WATER POLLUTION CONTROL RESEARCH SERIES
18050DOL 03/70
BACTERICIDAL EFFECTS OF ALGAE
ON ENTERIC ORGANISMS
U.S. DEPARTMENT OF THE INTERIOR • FEDERAL WATER QUALITY ADMINISTRATION
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WATER POLLUTION CONTROL RESEARCH SERIES
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On Cover:
Sunfish Lepomis gibbosus
Bullfrog Rana catesbeiana
Spotted salamander Ambystoma maculatum
Drawings By:
Alston Badger
Bears Bluff Field Station
National Marine Water Quality Laboratory
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BACTERICIDAL EFFECTS OF ALGAE
ON ENTERIC OBGAJHSMS
Ernst M. Davis, Assistant Professor
Earnest F. Gloyna, Professor
CENTER FOR RESEARCH IN WATER RESOURCES
Environmental Health Engineering Research Laboratory
Civil Engineering Department
The University of Texas at Austin
for the
FEDERAL WATER QUALITY ADMINISTRATION
DEPARTMENT OF THE INTERIOR
Program #18050 DOL
March, 1970
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ACKNOWLEDGMENTS
The financial assistance of the Federal Water Pollution Control
Administration has made this investigation possible.
Appreciation is extended to Dr. R. K. Guthrie, formerly of North
Texas State University for bacteria cultures and advice; also to C. Clifton,
J. H. Bandas, D. Trigg, C. J. Rogers, G. Sparks, A.Gravel, R. Morales,
and T. Mercer, all students at The University of Texas at Austin, Austin,
Texa s .
111
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ABSTRACT
A series of experiments involving the effects of blue-green and green
algae on the dieoff rates of selected bacteria have been conducted. The
algae were axenic cultures of Anabaena cylindrica, Anacystis nidulans ,
Gloeocapsa alpicola, Oscillatoria chalybia, O. formosa, Phormidium
faveolarum, Ankistrodesmus braunii, Chlorella pyrenoidosa, C . vulgaris,
and Scenedesmus obliquus. Cultures of enteric bacteria species (Alcali-
genes faecalis, Enterobacter aerogenes, Escherichia coli, Proteus vulgaris,
Pseudomonas aeruginosa, and Serratia marcescens) were added to the
axenic algal cultures during different periods of the algal life cycles.
Cultures of the normal blue-green contaminants were exposed to the
enterics to determine antagonistic effects toward the enterics. Filtrate
from actively growing algae was exposed to cultures of enterics to determine
whether any antibiotic compounds were imparted to the medium during lag
phase growth of algae. To determine aftergrowth of the enteric species,
the duration of the tests was extended to about 90 days. Mixed cultures
of green and blue-green algae were exposed to both single species of
enteric bacteria and mixed cultures. The results indicated that mixed
algal cultures cause a greater dieoff among the enteric bacteria than do
individual species of algae. The dieoff characteristics of pathogenic
species, namely, Salmonella typhosa, S. paratyphi, Shigella dysenteriae,
S_._ paradysenteriae, and Vibrio comma were also determined.
The pathogenic species did not survive as long as the enteric test
species under similar test conditions. Virtually no aftergrowth was detected
on the part of the pathogens .
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CONCLUSIONS
The following conclusions are derived from the results of this investi-
gation.
1. Dieoff coefficients for individual species of enteric bacteria
in the presence of axenic cultures of algae were comparatively low, the
majority of the coefficients being near or less than -0.1 per day. Chlorella
pyrenoidosa and Chlorella vulgaris caused the highest dieoff coefficients
among enteric bacteria. Chlorella spp. were substantially more effective
than Ankistrodesmus braunii or Scenedesmus obliquus in effecting acceler-
ated dieoff S .
2 . Mixed cultures of either the blue-green or green algae caused
significantly higher dieoff coefficients among the enteric test bacteria as
well as the pathogenic bacteria tested. The majority of the coefficients
were between -0.1 and -0.2 per day.
3. Effects exhibited by enteric bacteria on the growth of individual
algal species depended on the algal species in question. Constant patterns
of increased or decreased algal growth coefficients were uncommon. In
the majority of algae species, a slight inhibition of the overall growth
potential of the algae was observed.
4. Dieoff of enteric bacteria was more rapid under aerobic con-
ditions than anaerobic conditions.
