ATP i'!easureinents in Laboratory Cultures and
Field Populations of Lake Plankton

By

FRANCIS XAYIER HROIvNE

A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY Of FLORIDA IN PARTIAL

■Til,

T

THE REQlilREMENrS FO
OF DOCTOR OF PHILOSOPHY

;EE

UNIVERSITY OF 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
ass ist ance .

I v-Quld 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 Kish 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, Dr? . Patrick L.
Brezonik and Jackson L. Fox. I also thank the rest of
iny committee, Drs . Hugh Putnam and Frank Xordlie, for
their ideas and insights.

I also wish to thank my parents for all the help
and support they gave me thxroughout the years. For
without them, truly, none of this would have been possible

Finally, I thank my •\s'ifc, 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 p'^ssible througii the
financial support of the Department of Health, Education,
and V>'elfare, PHS Traineeship Grant No. 5 -TOl -EC-000 35-10 .

Ill

TABLE OF CONTENTS

Page

ACKNOIVLEDGMENTS ii

LIST OF TABLES vi

LIST OF FIGURES viii

ABSTPACT X

CHAPTER I . INTRODUCTION 1

CHAPTER II. MECHAA'ISM OF ATP METABOLISM IN ALGAE ... 4

CHAPTER III. LITERATURE REVIEW 9

Introduction 9

Biomass Paranieters 10

Pigrient Concentration 10

Particulate Organic Carbon 14

Cell Numbe r IS

Packed Cell \'olunie and Drv IVeight 16

DNA '. : 17

Liiniting Nutrient Bioassay Methods 18

Carbon-14 Uptake '. 19

Enzymatic and Extractive Techniques 21

ATP 24

CHAPTER IV. EXPERIMENTAL METHODS AND >LATERIALS 36

Analytical Techniques 36

Experinental Materials 58

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 6 8

ATP vs . Cell Number 76

ATP vs . Absorhance ■. . 76

-coplankton Studv 80

Page

ATP - Response Results 80

Light-Dark Study 81

pH Studies 83

Toxicity Studies • 88

Nutrient Studies 93

CHAPTER VII. DISCUSSION 110

CHAPTER YIII. CONCLUSIONS AND RECOM^.IENDATIONS US

Conclusions 115

Recoinnendations 117

BIOBLIOCRAPHY 118

BIOGRAPHICAL SKETCH 1 30

LIST OF TABLES

Page

TABLE 1. STANDARD FREE ENERGY OF HYDROLYSIS OF
COMMON INTRACELLULAR PHOSPHATE
COMPOUNDS 4

TABLE 2. COMPARISON OF BIOM^SS ESTIMATED BY ATP,

CHLOROPHYLL, DNA AND ORGANIC CARBON 18

TABLE 3. ATP AS PERCENT DRY WEIGHT 30

TABLE 4. ATP TO CHLOROPHYLL R.\TIO FOR VARIOUS
ALGAE UNDER DIFFERENT ENVIRONMENTAL
CONDITIONS 32

TABLE 5. EFFECT OF INCUBATION ON FIREFLY EXTR.ACT

AND ATP LIGHT EMISSION C6-SEC COUNT) 50

T\BI.E 6. ATP VS. CHLOROPHYLL .4 CORRELATION

COEFFICIENTS FORUNIALGAL CULTURES 60

TABLE 7. CHEMICAL AND BIOLOGICAL CHAP.ACTERISTICS

OF EXPERIMENTAL LAKES 6 3

TABLE 3. ATP VS. CHLOROPHYLL A CORRELATION

COEFFICIENTS FOR LAKE PHYTOPLANKTON 65

TABLE 9. ATP TO CHLOROPHYLL /. R.\TIOS FOR ALGAL

CULTURES AND LAKE PHYTOPLANKTON 66

TABLE 10. ATP VS. DRY IVEIGHT CORRELATION

COEFFICIENTS FOR UNIALGAL CULTURES 71

TABLF 11. ATP TO DRY IvEIGHT CORRELATION

COEFFICIENTS FOR LAKE PHYTOPL.ANKTON 74

TABLE 12. ATP TO DRY WEIGHT RATIOS FOR ALGAL

CULTURES AND LAKE PHYTOPLANKTON 75

TABLE 13. COMPARISON OF ATP/DRY WEIGHT R.ATIO WITH

AVEP..AGE TURBIDITY OF EXPERIMENTAL LAKES 75

Page

TABLE 14. ATP TO ABSORBANCE CORRELATION

COEFFICIENTS FOR UNIAIGAL CULTURES 79

TABLE 15. PERCENT REDUCTION AFTER ONE-HOUR

INCUBATION WITH MERCURIC CHLORIDE 90

TABLE 16. RESPONSE OF CELLULAR ATP 10 ADDITION

OF PHOSPHATE 99

TABLE 17. RESPONSE OF CELLULAR ATP TO NITR_^TE

ADDITION . 108

TABLE IS. ATP TO DRY IVEIGH'T R.\TIOS OF VARIOUS

MARINE ALGAE HI

TABLE 19. ATP TO DRY IVEIGHT PATIOS OF SELEILASTRUM . m

VI 1

FIGURE 1.
FIGURE 2.

FIGURE 3.
FIGURE 4.
FIGURE 5.

FIGURE 6.

FIGURE 7.

FIGURE 8.

FIGURE 9.
FIGURE 10.
FIGURE 11.

FIGURE 12.

FIGURE 13.

FIGURE 14.
FIGURE 15.

LIST OF FIGURES

Page

GHEMIGAL STRUCTURE OF ATP 5

CELLULAR CONTENTS OF ATP IN EUGL^llA

GRACILIS DURING ALTERNATING PERIODS OF

L I GHT AND DARK 2 7

ATP CONCENTR.ATION VS. CELLULAR ORGANIC

CARBON 2 9

LUMINESCENCE DECAY OF ATP FIREFLY

LANTERN EXTRACT REACTION IVITH TIME 43

DEPENDENCE OF QUANTITATIVE ACTIVATED
SLUDGE ATP EXTPv.^CTION ON TEMPEP.ATURE OF

EXTR.\CTION SOLUTION 51

STANDARD ATP CURVE 5 3

CORRELATION OF ATP WITH 3ELEUA37EuM

CONCENTR-VnON 54

ATP VS. CHLOROPHYLL A OF SELENASTRUM AND

CELORELLA 5 7

ATP VS. CHLOROPHYLL A FOR AJIABAEHA 5 8

ATP VS. CHLOROPHYLL A FOR MICROCYSTIS 59

ATP VS. CHLOROPHYLL A OF DILUTED

MICROCYSTIS 6 2

ATP VS. CHLOROPHYLL A FOR LAKE

PHYTOPLANKTON 6 4

VARIATION OF ATP/CHLOROPHYLL /. Pv.-\TIO WITH

CELL AGE 69

ATP VS. DRY WEIGHT OF UNIALGAL CULTURES .. 70

ATP VS. DRY WEIGHT FOR LAKE

PHYTOPLANKTON 7 3

Page

FIGURE 16. ATP VS. CELL C0U::T FOR MICROCYSTIS 77

FIGURE 17. ATP VS. ABSORBANCE OF UNIALGAL CULTURES .. 7 8

FIGURE 18. RESPONSE OF ATP TO PERIODS OF LIGHT AND

DARK 82

FIGURE 19. EFFECT OF pH ON ATP CONTENT OF SICLEIUSTRVM

CELLS S4

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 8 7

FIGURE 22. ATP POOL RESPONSE TO INCUBATION WITH

VARIOUS CONCENTR.ATIONS OF MERCURY 89

FIGURE 23. RESPONSE OF MERCURY- POISONED CHLORELLA

AFTER TR.'\NSFER TO FRESH MEDIUM 9 2

FIGURE 24. RESPONSE OF BIOMASS PARAMETERS IN BIVENS

ARM TO ADDITION OF COPPER 94

FIGURE 25. ATP RESPONSE IN AI^ABAEKA TO ADDITION OF

CARBON DIOXIDE 96

FIGURE 26. ATP RESPONSE OF BIVENS AR.M TO ADDITION

OF CARBON DIOXIDE 9 8

FIGURE 27. RESPONSE OF ATP IN AUABAEUA TO ADDITION

OF PHOSPHORUS 101

FIGURE 28. RESPONSE OF ATP IN iUCROCYSTIS TO

ADDITION OF PliOSPHORUS 102

FIGURE 29. RESPONSE OF ATP IN SELENASIRUM TO

ADDITION OF PHOSPHORUS 10 3

FIGURE 30. RESPONSE OF ATP IN CHLORELLA TO

ADDITION OF PHOSPHORUS 104

FIGURE 31. RESPONSE OF ATP IN AHABAEIJA TO

ADDITION OF NITROGEN 106

FIGURE 32. RESPONSE OF ATP IN SELEIJASTRUM TO

ADDITION OF NITROGEN 10 7

IX

Abstract of Dissertation Presented to tlie Graduate Council

of the Univ'ersity of Florida in Partial Fulfillment of the

Requirements for the Degree of Doctor of Philosophy

ATP MEASUREMENTS IN LAB0P..\10RY CULTURES AND
FIELD POPULATIONS OF LAKE PLANKTON

By

Francis Xavier Browne

December, 19 71

Chairman: Patrick L. Brezonik, Ph.D.

