DETERMINATION OF ALGAE GROWTH POTENTIAL

IN NATURAL ENVIRONMENT

by
SYED AZHAR MAQSOOD, B.S. in Engin.

A THESIS
IN
CIVIL ENGINEERING

Submitted to the Graduate Faculty

of Texas Tech University in
Partial Fulfillment of
the Requirements for
the Degree of

MASTER OF SCIENCE
IN
CIVIL ENGINEERING

Approved

Accepted

May, 1974

^^M TECH LIBRAKY

V \^ >y

73 l^--1^,71-

' ^}' ACKNOWLEDGMENTS

The author wishes to express his deep and sincere appreciation

to Dr. Robert M. Sweazy for his guidance, patience, and suggestions
throughout the course of this study.
The author also wishes to thank Dr. Russell C. Baskett and
Dr. Dan M. Wells for their interest in the problem, encouragement,
and their suggestions concerning laboratory techniques, and
completion and presentation of the data.
Marcia Headstream and Edgar D. Smith performed a great
percentage of laboratory analyses presented in this thesis. Their
assistance is greatly appreciated.
Finally the financial assistance of the Water Resources Center
at Texas Tech University is sincerely appreciated.

n
 TABLE OF CONTENTS

ACKNOWLEDGMENTS ii
LIST OF TABLES iv
LIST OF FIGURES v
I. INTRODUCTION 1
II. LITERATURE REVIEW 4
Batch Cultures 5
Continuous Cultures 6
In Situ Techniques 9
Dialysis Culture Technique 10
METHODS FOR MEASURING GROWTH 12
FACTORS AFFECTING ALGAL GROWTH 12
III. EXPERIMENTAL PROCEDURE 18
IV. RESULTS AND DISCUSSIONS 26
V. CONCLUSIONS AND RECOMMENDATIONS 44
LIST OF REFERENCES 46

m
 LIST OF TABLES

Table
1. Well Water Quality 27
2. Laboratory Study Results 34
3. Field Growth Rate Constants 43

TV
 LIST OF FIGURES

Figure

1. Buoyant algae culture apparatus 19

2. Common wall tanks 21

3. Standard curve for percent transmittance,

dry weight, and cell numbers 24

4. Laboratory algae growth curve 31

5. Laboratory algae growth curve 32

6. Laboratory algae growth curve 33

7. Field algae growth curve 36

8. Field algae growth curve 37

9- Field algae growth curve 39

10. Field algae growth curve 40

11. Field algae growth curve 41

12. Field algae growth curve 42

 CHAPTER I

INTRODUCTION

The survival and behavior of microorganisms in their natural

habitat should be examined when studying a natural aquatic system.

Valuable information regarding the ecology of a species can be gained

in laboratory cultures, but field studies are essential to achieving

a full understanding of a natural system because the behavior of a

species is very often different in nature than under controlled

laboratory conditions. Laboratory testing of a single species cannot

account for important biological variables such as interspecies

competition or nutrient and energy flow through the ecosystem.

Therefore, one should be cautious in applying laboratory results to

the interpretation of events occurring in the natural system.

Most recent studies concerned with microbial growth have been

performed in vitro with pure or axenic cultures under controlled

conditions, thus ignoring natural conditions with which the species

is associated and interacting. A recent review by Brock describes

a variety of methods for measuring microbial population growth rates

in natural habitats (1). However, these techniques are applicable

only to bacterial systems and, to date, there exists no suitable

technique which can be applied to algal systems.

Published works concerning the growth kinetics of algae have

been based on cultures grown in either batch or continuous laboratory

1
systems. Many investigators have used such algal cultures to measure

the productivity of different water samples and have tried to corre-

late this productivity with different physical, chemical and

biological factors.

Algae can be used as indicator organisms in evaluating water

quality. Since their nutrition is derived from dissolved chemicals

and they are sensitive to physical environmental factors, their

presence, absence or abundance can be indicative of the water's

quality.

Algal assays can be used in evaluating the algal growth

potential (AGP) of waters. Algal growth potential can be determined

by correlating different factors such as nutrient supply, temperature,

and light with algae growth. Knowing the algal growth potential of

a given body of water, insight into the degree of its eutrophication

can be gained.

In all instances, however, because the growth responses of a

given species of algae are apt to be very different in natural

conditions from those exhibited in controlled conditions, a method

of measuring algae growth which can be utilized in a natural aquatic

system will prove to be an invaluable research tool.

It is the objective of this research to modify, in order to

measure algae growth in situ, a technique developed by Baskett and

Lulves (2) for measuring in situ growth rates of aquatic bacteria.

This study will serve several important functions. Since this

technique can also be used as a bioassay system, the potential of a

body of water to support algal growth under prevailing conditions

can be determined. It will also provide information about different

parameters which control algal growth and multiplication in specific

aquatic environments.

Prediction of the algal growth potential of a given water will

determine, to a great extent, its usefulness with regard to serving

as a municipal, industrial or recreational water supply. Such

results will be of immediate concern to the City of Lubbock, which is

planning to use reclaimed percolated sewage effluent as a water

source for the Canyon Lakes Project, and to other cities contemplating

the reuse of reclaimed wastewaters. This study will also help in

determining the degree of eutrophication potential of a water supply

source.
 CHAPTER II

LITERATURE REVIEW

Because of the adverse effects of eutrophication on water

quality, the public's concern and awareness regarding it are

increasing. Many authorities consider eutrophication to be today's

major water quality problem. Lakes such as Lake Zoar, Lake Washington,

and Lake Erie offer good examples of the problems eutrophication can

cause (3).

Eutrophication is defined as the process of enrichment of a body

of water with nutrients. It is a slow process accelerated by man's
activities (4). Many field studies and research projects have been
carried out in past years in efforts to understand more clearly
enrichment and biological production in lakes, streams, and estuaries.
Despite these efforts, no standard method has been developed to
measure the enrichment level or fertility potential of various bodies
of water (5).

A striking phenomenon associated with eutrophication is algal

blooms. Therefore, the presence and abundance of algae and the
occurrence of algal blooms may be considered excellent indicators of
eutrophication potential. In recent years attention has been given to
algal assays as a method of determining the potential fertility of
water. Johnson (6) and Skulberg (7) concluded that bioassays using
algal cultures can be utilized successfully in measuring the

enrichment level of lakes.