5. Aftergrowth of Escherichia coli, Pseudomonas aeruginosa,
and Serratia marcescens occurred in axenic blue-green algal cultures as
well as in waste stabilization pond effluent. Alcaligenes faecalis,
Enterobacter aerogenes , Proteus vulgaris, Vibrio comma, Salmonella typhosa,
Salmonella paratyphi, Shigella paradysenteriae exhibited no aftergrowth
potential under similar conditions. Serratia and Pseudomonas exhibited
a greater aftergrowth potential than did E. coli.
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6. As the algal species reached their stationary and/or log death-
growth phase in the laboratory, quantities of organic carbon were released
to the medium; up to 200 mg/1 was not uncommon. Prolonged survival
periods and/or aftergrowth by some of the enteric bacteria were attributed
to this nutrient source.
7. Consistent dieoff effects on enteric bacteria in laboratory and
field waste stabilization ponds were achieved only after appropriate periods
of acclimitization of the pond microcosms. Those periods were observed
to be as long as 30 days, or more in some instances. Dieoff coefficients
for early stages in pond treatment units were higher than those obtained
for secondary stages such as maturation ponds. Higher coliform concentra-
tions and increased competition for nutrient sources in early treatment
sequences were attributed to that rapid dieoff.
8. Compared to axenic algal culture experiments and laboratory
scale ponds, the most rapid reduction in enteric bacteria occurred in the
waste stabilization ponds located in the field.
9. In laboratory ponds, E. coli exhibited a greater resistance to
dieoff than did Pseudomonas aeruginosa or Serratia marcescens; but in the
field ponds, E. coli exhibited the highest rate of dieoff of any enteric
bacterial species tested.
10. Occasional increases in concentrations of Pseudomonas and
Serratia were noted in laboratory and field ponds. Short-circuiting was not
considered to be the causative factor, but an association of these two
genera and other enteric bacteria with clumps of algae might have been
responsible for this increase. Pseudomonas spp. exhibited increases
in numbers when the total algal concentrations were lowest in both the labo-
ratory and field ponds.
11. Total coliform bacteria counts decreased significantly during
periods when the pond phytoplankton population was highest, and vice
versa.
12. The vast majority of bacteria in all pond effluents were of
the group of bacteria referred to as the chromagens; included in the group
are Flaveobacterium and Brevibacterium. Cultures of these two separate
vi
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genera were shown to exert marked antagonistic effects on enteric bacteria
when together in culture. Flaveobacterium was more antagonistic to enteric
bacterial species than Brevibacterium.
13. On several occasions extended periods of incubation were
necessary to produce any recordable growth of Pseudomonas spp. from
waste stabilization pond samples using either nutrient, trypticase soy, or
Endo agar plates. Special consideration should be given this factor when
total or enteric counts are made from wastewater environments.
vii
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TABLE OF CONTENTS
PAGE
ACKNOWLEDGMENTS iii
ABSTRACT iv
CONCLUSIONS v
TABLE OF CONTENTS viii
LIST OF TABLES **
LIST OF FIGURES x
CHAPTER
1 INTRODUCTION 1
Bacterial Characteristics 1
Review of Literature 3
2 MATERIALS AND METHODS 9
Algal and Bacterial Cultures 9
Laboratory Investigation Series Identification 13
Laboratory Data Analyses Methods 15
Laboratory and Field Waste Stabilization Pond
Studies 16
3 LABORATORY CULTURE DIEOFF EXPERIMENTS 17
Enteric Bacteria Dieoff Studies 18
Pathogenic Bacteria Dieoff Studies 25
Aftergrowth Potential Measurements 25
Organic Carbon Production by Algae 27
4 LABORATORY AND FIELD WASTE STABILIZATION
POND STUDIES 30
Laboratory Waste Stabilization Pond Studies 30
Field Waste Stabilization Pond Studies 44
BIBLIOGRAPHY 54
APPENDIX A: STATISTICS OF ALL LABORATORY AXENIC 59
CULTURE STUDIES
APPENDIX B: BACTERIOLOGICAL DATA FROM LABORATORY
