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
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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. II.
INTRODUCTION
1
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
CONCLUSIONS AND RECOMMENDATIONS
44
V.
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 correlate 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 eutrophication 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. Assay Procedures or PAAP.
They are known as Provisional Algal
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. accomplish one of two things:
All of these techniques attempt to
(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 concentrations 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 mathematical 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 characteristics 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 biological 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 translucent 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 microorganisms 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. interfere with this technique.
More clumping of algae can
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 determination 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:
and where
dlnV dt
^
dv dt
^
K & a =
a KIV K + al K_ In eD
(1) 1 + li K
(2)
I J II 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, therefore, \/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 anaerobes both as sources of carbon and as vehicles for transport for nutrients. In general, there are four sources of carbon in aquatic ecosystems; 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 biological 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 algaeHowever, 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 heterotrophic 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 approximately 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. employed for this study.
Ponds numbers 1, 4, and 7 were
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. also taken.
Temperature and pH measurements were
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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i
t
i +-> c
8
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Fig. 2
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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. 4, 5, and 6. temperatures.
The resulting growth curves are shown in Figures
These tests revealed increased growth at higher 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 sCU
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 Intermittent Light
6.6
6.4
6.2 to r— (U C_J
6 .0
o s(U J3
5 .8
E 3
z: C7> O
6
7
8
9
Number of Days Fig. 5
Laboratory algae growth curve.
10
12 13 14 15
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
Number of Days Fig. 6
Laboratory algae growth curve.
15
34 TABLE 2 LABORATORY STUDY RESULTS Percent Transmittance Date
Temperatu re 17° C Continuous Intermittent Light Light
Temperatuire 26° C Continuous Intermittent 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. was observed in all the dialysis bags.
This time some growth
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 responsible 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. temperature increased in pond 4, growth began.
However, as the
As shown in Figures
36
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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 calculated from the collected data, the reported growth rates are averages covering the entire experimental period.
39
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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. 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
Bacterial Rev.,
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. 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. 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.
Charles
Ed. by Zajic, J. E., Plenum
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.