Biological assays are being used mostly in studies of toxicity,

the effects of pollution on biological activity, and the eutrophi-

cation potential of a body of water. Bioassays, if they are carried

out with care, will yield valuable results relating to eutrophication

According to Skulberg (5), the bioassay methods are supplementary to

physical and chemical analyses. They are of value in determining the

quality of bodies of water and in assessing the effects of pollution

on eutrophication.

The need for a standard algal assay procedure is increasing

with the growing problem of eutrophication. Oswald and Golueke (4)

presented a simple inorganic bioassay procedure to evaluate algae

growth potential and the joint government-industry task force (5) has

proposed the use of bioassay procedures for assessing algal growth

potential of a body of water. They are known as Provisional Algal

Assay Procedures or PAAP. These include batch or bottle tests,

continuous culture techniques, and in situ tests. All of these

techniques are tentative and a great deal of research is still needed

to develop a standard procedure. All of these techniques attempt to

accomplish one of two things: (1) to duplicate natural conditions as

nearly as possible or, (2) to provide a set of artificial conditions

which are suitable for growth and which can produce desired results.

Batch Cultures

Batch culture algal assays can be useful in studying the

productivity potential of aquatic habitats. Algal batch cultures

were employed by Lackey, Rozich, Palmer, Eyster, Oswald and many

others (5) as bioassays. Such cultures were used to appraise

algacides and river fertility, to examine trace nutrient requirements

of algae, and to appraise various wastes for their potential as

nutrients for the cultivation of algae. Lake fertility, and taste

and odor relationships were also demonstrated.

Static or batch type procedures suggested in Provisional Algal

Assays Procedures are similar in principal to earlier batch culture

tests performed by Oswald (6) and Skulberg (7), but are more complex

in the degree of procedural detail (5). This type of technique

involves a limited volume of medium containing the necessary organic

and inorganic nutrients. The medium is inoculated with a small

number of cells and then exposed to suitable light, temperature and

aeration conditions. The resulting growth will follow the standard

growth curve with lag, exponential, stationary, and death phases.

Continuous Cultures

Continuous culture techniques were applied by many investigators

to study various problems related to microbiology. Their full

development for the study of microorganisms was followed by the

mathematical theory provided by Monod, Novick and Szilerd (8). The

theory was further developed by Herbert, Herbert, et_ ^ , Gader, Moser,

Fencel, et^ aj^ and many others (8). A complete publication on

continuous flow cultures was presented by Malck and Fencel (8).

Continuous cultures are considered to be the best models for

studying an ecological system (12).

 Phillips, Myers, Pipes, Maddox and Jones (5), and many others

have used continuous flow systems in analyzing the effects of

different parameters on algal growth. Phillips and Myers (5, 23)

found that the growth of algae is a function of light intensity and

intermittency of illumination. Maddox and Jones (5, 24) studied

temperature effects on growth and its interrelationship with light

intensity and nutrient supply. They concluded that minimum growth

rates in a daily light and dark cycle were lower when a medium with

nitrate and phosphate concentrations similar to those found in

natural waters was used than when a medium having higher concen-

trations of these substances was used. Pipes (5, 25) studied the

effect of COp on the growth of Chlorella pyrenoidosa at various

residence times. He concluded that (1) at a constant rate of CO2

addition, the equilibrium population density is directly proportional

to retention time and (2) the production rate is directly proportional

to the rate of COp supply. Bacterial studies have also been carried

out by many researchers.

Continuous flow systems can be classified according to the type

of operation. Two common systems, the chemostat and the turbidostat,

are utilized for studying microbial growth. Constant flow rate and

turbidity are maintained in chemostats and turbidostats, respectively.

The results obtained from these two systems are theoretically the

same but the chemostat is more economical and less emperical (7).

The term chemostat was established concurrently by Novick and

Szilerd (8, 9 ) , while Monod (8) independently developed its mathe-

matical theory. Fujimoto and his collaborators (9) have grown

 8

Chlorella ellipsoidea by this method and have determined the relation

of the limiting value of flow rate to light intensity. He concluded

that population density is directly proportional to retention time in

that production rate is independent of retention time in cultures in

which light is a limiting factor.

In the chemostat system, the flow rate is maintained to produce

a desired residence time in the reactor and the organisms themselves

establish their own concentration according to the capabilities

reflecting the given conditions (8). Growth is dependent on both

energy and non-energy yielding substances. Any one of them may be a

limiting factor. Therefore, identification of the limiting factor is

very important. Myers and Clark, Malek and Fencel, and Shelif, et al

(5) suggested many reactor designs considering nutrient as a limiting

factor. A yery simple design has been suggested in PAAP for the

growing of algae in the development of algal assay procedures, with

an attempt to satisfy all the requirements, i.e., it is as simple

as possible, yet is consistent with the basic concept that other

factors do not limit growth or interfere with operation of the

chemostat (5).

The turbidostat which was first introduced by Myers and Clark

(9), has been used frequently in algal cultures. Its theoretical

foundations were derived by Anderson (8). In the turbidostat system,

a constant concentration of cells is maintained by adjusting the flow

rate with the aid of a control device. It operates most effectively

in the range close to the critical dilution rate (8).

 9

Myers and Phillips (9) used a turbidostat to study the relation

of photosynthesis and culture characteristics of Chlorella pyrenoidosa

to light intensity. It has been proven successful for the culture of

chlorella, euglena, anabaena and anacystis in studying cellular char-

acteristics as a function of some single environmental factor which

is purposely varied. Using this method, Myers (9) found that with

respect to relative growth rate, Chlorella pyrenoidosa was insensitive

to changes in major salt concentrations involving variation in

nitrate-nitrogen concentration from 340 down to 17 mg/1. Jones (9)

has found a pronounced interaction between nutrient concentration and

light intensity in their effects on the growth rate of Carteria. At

low light intensities the concentration of nutrients exhibited no

effect on growth, but at medium light intensities higher concentrations

yielded higher growth rates than low concentrations.

In Situ Techniques

In situ techniques utilize isolated samples of natural bio-

logical communities. The isolated samples were resuspended or fixed

in some way in their natural environment in an attempt to simulate

natural environmental conditions. Translucent plastic bags were

utilized for in situ studies by C. F. Powers, e t _ ^ (10) for the

analyses of algal responses to nutrient additions in natural waters.

These bags were open at the top and were suspended in Shagawa Lake

from wood framed polystyrene floats. In situ experiments for

measuring algal growth assessment by fluorescence techniques were

carried out by Bain (7) using floating amberglass bottles or trans-

lucent containers to prevent excessive solar illumination (5). Glass

 10

bottles, plastic bags, cylinders and vertical glass cylinders have

been used by many researchers (11). Thomas (11) used a vertical

plexiglass tube of 5 cm inner diameter and 6-8 m length in studying

the diffusion kinetics in the epilimion and population dynamics of

phytoplankton with and without additional mineral nutrients.

