably not significant because of the relative

abundance of cellular ATP with respect to these other

compounds.

Since one quantum of light is emitted for each

molecule of ATP that is hydrolyzed (Seliger and McElroy

1960), the amount of ATP in a sample is directly proportional

to the total amount of light emitted by the enzyme mixture.

ATP was measured based on this principle.

Lyophilized aqueous extracts of firefly lantern'

were stored desiccated at -200 C until used. Each vial

contains the extract from 50 mg of firefly lanterns with

magnesium arsenate buffer added. The contents of each vial

was rehydrated with 35 ml of deionized distilled water.

After standing at room temperature for 1/2 hour, the enzyme

mixture was filtered (Whiatman No. 3) and the filtrate

incubated in ice water for 3 to 4 hours. The crystalline

disodium salt of ATP was used to prepare standard solutions.

A stock solution was prepared in tris buffer (0.02S M, pH

7.75), and poured into individual test tubes which were

capped and stored at -20 C until needed. Storage for as

long as two months showed no change in standard ATP



FLE-50, Sigma Chemical Company, St. Louis, Missouri.









concentration (Patterson 1970). When ATP standards were

needed a test tube of stock solution was thawed and diluted

with t ris buffer to the desired concentrations.

Samples to be analyzed for ATP were filtered through

47-mm membrane filters (pore size 0.80 1). As soon as no

liquid remained above the filter, the filter was quickly

removed and placed in a test tube containing 9 ml of boiling

tris buffer (0.025 M, pH 7.75). The test tube was held in

a boiling water bath for 10 minutes, with occasional shaking

to disperse clumped cells. The test tube was rapidly cooled

and brought up to a volume of 10 ml with additional tris

buffer. The test tube was centrifuged (10 minutes at

2,250 rpm) to bring down cell debris, and the supernatant

was poured into a clean test tube. The test tube was then

frozen and stored at -200 C for future analysis.

This method of ATP extraction depends upon bringing

all of the ATP into solution. Other extraction methods

have been used. Holm-Hansen and Booth (1966) tested ATP

extraction using perchloric acid, boiling ethanol, boiling,

water, and boiling tris buffer. Perchloric acid extraction

inhibited the luciferase reaction because of its neutral

pH. Extraction by the last three solvents all gave satis-

factory results. Forest (1965) used sulfuric acid to

extract ATP and D'Eustachio and Levin (1967) used sonic

disruption to extract ATP. Patterson (1970) compared

sonication to boiling tris extraction and found it was









equally effective in extracting ATP. DuPont de Nemours

Company (Inc.) recommends ATP be extracted with either

butanol or dimethylsulfoxide (DMSO) for use with their

Luminescence Biometer.2

A sensitive method of measuring light emission is

required in the ATP assay. Holm-Hansen and Booth (1966)

used a photomultiplier tube and amplifier connected to a

recorder. Others (Forrest 1965; Lyman and DeVincenzo 1967)

have also used similar instrumentation. Another instrument

used to measure ATP is the DuPont Luminescence Biometer.

The DuPont Luminescence Biometer consists of a photomulti-

plier tube and solid-state electronics that converts the

analog signal to a digital readout proportional to the

maximum intensity of the light flash. The instrument is

calibrated for each reaction mixture so that the ATP con-

centration is read out directly. Purified luciferin-

luciferase is used without arsenate buffer to give a high

initial light flash and a rapid luminescent die-off. At

the start of this research the Biometer was used to measure

ATP concentration. During a two month investigation

period difficulties were encountered with calibration of the

instrument and read-out reproducibility. The Biometer is

also sensitive to fluctuations in line voltage entering the

instrument. Lack of reproducibility was probably caused by

by a combination of the complex solid-state electronics



2
DuPont 760 Luminescence Biometer, DuPcnt de Nenours
and Company, Wilmington, Delaware.









and the sample size and injection procedure. The DuPont

procedure calls for injection of a 0.01 ml aliquot of sample

into a vial containing 0.1 ml of enzyme mixture. Although

a precision microliter syringe was used to inject the

sample, it is doubtful whether exactly 0.01 ml was delivered

each time at the same rate. Small droplets would sometimes

cling to the needle point, indicating a lack of precision

in the injection procedure. In general, the Biometer was

found to be an unsatisfactory method of measuring ATP.