AND FIELD WASTE STABILIZATION POND STUDIES 97
APPENDIX C: PROGRAM BETA FORMAT 126
vm
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LIST OF TABLES
TABLE PAGE
2-1 Composition of Algal Culture Medium 10
2-2 Element Concentrations in Algal Culture Medium 11
2-3 Algae and Bacteria Test Species 12
3-1 Dieoff Coefficients for Series Utilizing Axenic 19
Algal Cultures and Enteric Bacteria
3-2 Dieoff Coefficients for Series Utilizing Pathogenic 26
Bacterial Species
3-3 Aftergrowth Characteristics of Enteric Bacterial 28
Species with Single Species of Algae (Series I)
3-4 Total Carbon and Total Organic Carbon Content of 29
Biomass After Ninety Days (Series BG-I, G-I,
BG-VIII, and G-VIII, in mg/1)
4-1 Bacteria Inoculated Into Selected Stations In Laboratory 33
Waste Stabilization Ponds
4-2 Dieoff Coefficients of Inoculated Bacteria in Laboratory 34
Scale Waste Stabilization Ponds
4-3 Phytoplankton Found in Laboratory Waste Stabilization 36
Ponds
4-4 Total Phytoplankton Concentrations Found In Laboratory 37
Waste Stabilization Ponds
4-5 Bacteria Inoculated Into Selected Stations of Waste 46
Stabilization Ponds
4-6 Phytoplankton Found in Waste Stabilization Ponds 51
4-7 Phytoplankton Concentrations Foundin Waste Stabiliza- 52
tion Ponds
A-l through A-72
Appendix A: Statistics of All Laboratory Axenic 59
Culture Studies
B-l through B-28
Appendix B: Bacteriological Data from Laboratory 97
and Field Waste Stabilization Pond Studies
IX
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LIST OF FIGURES
FIGURE £££J
4-1 Schematic of Laboratory Ponds 31
4-2 Total Bacteria, Laboratory Ponds 38
4-3 Total Coliform, Laboratory Ponds 39
4-4 £_._ coli, Laboratory Ponds 40
4-5 Phy to plank ton Densities in Laboratory Ponds 41
4-6 Schematic of Waste Stabilization Ponds 45
4-7 Total Bacteria in Waste Stabilization Ponds 48
4-8 Total Coliform in Waste Stabilization Ponds 49
4-9 Phytoplankton Densities in Waste Stabilization Ponds 50
x
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CHAPTER 1
INTRODUCTION
An important reason for the treatment of domestic wastewaters is the
reduction or elimination of the enteric bacteria from these wastewaters;
in this connection, waste stabilization ponds have been used successfully.
In established ponds the most obvious population consists of various species
of algae as evidenced by their pronounced color. These algae, under
proper pond design, can produce the greater percentage of required dissolved
oxygen and can interact with the entire biological community. As yet,
however, the specific role that algae play in the overall reduction of enteric
bacteria in waste stabilization pond systems has not been firmly established.
The purpose of this investigation was to determine the degree of
toxicity exerted by typical species of blue-green and green algae on
representative bacteria found in wastewaters. The scope of this investiga-
tion included: (a) long-term studies involving selected species of algae,
coliform bacteria, and pathogenic bacteria; (b) bactericidal and bacteri-
static effects; (c) algal culture filtrate effects on bacteria test species;
(d) aftergrowth capabilities of test bacteria by extension of study periods;
and (e) enteric bacteria dieoff investigations in both laboratory waste
stabilization ponds and field ponds having different design characteristics.
Bacterial Characteristics
A brief description of some of the important characteristics of the
bacteria studied in this investigation is appropriate in order that their
complexity and the significance of their reduction in wastewaters can be
fully appreciated. Coliform bacteria are, by definition and description,
inclusive of all aerobic and facultative anaerobic, gram-negative, nonspore-
forming rod shaped bacteria which are capable of fermenting lactose with
gas formation within forty-eight hours at a temperature of 35 degrees
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Centrigrade (27). They have been variously named the _B.. coli group and
the coli-aerogenes group in past years with no change in the specifications.