Stepanek and Zelinker (11) studied the development of phytoplankton

population using larger containers made from transparent plastic

film.

Dialysis Culture Technique

The first use of dialysis techniques for culturing micro-

organisms was in the late 19th century. In 1896, Metchnikoff, et ^

(12) used this technique for determining the existence of diffusable

cholera toxin. Very little attention was given to this technique for

culturing autotrophic organisms. It was first applied by Trainor (13)

to grow the fresh water algae, Scenedesmus. Recently, it has been

applied by Jensen, et^ £ 1 (13) to grow a number of phytoplanktonic

species.

The kinetics of algae growth in dialysis culture have not been

adequately studied, but it seems that for the most part, they

approximate the kinetics of continuous culture techniques. Schultz

and Gerhardt (12), who have dealt with theoretical and quantitative

aspects of bacterial growth in dialysis culture, have proposed

mathematical models to relate dialysis kinetics to growth kinetics.

Such models may be equally applicable to algal growth, provided

relationships between the rate of nutrient utilization and the rate

 n
of cell production can be determined with sufficient precision for
different algae species.

The principle involved in this technique is that microbial

populations are kept on one side of the diffusion barrier, while on

the other side is kept the enriched medium which contains the

nutrients for metabolism and growth. These nutrient diffuse through

the barrier into the culture compartment, and diffuseable metabolic

products diffuse away from the culture. Exchange dialysis is thus

occurring (12). The production of cells in a dialysis culture is

maintained as long as the rate of exchange of chemicals across the

membrane is constant and the culture growth is not affected by

density dependent factors. If these conditions are not satisfied,

the growth pattern of the culture approaches that of a batch

culture (13).

A dialysis culture can be operated as a batch culture or as a

continuous culture. Continuous operations are directly amenable to

mathematical analyses (12). Parkash, et_ £ 1 (13) have used batch and

continuous dialysis cultures in studying the growth of planktonic

algae.

Three main types of membranes applicable to dialysis cultures

are presently available commercially. The first type is made of

regenerated cellulose by the visking process. This type retains

large molecules such as enzymes and toxins but permits the passage of

small molecules, such as sugars and salts. A second type of membrane,

microporus, is usually made from cellulose acetate. It can retain

particles such as bacteria but permits the passage of most solutes

 12

including macro molecules. The third type, made from materials such

as silicon rubber or teflon, has a more restricted applicability in

dialysis culture because only gases can penetrate it (12).

METHODS FOR MEASURING GROWTH

The Proceedings of the Eutrophication Biosimulation Assessment

Workshop (5) stated that algae crops are measured by a variety of

techniques including cell counts, absorbance, gravimetric, carbon-14,

fluorescence, and volumetric. Each technique has certain advantages

and disadvantages over the others. Counts have the major advantage

of being determinant at concentrations far below those which are

measurable gravimetrically. Fluorescence is an excellent technique

for measuring algal crops which does not involve destruction of the

samples. However, results are not relative and therefore do not meet

the gravimetric requirement of PAAP. More clumping of algae can

interfere with this technique. Absorbance measurements are also

virtually useless when clumping of algae has occurred. The radio

carbon technique as set forth in PAAP seems needlessly complex,

delicate, and subject to error in the hands of inexperienced

personnel. A rational system might utilize counting for AGP deter-

mination of 0.1 to 10 mg/1, fluorescence for AGP's of 10 to 100 mg/1

and gravimetry for AGP's of 100 to 1,000 mg/1 (5).

FACTORS AFFECTING ALGAL GROWTH

The rate of growth of algae is dependent on four main factors;

(1) Quantity of available light, (2) Temperature, (3) Concentration

of nutrients, and (4) Availability of CO^ (14). There are some other
 13

factors which also affect the algal growth potential of a body of

water, e.g., pH of medium, autoinhibition, retention time, and

concentration and type of organisms present. Each species has its

own behavior patterns under given conditions, and these patterns may

not resemble the behavior patterns of other species. The growth

pattern of a given species may be entirely different in the natural

environment than in the laboratory.

Temperature and light requirements differ with different

species and its is possible that a particular species may predominate

because of favorable existing weather conditions at that time. It

was found that under a light-saturation condition, the growth rate of

Chlorella pyrenoidosa is higher at 25° C than at either 20° C or

30° C (9). Miller (9) showed that in sunlight, maximum growth occurs

at a high temperature, while cells held at low temperatures exhibit

little or no growth. The optimum temperature for maximum growth of a

particular species varies with other factors. Hammer (14) noticed

the maximum development of species during a certain range of

temperatures. Spring blooms of Anabaena flos aquae appeared when

the water temperature was 14° C and higher. Aphanizomenon flos

aquae was most predominant when the temperature ranged from 22.5 to

26.5° C. Microcystis showed the widest temperature tolerance range,

i.e., from 0° C under the ice to 26° C in the summer (14).

The amount of light reaching the water surface is by no means

the same as that available to algae at different depths. Clark and

Oster (15) observed that the depth at which photosynthesis balances

respiration for certain planktonic algae was 7 to 20 meters in turbid

 14

waters and about 30 meters in clear water. Birge and Juday (15) have

estimated that the photosynthesis zone in clear-water lakes is

confined to the upper ten meters, and that in more turbid or colored

water, it may extend less than two meters below the surface. Ryther

(16) observed that photosynthesis was light saturated at intensities

of 5000 to 7500 lux for green algae. Inhibition of photosynthesis

was noted at intensities exceeding the saturation values by 10,000

lux or more. Nielsen (9) working with Chlorella vulgaris in batch

cultures, found that the cells grown in low light were more efficient

at low intensities but that they became saturated at a lower level

than did the cells grown in high-intensity light. Work done by

Myers (17) and his colleagues on the growth of Chlorella confirmed

the effects of light and temperature variation on algae growth.

Maximum yields were obtained with sunlight and 25° C during the day,

while the temperature at night was kept at 15° C.