Co-Chairman: Jackson L. Fox, Ph.D.

Miajor 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 grov/th. 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 betv;een ATP and
chlorophyll a, dry weight, and cell number. ATP to chloro-
phyll a ratios were relatively constant, ranging from 0.09
to 0.55. These values agree with those reported by other
inA'estigators . ATP to dry v.eight ratios \;ere 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. Maxiirium
cellular ATP concentrations occurred at a pH 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 experim.ent 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 nut rient- de f icient 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 biom.ass param.eter for
aquatic systems. It is also a sensitive activity parameter
v,'hich 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
serioii.s 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
biom.ass and activity are needed. The biom.ass 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
param.eters are discussed in Chapter III, but it suffices to
say here that better measures of phy toplankton biomass and
activity are needed.

The deficiencies of present methodologies are
evidenced by the -laany conflicting reports in the literature
concerning lim.iting nutrients and the effects of m.unicipal
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 Avaters. However, this procedure has
not been v/holly successful probably because it utilizes
inadequate biomass parameters.

Aji 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 biom.ass and metabolic actiA'ity. Biomass
parameter evaluation was divided into two phases. First, the
ATP content of batch unialgal cultures A\-as measured and
correlated with traditional biomass parameters. In the second
phase, the ATP content of natural lal:e phytoplankton w^as
measured ai'xd correlated with biomass param.eters. ATP as a
measure of metabolic activity \vas 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
biomiass parameters would indicate the validity of using ATP
to measure phytoplankton biojnass. The lack of response of

cellular ATP to various environmental conditions v/ould
indicate tlie stability of cellular ATP to minor environ-
mental changes. The response of cellu.lar 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 hss been called the "energy currency" of living
cells. It occurs in all living cells. ATP is a nucleotide
containing adenine, a 6-ainino derivative of purine, D-ribose,
a 5-carbon sugar, and three phosphate groups as shown in
Figure 1. The ATP molecule Avithi.n the cell is highly
charged with negative charges concentrated around the
polypjiosphate structure. Most of the ATP in cells is
present as a Mg"^^ complex.

It is common practice to refer to ATP as a "high
energy" phosphate compound. However, as shov;n in Table 1,
ATP has an intermediate energy value when compared with
other phosphate compounds. It is this intermediate position
which laakes ATP so important.

TABLE 1

STANDARD FREE ENERGY OF HYDROLYSIS OF
C0\'3!0N INTR^^CELLULAR PHOSPHATE COMPOUNDS

AG°
Kcal/Mole

Fhcsphoenolpyruvate -14 . 80

1 , 3-Diphoyphoglycerate -11.80

Phosphocreative -10.30

Acelyl phosphate -10.10

Fhosphoarginine . 7 70

ATP - 7.30

Glucose 1-phosphate - 5,00

Fructose 6-phoaphate - 3.80

Glucose 6 -phosphate - 3.50

Glycerol 1-phosphate ' - 2.20

c

U

H
m

<
u

s:

The function of the ATP-ADP (adenosine dipliosphate)
system is to act as an intermediate between high-energy
ph.osphate compounds and low-energy phosphate compounds. One
set of enzymes helps transfer pliosphate from high -energy
compounds to ADP, forming ATP; while another set of enzymes
help transfer the terminal phosphate grou.p of ATP to low-
energy phosphate acceptors, Xo 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 v/ork.
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, Er.caryotic algal cells
contain chloroplasts , where photosynthesis occurs, and
mitochondria, where respiration occurs. Procaryotic cells,
like the blue-green algae, do not have chloroplasts and
Fiitochondria . Although eucaryotic and procaryotic cells
differ in sti'ucture, the chemdcal 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. TJie dark reactions use carbon dioxide
to fonr. si]:jple sugars, some of which are converted into
glucose as the product of photosynthesis. In photosynthesis,
chlorophyll inolecules 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 AlP. The electrons, after losing their energy, return
to the chlorophyll molecule again. T]:is 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 chloropliyll 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
m.olecules, 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 forir. 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. Som.e believe it is a shunt mechanism used to

form ATP without formation of reduced NADP, Tt appears that
1 ight- 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 term.inate 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. LITER.VrURE REVIEW

Introduction

Yollen^•eider (19 70) defines eutropliication as any-
thing that accelerates the nutrient loading, increases the
nutrient level and directly increases iv-ater productivity.
Me 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. Vollenv/eider ' s
definition emphasizes the tv,"o primary constituents of
eutrophi cation - 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. V^hile commonly
used each suffers from certain deficiencies. The following
sections v.'ill briefly describe the merits and drau'backs of
e ach .

10

Biomass Parameters

Pit-iri.ent Concentration

The algal pigments consist primarily of the chloro-
phylls, th.e carotenoids, and the phycolilins. Chlorophylls
are lipo-soluble inolecules characterized In- strong absorp-
tion of red (650 - 680 mu) and blue light (400 - 450 my)
and by their red fluorescence in organic solvents. Chloro-
phylls are divided into three groups: chlorophyll a which
is present in all photosynthesi zing cells; cnlorophyll b,
an oxidized derivative of chlorophyll a, which is found in
green algae; and chlorophyll a which is found in brown
algae and diatoms. Carotenoids are C^o hydrocarbons with
numerous conjugated double bonds. There are tis'o major
groups of carotenoids: the carotenes, which are pure
hydrocarbons, and the xanthophylls , vrhicli contain from 1
to 8 hydroxyl groups making them more polar. The carote-
]ioids 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 photosynthesi zing cells contain
carotenoids. Odum and N'ixon (19 70) have studied the role
of carotenoids in photorespiration . Odum et al (In Press)
note that tlie carotenoid-chiorophyll ratio may help measure
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

11

chlorophylls. They consist of an open conjugated s>stei:i
g£ four p;'rrol 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 (I^^hodophyceae)
and the primitive blue-green algae (Cyanophyceae) .
Phycoery thrin , present primarily in red marine algae, absorbs
light in tlie middle of the visible spectrum. Phycocyanin,
present primarily in blue -green algae, absorbs red light at
about 6 30 my.

Chlorophyll is the major pigm.ent of algae and occurs
in all species. Because of its universal distribution,
chlorophyll a. is often used to estimate phy top lank ton
niomass. Xeber and McFarland (1969) and Keup and Stewart
(1966) used chlorophyll a as an estimate of the phy tcplankton
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
chlorop]iyll content as percent of dry \;eight. Usually the
chlorophyll content is about 0.5 t6 l.S percent of the dry
weight (Round 1967). Studies on algae cultured under
controlled laboratory conditions denions trate that mineral
nutrition, light intensity, and cell age affect the cellular

12

chlorophyll concentration. Emerson (1929) found a direct
correlation between photosynthetic activity and chlorophyll
content in Chlovella pyrenoidcsa 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" Chlovella 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 phytoplanhton
at light saturation has a reasonably constant assimilation
ratio of 3.7 gram.s of carbon assimdlated 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 li ght-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 at
1958).

The analytical technique for extracting and calcu-
lating chlorophyll concentration suffers from many weak-
nesses. Strickland and Parsons (1968) note that the only

13

rapid chemical method kno'cn for esti:nating living plant
matter in the particulate organic matter of sea water is
to determine the characteristic pigments. Hoisever, 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 chlcrophyll a concentra-
tion to total plant carbon. According to Strickland and
Parsons (1968] extraction v.-ith 90 percent acetone gives
results undoubtedl)^ loi\' 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 miore of the pigments in their cells. Bogarad (1962)
notes that it is generally more difficult to extract
cjilorophylls from algae than from higher plants; he adds
that extraction with hot or cold methanol is usually more
effective than acetone. Another v;eakness of the extraction
process is that inactive chloropliyll and degradation products
may be determined along with the active chlorophyll of the
living phy toplanktons . Glooschenko and Moore (19 71)
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 ion percent measured chlorophyll a in some regions
of the lake. These studies indicate the necessity of
correcting chlorophyll a data for degradation products.

14

Particulate Oraanic Carbon

Particulate organic carbon is often used as a
TTieasure of phytoplankton biomass. >!enzel and Ryther (1964)
ineasured the composition of particulate matter in the
western North Atlantic to determine the relationship
befi\een carbon, nitrogen, phosphorus, and clilorophyll .
Regression of phosphorus or chlorophyll vs. nitrogen or
carbon, when extrapolated back toward tJie 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,
vrhile carbon and nitrogen are more refractory. Menzel and
Gosring (1966) studied the decomposition of particular
matter from surface and deep Atlantic waters. Changes in
carbon \;ere 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) , Holn.-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 biomass is
questionable .