By this definition, therefore, only part of the bacterial flora inhabiting the
gastrointestinal tract of animals are coliforms. The total number of genera
which are capable of living in those conditions is largely unknown. Invari-
ably, species and varieties of Escherichia, Streptococcus , Clostridium,
Aerobacter (renamed Enterobacter), Paracolobactrum, Salmonella , Shigella,
Proteus, Pseudomonas, Alcaligenes, Serratia, and Bacteriodes are among
those found. In this sense any species which has the capability to sur-
vive and multiply in any intestinal tract could be called an enteric bacterium
Taxonomically, the enteric bacteria follow this classification (26):
Order Eubacteriales
Family Enterobacteriacede
Genus Escherichia
Genus Aerobacter
Genus Klebsiella
Genus Paracolobactrum
Genus Alginobacter
Genus Erwinia
Genus Serratia
Genus Proteus
Genus Salmonella
Genus Shigella
Other genera which are found routinely in domestic wastewaters have the
following classification:
Order Eubacteriales
Family Achromobacteraceae
Genus Alcaligenes
Order Pseudomonadales
Family Pseudomonadaceae
Genus F*seudomonas
To assume that all of the species of the genera listed in the above classi-
fications are nonpathogenic would be erroneous. Several species in the
Family Enterobacteriaceae, for example, have been known to be pathogenic
to man, producing various intestinal diseases and septicemic infections.
For this reason alone their elimination from wastewaters is of utmost
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importance, Alcaligenes faecalis has been isolated from infections of
bacteremias, gall bladder infections, eye infections, and has frequently
been incriminated in cases of enteritis . Species of Pseudomonas, by the
same token, are frequently encountered in eye and ear infections as well as
urinary tract infections (26). This genus is universally treated with a great
deal of respect, especially in facilities in which burn patients are housed.
A septicemia caused by Pseudomonas may occur as frequently as staphy-
lococcal septicemia in severely burned patients and in persons who have
leukemia. The outcome is usually fatal.
The genus Vibrio, which has several nonpathogenic water-borne species,
is also found in the Family Pseudomonadaceae. Most dangerous of the
species are Vibrio comma and Vibrio El tor which are the causative agents
of the well-known Asiatic cholera.
Review of Literature
The mechanism by which populations of undesirable bacteria are reduced
in numbers has been the subject of many investigations. In waste treat-
ment facilities the bacterial dieoff is affected by several factors. In lakes,
reservoirs, impoundments, and streams the bacterial dieoff may be assumed
to be similar insofar as these factors are concerned. The principal difference
between the aquatic environments is one of bacterial concentration. Some
of the factors which undoubtedly play an important part in the bacterial
dieoff mechanism are sunlight, pH changes, changes in oxygen tension,
predation by other organisms such as rotifers, changes in organic content
of the water, temperature, and antagonistic effects of other bacterial
species and other faunistic species such as fungi and algae. Gravel,
et al. (2) found temperature, pH, and dissolved oxygen concentration to
be important, in that order, in dieoff rates of reservoir coliforms. Gameson
and Saxon (3) attributed the dieoff primarily to sunlight effects.
Bacteria must have certain quantities of organic carbon present for
their survival or multiplication. Ward and Moyer (14) reported that organics
excreted by algae during growth could serve as bacterial nutrient sources.
This source of carbon may reach appreciable concentration levels. Hellebust
(13) reported that some phytoplankton are capable of excreting up to 25
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percent of their photoassimilated carbon during their log growth phase.
Therefore, when large populations of algae are present, adequate supplies
of carbon should be present for the survival of some of the coliform bac-
teria. Data presented by McGrew and Mallette (10) stated that some bac-
teria of intestinal origin, including Escherichia coli, could survive and
even multiply at concentration levels of glucose less than 5 micrograms
per milliliter.
The literature contains various reports of interactions between coli-
form s and other faunistic species such as algae. McLachlan and Yentsch
(17) and Nakamura (18), respectively, found that certain bacteria enhanced
the growth of Dunaliella and Chlorella. Ward and Moyer (14) and later
Ward, Moyer, and Vela (25) demonstrated that there was significant reduc-
tion in growth of Chlorella pyrenoidosa when in the presence of Pseudomonas
aeruginosa. Opposing opinions can be found regarding the antagonism of
microorganisms to one another. Guthrie et al. (11) and Geldreich and
Clarke (4) have identified inhibition characteristics between Pseudomonas
aeruginosa and Escherichia coli under different environmental conditions.
