determination of algae growth potential in natural
TRANSCRIPT
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)
where K & a = Constants dependent on light intensity "i I J II
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
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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
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o CTi
o 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. AQ 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) Q. E Qi
o O
^
J 3 S-
o cni o !
CQ
<: :z> cy
Of UJ
a .
o
o Q.
-a <D >
1 —
o to (/)
" p -
c: <u o> >> X
Q O
en
«3 I sz
O Q-Jir to +J o
O Q.
C7)
I CO CT O S
o o c o < s O < ^ c o L n r o L O L n c o c o c \ J c y > C T >
I LD CM CO C\J
•=*• •=d- o CM C\J CM
CO CM
IT) CO
O O CM in
LO LO Lf) IT)
LO
un CM Lf)
00 o o «* • • • •
UO ( 00 vo
o o o «?i- 00 Ln o o o
LD LO VO CM 'd- "5i- «d- CM
o o o o
LO CM O
'5i-oocr»"!*-"5d-r>*u3<x>
<u +J fl3 Q
to +-> • r -
c rD
CO r>.
1 t ^
1 —
CO r^
1 «st-t—
1 r—
CO r*
1 cr> — 1
1 —
CO t ^
1 af^ CM
1 n—
CO t ^
1 vo — 1
CM
«cj-
r>» 1
CM 1
•sJ-
r^ 1
0 0 1
^ r^
1 ^D — 1
^ r^
1 CO CM
1
">f r^
1 o CO
1
• * r>»
1 <£)
1 CM
«^ r^
1 CM 1 —
1 CM
•sl-
r*. 1
• ^ CM
1 CM
«* r^
1 CM
1 CO
^ r*
1 0 0
1 CO
28
Qi
+J
S-O) Q.
E
o
o
5-I—
CM -o al o
o o
< ID
-— <:
OQ •St
O
o
o
o Q.
Q.
T3 (U > r— d O OJ to CD to >, •I- X a o
03
en
I o a. sz to +-> o s- ^ O Q_
cn
I CO C71 O !
00 "?j- LO CT LO CM CM CM I—
CM
<^ O ^O CM I— CM
O LO CM LO o
LO
CM LO
CM CO to
LO 00 o o
cn
to <^ 00 to • • • •
to r»* 00 to
LO o o 1— LO LO
o o I—
O LO LO LO LO CM CO O «* 1— r— O O O
C M « ! d - t O « v f « ^ C O L O O
v o t o t o t o ^ ^ ' ^ ^ o v o
CU -M 03 O
to +-> •r— C =)
CO r>. 1 r 1 ^—
CO p- 1
«>i-^— 1
1 —
CO 1^ 1
CT> r— 1
^—
CO 1^ 1 cy» CM 1
r—
CO r». 1 to I —
1 CM
^ r->. 1
CM 1
•=d-r* 1 00 1
«;!-
r* 1 to 1 —
1
^ r 1
CO CM 1
«* r«» 1 o CO 1
^ r 1 to 1
CM
«:d-r 1
CM r— 1
CM
"* r** 1
^ CM 1
CM
^ r 1
CM 1
CO
^ r 1 00 1
CO
29
1—1
h-"Z. O C_J 1 1
r—
LU _l CQ
<c ZD cy
a: UJ 1— <:
o •z. o
r •
o 2:
Q 2r 0 0.
CU
s -4-> 03 S . (U Q. E (U
o O
5-
3
LO CO CO CM O •— O I— r— CO CO
CM
CM " O Oil
CM
• o CU >
1— E O OJ to CD to >5
•<- X Q O
(U 4-> 03
CD
E
I O Q.