15

C_e J_l_ j\' uTTib e r

Early i;ork on algal pliysiology utilized the cell
number, determined by counting cells with a hemocy tometer ,
Whipple disc, or a Segwick- Rafter Cell (Myers 1962). With
unialgal cultures the cell count can be relativ^ely 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
Gacnkar (1969) in a review of the Provisional Algae Assay
Procedure (Joint Industry-Government Task Force on Eutrc-
phication 1969) note the difficulty in counting cells in
mixed populations: taking the size of Chlor-ella -pyrenoidcsa
as unity, the size of Soenedesnus obliguv.s is 1.2,
C''nla!riydor:o-uas 6.9, Englena gracilis 54, Euglena viridis 84,
and a Fhaeus sp. 71. Besides this variability among species,
cell size differs with both age and nutritional state of
each species. Euglena cells tend to become larger with age
while Chlorslla and Saendesmus cells become smaller.
Nutrient starved Euglena usually decrease in size while
nutrient starved Chlovella increase in size. There is also
s. difficulty in counting filamentous and agglomerated cells
like Anahaena and Miarocystis. Oswald and Gaonkar also
note that miany algae are photo regulated. Cklcr-ella , for
exam.ple, accumulates nutrients during the day and divides
during the nigh.t; thus, unless cells are counted at the same
time each day, the number of cells could vary significantly.

15

AsiJe from the probleni of size, there are difficul-
ties with counting technique. llee (1954) studied varia-
bility in cell counts using tiie herr.ocytoi'ieter , a popular
and simple cell counting device. He found that for Euglena
the variation in cell counts exceeds 30 percent when the
nu:nber of cells counted in the hemocytoiietcr 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 wlien 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 ^'olume and D ry IV eight

OsAvald and Gaonkar (1969) recommended the volum.etric
technique, com.monly known as the packed-cell volume, be
added to the Provisional Algal Assay Procedure as a method
for evaluating algal growth. As Oswald (196 7) noted, the
volumetric (packed- cell volum.e) technique is a non-
destructi've procedure in v/hich the sample need not be dis-
carded after analysis and can be used for fui ther analysis.

According to Osv.-ald, the packed volujne usually contains
about 14 percent dry weiglit of algae and the approximate
algal content in dry weight is 1400 tiihes the paclced cell
volume plus or minus 400. Myers (1962) observed that
sufficient ceiitrifugal force and ti'Te 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 m.easures detrital as well as living matter. Lee
et al (1971) found a significant amount of detrital material
in unialgal cultures of Selenas truri aapricor-frutum after only
four days of growth in batch systems. Thus, quantitative
interpretation of packed cell volume results can be mis-
leading.

D>]A

Deoxyribonucleic acid (DXA) consists of two poly-
nucleotide chains twisted upon each otiier 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 phytopiankton biomass has been investigated by
Hclm-Hansen and colleagues (1968). After mieasuring concen-
trations in t?ie Atlantic and Pacific Oceans and comparing
them v.'ith measurements of chlorophyll and organic carbon,
th.e 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-
iiansen (1969) measured ATP, chloropJiyll , DNA, and organic

IS

carbon off the coast of California; he converto.; ATP,

chlorophyll, and DNA concentrations to organic carbon using

conversion factors of 250, 50, and 100 respect i\cly as
shown in Table 2 •

TABLE 2

COMPARISON OF BIOMASS ESTIMATED BY ATP, CHLOUOPHYLL, DNA
AND ORGANIC CARBON

Bioi'ass (ug c/ liter)

Direct Examination

ATP

Chlorophvll

DNA

Depth (m)
50 100

200

13

25

.^.8

1.1

24

22

S.6

1.9

14

22

S.O

0.2

250

200

9 5

50

The bicFiass estimates as determined from DNA measurements
are, according to Holm-Hansen, "impossibly high." He
concluded that DNA is an excellent biomass paraiuoter for
laboratory cultures but not for Avork in the natural environ-
ment .

None of the methods discussed in this section are
very good estimates of biomass, U'ith biomass, ii.othing is
constant or absolute; all parameters vary and tliis variation
m^akes it difficult to say which is the best measure of
biomass .

Limiting Nutrient Bioassay Methods

Many miethods are used to determine which physical or
chem.ical conditions limit phy toplankton growth. One general
approach is to m.onitcr the phytoplankton response ever a

19

period of liiTie after enriching the sample ivith sorie nutrient.
Although popular, this method depends on the response para-
meter measured an.d may giA.'e misleading results if another
nutrient or condition becomes limiting. Response parameters
include th3 various biomass measures and ^'*C assimilation
rates. The former are usually insensitive to short term
ci^.anges v.'hile insufficient time for response to enrichment
may give inaccurate results with the latter. Other nutrient
bicassay procedures are based on analysis of planktonic
constituents (e . g. , adaptive enzymes, percent nitrogen or
phosp'iorus) which vary in response to nutritional conditions.

Carbon- 14 Uptake

Carbon assimilation rates, as m.easured by the fixation
of carbon-14, are often used as a m.easure of algal grov;th
in bioassays (Goldman 1960, 1961, 1962, 1964, 1965, 1967;
Goldman and Carter 1965; McAllister et at 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 tim.e 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
formaldeliyde may affect the excretion or loss of organic
material from delicate algae. In situ measurements of
productivity requiie two to three 'nours of incubation, thus

20

liiTiiring the number of sanpling stations that can be tested
in one dav. Cn the other n.and, laboratory incubation using
a constant light intensity gives more precise but inaccurate
results. Oswald and Gaonkar (1969) state that the technique
of radiocarbon nieasurenents as set foith in PAAP seems
needlessly co]-tplex, delicate, and subject to error in the
hands of inexperienced personnel.

Goldman (1960, 1962, 1965) used carbon-14 uptake for
bioassays of limiting nutrients. Carbon-14 is"as 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 m.easaring 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
m.easuring 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?

>!any basic questions concerning the carbon-i4 method
must be answered. Is there any luxury uptake of carbon;
hcH' miuch fixed ^ '* C is excreted, how miuch is internally
recvcled? Uliat is the difference between ^^C and ''*C

21

uptake? Strickland and Parsons (196S) use the factor of
1.05 to account for this difference, buc note that this
value is uricertain.

Enzymatic and _Extractive Techniques

Enzvmatic 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 'vater 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
vhile algae grown with surplus phosphorus released more than
0.08 mg PGi^-P/100 mg algae. Gerloff and Skoog (1954, 1957)
used a tissue analysis method to evaluate nutrient availa-
bility in IVisconsin lakes for the growth of y.icr-ocys tis
aer-y.gincsa. Tissue analysis was also used to measure the
nutrient availability for the growth of angiosperm aquatic
plants (Gerloff and Kromliolz 1966). The tissue analysis
method requires the establishment of a critical level for
each element. The critical level is chc minimum tissue
content in a particular species that is required for maximum
growth. Tissue contents below the critical concentration
are associated with deficiencies of tliat element resulting
in less than maximum, yields. This m.ethod requires the

2 2

determination of the critical level f^u each species
encountered; the naxinum grov:th 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 phy topi ankton populations the complexity
of critical levels tend to make the r.ethod imipractical .
Gerloff and Skoog (1954, 1957), using tissue analysis on
l-licToystis aeruginosa in Vv'isconsin lakes, concluded that
nitrogen v/as more likely to become growth limiting than was
phosphorus. However, Gerloff and Krombliolz (1966), using
tissue analysis on angiosperm aquatic plants in the same
Wisconsin lakes, concluded that phosphorus was more likely
to lim.it higher aquatic plant growth than nitrogen. These
results demonstrate the species -dependency of the tissue
analysis technique, and highlight t]:e qualitative and
relative nature of the method.

Alkaline phosphatase activity is another m.easure of
algal phospliorus nutrition. V.'hen algae are phosphorus
limited, the alkaline phosphatase activity per unit weight
is as m.uch as 25 times that of algae grown with surplus
phosphate (Fitzgerald 1966, 1969). Fitzgerald cautions that
the effect of tlie local environment such as recent rains
or unusual circulation patterns in lakes cause changes in
the disxribution of algae and must be considered when
interpreting nutritional data. He also notes that results
x'ary according to the species of algae under consideration.

23

ritzgerald (1968) found that tho rate of NH^-N
absorption by algae in the dark is 4-5 tinges greater for
algae v.-hich are nitrogen limited compared to plants with
available nitrogen. The comparative rate of ammonium
nitrogen absorption in the dark of algae contalnin.g 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 m.edium, into 10-30 ml of
Gorham's medium (minus N) and adding 0.1 mg ammonium nitro-
gen. After one hour incubation at 25° C in the dark the
ammonium nitrogen content of the supernatant was comipared
to controls not containing algae. The procedure is based
on the fact tb.at nitrogen-starved cells can assimilate
ammonium-nitrogen in the dark while normal cells require
light and carbon dioxide (IVetherell 195S). It is believed
that nitrogen-starved cells have a carbohydrate reserve
which is lacking in normal cells and nitrogen-deficient
cells assir.ilate ammonium-nitrogen until their carbohydrate
reserves are exhausted (Syrett 1962).