The interactions which occur between bacteria and algae may affect
the physiology and productivity of an aquatic community. Stimulation of
bacteria by algae or algal exudates has been reported by the following inves-
tigators. Recent work by Vela and Guerra (52) and Ward, et al. (25)
furnished evidence that, in some cases, the proliferation of bacterial
species may be a function of algal growth. In tests involving Shigelia,
Proteus, Staphylococcus, Streptococcus, and Corynebacterium they found
rapid dieoff patterns of these bacteria when exposed to Chlorella. Yet,
it was also reported that Salmonella typhi and Salmonella paratyphi grew
well in the presence of Chlorella. In extensive works on toxic blooms of
blue-green algae, Gorham (36) found that Microcystis produced a toxin but
stated that it did not inhibit bacteria such as Bacillus subtilis , Staphylo-
coccus aureus, Escherichia coli, and Pseudomonas hydrophila. It was
hypothesized that the age of the culture may have been an important factor
in the results and should be considered when analyzing future data. Fogg
(15, 16) also reported stimulatory effects to bacteria when associated with
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algae. He attributed those effects to extracellular by-products of the algae.
Lefevre (37) substantiated earlier hypotheses that the extracellular products
did exist.
The numbers of reports which conclude that algae may be inhibitory to
bacterial growth are in the majority. Geldreich and Clarke (4) reported that
fecal coliform bacteria were influenced adversely by Schizothrix calcicola
within 24 hours. Telutchenko and Fodorov (5) concluded that their algae
affected the test bacteria by using the carbon dioxide and, thus, shifting
the pH, by releasing antibacterial substances, by inhibiting the bacterio-
phage which Lyse bacteria, and by increasing the organic content which
stimulated the growth of the bacteria. They further concluded that Chlorella
vulgaris was more efficient than Scenedesmus obliquus in killing E... coli
and Salmonella typhimurium. Chlorella vulgaris was the test algal species
used by Pratt and Fong (19). Their conclusion was that that species of
Chlorella was capable of inhibiting the growth of associated bacteria.
Birge and Judey in 1929 (28) indicated that algae may have a role in
reducing the numbers of bacteria in water. To date only "Chlorellin"
has been named specifically with regard to its antibacterial characteristic
by Caldwell (30), Pratt (46), and Spoehr, et al. (50). Flint and Moreland
(35) were able to demonstrate that metabolic exudates of certain blue-green
algae were toxic to bacteria but carried the report no further. Neel and
Hopkins (43) observed the reduction in numbers of types of coliforms during
seasons of the year in which prolific algal growth occurred in the ponds.
Work by Vladimirava (53) reported that cultures of Chlorella pyrenoidosa
were definitely capable of suppressing bacterial growth. Prescott (47)
cited two genera, Microcystis and Chlorella, as being capable of producing
and secreting substances active against two bacterial genera, Staphylococeus
and Clostridium. Oswald and Gotaas (45), in an extensive work dealing
with pilot-plant waste stabilization ponds, proposed that no specific
anticoliform activity could be credited to an algal culture tested in the
laboratory. However, they did not discount the possibility of antibacterial
properties of algae.
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The problem of bacterial contamination has been overlooked by some
investigators and this is of importance when testing single species of
algae. It is doubtful that there are many bacteria-free cultures of filamen-
tous blue-green or green algae. Unicellular or diplo forms of blue-greens
and greens are relatively easy to grow in a bacteria-free state. In naturally
contaminated cultures, Ward and Moyer (14) reported that the bacterial mass
was less than one percent of the algal mass. Yet, the numbers of bacteria
were shown to exceed a million per milliliter; figures approaching a billion
per milliliter were not uncommon. As to the contaminants themselves,
Krauss and Thomas (20) reported Flavobacterium to be the most common and
persistent genus in cultures of Scenedesmus obliquus. Levinson and Tew
(21) also reported Flaveobacterium as a contaminant of their research cultures
of algae. Their test species was Chlorella vulgaris .
Numerous reports are available which quote reductions in the coliform
numbers through waste stabilization ponds. The reduction percentages are
usually impressive; however, as Geldreich (1) pointed out, even with
reductions of from 90-99 percent the remaining 1-10 percent of the coliforms
may easily constitute numbers of from 4x10 to 10x10 per 100 milli-
liters. These values are not acceptable for more effluent standards. Most
of the species incorporated in the coliform group obviously have a similar
metabolic pattern of growth and development. Geldreich (7) and Gallagher
and Spino (8) have observed similar survival characteristics (or death
rates as the case may be) among the more abundant coliform species. In
making observations on streams, Churchill (9) reported that the slopes of
die-away curves for the total and fecal coliforms were essentially the
same. While figures are usually the best measure of coliform dieoff when
describing their functions, it must be remembered that a rate number does
not relate the environmental conditions. In reports dealing with enteric
bacteria reduction in ponds and series of ponds in South Africa and Zambia,
Marais and Shaw (40), and later Marais (38, 39) used a value of K = 2.0
for £_._ coli and K = 0.8 for Salmonella typhi. These differences between
only two species indicate the need for further data . Projections made by
use of the modifications of Chick's Law as reported by Marais (38, 39)
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may be used effectively to provide an insight into what general bacterial
pollution control may be required. Hanes, _et al. (6) reported log death
rates of 0.134/day, 0.29 I/day, and 0.355/day respectively at temperatures
of 10°, 20° and 30°.