The adverse effect of shading was also observed on the growth

of algae (18). Light absorption by a Chlorella culture approximately

follows Beer's law (9). On the basis of this assumption, Tamya,

et_ al (17) derived the following equations for growth ratio of algae

with continuous illumination:

dlnV ^ a KIV
dt K + al (1)

and dv ^ K_ In 1 + li
dt eD K (2)
I J II
where K & a = Constants dependent on light intensity "i
 15
X = Distance from surface

I = Incident Light

e = Extinction co-efficient for algae suspension

V = Population density

D = Depth of sample

Equation (1) is the expression of exponential growth rate to be

observed at lower population densities, and Equation (2) represents

the linear growth rate to be observed at higher population densities.

Another important factor which affects algal growth is nutrient

supply. It has been suggested by many investigators that by con-

trolling the nutrient input, the algal growth potential of a body of

water may possibly be neglected (5). Inorganic compounds of nitrogen

and phosphorus are the nutrients that are considered most important

in eutrophication studies, but trace elements can also play major

roles in algal growth. These elements include iron, manganese,

copper, zinc, molybdenum, vanadium, boron, chloride, cobalt and

silicon. Substances such as calcium, magnesium and potassium are

also required but they exist in sufficient amounts in most natural

waters (5).

Exclusive of carbon and oxygen, phosphorus and nitrogen are

considered to be the most important nutrients in a natural water.

Municipal, industrial and agricultural wastewaters are major sources

of these nutrients in natural waters. Nitrogen occurs in the form

of ammonia, nitrite, nitrate, and organic compounds, and is, there-

fore, \/ery difficult to remove by a single treatment method. In

addition, a number of blue green algal species can fix Np from the
 16
atmosphere. Because of this, phosphorus has received the most
consideration as a controllable nutrient in algal growth.

Some investigators think the N/P ratio is responsible for

controlling algal growth, but Chu (18) concluded it was not the N/P
ratio but only the concentrations of nitrogen and phosphorus which
control growth. He pointed out that a deficiency in either may
limit growth.

A nutrient which is of primary importance in the production of

any cellular material is carbon. The role of carbon in eutrophication

has been mentioned numerous times over the years (1911-1970).

Wright and Mills (17) found carbon to be somewhat limiting in their

productivity studies on the Madison River. Kerr, Lange and

Kuentzel (17), claim that bacterial oxidation of organics is

necessary to provide the carbon necessary for algal blooms. Ohle

and Conger (17) stressed the importance of bubbles produced by anae-

robes both as sources of carbon and as vehicles for transport for

nutrients.

In general, there are four sources of carbon in aquatic eco-

systems; the atmosphere, carbonates, allochthonous inorganic carbon,

and carbon resulting from biological cycling of autochthonous and

allochthonous materials (18). Availability and rate of supply of

specific forms of carbon can regulate the extent and rate of bio-

logical activity. Dissolved carbon in the form of simple organic

compounds can be used by many kinds of algae. Carbon dioxide and

bicarbonate ions usually serve as a source of carbon for the algae-

However, the lack of sufficient CO^ and bicarbonate ion could limit
 17

algae growth (17). Hes (18) reported that CO^ is essential for the

normal functioning of the oxidation-reduction catalysts in hetero-

trophic cells, but high CO2 levels serve as growth inhibitors of

some algae through toxic effects on photosynthesis.

Other nutrients previously mentioned can also limit algal

growth. Addition of any one of these nutrients, as well as certain

vitamins which may be limiting, could cause explosive algae growth.

Factors such as the pH of the medium, autoinhibitors, retention time,

and turbulence may also become limiting under certain conditions.

A factor which is limiting in one condition may not become

limiting in other conditions. It can be concluded then, that it is

wery difficult to determine which factors should be considered to be

limiting when attempting to eliminate algae growth problems. Each

aquatic system must be analyzed to determine the limiting factor

considering the given conditions. Methods should then be employed to

regulate that factor in a range which will eliminate excessive algae

growth.
 CHAPTER III

EXPERIMENTAL PROCEDURE

A special buoyant, suspending apparatus was designed to which

a dialysis bag containing algae and their growth medium was attached
as shown in Figure 1. The apparatus consisted of a light steel
structure attached to two 2" x 4" x 1' styrofoam floats. The ends
of a four foot length of three inch diameter dialysis bagging were
sealed with enclosed plexiglass cylinders, approximately three
inches in diameter. Waterproof tape was then wrapped around the
joints to make the system leakproof. Plastic tubes penetrating
through the cylinder into the bagging were used at one end of the
apparatus for periodic removal of samples and for inoculating the
contained media with algae. Three such units were constructed.

The dialysis bagging used in this experiment was made of

regenerated cellulose by the Visking process. The average rated

pore size of this membranous material was on the order of 5 nm

which allows molecules with molecular weights smaller than approxi-

mately 12,000 to diffuse freely in and out of the bagging. However,

large molecules and cells are denied entry or readily retained

within the bagging. In this way, necessary nutrients diffuse into

the bagging and waste products are removed.

The dialysis bags were filled with water from a well located

near the sewage effluent holding ponds on the Texas Tech campus.

18
 20

The well water was filtered to remove suspended solids, bacteria,

sand or any other filterable material. The dialysis bags were then

inoculated with five liters of well water containing approximately

1.0 X 10 cells of Chlorella vulgaris. The Chlorella inoculum was

cultured in modified basic ASM media as suggested by PAAP (20) and

was in the exponential growth phase when transferred. The entire

apparatus was then suspended and anchored in common wall concrete

ponds filled with percolated sewage effluent from the previously

mentioned well.

As shown in Figure 2, a series of nine ponds, each

16' X 8' x 6', were constructed on the Texas Tech farm under an OWRR

contract to study the recreational reuse potential of percolated

sewage effluent. A six inch layer of soil was placed on the concrete

bottom of each pond. The ponds, operated on a continuous flow basis,

were equipped with valves which enabled the influent distributed

through a header pipe to the three western most ponds to flow through

the system via numerous routes. Ponds numbers 1, 4, and 7 were

employed for this study. The system, intended to simulate the

Canyon Lakes Project, provided an excellent quasi-natural environment

in which to study algal growth.

Analyses were performed to determine the concentrations of

nitrogen, phosphorus, carbon dioxide, dissolved oxygen, and

turbidity in the pond water. Temperature and pH measurements were

also taken. These analyses were performed on days when 20 ml

aliquots were removed from the dialysis bags for the purpose of
 21

T
00

t i i
+->
c
8 Z3
00

+ + i
i
PLAN

T-
u3

1 16 -vi-^ 16' -W<- 16' • > !

ELEVATION

Fig. 2 Common w a l l tanks

 22

algae enumeration. This procedure should enable one to correlate

physical and chemical parameters with corresponding algae growth.