Conventional liquid scintillation spectrometers have

been used to measure luminescence (Cole, Wimpenny, et aZ.

1967; Patterson 1970). A liquid scintillation spectrometer3

was used in this study. Instrument gain was set at 53, with

a window opening of 50 to 1,000. The spectrometer was set

in a repeat count mode, with each 6-second counting interval

separated by a 7.5 second data print-out sequence.

Prior to each ATP analysis the background light

emission from the luciferin-luciferase firefly extract was

measured. Exactly 1.0 ml of the enzyme preparation was

pipetted into a scintillation vial. Normal background

emission was 10 to 20 counts in the 6-second counting

interval. Patterson (1970) found that higher counts

indicated glassware contamination. All glassware was washed



3Packard Tri-Carb Model 2002, Packard Instrument
Company, Downers Grove, Illinois.









in an automatic dishwasher using hot soapy water, a hot

rinse, and a final rinse with distilled water. Following

this, the glassware was boiled for at least one hour in an

acid bath and rinsed three times in deionized, distilled

water.

After determining the background emission, exactly

0.5 ml of ATP standard or sample was pipetted into the

enzyme mixture and the vial was swirled to thoroughly mix

the contents. Exactly 11.0 seconds after ATP addition, the

vial was placed into the scintillation counting chamber.

This procedure was followed because of the exponential decay

of the luminescence, necessitating careful control over the

addition of ATP to the enzyme mixture and initiation of the

counting sequence.

Patterson (1970), using the scintillation counter to

measure ATP, analyzed the data graphically because of the

random variability of any 6-second count. The 6-second

emission counts were plotted on semi-log paper versus

elapsed reaction time. Good correlation between counts and

standard ATP concentrations were observed for reaction times

of one minute or more. The line of best fit to the data

points was extrapolated back to the initial data point and

the count at one-minute was read from this line. This

graphical technique was used in this research. Figure 4

shows lumincence decay curves for several ATP concentrations.

















200 e


o -TP = 200 jg/1
00









ATP = 60 pg/1
0


0 0







TP 40 pg/l


^^ o-^


1.0


2.0


2.5


3.0


0.5


Elapsed Reaction Time, Minutes
FIGURE 4. LUMINESCENCE DECAY OR ATP FIREFLY LANTERN
EXTRACT REACTION WITH TIME (Patterson 1970)


100


6
5
'o 4

3


2


3.5













The effects of aging on both ATP standard solutions

and firefly lantern extract were studied (Patterson 1970).

As shown in Table 5, the background emission of the luciferin-

luciferase mixture decreased rapidly during a 24-hour incuba-

tion period. Two sets of ATP standards were also made up:

one set was frozen, then thawed just prior to analysis;

the other was held in an ice water bath with the firefly

mixture. The light emission from the freshly thawed

standards decreased with the time of incubation, indicating

a slight less of enzyme activity over the 24-hour period.

Light emission from the ATP solution kept in an ice bath

decreased rapidly, indicating a loss of ATP from the

standard solutions during the incubation period. In light

of these results, standards were always measured immediately

after preparation, and samples were thawed just prior to

analysis.

The effect of tris buffer temperature on ATP extraction

was also studied by Patterson (1970). He found (Figure 5)

that a temperature reduction of a few degrees below boiling

caused a significant decrease in the amount of ATP extracted.

For this reason, all extractions were performed in a rapidly

boiling water bath. However, it was often found that water

which appeared to be boiling actually had a temperature of

90-950C. Thus a thermometer was always kept in the boiling

tris buffer to assure that the temperature remained at 100C.






