All of the above techniques whether tliey be extrac-
tive or enzymiatic 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 num.ber
by itself rieans little and interpreting \shat it means is no
easy task.

24

ATP

In t?ie past, most studies on the aTP pool in micro-
organisins 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
r.o study the role of ATP in the bioenergeti cs of the cell.
Recently, hov/ever, some researchers have proposed that ATP
could be used to measure m.icrobial biomass. Determination
of microbial biomass by measurem.cnt of ATP depends upon
•che 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
varv substantially under different environmental conditions.

Forrest and IValker (1965) observed that the ATP pool
in starved cultures of Sti^eptoaoaaus fasaalis 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, shewed that under anaerobic conditions the
amount of ATP synthesized was proportional to new cell
yield. Elsden (1965) reported that the "ATP growth

25

coefficient," that is, the grams of nev; cells produced per
nole of ATP synthesized, was approximately constant for all
the organisms studied. D'Eustachio et al (1968) reported
that cell counts based upon ATP concentration were linearly
correlated to standard plate count. D'tustachio and Levin
(1367), studying the ATP pool in three aerobic bacterial
species during lag, exponential and stationary growth phases,
fciund that E soli had a relatively constant level of ATP
througliout all growth phases. Vseudomonc.s flucvesaens and
Bacillv.s suhtillis were also fairly constant except for a
sniall increase in ATP pool during exponential growth.

Holm-Hansen and his co-workers have used ATP to
measure phy toplankton in the ocean (Holm-Hansen and Booth
1966; Hamilton and Iiolm-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 lieat, 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 rhe content in eight species of algae ranged
from 0.00 3 to 0.016 percent of the dry weight. (Holm-Hansen
and Booth 1966.) These Avere maximum variations in ATP over
a wide variety of growth conditions and stages in batch
cultures .

H?r.iltcn and Holin-I'ansen (196 7) deteriiiined the ATP
content of seven marine bacterial isolates cultured in both
batcii and chenostat conditions. The range of ATP in the
chemostat grown cells was 0.5 to 6.5 x 10'^ yg 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"^ yg 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-IIansen (19 70)
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 peel 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-Iiansen (1969) measured the total particulate
carbon, nitrogen, ATP, DNA, and chloropliyll 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 agreem.ent with those
based on clilorophyll data and direct microscopic measurements

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KolFi-Hansen concluded that there is a considerable uniformity
in algal MP 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 50 different algal cultures under different
environmental conditions. As shov,-n 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
Skeletonema aostatum, to 46 percent for I-^onochn-^sis lutheri ,
and to 14 percent for Dunaliella tevtioleata. Phosphate
deficient cultures of M. ly.thari showed that ATP dropped
to about 0.0 5 percent of the carbon content, but that it
increased to 0.15 percent one day after phosphate was added.
Hol-n-Kansen j-iaintains 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

29

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51

value may be ineaningless because of t.]\r presence of detrital
inaterial. llolm-IIansen (private coranunication) does not
believe it is feasible to report ATP/dry v:eight 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
seeins 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 m.ore 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 al (1971), working with
batch cultures of Selenas trv.m aapvicornutum , only calculated
the AlP 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 (19/1) estimated that the turnover time
of the E. aoli ATP pool is only a fraction of a second.
Foriest and Walker (1965) have found the turnover time in
St-r-evtococous faesalie 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
faeoalis increased after addition of glucose and later
returned to its endogenous level when the substrate was

32

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used up. Ivorking with E. cell, IVinpenny (1967) also noted
an increase in ATP pool size vdien glucose or pyruvate was
a dded.

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 al (1970)
reported 15 to 20 percent viability in a full scale activated
sludge unit and 35 to 40 percent in a laboratory unit.
Frezonik and Patterson questioned wliether the increased ATP
pool levels follov-ing 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, shoved that substrate addition can effect an
increase in ATP per cell.

Coomb e.t al (1967a) subjected cultures of the marine
diatom Cylindvofheca fusi forms to 24 hours of darkness
follovred by re - illuinination at a high light intensity and
addition of silicon. During the time of silicon uptake,
the ATP pool size decreased v.'ith 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
plienomena using Uaviaula -pellicuZooa , 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. l\1ien silicon was added, a rapid
tem.porary increase in the ATP pool occurred followed by a
sharp decrease. The ATP pool increased slowly throughout
the remainder of the silicon uptake period. Sim.ilar changes
occurred in a synclironous 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 chloroplas tic and a residual fraction.
IVhen 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

35

in a sir.il ar inanner in the cytoplasnic fr:5ction. The ADP
change was opposite to the ATP change and the AMP change
follov.-ed 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
ra-Mo of ATP to ADP rather than a drop in the orthophosphate
concentration .

Because of the energy balance occurring within cells,
ATP seer.is to be relatively constant under normal environ-
m.ental conditions. This constant ATP pool may allow the
use of ATP as a measure of m.icrobial biomass. On the otlier
hand, the rapid turnover time and sensitivity of ATP to
environmental stress may permit the use of ATP analysis
as a rapid bioassay m:ethod.

CHAPTER IV. EXPERIMENTAL METHODS AND ^L\TERIALS
Analytical Techniques

The procedui-es used in this study v.'ere basically
those coiamonly used to anal)'ze the 1)iological and chemical
constituents of aquatic systems. Analysis of ATP is
discvissed separately in Chapter V.

Chemical oxygen demand (COD) was measured using the
dichromate reflux method described in Standard Metliods
(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 v/as determined
using tiie 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 90 '' C in an oven, then
they were placed in a desiccator to cool. The filters
were weighed on a Mettler balance.

6o

37

Tuibiclity 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 Eut rophication
1969). A Bausch and Lomb Spectronic 20 spect ropliotometer
at a wavelength of 600 nm ".'as used. Conductivity \<as
measured as described in Standard Methods (APHA 19 71) using
a Becbnan conductivity bridge.

Ortho-phosphate was measured by the single reagent
molybdenum blue method of Murphy and Riley (1962) adapted
to the Technicon Autoanaly zer . 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-
Sum.erson 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 H2SO4 as described in Standard Methods
(APHA 19 71) .

Chlorophyll a was extracted and measured by the
procedure originally described by Richards and Thompson

58

(1952) and Crietz and Richards (1955). The equations of
Parsons and Strickland (1963) were enployed to calculate
the chlorophyll a concentration.

A modification of the carbon- 14 procedure deA^eloped
by Steeman-Nielsen (1951) was used to measure priiiiary
production. Rather than inoculating 300 ml samples with
5 micro-caries of ^"^C, 30 ml samples were inoculated with
0.5 micro-curie of ^"^C. After incubation for three hours
in a constant tem.perature 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 relatii^e standard deviation of five
percent .

Alkaline phosphatase activity of the algal cultures
was measured using a m.odi fication of the method of
Fitzgerald and Nelson (1966). Instead of using Gorham's
medium as suggested, PA.A.P medium was used since all algal
cultures v/ere grown on PAAP medium.

Cell numbers \vere determined using a hemocyto-
meter.

Experimental Materials

Four laboratory batch unialgal cultures v;ere
operated for a period of seven months. The following

algal species were grown: Anabaena flos-aqu'2e ^"^ Micro-
systis aeruginos a^ Selenas trum aapj'-iocrni-'.ti'im^ and Chloretla
sp. Initially, chemostat units were set up using Anabaena
fZos-aq-uaej Selenastrum eapricovnuturi^ and Naviaula minima, "^
a diatom. A detention time of three days v/as maintained
by feeding three liters of PAi\P 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 v^ei-e contaminated with Chloi-ella and new culture
material had to be ordered from the National Eutrophication
Research Center in Oregon and from tb.e Starr Collection in
Indiana. At this time a decision was made to change to
batch cultures.

Batch unialgal cultures of Anabaena, llicrocystis,
oelenas trum, and Naviaula were grown. However, Naviaula
continued to be contaminated with Chlpvella . Finally,
Naviaula cells declined in numbers and had to be discarded.
A unialgal culture of Chlorella was substituted for the
diatoir..

The batch units contained four liters of culture
material which were kept well-mixed with magnetic stirrers.
Gro-Lux~ fluorescent lamips were used to illuminate the

^Obtained from the National Eutrophication Research
Program, Pacific Northwest V/ater Laboratory, Corvallis,
Oregon 9 7 350.

'Obtained from Dr.R.Starr, Dept. of Botany, Univ. of
Indiana, Bloomington, Indiana.

Sylvania Electric Products Inc., Danvers, Mass.