All laboratory analyses of the pond water were performed

according to procedures given in Standard Methods for the Examination

of Water and Wastewater (21). Nitrate determinations were performed

by the ultraviolet spectrophotometric method because this method is

not subject to interference from iron and chlorides, substances apt

to be present in relatively high concentrations in the well water.

The stannous chloride method was used for phosphate determinations.

The azide modification of the Winkler procedure was used for dis-

solved oxygen analyses. COp was monitored at the site by application

of the titrimetric method using 0.0279 normal NaOH. The temperature

was taken at the site and pH was determined electrometrically in the

laboratory.

Percent transmittance of the cell suspension in the bags was

employed as a measure of algal growth. Chlorella vulgaris which was

used in this study is a unicellular, non-filamentous green algae

which forms a homogeneous suspension, thus permitting a turbidimetric

determination of growth. The transmittance or absorbance of an

algal suspension is a function of both light scattering and

absorption due to cell pigments. In order to minimize the effect of

cell pigments, a set of turbidity measurements using a Bausch and

Lomb Spectronic 20, were taken to measure the wavelength at which

minimum absorption by chlorophyll occurs. Wavelengths ranging from

500 nm to 600 nm were used, and it was determined that at 580 nm the

pigments exhibited their minimum absorption effect.

 23

To provide a means of converting percent transmittance to

number of cells per ml, or dry weight of cells per ml, a calibration

curve relating these measurements was developed. The relationship

between the dry weight of algae cells and corresponding transmittance

was determined by diluting 1, 4, 6, 8, and 10 ml aliquots of a

concentrated algal suspension to 10 ml volumes. The percent trans-

mittance of the resulting suspensions was measured at 580 nm. The

cellular dry weight was then determined by vacuum filtration of each

algal suspension through a 0.45u pore diameter membrane filter. Two

superimposed filters, matched in weight to within 0.1 mg, were

assembled in a pyrex filter holder. Ten ml sample aliquots were

passed through both filters while carefully avoiding contact of the

cell suspension with the walls of the filter holder. In this way

both filters were subjected to the same fluid flow, but all algae

cells were retained on the upper filter. After drying both filters,

the weight of the lower filter was subtracted from that of the test

filter to determine the weight of dry cells. Correlation between

percent transmittance and corresponding cell counts was made with

the aid of a Whipple eye piece and a Sedgewich Rafter counting cell.

Percent transmittance versus cell counts/ml and dry weight/ml was

plotted and the linear relationship shown in Figure 3 was obtained.

Growth responses in terms of the log number of cells were

plotted against the number of days. Line of best fit was developed

using linear regression analysis.

The expression is of the form

Y = A^ + A^X,
 24

O
-o
c
n3

C7>

XJ

to +->
c
o
to
C
03
•1- vo S-
+->

to

o
C_3 s-
(U
Q.
s-
O
o

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O
-a '
s- to
ro i-

<o E
4-> =3

CO

o o o o
LO
o
K£>
CD o
00
o
CTi
o
CO CO "sj- r^ o

a3ue:;:^_LUisupji :^uaDj9d
 25

where

Y is the estimate of the true value of the dependent

variable, i.e., log number of cells/ml. A Q represents a constant,

A, is the coefficient of correlation and X-. represents the independent

variable, i.e., time in days. Growth rates (K values) were calcu-

lated by using the following equation.

0.301 X t
where
K = Number of generations/day

N-j = Initial cell concentration

Np = Cell concentration after time t

t = Number of days
 CHAPTER IV

RESULTS AND DISCUSSIONS

Prior to initiation of the experimentation, pertinent physical

and chemical parameters depicting the quality of the water in the

experimental ponds were measured. The results of the initial and

subsequent water quality determinations are presented in Table 1.

As a result of these analyses and literature consultation, it was

concluded that the nutrients C, N, and P were present in concen-

trations adequate to support significant algae growth. In the pond

water, average concentrations of COp, NOZ* P07 were 20 mg/1, 6.4

mg/1, and .05 mg/1 respectively. Concentrations did not vary

significantly from pond to pond with regard to COp and nitrogen, but

phosphate concentrations occasionally approached concentrations

considered by some to be limiting. According to Mackenthun (18) the

inorganic phosphorus concentrations should be limited to 100 ug/1 for

flowing water and should be less than 50 ug/1 for standing waters to

prevent algae growth from becoming excessive. Malony (18) concluded

that a water should have less than 0.1 ug/1 phosphorus with

essentially no iron present in order for nutrient scarcity to

prevent algae growth.

Carbon dioxide is considered by many to be a limiting factor

with regard to primary production in bodies of water. The high

26
 27

s-
13
o ooco<sO<^coLnroLOLncococ\Jcy>CT>
O
O)
Q.
E
Qi

I LD CM CO C\J
J3
S-

•=*• •=d- o CO
o cni CM C\J CM CM
o !

<:
:z> IT) O O CM LO LO CM
cy CO LO Lf) IT) Lf)
in un
Of a.
CQ UJ

-a
o <D
Q. >
1 —
c:
o <u
to o>
00 o o «*
en • • • •
(/) > >
"p- X UO (^ 00 vo
Q O

«3 LD
O
I sz
Q-
o o o LO V O CM
«?i- 0 0 L n 'd- "5i- «d- CM
LO
CM
Jir to C7) o o o o o o o O
+J o
O Q.

'5i-oocr»"!*-"5d-r>*u3<x>
I CO CT
O S

CO CO CO CO
CO r^1 r*^ t^ t^ ^ ^ ">f «^ •sl-
r>. 1 1 1 «cj- •sJ- r^ r^ r^ •* r^ r*. «* ^
to 1 «st- cr> af^ vo r>» r^ 1 1 1 r>» 1 1 r^ r*^
<u +-> t^ t—
^— CM ^— 1 1 ^D CO o 1 CM • ^ 1 1
+J •r- 1 1 1 1 1 CM 00 ^— CM CO <£) 1 — CM CM 00
fl3 c ^— r— 1 — n— CM 1 1 1 1 1 1 1 1 1 1
Q rD CM CM CM CO CO
 28

Qi

+J
S- o 00 "?j- LO CT^ LO CM CM CM I—
O) o
Q.
E

5-
I— CM

CM - <^ O ^O
o
o al CM I— CM

<
ID O LO CM
o CM CM CO LO o o
o o
LO LO to 00 cn
LO
o Q.