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<-









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r-



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SZ LO 0





c P


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3.0

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/

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75 80 85 90 95 100 105
Temperature of Extraction, OC
FIGURE 5. DEPENDENCE OF QUATITATIVE ACTIVATED SLUDGE
ATP EXTRACTION ON TEMPERATURE OF EXTRACTION SOLUTION
(Patterson 1970)









Rhodes and McElroy (1958) investigated the sensitivity

of the firefly reaction to pH and found that luminescence

increased rapidly from pH 2.0 to 3.8, decreased from pH

2.8 to 3.8, and increased from pH 4.8 to 7.0. The rate of

light emission is much more rapid at pH 7.6 than at pH 9.4

(Seliger and McElroy 1960). Holm-IIansen (1966) used a

reaction mixture buffered at pH 7.75. For this research,

tris buffer at a pH of 7.75 was used.

Figure 6 shows a typical standard ATP curve.

Depending on the enzyme age and concentration, the standard

curve may be linear or non-linear. Results of recent

measurements of ATP standards indicate that plotting the

points on log-log graph paper gives a straight line of

best fit.

A sample of Selenastrum was diluted into five

portions and the ATP of each portion was measured. This

experiment was run to determine whether the sampling proce-

dure was valid. As shown in Figure 7, excellent correlation

between ATP content and SeZenastrum concentration was

observed.

Using the method of ATP analysis described in this

chapter, replication of ATP samples gave results with a

relative standard deviation of less than 5 percent.
















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300


250 -





200 -





3- 50
, 150 .





100






50





I t 1 I !
0.125 0.25 0.5 1.0
Fraction of Selenastr:im Sample

FIGURE 7. CORRELATION OF ATP WITH SEL.,EAS TU.ii CONCENTRATION














CHAPTER VI. EXPERIMENTAL RESULTS


The study of ATP in phytoplankton was divided into

two phases. The first phase consisted of monitoring the

ATP pool in batch grown unialgal cultures and natural lake

populations. In the second phase, samples from the unialgal

cultures and from lake waters were subjected to various

environmental conditions such as pH, light and addition of

nutrients and toxic substances. The ATP response to these

conditions was measured.


ATP Biomass Results

Batch cultures of Anabaena flos-aque, Selenastrum

caprico'rnutum, Microcystis aeruginosa, and Chlorelta sp.

were grown in PAAP media for a period of approximately three

months. Twice each week a 200 ml sample was taken from each

culture and analyzed for ATP and other biomass parameters

including chlorophyll a, dry weight, and absorbance. These

experiments were performed to determine whether the cellular

ATP concentrations in batch unialgal cultures would correlate

with current biomass parameters (chlorophyll a, dry weight,

etc.). Samples of phytoplankton from local lakes were also

collected and analyzed for ATP, chlorophyll a, dry weight,

and absorbance. The correlation of ATP with current biomass









parameters wqs determined to test the usefulness of ATP

measurements in natural phytoplankton populations. No

attempt was made to quantitatively measure the bacteria

population in the batch cultures or the lake samples.

ATP vs. Chlorophyll a

Figures 1 to 3 present ATP vs. chlorophyll a values

for the batch algal cultures. The slopes of the linear

regression lines for Selenastrum and ChZorelZa are similar;

thus Figure 8 contains the data for both these organisms.

Although the slopes of the linear regression lines are

similar for Anabaena and Microcystis, the ATP and chlorophyll

a values are plotted separately for reasons of clarity and

because of the much larger range of chlorophyll a values

observed in the Anabaena culture.

Although there is a wide degree of scatter about the

regression lines for all algal species, a definite relation-

ship exists. Correlation coefficients for ATP vs. chlorophyll

a concentrations are relatively high as shown in Table 6.

Correlation is a measure of the degree to which variables

vary together, that is, it is a measure of the intensity of

association. The sample correlation coefficient, r, given

in Table 6 is an estimate of the population coefficient, p.