40

cultures. 3£lenastr--iiiri and Chlorella received abcut 350
ft-c Khile Anahaena and Microoi/s tie received about 200
ft-c. Each day 200 ml o£ culture material was removed
from each unit and 200 ml of fresh PAAP medium was added.
The PAAP medium v/as sterilized before it was fed to the
units. Because of the poor buffer capacity of P.-'V.AP medium,
the pK of the cultures often increased to a pH of 10 or
greater, indicating a carbon-limiting condition. Carbon
dioxide was occassional ly bubbled into the units to lower
the pH and add carbon. Frequent visible changes in the
algal pigments of the units occurred, especially when the
pH increased. At no tine 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. Eight production by
firefly lantern extract (FEE) depends on the presence of
luciferin, the enzyme luciferase, oxygen, magnesium ions,
and ATP (McElroy 1947; McElroy and Strehler 1954). iiastings
(1968) has reviev.'ed the biochemistry of luminescent reactions
in detail. Equations 1-3 illustrate the chemical reactions
involved in the firefly light production process.

Lh2 + ATP + E ^ E - LH2 - AMP + PP. (1)

E - LH2 - AMP + O2 Z E - L - 0=^- + Product (2)

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

Luciferin and ATP, catalyzed by luciferase, react to form an
enzyme- 1 uci fe rin- adenosine monophosphate complex (luciferyl-
adenylate) and inorganic pyrophosphate (PP.)- Rapid oxida-
tion to oxyluciferyl- adem-late 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 al 1968), One light quantum is emitted for each luciferin
molecule oxidized (Seliger and McElroy 1959, 1960),

41

4 2

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 phosphiate buffer
to the firefly reaction decreases the initial light flash
and produces an intermediate level of light ^chich 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 v.-ith purified extracts of firefly lantern to produce
luminescence. The crude extracts of firefly lantern,
however, contain trarisphosphorylase enzymies , resulting in
light emission in the presence of high energy phosphate
molecules other than ATP (Balfour and Samson 1959). To
determine which Iiigh energy phosphate m.olecules riight cause
light emission with firefly extract, Molm-Iiansen and Booth '
(1966) tested thirteen intracellular comipounds including
adenylic acid, guanylic acid, cozymase, glucose- l-phosphate ,
fructose -1- 6 -diphosphate , phosphocreati ve , adenosine, thia-
m.ine pyrophosphate, coenzyme -A, sodium, glass, adenosine
diphosphate (-XDP), cytidine - 5 - triphosphate (CTP) and inosine'
5-triphosphate (ITP). Of these conr)o\xrLds , only ADP , CTP,
and ITP affected light em.issions. The amount of light
resulting from addition of ADP v/as less tiian 1 percent of
an equivalent amount of ATP. Both CTP and ITP stimulated
liglit production equivalent to that of ATP. Franzen and

43

Binkley [1961) reported cellular ATP values 10 to 25 per-
cciTt higher using the firefly method than v;hen determined
by chromatographic techniques. The fact that some light
em.ission 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 otiier
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 -20° C until used. Each vial
contains the extract from 50 mg of firefly lanterns with
magnesium, arsenate buffer added. The contents of each vial
v;as rehydrated with 35 ml of deionized distilled water.
After standing at room temperature for 1/2 hour, the enzyme
m.ixture was filtered (V.Tiatman 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 (0.025 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 m.onths shov;ed no change in standard ATP

'1
FLE-50, Sigma Chemical Company, St. Louis, Missouri.

44

copcentration (Patterson 1970J. Khen ATP standards were
needed a test tube of stock solution ivas thavv-ed and diluted
■A'ith tris buffer to the desired concentrations.

Samples to be analyzed for ATP v.'ere filtered through
47-miii membrane filters (pore size 0.80 y) . 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 occassional 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 in.to a clean test tube. The test tube was then
frozen and stored at -20° 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-llansen 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
pK, 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 (19 70) compared
sonication to boiling tris extraction and found it w^as

4:

equally effective in extracting ATP. DvPont de Xeinours
Cornpany (Inc.) recommends ATP be extracted v.'ith either
butanol or dimethylsul foxida (DMSO) for use with their
Luminescence Biometer.^

A sensitive method of measuring light emission is
required in the ATP assay. Holm-Hansen and Booth (1966)
used a pliotomultiplier tube and amplifier connected to a
recorder. Others (Forrest 1965;' Lyman and UeVincenzo 1967)
have also used similar instrumentation. Another instrument
used to measure ATP is the DuFont Luminescence Bioioeter.
The PuPont 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

^PuFont 760 Luminescence Biometer, DuPcnt de Aemours
and Com.pany, V.'ilmington , Delaware.

46

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 enzym.e mixture. Although
a precision m.icroliter 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, U'impenny, et al .
1967; Patterson 1970). A liquid scintillation spectrometer^
was used in this study. Instrument gain was set at 53, with
a window opening of 50 to 1,000. The spectrometer v.'as 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 vras 10 to 20 counts in the 6-second counting
interval. Patterson (19 70) found that higher counts
indicated glassware contamination. All glassware was washed

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

47

in an autoiiiatic dishwasher using hot soapy water, a hot
rinse, and a final rinse v;ith distilled v/ater. Following
this, the glassware was boiled for at least one hour in an
acid bath and rinsed three times in deionized, distilled
wMter.

After determining the background em.ission, exactly
0.5 ml of ATP standard or sample was pipetted into the
enzyme mixture and the vial w^as 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 w^ere 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 w-as extrapolated bade to the initial data point and
the count at one-minute was read from this line. This
graphical technique w^as used in this research. Figure 4
shows lumincence decay curves for several ATP concentrations

4 8

ATP = 20 0 yg/1

0.5

1.0 1.5 2.0 2.5

Elapsed Reaction Time, Minutes

FIGUP'E 4. LUMINESCENCE DECAY OR ATP FIREFLY LANTERN
EXTPvi\CT REACTION WITH TIME (Patterson 19 70)

3.5

and firefly lantern extract v.'ere studied (Patterson 19 70).
As s]iov;n 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 v;ith the time of incubation, indicating
a slight loss 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 tem.perature reduction of a few degrees below boiling
caused a significant decrease in the am.ount of ATP extracted.
For this reason, all extractions were performed in a rapidly
boiling water bath. However, it i.'as often found that ;vater
which appeared to be boiling actually had a tem.perature of
90-95°C. Thus a tlierm.ome ter was alv;ays kept in the boiling
tris buffer to assure that the temperature remained at 100°C.

H

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Tejaperature cf Extraction, °C

FIGURE 5. DEPENDENCE OF QUANTITATIVE ACTIVATED SLUDGE
ATP EXTRACTION ON TE^IPEPATURE OF EXTRACTION SOLUTION
(F-itterson 1970)

52

Rhodes and McElroy (1958) inves t: igated the sensitivity
of the firefly reaction to pH and found that luminescence
increased rapidly from pH 2.0 to 3.S, 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 Has used.

Figure 6 shews a typical standard ATP curA/'e,
-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 Selenastrun concentration was
observed.

using the m.ethod of ATP analysis described in this
chapter, replication of ATP samples gave results with a
relative standard deviation of less than 5 percent.

5 3

>

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rH Di
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to Q
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01 X s;unc3

54

300 t

250 -

200 .

150

100

0.125 0.25 0.5

Fraction of Sc-lenas tryj:

FIGURE 7. CORRELATION OF ATP IVITH SEI.

1.0

Sample

EHASTEUM CONCENTR.\TION

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 uni algal 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-aqv.e, Selenastr-wv
aapriaorrruturij Microcystis aeruginosa^ and Chlorella 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,
etcl. 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

55

56

parameters wis determined to test the usefulness of ATP
measurements in natural phytoplankton populations. No
attem.pt was miade to quantitatively measure the bacteria
population in the batch cultures or the lake samples.

ATP vs. Chip rophyll 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 Chlorella are sim.ilar;
thus Figure 8 contains the data for both these organisms.
Although the slopes of the linear regression lines are
similar for Anabaena and Miovoaystis , 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

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61

at a particular confidence level, the confidence interA'al
must not contain zero. As shov:n in Table 6, the sample
correlation coefficients for all algal species are signifi-
cant at the 99 percent confidence level, but the correlation
for 'Helenas tr-um, which had the lov;est 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
clilorophyll a, a dense culture of Micr-ocystis 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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66

three lakes is significant at the 95 percent confidence
level. Eivens 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 vith 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 i^'ith chlorophyll a in lake phy toplankton.

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 Miarccystis have lower
ATP to chlorophyll a ratios than Selenastvum and Chlorella.
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

Sslenastyum 0.35

Chlorella 0.29

Bivens Arm 0.09

Newnan's Lake 0.11

Anders on- Cue Lake 0.69

All Algae 0.20

All Lakes 0.25

Algae and Lakes ^ " 5-ili.

67

Mici'ca-jstis , both blue-green algae, are procaryotic cells
Kith a pigment system containing chlorophyll a, phycocyanin,
and trace aiaounts 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
chloropl asts . 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 tl^at Bivens Arm and
Ne\;nan'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.