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o
O Q.
OQ T3
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>
r— d
O OJ to <^ 00 to
to CD en
to >, • • • •
•I- X to r»* 00 to
a o

03
I LO o o O LO LO LO
o a. LO CM CO O «*
sz to cn 1— LO LO 1— r— O O O
+-> o o o I—
s- ^
O Q_

CM«!d-tO«vf«^COLOO
I CO C71
O ! votototo^^'^^ovo

CO CO CO CO
CO p-^
1
1^
1
1^
1
r»1. ^ •=d- «;!- ^ «* ^ «:d- "* ^ ^
r>1. «>i-
r*^ r^ r«» r^ r*1*
CU
to
^—
CT>
r—
cy» to r->.
CM I — 1
r*1^ 1 CO1 1 r^ 1
1
CM ^
r^
1
r^
1
-M
+->
•r—
r^1 1 1 1 1 CM 00 1 —
to CM CO
o r— CM CM 00
03 C ^— 1— ^— r— CM 1 1 1 1 1
to
1 1 1 1 1
O =) CM CM CM CO CO
 29

CU
s-
4->
03 o LO CO CO CM O •— O I— r— CO CO
S. O
(U
Q.
E
(U

5-
CM

CM " CM o <x>
O Oil CM f— CM

1—1
<c
ZD
r^
h- 00 00 CM O o CO cr» CM
"Z. cy • 00 to CO 00 to
O o
C_J 2:
1 a:
1 UJ
1— Q
r— <: 2r
0
LU 0.
_l o
CQ •z. •o
o CU
>
1— E
O OJ O ">1- CM ^
to CD CD • • • •
to >5
•<- X E 00 00 O VO
Q O

(U
4->
03
I O O CM O O O CO
O Q. "sd- I— p— 1— O CM CM
JC to CD 0 0 0 0 0 0 0
4-> O
s- x : E
O D-

O O L O O O O i — 00
I CO cn
O E t O t O t O t O t O t O t O L O
2:

CO CO CO CO
CO r^ c^ r>1. r«1^ «=J- ^ •v^ «:a- ^ «d- ^
1^ 1 1 r*1- r>1. rv1. "sd- r«1* r*«. ^ «d-
(/) 1 "!d- P^ 1
(L) -M n— ^- cr> r—
cn CM to r-1^ r^ 1 CO 0 CM «* r«1» r**.
1
4-> •I— r-1^ 1 1 1 1 CM 00 to 1— CM CO
1
r— CM CM 00
«U C i~~ p— I— t— CM 1 1 1 1 1
vo
1 1 1 1 1
0 ZJ CM CM CM CO CO
 30
concentration of CO2 in each of the ponds, however, precludes any
consideration of its limiting growth.

The pH of the pond water was in the range of 7.3 to 7.55, 7-4

to 7.9, and 7.5 to 7.9 in ponds 1, 4, and 7 respectively. These pH

values are in the optimum reported range for green algal growth.

The first experiment was initiated on November 21. One

culturing apparatus inoculated with algae, was placed in each of the

designated ponds and the percent transmittances of the contents of

each dialysis bag were measured. It was observed during the

following week that no growth had occurred.

In an attempt to determine which environmental factors were

limiting algae growth, a parallel laboratory study utilizing the

pond water as the growth medium was performed. Algae were cultured

at different temperatures and under continuous and intermittent

light conditions. The resulting growth curves are shown in Figures

4, 5, and 6. These tests revealed increased growth at higher

temperatures. The effect of light was observed to be less signifi-

cant (Table 2 ) . Therefore, it appears that the low temperatures

experienced during the first experiment were primarily responsible

for the lack of growth- Hammer (14) has shown temperature to be a

controlling factor in influencing microbial growth.

During the next experimental period (November 28 - January 10)

the weather conditions improved. The water temperature ranged from

16° C in the first pond to 12° C in the last pond. The dialysis

bags were reinocculated with Chlorella vulgaris of essentially the

same nutrient state and age as before. The apparatus were again
 31

7.Or

K = 3.82
Temperature = 26° C
Continuous Light

to

CU
C_>
^-
o
s-
CU

cn
o

3 4
Number of Days

Fig. 4 Laboratory algae growth curve.

 32

7.OF

6.8

K = 3.60 G/day
Temperature = 26° C
6.6
Intermittent Light

6.4

6.2

to
r—
(U 6 .0
C_J

o
s-
(U
J3 5 .8
E
3
z:
C7>
O

6 7 8 9 10 12 13 14 15
Number of Days
Fig. 5 Laboratory algae growth curve.
 33

K = 2.0 G/day
Temperature = 17° C
6.5 _ Continuous Light

r- 6.0
to
CU
o
O

CU

O 5.5

5.0 -

4.7
6 7 8 9 10 15
Number of Days

Fig. 6 Laboratory algae growth curve.

 34

TABLE 2
LABORATORY STUDY RESULTS

Percent Transmittance

Date Temperatu re 17° C Temperatuire 26° C

Continuous Intermittent Continuous Intermittent
Light Light Light Light

12-14-73 99-5 99.5 99.5 99.5

12-15-73 99.0 99.25 85.0 93.25
12-16-73 97.25 98.75 81.0 88.75
12-17-73 96.25 99.25 78.5 82.5
12-18-73 93.75 98.75 74.75 77.25
12-19-73 92.25 98.25 72.0 73.0
12-20-73 90.75 98.75 71.75 72.00
12-21-73 89.0 98.75 70.00 69.25
12-22-73 85.0 98.50 69.00 66.00
12-23-73 83.5 97.50 72.5 66.5
12-24-73 82.5 97.25 77.25 67.0
12-25-73 80.5 96.0 81.5 66.5
12-26-73 78.5 95.75 88.0 65.5
12-27-73 78.5 95.75 91.0 69.0
12-28-73 77.25 94.75 89.5 70.25
12-29-73 77.25 93.50 93.75 72.25
12-30-73 76.50 91.50 95.25 72.75
12-31-73 76.00 90.75 94.50 76.25
1-1-74 78.00 93.00 97.0 80.5
1-2-74 77.50 92.5 96.0 82.50
1-3-74 78.00 93.0 96.0 83.25
1-4-74 81.25 94.0 96.5 89.5
1-5-74 81.0 94.25 97.0 84.0
1-6-74 82.0 95.0 95.5 86.0
 35

suspended in the ponds and the initial percent transmittance for

samples from each dialysis bag was recorded. This time some growth

was observed in all the dialysis bags. Extensive growth was

encountered in the bags suspended in ponds 1 and 4. However, the

bag in pond 4 exhibited less growth than the bag in pond 1. In

pond 7, growth was negligible although the cells survived for three

weeks. The water temperature was within the range of 16° C - 13° C

for the first three weeks of experiment in all the ponds. Weather

conditions again changed and water temperatures of 13° C, 12° C, and

7° C were observed in ponds 1, 4, and 7 respectively. This cold

weather lasted for almost fifteen days, during which reduced growth

of algae was again observed.