Table 6 gives the 95 percent and 99 percent confidence

intervals for the population correlation coefficient. Note

that a correlation does not exist if the population

correlation coefficient is zero. Thus, to be significant






57






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at a particular confidence level, the confidence interval

must not contain zero. As shown in Table 6, the sample

correlation coefficients for all algal species are signifi-

cant at the 99 percent confidence level, but the correlation

for .elenastrum, which had the lowest correlation (r = 0.539)

is barely significant at the 99 percent confidence level

since its confidence interval approaches zero at the lower

limit.

To study further the relationship between ATP and

chlorophyll a, a dense culture of Microcystis was diluted

into 10 portions, incubated for 24-hours, and analyzed for

ATP and chlorophyll a. A nearly perfect linear relationship

was obtained (Figure 11). These results demonstrate a strict

ATP to chlorophyll a relationship in the absence of varying

conditions of pH, light intensity, and nutrient state.

To determine the relationship of ATP to chlorophyll

a in natural lake phytoplankton, measurements were made on

three Florida lakes: Bivens Arm, Newnan's Lake, and

Anderson-Cue Lake. These lakes were selected to provide a

wide range of biomass values. Bivens Arm is a hypereutro-

phic lake, Newnan's Lake is eutrophic, and Anderson-Cue

Lake is oligotrophic. Chemical and biological character-

istics of these lakes are given in Table 7. Results of the

lake measurements are shown in Figure 12, and the correla-

tion coefficients and confidence intervals are given in

Table 8. Correlation of ATP with chlorophyll a for all















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three lakes is significant at the 95 percent confidence

level. Bivens Arm and Newnan's Lake are significant at

the 99 percent confidence level. However, the correlation

is not very good for Anderson-Cue Lake alone as evidenced

by its large confidence interval and its not being signifi-

cant at the 99 percent confidence level. The poor correla-

tion of ATP with chlorophyll a in Anderson-Cue is probably

a result of the small range of values measured. The

correlation coefficient of the combined lake values is 0.830.

These high correlation values indicate the excellent associa-

tion of ATP with chlorophyll a in lake phytoplankton.

The ATP to chlorophyll a ratios for the laboratory

algal cultures and the lake samples were calculated and

are given in Table 9. Anabaena and Microcystis have lower

ATP to chlorophyll a ratios than Selenastrum and Chiorella.

This could be explained by the fact that Anabaena and

TABLE 9

ATP TO CHLOROPHYLL A RATIOS FOR ALGAL CULTURES
AND LAKE PHYTOPLANKTON

ATP/Chlorophyll a
Anabaena 0.09
Microcystis 0.09
Se enastrum 0.35
ChloreZZa 0.29
Bivens Arm 0.09
Newnan's Lake 0.11
Anderson-Cue Lake 0.69
All Algae 0.20
All Lakes 0.25
Algae and Lakes 0.21









Microccstis, both blue-green algae, are procaryotic cells

with a pigment system containing chlorophyll a, phycocyanin,

and trace amounts of phycoerythrin. A very efficient energy

transfer, approaching 100 percent, occurs from phycocyanin

to chlorophyll a (Brock 1970). Thus the amount of chlorophyll

a required by a blue-green alga may be less than that required

by a green alga. In blue-green algae the photosynthetic

pigments occur in organized internal membranes unlike

eucaryotic algae where the pigments occur in membrane-bound

chloroplasts. This difference in cellular organization could

also account for the observed differences in the ATP to

chlorophyll a ratio.

Bivens Arm and Newnan's Lake both have lower ATP to

chlorophyll a ratios than Anderson-Cue Lake. One would

almost expect the opposite, considering that Bivens Arm and

Newnan's Lake probably contain a larger bacterial population

than Anderson-Cue Lake. However, other factors must be

considered. For instance, both Bivens Arm and Newnan's

Lake contain large populations of blue-green algae while

Anderson-Cue Lake does not. A large population of blue-

green algae, according to the above data, would give a

lower ATP to chlorophyll a ratio. Also, the much higher

turbidity and color in Bivens Arm and Newnan's Lake probably

induce a higher cellular chlorophyll content in order to

utilize the subdued light entering the water.