JC to 4-> O s- x :
O D -
CD
E
I CO cn O E 2 :
o <x> CM f— CM
0 0 0 0 0 0
CM O t o CO
o CO 0 0
cr» t o
CM
O ">1- CM ^ • • • •
00 00 O VO
O O CM O O O CO "sd- I— p— 1— O CM CM 0 0 0 0 0 0 0
O O L O O O O i — 00
t O t O t O t O t O t O t O L O
(L) 4-> «U 0
(/) -M •I—
C ZJ
CO 1^ 1 r- 1
i~~
CO
r 1
"!d-n— 1
p —
CO c 1 cn ^-1
I —
CO r>. 1 cr> CM 1
t—
CO r« 1 to r— 1
CM
«=J-
r- 1 CM 1
^ r 1 00 1
•v^
r*-1 to 1 —
1
«:a-r>. 1
CO CM 1
^ rv. 1
0 CO 1
"sd-P^ 1 vo 1
CM
«d-r«* 1
CM r— 1
CM
^ r*«. 1
«* CM 1
CM
^ r«» 1 CM 1
CO
«d-r**. 1
00 1
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
to
CU C_>
^-o
s-CU
cn o
K = 3.82
Temperature = 26° C
Continuous Light
3 4
Number of Days
Fig. 4 Laboratory algae growth curve.
32
7.OF
6.8
6.6
6.4
6.2
to r— (U
C_J
o s-(U
J 3 E 3
z: C7> O
6
5
.0
.8
K = 3.60 G/day
Temperature = 26° C
Intermittent Light
Fig. 5
6 7 8 9 10
Number of Days
Laboratory algae growth curve.
12 13 14 15
33
6.5 _
r- 6.0
to
CU
o
O
CU
O 5.5
5.0 -
K = 2.0 G/day
Temperature = 17° C
Continuous Light
4.7 6 7 8 9 10
Number of Days
15
Fig. 6 Laboratory algae growth curve.
34
TABLE 2
LABORATORY STUDY RESULTS
Date
12-14-73
12-15-73
12-16-73
12-17-73
12-18-73
12-19-73
12-20-73
12-21-73
12-22-73
12-23-73
12-24-73
12-25-73
12-26-73
12-27-73
12-28-73
12-29-73
12-30-73
12-31-73
1-1-74
1-2-74
1-3-74
1-4-74
1-5-74
1-6-74
Temperatu Continuous
Light
99-5
99.0
97.25
96.25
93.75
92.25
90.75
89.0
85.0
83.5
82.5
80.5
78.5
78.5
77.25
77.25
76.50
76.00
78.00
77.50
78.00
81.25
81.0
82.0
Percent Transmittance
re 17° C Intermittent
Light
99.5
99.25
98.75
99.25
98.75
98.25
98.75
98.75
98.50
97.50
97.25
96.0
95.75
95.75
94.75
93.50
91.50
90.75
93.00
92.5
93.0
94.0
94.25
95.0
Temperatui Continuous Light
99.5
85.0
81.0
78.5
74.75
72.0
71.75
70.00
69.00
72.5
77.25
81.5
88.0
91.0
89.5
93.75
95.25
94.50
97.0
96.0
96.0
96.5
97.0
95.5
re 26° C Intermittent
Light
99.5
93.25
88.75
82.5
77.25
73.0
72.00
69.25
66.00
66.5
67.0
66.5
65.5
69.0
70.25
72.25
72.75
76.25
80.5
82.50
83.25
89.5
84.0
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
r—
• o
™ » ^ *
• o c o Q .
CM
• o
2 :
-M C (U E
•r—
s-CU CI. X
LxJ
•v^
r>» i to
0 4->
CO r
1 0 0 CM
1 —
n—
. . CU
4-> 03
Q
> . 03
- 0 ^ • ^
CU
CO to 0 —
. 0
II
i>^
-o CU > s-(U <A
JH 0
• o (U
4-> 03 — 3 0
1 —
03 0
O
to CO
CM CO
0 0 CM
«v^ CM
^
0 CM
to
CM
0 0
ays
Q
l i
ber
0
E 3
2 ^
urve
. CJ
rowt
h
cn
alga
e el
d
CD
O 00
to
to
to to
LUi/SLL^O J-O •>i9qiiJnN 6o"i
37
CM
C CU
o
X
•a Qi > S-CU to
j a o
-a Qi
4J 03
r ^ 3 o
r— 03
O
S-. cu d . X
UJ
• • CU
-«-> 03
Q
O
II
>^
^ ^
o
to CO
CM CO
0 0 CM t/)
03 Q
CM
. O ^ CM
to
CM
CU J3
_L
0 0
CU
> 3 o
o s-CD
CU 03 CD
03
-o CU
00
CD
cn
to to
LO
to
CO
to to
CT>
LO
p>.