68

Other experiments \<ere performed to determine whether
ihe ATP to chlorophyll a ratio varied with nutritional state
and cell age. Aliquots from each of the unialgal cultures
were transfered to flasks containing fresh media. One flask
contained PAAP medium with all nutrients present, one
contained PAAP medium without phosphorus, and one contained
PAj\P medium without nitrogen. Tlie 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 coefficients for A:iabaena,

69

1
1

0

0

« 0

1—1

rH 0

n
8

6

4 .

'^ ■

, 2
,0
,8
.6
,4

CHLQFELLA

12 3

MICBOCYSTIS

■^ K Deficient
o P Deficient

Control

.0 P Deficient
Control
N Deficient

\T Deficient

o P Deficient
Control

Control

N Deficient
■^ P Deficient

12 3456 78

Time , Days
FIGURE 13. VARIATION OF ATP/CilLOROPHYLL A RATIO WITH CELL AGE

70

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Miarccystis ^ and Cnlcvslla 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
Sele-nastvum is poor and not significant even at the 95
percent level. The poor correlation of Selenastv-j.r^, probably
resulted prinarily 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 v^eight 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 yg/mg. However, the
ATP to dry weight ratios for the three lakes vary from 0.12
to 0.96 yg/mg. Bivens Arm and Newnan's Lake have a much
lower average ATP to dry weight ratio than Anc^erson-Cue Lake,
These low ratios are evidently the result of a high
concentration of detrital matter which also explains the

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75

TABLE 12

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

ATP/dry weight

Ar.abaena 0.26

l-i'Lcvcay stts 0.36

Selenastvum 0.24

Chlorella 0.38

Bivens Arm 0.13

NeKnan ' s Lake 0 . 12

.Anders on -Cue Lake 0.9 6

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
Xewnan's Lake have large amounts of turbidity. Bivens
Ann, 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 AVEP^AGE
TURBIDITY OF EXPERIMENTAL LAKES

Average
Turbidity^
Lake ATP/Dry Weight (JTU)

Bivens Arm 0.13 10.2

Newnan's Lake 0.12 4.2

Anders on -Cue Lake 0.96 1.0

-Shannon, 19 70

•75

ATF vs . Cell Number

Another common biomass measurement is the cell
num.ber. A dense culture of hiici-'ooys tis was diluted into
ten portions, incubated for 24-hours, and analyzed for ATP
and cell nuiaber. Figure 16 sliows that a linear correlation
exists between ATP concentration and cell numiber. The ATP
per cell for Microcystis ranged from 5.0 x 10"^ to 6,25 x
10"^ ]jg/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
vveight 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 Chlorella. The reason
lies partially in the small range of ohlorcphyll a values
observed.

77

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80

Z_o 0 plankton 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 ?\eu'nan's Lake was collected using a zoo-
plankton net. The zooplankton consisted almost exclusively
of Cyclops v.-hose average diinensions v/ere 1.4 mm by 0.5 mm.
Using these average dimensions and assuming a specific
gra"\4ty of 1.05, the ATP to dry weight ratio for zoor)] a^ikton
was calculated to be 0.1 yg/mg. This value is slig?it]y lower
than the average value reported in tliis research for algal
cultures and much lower than the average value reported for
bacterial cultures (1.5 yg/mg). Based on the lovrer ATP to
dry weight ratio of zooplankton and their relatively small
number in most lakes, it seems that the ATP in zooplankton
v.'ould 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
estijnated 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 bioinass. Laboratory algal

cultures and lake phytoplankton were subjected to varying
light periods, different pM levels, and additions of
m:trients and toxins.

Light-Dark Study

In order to be used as a reliable estimate of phyto-
plankton biomass, the cellular AT? 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 m.onitored. Tlie response of ATP in
Selenastrum 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 m.aintained at fairly uniform levels in both light and
dark. Holm.-Hansen ' s experim.ent only covered a tine 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

8 2

l/o< 'dlV

83

and respiratory mechanisins of the cell. AT? occurs in trace
amounts in the cell and has a rapid 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 iranediately 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 Selenastr-um 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.

84

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Tlie above experiment was repeated using lake v/ater,
in order to determine whether maximum ATP content always
ccurred 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. ^!aximum ATP concentration
occurred at a pH of 4.6, the original pH of tlie lake water.
The greatest reduction in ATP concentration occurred in the
pll ra;;ge 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.
^!aximu]n reduction in ATP concentration occurred at a pH
of 3.0.

It should be noted tliat the pH values reported are
those of the incubation media or lakewater. It is probable
that the cellular pH changed less than the m.cdia 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 organism.s normal pH
range causes a reduction in the ATP level, v.-hether acidic,
neutral, or basic.

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88

Toxicit y_ _S_t; u dies

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 Avastes. IVastes containing
concentrations of heavy metals may be toxic to aquatic
orgaiiisms 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 m.edicai treatment. They have been used extensively to
control slimes in paper mills. To study the effects of
mercuric ion on phytoplankton , aliquots of Selenastrum v:ere
incubated with various concentrations of mercuric chloride
and the response of ATP, carbon-14 uptake, chlorophyll a,
and suspended solids was mieasured. Figure 22 shows the ATP
content in Selenastrum samples after three hours of incuba-
tion in the presence of various concentrations of mercuirc
c?iloride. The ATP toxicity pattern in Figure 22 shows a

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90

rapid reduction of ATP for mercuric chloride concentrations
of 50 ppb and 100 ppb , with a gradual further reduction
for concentrations above 100 ppb. Table 15 shows the per-
cent reduction in ATP, carbon-14 uptake, chlorophyll a,
and suspended solids after one-hour inciibation with 500
ppb of mercury.

TABLE 15

PERCENT REDUCTION AFTER ONE-HOUR INCUBATION WITH
MERCURIC CHLORIDE

Parameter Percent Reduction

ATP 54

Carbon-14 Uptake 56

Chlorophyll a 20

Suspended Solids 10

From Table 15 it is apparent that ATP is a m.uch more sensi
tive bioassay response parameter than chlorophyll a or
suspended solids. However, ATP is about equal to or a
little less sensitive than carbon-14 uptake as a response
parameter.

The observed reduction in ATP may result from a
reduction in the viable cells or it may result from a
reduction in the cellular ATP pool. In an attempt to
explain which m.echanism was responsible for the reduction
in ATP, a sample of Chlorella was incubated with 100 ppb
of m.ercury for 80 minutes at which time half of the sample
was centrifuged and the Chlorella cells were transferred
to fresh PAAP media without mercury, .The final volumes

91

cf both saTT'ples were the same. As shovrn in Figure 23, the
ATP content of the cells transferred to fresh media immediately-
increased from 104 yg/l to 192 yg/1 while the ATP of the
sample with mercury remained constant. After five minutes
the ATP content of the transferred cells decreased to 125
!Jg/l, a concentration slightly below the initial concentra-
tion of the culture before addition of mercury. These
results indicate that most of the ATP reduction results from
a reduction in the cellular ATP pool rather than a reduction
in t?Le viable cell population. The difference between the
initial ATP concentration (135 yg/l) and the final concentra-
tion of the transferred cells (125 yg/l) indicates that a
fraction of the algal cells were killed by the addition of
mercury. Eighty minutes was selected as the time to transfer
the poisoned Chlcrella cells to fresh media because previous
experim.ents showed that maximum ATP reduction occurred in
the first 1 to 1 1/2 hours after addition cf the poison. It
is probable that as the incubation time increases the
fraction of dead cells would increase. After two hours of
incubation the ATP content of the transferred cells decreased
slightly; this is probably a result of carry over of mercury
in the centrifuged cells.

Another important toxic material is copper. Copper
sulfate is used extensively in industry for tanning,
electroplating, engraving, and pigment m.anuf acture . It is
also used to control undesirable plankton growth in reser-
voirs and lakes. In order to study the effect of copper on

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93

natural phytoplankton populations, Eivens Arm water was
incubated with a concentration of 2 mg/1 of copper sulfate.
Bivens Arm, at the time of sampling, contained a very dense
bloom of Microcys tis . Figure 24 shows the response of ATP,
chlorophyll a, suspended solids, and COD to incubation with
copper sulfate. In the first three hours of incubation
the ATP content dropped by 45 percent while carbon-14 uptake,
suspended solids, chlorophyll a, and COD dropped 40 percent,
22 percent, 29 percent, and 21 percent respectively. The
ATP concentration decreased much more rapidly than all of
the other parameters with the exception of carbon-14 uptake.
Both ATP and carbon-14 uptake decreased rapidly after
addition of copper sulfate. The rapidity of the decrease
in ATP and its similarity to the decrease in carbon-14
uptake indicates that a decrease in cellular ATP occurs
initially rather than a decrease in living cells. These
results indicate that ATP analysis is a sensitive response
method that can be useful in routine toxicity bioassays.