Other experiments were performed to determine whether

the ATP to chlorophyll a ratio varied with nutritional state

and cell age. Aliquots from each of the unialgal cultures

were transferred to flasks containing fresh media. One flask

contained PAAP medium with all nutrients present, one

contained PAAP medium without phosphorus, and one contained

PAAP medium without nitrogen. The ATP to chlorophyll a

ratio increased as the nutritional content of the medium

decreased and the cell age increased (Figure 13). In most

cases, the ATP to chlorophyll a ratio remained relatively

constant for the first few days then increased. This

resulted from an initial increase in chlorophyll a and ATP

concentrations followed by a decrease in chlorophyll a as

the cultures aged and the nutrient content of the medium

decreased. Fogg (1965) observed that the cellular chlorophyll

concentration decreased with cell age and nutrient deficiency.

Thus, variations in the ATP to chlorophyll ratio may be a

result of natural fluctuations in cellular chlorophyll rather

than changes in cellular ATP.


ATP vs. Dry Weight

ATP and dry weight values of the batch algal cultures

are shown in Figure 14. All four algal species have similar

linear regression lines indicating a relatively constant

association between ATP and dry weight. Correlation

coefficients and their confidence intervals are given in

Table 10. The sample correlation ccefficients for Anaba na,











CHLOPELLA N Deficient

P Deficient

Control


,- --2 3 4 5 6----
1 2 3 4 5 6 7 8


SELENASTRUM


P Deficient

Control

N Deficient


1 2 3 4 5 6 7 .8
MICROCYSTIS


N Deficient


_---o P Deficient
Control


1 2 3 4 5 6 7 8


Control
N Deficient


P--) Deficient
0.


1 2 3 4 5 6 7 8


FIGURE 13.


Time, Days
VARIATION OF ATPi/CHLOROPHYLL A RATIO WITH CELL AGE


1.0
0.8

0.5
0.4

0.2


1.2

1.0


0.8
0.6


0.4L


0.2





1.0

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0.6

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Micrcc'ystis, and ChZcrZlla are relatively good ranging from

0.607 to 0.860, with all three significant at the 99 percent

confidence level. Correlation at ATP with dry weight for

SeZenastrum is poor and not significant even at the 95

percent level. The poor correlation of SeZenastrum probably

resulted primarily from the snail range of values measured.

All of the algae together have a correlation coefficient of

0.726 which is significant at the 99 percent confidence

level. In general, good correlation exists between ATP

and dry weight in the unialgal cultures.

The ATP to dry weight measurements for Bivens Arm,

Newnan's Lake, and Anderson-Cue Lake are shown in Figure 15.

Correlation coefficients for the three lakes are all

significant at the 99 percent confidence level (Table 11).

Good correlation exists between ATP and dry weight for all

three lakes, with Anderson-Cue Lake having the best

correlation.

ATP to dry weight ratios for the algal cultures and

the lakes were calculated and are presented in Table 12.

All four algal cultures have relatively constant ATP to dry

weight ratios ranging from 0.24 to 0.38 pg/mg. However, the

ATP to dry weight ratios for the three lakes vary from 0.12

to 0.96 pg/mg. Bivens Arm and Newnan's Lake have a much

lower average ATP to dry weight ratio than Anderson-Cue Lake.

These low ratios are evidently the result of a high

concentration of detrital matter which also explains the



















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TABLE 12

ATP TO DRY WEIGHT RATIO'S FOR ALGAL CULTURES
AND LAKE PHYTOPLANKTON


ATP/dry weight
Anabaena 0.26
Micrccys tis 0.36
Se enastrum 0.24
ChZoreZZa 0.38
Bivens Arm 0.13
Newnan's Lake 0.12
Anderson-Cue Lake 0.96


poorer correlation of ATP with dry weight in these lakes.