As shown in Figures 7 and 8, the growth rate, K, for the bag in

pond 1 was 0.1063 G/day as compared to 0.0797 G/day for the bag in

pond 4. Since measured parameters other than temperature were

relatively constant in the ponds throughout this experiment, it is

assumed that the lower temperatures recorded in pond 4 are respon-

sible for the decreased growth rate in pond 4.

For the third experiment, initiated on January 19 in the same

manner as the others, only two ponds, 1 and 4, were selected for

observation because the temperature in pond 7 was considered to be

too low to support significant growth. Immediate growth was observed

in the pond 1 bag but there was no growth in the pond 4 bag for

several days. The temperature in the fourth pond was well below

that of the first pond for the first several days. However, as the

temperature increased in pond 4, growth began. As shown in Figures

 36

•o
(U
-o 4->
CU 03 O
> ^—
s- 3
(U 0
<A 1 —
JH 03
0 0
to
CO

CM
CO

00
CM

ays
«v^ Q
CM
•v^ li-

ber 0
r>»i
to ^
0 E
0 CM 3

urve.
4-> 2^
CM >.
CO 03
• r^1 -0
o
2: 00
^ • ^

CU to CJ
CM
rowth
r— -M 1 CO
C ^— to
• (U n— 0
o E ^—
™»^*
•r— .
0 CM cn
s- . .
•o CU CU
eld algae

c CI. 4-> II
o X 03
Q. LxJ Q i>^

00

CD

O 00 to
to to to

LUi/SLL^O J-O •>i9qiiJnN 6o"i

 37

^
^

-a
Qi
•a 4J
Qi 03
>
S-
r^
3
o
CU o
to r—
ja 03
o O

to
CO

CM
CO

00
CM t/)
03
Q

CM

CU
J3

. O
^ CM

CU
>
to 3
CM
o

o
CM s-
C CD
CU
CU
03
S-. •• O CD
cu CU
d. -«-> II 03
X 03 00
o UJ Q >^ -o
CU

00

CD

X _L LO
CT> p>.
LO CO
cn lO
LO LO
to to to to to

LIU/SLL9D :^o JiaquinN b o i

 38
9 and 10, the growth rate for the twenty-three day experimental
period was higher in the first pond, K = 0.072 G/day, than in the
fourth, K = 0.060 G/day.

The fourth experiment, begun on February 17, again resulted in

a lack of growth in all the bags. During this period the temperature
ranged from 12° C to 10° C in ponds 1 and 4 respectively. At night
the ambient air temperature was below 0° C. After one week without
growth the dialysis bags were removed.

Accompanying warmer weather, the fifth experiment was begun on

February 28. Again, only ponds 1 and 4 were utilized. The
temperature ranged between 19° C and 16° C in pond 1 and 16° C and
14° C in pond 4 during this experiment. Continuous growth was
observed in bags in both ponds. The observed growth rates were 0.19
and 0.1090 G/day for pond 1 and 4, respectively, as shown in Figures
11 and 12. Again, the effect of temperature appears to be the
controlling factor.

From Table 3, it can be seen that throughout the entire study

higher growth rates were observed in those dialysis bags cultured at
the highest temperatures. Further reference to Table 2 indicates
that other parameters monitored throughout the course of study
remained relatively constant.

It should be pointed out that although maximum algal growth

rates for only a portion of the experimental period can be calcu-

lated from the collected data, the reported growth rates are

averages covering the entire experimental period.

 39

CM
CM

•a
T3
CU
4->
o
CU 03 CM
> 1 ^
3
s~
CU CJ
to t—
00
s:^ 03
0 0

to

to
CM

«ct-
r^
1
CM
f—
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co 4->
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o P^ -o
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CD
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LLU/sLL^O J-O uaqiunN 6 0 1

 40

CO
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CM
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CU CJ
t o I— cr>
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LUi/s|.L93 J-0 ueqiunM 6on

 41

CU
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LLU/SIL93 :^o uaquinN 6 o i

 42

-a
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00

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r - s . p - ^ r ^ t o v o v o t o t o t o ' ^ '^ ^o *^
LIU/SLL93 J-0 uaqiunM 5o"i
 43

to
CU
3 o o o o o
"— > l o o o o o
03 fO
> "O
p^ -*v.
^ CD

6
z^ CU
s- s-
T 3 CU 3
C 4-» + J
o 303: OJ CJ o O o O
a. $-
CU
0
p««.
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r—
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p>.
o
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C Q.
(O E
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:E I—
to o
CU CT> o cn
3 O p>. to o o
•— H o o o o
fCJ 03
> T3
i«i CD

CU
S- i .
\— T3 CU 3
+J +->
•z. o 03 03 CJ
o CJ
o o
2 %- o o
h- Qi CT> o CM o o
CO
e QJ CO
o 03 E o
o CU CU

CO
to CO
CU to CM
CQ 3 o o 1^ o cn
>— > J o o o
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> -a o
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CD :i<i CD

CU
s- s-
CU 3
4J +J C_) O CJ CJ
o 03 03 o o O o o
3 S- o LO CM
cu cn
C CL\
03 E
CU CU

o o
+-> O o
+->
CO CO 4->
28-74
12-74

CO p ^ r^ p^ p"^ p«-.
to r - 00 1^ I i I I I
CU CM CM I
00
o cn CM r^ to
I— CM
+03-> i I
CM I I I I 1 1
Q I CM CM CM CM CO

+J
C
CU
E
• ^ s- LO
s- cu CM CO
cu sn
O. E
X 3
 CHAPTER V
CONCLUSIONS AND RECOMMENDATIONS

This study was undertaken as an effort to develop a technique

which could provide information regarding algae growth in a natural
environment. This research has demonstrated that the dialysis bag
culture technique is suitable for studying algae growth. This
technique has an advantage in studying growth characteristics at low
nutrient concentrations, because the nutrient concentrations are kept
constant by continuous replenishment. In addition, the growth may
be allowed to accumulate over a period of time resulting in more
accurate and precise measurements.