Nutrient Studies

Besides bioassays for determining the effect of toxic
substances on aquatic communities, there is a need for
better limiting nutrient. Considerable controversy exists
today as to whether phosphorus, nitrogen, or carbon is the
primary limiting nutrient. These three nutrients were used
to determine the usefulness of ATP analysis as a rapid
bioassay m.ethod.

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The effect of adding carbon dioxide to carbon- limited
algae was determined by bubbling carbon dioxide gas into a
batch culture of Anahaena and measuring the response in the
ATP concentration. The Anahaena culture had been grown for
three days on V^AV medium. Initially the pH of the culture
\\a.s 7.6 but each day it increased until it reached 10,5
on the day of the experiment. At this pH tlie culture was
carbon- limited, very dense, and a dull green. Past
experiments indicated that carbon- limited Anahaena become
dull green, but change to a bright green color approximately
one day after adding carbon-dioxide. In this experiment
the carbon-dioxide was bubbled into the culture for five
seconds. The response of ATP to addition of carbon dioxide
v;as dramatic as shown in Figure 25. Cellular ATP increased
from 134 yg/1 to 312 yg/l in three minutes. The ATP content
was measured for 12 minutes and exhibited a tendency to
rapidly increase and decrease. After six hours the ATP
content still remained relatively constant at 312 yg/l.
The significant increase in ATP concentration and its
stability at the higher concentration may also indicate
that the original sample at pH 10.5 may have been inhibited
by the high pH. The higher ATP concentrations may be a
result of increased cellular activity, utilizing the
available carbon. Immediately after carbon dioxide addition
the pH dropped to 5.8, but after six hours the pH increased
to 8.6, indicating that the carbon was being utilized by
the algae.

96

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Another experiricnt on carbon dioxide addition -.vas
performed v;ith a natural population from Bivens Arm. The
Bivens Arm sample had a mixed algal population with a low
density. The initial pH of the sample was 9.0. Figure 26
slioAvS the ATP response in Bivens Arm water to which carbon
dioxide was added. Like the algal culture, an initial
increase in ATP occurred. However, unlike the algal culture,
the ATP concentration decreased shortly thereafter and
continued to decrease, indicating inhibition or die-off of
the phytcplankton. After three minutes the pH dropped to
6.7 and after 23 mijiutes it dropped to 5.7. The decrease
in ATP is probably a result of the lowered pH of the lake
water. An increase in the pH to 7.6 was observed after
tliree hours, an indication tliat the lake water was recovering
from the carbon dioxide addition.

The response of ATP in phosphorus -deficient cultures
to the addition of phosphorus A\'as also mLcasured. Batch
cultures of Anabaena^ Microcystis , Selenas trun^ and Chlorella
were grown in duplicate for a period of five days. One
culture of each algal species contained regular PA.\P medium
v.'ith 0.62 m.g/1 while the other culture contained a modified
PAAP medium without phorphorus . All cultures were relatively
dense having an absorbance ranging from 0.19 to 0,57. On
the fifth day 1.24 mg/1 of phosphorus was added to each
culture and the ATP response measured. Table 16 gives the
percent increases of ATP after addition of phosphorus. All

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phosphorus - deficient cultures exhibited a greater ATP
increase than the cultures with phosphorus. Anabaena
shelved the greatest difference in ATP response with the
phosphorus deficient culture increasing eight times as riuich
as the control culture. It is interesting to note t?iat al-
though both cultures of Microcystis had very low final
concentrations of phosphorus, the ATP of the phosphorus-
deficient culture increased approximately three times as
much as the pliosphorus -enriched culture. This would
indicate that even though the phosphorus concentration of
the phosphorus -enriched culture was low, the algal cells
were not phosphorus limited, that is, at least not to the
same extent.

The response patterns for each of the species are
different as shown in Figures 2 7 to 30. Anabaena (Figure
27) exhibited an initial drop in ATP followed by an increase
while Mic-rocus tis and Selenastrv.m (Figuies 28 and 29
respectively) showed a rapid initial increase. Chlorella
(Figure 30) displayed a gradual increase in ATP. All of the
species, hoAvever, responded essentially in the same m.anner,
that is, each exhibited an increase in ATP concentration.

To study the ATP response in nitrogen deficient
cultures, A.nahaena and Selenastrum were grown for five days
in duplicate cultures. One culture contained PAAP medium
v/ith 14.0 mg/1 of nitrogen as nitrate -while the other
contained a modified PAAP medium without nitrogen. On the

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fifth day, 23 mg/1 of nitrogen as nitrc.te v.-as added to each
culture. Percent increases in ATP concentration after
addition are given in Table 17. Like the phosphorus experi-
ment, A-nabaena exhibited the greatest difference in ATP
response which is surprising because Anabaey.a is a nitrogen
fixer while Selenastrum is not. The ATP response in
nitrogen- deficient Anahaena was 46 times as great as that
of the control. The ATP increase in the nitrogen-deficient
Sclenastri^m was only twice that of the increase in the
nitrogen-enriched culture. The response of the nitrogen-
deficient Anakaena showed a gradual increase in ATP while
the Anahaar2 control showed a rapid initial increase
followed by a gradual decrease to approximately the original
concentration (Figure 31). Both the nitrogen- deficient
Salenastry.r-i and the control responded rapidly as shown in
Figure 32. Essentially, the ATP response to the addition
of nitrogen was similar to that of phosphorus addition in
that an increase in ATP concentration always occurred.

The percent increases in ATP concentrations reported
here were calculated using the ATP values measured 70 to 80
minutes after the addition of nitrogen. This time interval
was selected because the ATP content of both the nitrogen-
deficient and the nitrogen-enriched cultures seemed to level
off by this time. Different percent clianges in ATP could
be obtained simply by choosing a shorter or longer interval.

106

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For example, if the initial increase was used, the same
response would be shown by both cultures in several

instances .

This criticism is valid for the phosphorus experi-
ments as well. The percent increases shown in Tables 16
and 17 for phosphorus and nitrogen respectively are
presented merely to illustrate the differences observed in
nutrient-deficient and nutrient-enriched cultures. The
values presented in these tables indicate that ATP could
be used as a rapid nutrient bioassay parameter. However,
to be of practical value, the ATP response to nutrient
addition should be measured over a period of a few hours
and plotted against time. Plots of ATP vs. time would be
a qualitative but relatively good tool in nutrient
bioassays .

CHAPTER YII. DISCUSSION

The expei-imental results presented in Chapter VII
".vere briefly discussed as they were reported. In this
chapter these results will be discussed in furtlier detail
and compared with work reported by other investigators.

Correlation of ATP with chlorophyll a for both
unialgal samples and lake phy toplankton was good. The
scatter of values around the linear regression line may be
a result of changes in the cellular ATP pool but it is also
likely that it may result from changes in cellular
chlorophyll content. The lake samples were collected at
different times of the day, during different seasons, and
under different atmospheric conditions. Thus, the cellular
chlorophyll may have varied diurnal ly. Ryther and Yentsch
(1957) and Odum et al (1958) reported diurnal variations
in chlorophyll concentrations. The chlorophyll concentra-
tions may also have varied with nutritional state as reported
by Fogg (1965) and others. Atmospheric conditions, such as
a clear sky or an overcast sky, would also affect tlie
chlorophyll concentration.

Holm-Hansen (1969) found good correlation between
ATP and chlorophyll. He found tliat both ATP and chlorophyll
gave good estimates of the cellular carbon in pliytoplankton
in seawater.

110

Ill

The ratios of ATP to chloroph}-!! a ranged from 0.09
to 0.35 for imialgal cultures and fron 0.09 to 0.69 for
lake phytopl ankton . These values are in excellent agreenent
v;ith those reported by Holni-Hansen (1969, 19 70). His
values ranged from 0.04 to 0.60 for five species of marine
algae and one species of freshwater algae, and from 0.2 2
to 0.36 for seav,"ater p'lytoplankton .

Good correlation of ATP with dry iveight of unialgal
cultures and lake phytoplankton was also observed. The
ATP to dry weight ratios ranged from 0.2 4 to 0.38 ug/ng for
unialgal cultures and from 0.12 to 0.96 yg/ng for la]:e phyto-
plankton. Holm-Hansen (1966) reported average ATP to dry
weight values ranging from 0.15 to 1.6 yg/mg dry weight
for various marine algae (Table 18).

TABLE 18

ATP TO DRY WEIGHT R^ATIO'S OF VARIOUS MARINE ALGAE
(Holm-Hansen 1966)

Organism

Skeletomma acstatum
Amp'nidinium carteri
Dunaliella tertioleota
Svraaosphaera elongata
Monochrysis lutheiH
CyaZotella nana
Ditylum hrightweltii.
Rhizosole7iia sp.