Table 13 compares the turbidity of each lake with the

measured ATP to dry weight ratio. Both Bivens Arm, and

Newnan's Lake have large amounts of turbidity. Bivens

Arm, for example, has an average turbidity of 10.2 JTU's

compared to Anderson-Cue's average of 1.0 JTU.


TABLE 13

COMPARISON OF ATP/DRY WEIGHT RATIO WITH AVERAGE
TURBIDITY OF EXPERIMENTAL LAKES
Average
Turbidityx
Lake ATP/Dry Weight (JTU)
Bivens Arm 0.13 10.2
Newnan's Lake 0.12 4.2
Anderson-Cue Lake 0.96 1.0


iShannon, 1970





76



ATP vs. Cell Number

Another common biomass measurement is the cell

number. A dense culture of Microcyetis was diluted into

ten portions, incubated for 24-hours, and analyzed for ATP

and cell number. Figure 16 shows that a linear correlation

exists between ATP concentration and cell number. The ATP

per cell for Microcystis ranged from 5.0 x 10-8 to 6.25 x

10-8 pg/cell. It should be noted that cell number, because

of differences in size between various algal species, would

not give a useful correlation with ATP in mixed populations.

These results are presented only to illustrate the good

correlation between ATP and cell number in unialgal cultures.

ATP vs. Absorbance

Absorbance is a common means of measuring relative

algal densities in laboratory cultures (Joint Industry-

Government Task Force on Eutrophication 1969). The absorbance

of the four algal cultures was routinely measured along with

ATP and the values are plotted vs. each other in Figure 17.

Correlation coefficients for ATP vs. absorbance are given

in Table 14. Except for Microcystis the correlation of ATP

with absorbance is poor. A high correlation between dry

weight and absorbance exists, indicating that scattering

rather than absorbance is really being measured. It is

surprising that better correlations were not obtained, espe-

cially for such dispersed algae as ChZorella. The reason

lies partially in the small range of chlorophyll a values

observed.




























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Zooplankton Study

In order to determine the effect of zooplankton ATP

on the total measured ATP in natural plankton populations,

the ATP of zooplankton from a local lake was measured.

Zooplankton from Newnan's Lake was collected using a zoo-

plankton net. The zooplankton consisted almost exclusively

of Cyclops whose average dimensions were 1.4 mm by 0.3 mm.

Using these average dimensions and assuming a specific

gravity of 1.05, the ATP to dry weight ratio for zoopliankton

was calculated to be 0.1 pg/mg. This value is slightly lower

than the average value reported in this research for algal

cultures and much lower than the average value reported for

bacterial cultures (1.5 pg/mg). Based on the lower ATP to

dry weight ratio of zooplankton and their relatively small

number in most lakes, it seems that the ATP in zooplankton

would not significantly add to the total ATP measured in a

lake. Thus, these results indicate that ATP from zooplankton

would not significantly affect the phytoplankton biomass

estimated using ATP concentration.


ATP Response Results


The response of ATP to different environmental

conditions was measured to determine the usefulness of ATP

analysis as a rapid bioassay technique and to assess the

stability of ATP in reference to its application to

measurement of phytoplankton biomass. Laboratory algal









cultures and lake phytoplankton were subjected to varying

light periods, different p-! levels, and additions of

nutrients and toxins.

Light-Dark Study

In order to be used as a reliable estimate of phyto-

plankton biomass, the cellular ATP concentration must not

vary under light and dark conditions. To determine whether

the cellular ATP concentration remained relatively constant

under light and dark conditions the following experiment

was performed. Aliquots of Selenastrum were placed alter-

nately in the light and the dark for a period of ten hours

and the ATP content was monitored. The response of ATP in

SeZenastrum cells to light and dark periods is shown in

Figure 18. Fluctuations in the ATP content occurred when-

ever the algae were changed from either light to dark of

from dark to light. Within 15 to 20 minutes after each

transition the ATP concentration returned to the initial

ATP level. Holm-Hansen (1970) obtained similar results

working with marine algae, and found ATP concentrations to

be maintained at fairly uniform levels in both light and

dark. Holm-Hansen's experiment only covered a time span

of 22 minutes while the above mentioned experiment covered

a span of ten hours, indicating the long-term stability of

ATP under light and dark conditions. The ATP pool in algae

is probably regulated by the interaction of the photosynthetic










































































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anld respiratory mechanisms of the cell. ATP occurs in trace