From the results of these studies, it is concluded that

temperature was the parameter most responsible for limiting algae

growth. When the water temperature was low (7° C - 10° C ) , zero

growth was observed. Conversely, when the temperature was in the

range of 12° C - 19° C extensive algae growth in the dialysis bags

was always observed. Within this latter range, increased growth

paralleled increased temperatures. Inherent in this conclusion is

the fairly well substantiated assumption that no other environmental

factors limited algal growth.

The growth rates observed in this study were low compared to

values reported in the literature. These low growth rates probably

44
 45
resulted from the fact that during the whole period of study the

temperature was not in the range necessary for optimum growth of

Chlorella vulgaris. M. J. Geoghegan (17) reported that the optimum

temperature at which to culture Chlorella vulgaris was 25° C. At

20° C and 30° C very poor growth was exhibited.

Some problems with the technique were encountered and they

should be corrected. Provision for continuously mixing the cultures
should be made in order to distribute the cells homogeneous in the
medium. Dialysis bags should be replaced eyery three or four weeks
to avoid any interference that may result from an alteration of the
diffusion characteristics of dialysis bagging. Slight movement or
agitation should be applied to the dialysis bags in order to avoid
settling of suspending material in the medium on the top of bags
which affects the penetration of light through the bags.

It is further recommended that mixed algal cultures be employed

in future studies of this sort. In this way through natural
selection one or more species will predominate. Dominance will
depend upon the ability of the species to compete with other species
and upon the prevailing environmental conditions. As a result,
environmental factors such as temperature are less apt to be
limiting, and a better indication of the true algal growth potential
of a particular water can be obtained.
 LIST OF REFERENCES

1. Brock, T. D., Microbial Growth Rates in Nature. Bacterial Rev.,

Vol. 35, 1971.
2. Baskett, Russell C. and Lulves, William J., A Method of
Measuring Bacterial Growth in Aquatic Environment Using
Dialysis Culture. Journal of the Fisheries Research
Board of Canada, Vol. 31, 1974.

3. Clark, J. W., Viessman, W., Jr., and Hammer, J. M., Water Supply
and Pollution Control. International Textbook Company,
Scranton-Toronto-London, 1971.
4. Sweazy, Robert M., The Exchange and Growth Potential of Phos-
phorus in Algae Cultures. Doctoral Dissertation of The
University of Oklahoma, 1970.
5. Proceedings of the Eutrophication Biostimulation Assessment
Workshop. Ed. by E. J. Middlebrooks, Maloney, T. E.,
Powers, C.E., Kaack, L. M., June 19-21, 1969.

6. Johnson, J. M., Ruschmeyer, 0. R., Odhug, T. 0., and Olson,

Y. A., Algal Bioassay Potential Primary Productivity
Studies of the Lower St. Louis River, Minnesota, in
Advances in Water Pollution Research, 5th International
Conference of the Water Pollution Control Federation,
Washington, D. C , 1970.
7. Skulberg, 0. M., Algal Cultures as a Means to Assess the
Fertilizing Influence of Pollution, in Advances in Water
Pollution Research, 3rd International Conference of the
Water Pollution Control Federation, Washington, D.C.,
1967.
8. Theoretical and Methodological Basis of Continuous Culture of
Microorganisms. Ed. by Ivan Malck and Zdenck Fencel.
New York, Academic Press Inc., 1966.
9. Fogg, C. E., Algal Cultures and Phytoplankton Ecology. The
University of Wisconsin Press, Madison and Milwaukee, 1965.

46
 47
10. Powers, C. F., Schultz, D. W., Malueg, K. W., Brice, R. M.,
and Schuldt, M. D., Algal Responses to Nutrient Addition
in Natural Waters II. Field Experiment. Proceedings of
the Symposium on Nutrient and Eutrophication: The
Limiting Nutrient Controversy. W. K. Kellogg Biological
Station, Michigan State University, February 11-12, 1971.
!"'• A Manual on Methods for Measuring Primary Production in Aquatic
Environments. Ed. by Vollenweider, R. A. International
Biological Programme, 7 Marylebone Road, London, N.W. 1,
1969.

12. Schultz, J. S. and Gerhardt, P. Dialysis Culture of Micro-

organisms Design, Theory and Results. Bacterial Rev. 33,
T969:

13. Parkash, A., Skoglung, L., Rystad, B., and Jensen, A. Growth
and Cell Size Distribution of Marine Planktonic Algae in
Batch and Dialysis Cultures. Journal of the Fisheries
Research Board of Canada, Vol. 30, 1973.
14. Stewart, Kenton M. and Rohlich, Gerald A. Eutrophication - A
Review. A Report to the State Water Quality Board,
California, Publication No. 34, 1967.
15. Tiffany, Lewis H. Algae the Grass of Many Waters. Charles
Thomas, Publisher, Illinois, U.S.A., 1958.
16. Physiology and Biochemistry of Algal. Ed. by Lewin, Ralph A.
Academic Press, New York and London, 1962.
17. Algal Culture from Laboratory to Pilot Plant. Ed. by Burlew,
John S., Carnegie Institution of Washington, Publication
No. 600, Washington, D. C., 1953.
18. Winn, Walter T., Jr., Recreational Reuse of Municipal Waste-
water. Master's Thesis of Texas Tech University, 1973.
19. Nutrients in Natural Waters. Ed. by Allen, Herbert E. and
Kramer, James R., John Wiley and Sons, Inc., 1972.
20. Properties and Products of Algae. Ed. by Zajic, J. E., Plenum
Press, New York, 1970.
21. Standard Methods for the Examination of Water and Wastewater.
Joint Publication of the American Public Health Associ-
ation, American Water Works Association, and Water
Pollution Control Federation, 1971.

22. Fouhrenbach, Jack, Eutrophication. Annual Review of Literature,

Water Pollution Control Federation, 1968.
 48
23. Phillips, J. N. and Myers, J. Measurement of Algae Growth
Under Controlled Steady State Conditions. Plant Physical,
Vol. 29, 1954.

24. Maddox, W. S. and Jones, R. F. Some Interactions of Temperature,

Light Intensity, and Nutrient Concentration During the
Continuous Culture of Nitschia Closterium and Tetraselims
Sp. Limnol. Oceanog., Vol. 9, 1964.
25. Pipes, W. 0., Carbon Dioxide - Limited Growth of Chlorella in
Continuous Growth. Applied Microbiol., Vol. 10, 1962.