Average ATP/Dry l\"eight (yg/mg)

0. 31

0.15

1.5

0.03

0.28

0.84

1.2

1.6

ii:

Lee 2t al (1971) reported ATP to dry \vei-ht values of
Selenast-i>um oapriaornuturn ranging from 1 . 4 to 3.4 yg/mg
dry weight. These values are much higher than those
reported for this research. However, the authors note that
they did not calculate the ATP to dry weight ratio after
four days of incubation since the bioriass after four days
was known to consist of dead as well as living cell material
Table 19 lists the ATP to' dry weight ratios reported by
Lee et al (1971). Three cultures were grown with three
different concentrations of phosphorus: 0.62 mg/1, 0.062
mg/1, and 3.1 mg/1.

TABLE 19
ATP TO DRY WEIGHT R.\TIO'S OF SELEiJASTBUi; (Lee et al 19 71)

Phosphorus Content
(mg/1)

0.62

0,062
3.1

Incubation

Time

ATP/D

rv

'A'eight (ug/mg)

4

3.1

8

2.4

16

0.7

32

0.57

4

1.4

8

0.47

4

3.4

The res ilts in Table 19 indicate that the ATP to dry
weight ratio varies with nutritional state. It is question-
able whether the decrease in the ATP to dry weight ratio
results primarily from a build-up of detrital material or
from a pliysiological change due to a decrease in nutrition.

115

Correlation of ATP with cell nur.i.ber was also good.
Holm-Hansen (1970) reported excellent correlation between
ATP/cell and organic carbon/cell. Lee et at (1971) reported
ATP contents ranging from 3.9 x 10"^ to 6.S x 10"^ ug/cell
for Selenastvum capricorn-utujri grown with 0.62 mg/l phosphorus
and 1.9 x 10"2 to 2.1 x 10"^ pg/cell for Selenasti^um
cavvioovnuturn grown with 0.062 mg/l phosphorus.

Cellular regulation of ATP was demonstrated by
subjecting algal cells to alternating periods of light
and dark. The ATP concentration fluctuated but always
returned to its initial level. Santarius and Heber (1965)
showed that ATP increased rapidly when an isolated chloro-
plast v;as exposed to light. Ulien placed in the dark, the
ATP decreased rapidly. They concluded that the ATP to ADP
ratio controlled respiration in plants. Is'hen photosynthesis
increases under high light intensity, it is believed that
photorespiration occurs to stabilize the cell (Lehninger
1970). According to Lehninger, photorespiration does not
occur in the mitochondria like regular respiration; it
seems to be a wasteful way of regulating cells which are
photosynthesizing too fast. Thus, evidence indicates that
ATP is regulated by an intricate balance between photo-
synthesis, photorespiration, and respiration.

The response of ATP to various incubation pH levels
indicates that cellular ATP decreases when an organism is
shifted to a pH level different than its norma] level.

114

Patterson (19 '^0) reported siipilar results for activated
sludge. He found tlie optimum ATP pool size occurred in the
pH range of 7.5 to S.O, the normal operating range of the
activated sludge process.

Addition of toxic chemicals to unialgal cultures
and lake phytoplankton caused a rapid decrease in ATP
content. Patterson (19 70) reported rapid decreases in ATP
of activated sludge incubated with various toxins including
nickel, chromium, chromate, mercury, copper, and cyanide.

Nutrient-deficient cultures of algae exhibited an
increase in ATP content when nutrients were added. Holm-
Hansen (1970) reported similar results for marine algae.
He found that the ATP content increased when nitorgen was
added to nitrogen-deficient cultures of Monoalrrysis lufheri
and Skeletoy^erTia aostatum. He also leported an increase in
ATP content after addition of phosphorus to pliosphorus-
deficient cultures of Vunaliella tertiolesta and y.cncchrysis
liithari. Patterson (1970) reported an increase in ATP pool
size after addition of substrate to a starved activated
sludge. Brezonik and Patterson (1971) reported increased
ATP per cell upon addition of substrate to activated sludge.
The rapid increase in ATP content shovm in Figures 2 7 to 32
indicate an increase in ATP per cell rather than an increase
in biomass. These results agree with those of Brezonik
and Patterson (1971).

CHAPTER VIII. CONCLUSIONS AND RECOMMENDATIONS

Conclus ions

It is difficult to evaluate ATP as a biomass para-
meter since it must be compared to current biomass para-
meters vhich, it is believed, are inadequate. There is no
absolute biomass parameter to which ATP can be compared.
However, working with the available biomass estimates,
som.e conclusions can be made.

Good correlation was observed betv.-een ATP and three
biomass parameters: chloropliyll a, dry weight, and cell
number. Cellular ATP did not vary under light and dark
conditions, and it was relatively constant under normal or
ambient pH conditions. It is proposed, therefore, that
ATP could be used as a biomass parameter in studies o£
aquatic systems. One problem with ATP as a biomass para-
meter is that it is a measure of total biomass, not just
algal biomass. In measuring the ATP content of the unialgal
cultures and the lake phytoplankton , no account was taken of
the bacterial population. However, in many instances the
total biomass is ^is important or even more important than
algal biom.ass alone. The plankton coinmunity consists of
phytoplankton, bacteria (free- floating and attached to the
phytoplankton), and zooplankton. It seems reasonable that

115

116

in pollution or trophic-state studies the biomass of the
total community is im.portant, especially considering the
symbiotic relationship between bacteria and algae. Too
often, the phy toplankton , the bacteria, and the zooplankton
have been studied as separate entities when the total
community should have been studied.

If a distinction between algal and total biomass is
required, then the ATP to chlorophyll ratio can be
determined and a rough estimate of the autotrophic nature
of a water can be made. Taylor (1967) coined the term
"Autotrophic Index" as a m.easure of the self- feeding or
food-producing organisms. The "Autotrophic Index" is
defined as the ratio of biomass to chlorophyll. Although
the chlorophyll concentration varies considerably under
different conditions, it can be used to give a rough
estimate of the autotrophic le^^el of a particular lake or
stream. Each water, however, because of differences in
color, turbidity, and nutritional state, should be monitored
to deterinine the average ambient ATP to chlorophyll ratio.
In many '.vaters, the relative abundance of algae compared
to bacteria make the distinction between total biomass and
algal biomass almost insignificant.

The rapid response of ATP after the addition of toxic
substances indicates tbie feasibility of using ATP in toxicity
studies. The data presented in this thesis dem.onstrates
that cellular ATP responds almost immediately. It is

II'

suggested that ATP is an excellent bioassay parameter for
toxicity studies.

The response of ATP in nutrient - de ficient cultures
after addition of nutrients suggests that ATP could be
used in limiting nutrient bioassays. liois-ever, the qualita-
tive nature of the response must be considered. Tike
alkaline pliosphatase activity, ATP activity is a relative
measure of deficiency. Unlike alkaline phosphatase activity
ivhich is limited to phosphate- deficient cells, ATP activity
can be used for many nutrients. It is suggested that ATP
as a limiting nutrient bioassay parameter is an important
tool which can be used in a qualitative fasliion in nutrient
bioassays .

Recommendations

The first recommendation of this report is that
more theoretical work be performed to determine mere about
t'ne role and response of ATP under different conditions
and in different species. Chemostats should be used in
order to maintain algal cultures in a defined state of
growth.

Secondly, ATP analysis should be used in routine
laboratory and field studies. ATP analysis has a great
potential in research and in solving every day problems ,
but unless it is used in routine studies, it will never
be anything but a potentially good tool.

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BIOGMPHICAL SKETCH

Francis Xavier Browne was born October 11, 1943,
at Brooklyn, Xew York. In June, 1961, he was graduated
froir, Xaverian High School. In June, 1966, he received
the degree Bachelor of Civil Engineering from Manhattan
College. In Septenber, 1966, he enrolled in the Graduate
School of Manhattan College. He worked as a research
assistant in the Department of Civil Engineering until
January, 1967, when he received a Graduate Fellowship.
In January, 1968, he received the degree of Master of
Civil Engineering with a major in Sanitary Engineering.
From 1967 to 1968 he was employed as a consulting sanitary
engineer at Hydrotechnic Corporation, New York. From
September, 1968, until the present time he has been
enrolled in the Graduate School of the University of
Florida vhile he has pursued his work toward the degree
of Doctor of Philosophy.

Francis Xavier Browne is married to the former
June Marie Kendall. He is a member of the Water Pollution
Control Federation, and the American Society of Limnology
and Oceanography.

130

I certify that I have read this study and that in my
opinion it conforiiis to acceptable st&ndards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy,

I ajtu^ck J^-

Patrick LT brezon'ik/, Cna i r laan
Associate Professor of

Environmental Engineering

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.

Co- Chairman
Assistant Professor of

Environmiental Engineering

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy,

Professor of Environmental
Engineering

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.

Frank G. Nmrcllle'

Associate Professor of Zoology

This dissertation was subrp.itted to the riean of the Colle;
of Enf;iiieering and to the Graduate Council, and vas
accepted as partial ful filliaent of the requirements for
the decree of Doctor of Philosophy.

Dece-iLDer, 19 71

'Man

Dean, Gr'aSUaTe School

UNIVERSITY OF FLORIDA

3 1262 08666 936 2