amounts in the cell and has a raDid turnover rate. It is

probable that ATP is primarily regulated by respiration

rather than photosynthesis. In photosynthesis light energy

is used to form ATP which is almost immJediately used form

simple sugars. These simple sugars enter the carbon cycle

and eventually supply all che energy needs of the cell.

However, the fluctuations in ATP levels observed at light

to dark transitions indicate a definite interaction and

dependence between photosynthesis and respiration.

pH Studies

The pH of natural waters is an important environ-

mental variable. The biota, productivity and solubilities

of nutrients and toxic substances are all somewhat dependent

on the pH of a specific water. To use ATP as a measure of

biomass, the relationship between ATP concentration and pH

must be known. Thus, the response of cellular ATP level to

different pH values was measured. Figure 19 shows the ATP

concentration measured in Selenastrum cells after incubation

for one hour at various pH levels. Maximum ATP concentration

occurred in the pH range of 7.5 to 8.0, the normal growing

range of the laboratory cultures. The ATP concentration

was greatly reduced in the pH range of 4.0 to 5.0. Moderate

reduction in ATP concentration occurred at a pH of 10.0.








































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The above experiment was repeated using lake water,

in order to determine whether maximum ATP content always

occurred within a neutral pH range such as pH 7 to 8, or

whether the optimum ATP occurred at the normal ambient pH of

the organisms. Figure 20 shows the ATP concentration

measured in Anderson-Cue Lake water after incubation for

one hour at various pH values. Maximum ATP concentration

occurred at a pH of 4.6, the original pH of the lake water.

The greatest reduction in ATP concentration occurred in the

pH range of 9.0 to 11.0. A high pH lake water was tested

in the same manner. Figure 21 shows the ATP concentration

measured in Bivens Arm lake water after incubation for one

hour at different pH levels. Maximum ATP concentration

occurred at a pH of 9.0, the original pH of the lake water.

Maximum reduction in ATP concentration occurred at a pH

of 3.0.

It should be noted that the pH values reported are

those of the incubation media or lakewater. It is probable

that the cellular pH changed less than the media or lake

water since the cell can control intracellular pH to some

extent. The reduced ATP concentrations may indicate a

reduction in viable cells or a shift in the energy balance

of the cell as it attempts to maintain homeostasis. These

results indicate that a shift from the organisms normal pH

range causes a reduction in the ATP level, whether acidic,

neutral, or basic.






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Toxicity Studies

One of the greatest problems today is the intro-

duction of toxic materials into our waterways. In 1969

almost 70 percent of the reported fish kills were caused

by the discharge of industrial wastes. Wastes containing

concentrations of heavy metals may be toxic to aquatic

organisms and severely affect the water community. The

bioassay is an important tool in the investigation of

toxic materials because the results of such a study indicate

the degree of hazard to aquatic life of particular toxins.

From bioassay studies interpretations and recommendations

can be made concerning the level of discharge that can be

tolerated by the receiving water.

Mercury compounds are highly toxic to aquatic

communities. Mercuric chloride is used in disinfecting,

preserving, tanning, electroplating and many other processes.

Mercuro-organic compounds are used in herbicides, fungicides,

and medical treatment. They have been used extensively to

control slimes in paper mills. To study the effects of

mercuric ion on phytoplankton, aliquots of Selenastrum were

incubated with various concentrations of mercuric chloride

and the response of ATP, carbon-14 uptake, chlorophyll a,

and suspended solids was measured. Figure 22 shows the ATP

content in SeZenastrum samples after three hours of incuba-

tion in the presence of various concentrations of mercuirc

chloride. The ATP toxicity pattern in Figure 22 shows a






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