LO
LO
l O
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
r-^
• o
'^Z.
-a c o
O-
co
• o
2 :
+ J e Qi E
• 1 —
s-CU 0 . X
LxJ
«ct-r^
1 CM f —
1 CM
0 4->
«:d-P^
1 cn r—
1 p —
. * Qi
+ J 03
Q
>^ 03
-o • v ^ CD
CM P< 0
• 0
II
^
p-^
J .
T3 CU > s~ CU to
s:^ 0
• a CU
4-> 03
1 ^
3 CJ
t—
03 0
CO
CM CM
o CM
0 0
t o
CM
to
0 0
t o
CM
Qi
> 3
o s-CD
O) 03 CD
03
T3
CD
LLU/sLL^O J-O uaqiunN 6 0 1
40
-o Qi •a 4-> CU 03 > r— S- 3 CU CJ
«=J-
• o z: T5 C o Q .
CO
• o z^
+J E CU E
• r -
s-CU Q . X
LJJ
^ P>.
1 CM 1 —
1 CM
O 4->
• ! * • P>.
1 <Ti t—
1 1 ^
. . CU
4-> ns o
>» 03
T5 • ^ ^ ^
CD
O t o O
• o II
^
CO CM
CM
to I— .n 03 o o
± CO
p->»
C M
p>«.
o en to
00
to
cr>
LO
CO to 03
Q
O
s -CU
cn
LO
CU
> 3 o
o cn CU 05 CD
03
CU CO
CD
to
LUi/s|.L93 J-0 ueqiunM 6on
41
-o CU > s-<u to
X ) o
CU +J 03 — 3 O
r ^ 03
O CM
0 0
to >> 03 Q
S-cu
X I
t o
CU
> 3 o
o s-CD
CU 03 CD
03
-a
CM
CD
to to
LLU/SIL93 : o uaquinN 6oi
42
-o CU > &-CU to .a o
-a Qi
•<-> 03
p —
3 o r— 03
O
o O-
LO
CU
s-cu ex. X
LU
J . LO
CM
CM
0 0
to
03
CU J:2
- to
CM
CU > 3 O
o s-
CD
CU
CD
03
•o CU
CM
CD
LO
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
p^
6 z^
T 3 C o a.
to CU 3
"— > l 03 fO
> "O -*v.
^ CD
CU s- s-CU 3 4-» +J 03 OJ
3 : $-CU
C Q. (O E 0) Q)
:E I—
o o
o o
o o
o o
o o
\—
•z. h-CO
o o CO
CQ
o CD
T3
o
CJ 0 p««.
o 0 O
O o r—
o o p>.
O o CO
o
to CU 3
•— H fCJ 03 > T3
i«i CD
CU S- i . CU 3 +J +-> 03 03 2 %-
Qi e QJ 03 E CU CU
to CU 3
>— >J OJ 03
> -a :i<i CD
CU s- s-CU 3 4 J +J 03 03
3 S-cu
C CL\ 03 E CU CU
O
o
CT> p>. o
o to o o o
o cn o
CJ o CT>
o o
o o CO
CO to o
CJ o CM
o o
o
o o
CM 1 ^ o o o cn
o
to CU +-> 03 Q
+ J C CU E
•^ s-cu
s-cu sn O. E
X 3
o o
C_) o LO
O O
CJ o CM
CJ o cn
o +->
CO CO
r - 0 0 CM CM
i I
o 4->
CO 1 ^
I 0 0 CM
I
p^ I
o r^ p^
i I cn CM
I I CM
O
p"^ p«-. I I
r^ to I— CM
I I CM CM
o +->
28-7
4 12
-74
1 1 CM CO
CM CO LO
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 Phosphorus 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 Microorganisms 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 Wastewater. 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 Association, 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.