It is apparent that not enough information is available on pathogenic
bacteria such as Salmonella, Shigella, and Vibrio. With large numbers
of coliforms present, the probability of finding Salmonella, for example,
increases. Of course, with several hundred serotypes of Salmonella in
existence, it would be difficult to establish the exact nature of the type
found in the sample. Geldreich (12) reported that when the fecal coliform
count exceeded 1,000, the Salmonella also increased. Periodic reports
of isolation of various species of Salmonella are routine (22). In March
1969 , 1,165 isolations of Salmonella were reported for humans , an average
of 291 per week. This was an increase of 13.2 percent over the average
for February 1969, and an increase of 7.0 percent (weekly) for March 1968.
At the same time, 738 non-human isolations occurred during March 1969.
These figures indicate that, even through few outbreaks of disease caused
by Salmonella are occurring in this country, the causative agents are ever-
present. Ward and Moyer (14) reported that Salmonella typhi and Salmonella
paratyphi grew well in algal cultures for periods of time extending through
seven days. Sidio (48) reported up to 99 percent removal of coliforms
along with complete removal of the pathogenic genus, Salmonella. There
is additional evidence to indicate that Salmonella typhi survival is dependent
on the available supply of nutrients. Increased loadings with shorter
retention times were seen to support the survival of the typhoid bacillus.
This was reported by McGarry and Bouthillier (41). They also reported
that ponds with longer detention times and reduced nutrient concentrations
provided a more antagonistic environment. Goetzee and Fourie (31)
showed in field studies that waste stabilization ponds operating in series
were capable of reducing Salmonella spp. by at least 99.5 percent. The
total reduction of E. coli was similar to that found for other bacteria.
These investigators reported that Salmonella spp. was more resistant,
as compared to E. coli, in highly polluted waters.
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Generally, the operating data on waste stabilization ponds are well
documented (54, 55, 56, 57, 58, 59). Smallhorst and Walton (49) and
Towne, et al. (51) have observed and reported the reduction of enteric
organisms in waste stabilization ponds. They attributed this reduction
primarily to detention. Towne, et al. (51) also reported that the reduction
in coliform numbers was not appreciably different for the seasons despite
variations in algal concentrations. Detention coupled with short-circuiting
was considered by van Eck (32 , 33, 34) and Bolitho (29) to be the most
important parameter which influenced bacterial concentration. Reductions
in coliform bacteria of above 90 percent routinely occur in ponds which
are functioning in an acceptable manner (42 , 48, 54). Gann, et al. (23)
found Achromobacter 65 percent of the total population of pond bacteria,
Pseudomonas 25 percent, Flaveo-bacterium 5 percent, and the coliforms
less than (or rarely equalling) 10 percent.
One aspect of the dieoff of bacteria in treatment facilities which has
received very little attention has been the aftergrowth phenomenon. After
treatment and discharge, the surviving bacteria, including those which
have been exposed to chlorine, may find suitable growth conditions in
the receiving waters and continue to multiply. This aftergrowth has been
reported by Orlob (44), Geldreich (12), Eliassen (24) and others. Under
certain conditions coliform bacteria were found to increase in numbers
to peak values within 30 hours up to 10-40 times the original number (24).
Even with chlorination of 15 minutes duration, a lesser increase in after-
growth occurred of 1-12 times the original number of bacteria. Clearly
a greater understanding of the ability of these organisms to reproduce
and the accompanhing necessary conditions is needed.
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CHAPTER 2
MATERIALS AND METHODS
Algal and Bacterial Cultures
Standard microbiological laboratory procedures were incorporated
during all phases of this investigation. Algal species were maintained as
bacteria-free as possible under normal laboratory conditions prior to addi-
tions of the test bacteria. The axenic algal cultures were subjected to
transfer from a solid algal growth medium to a liquid medium, and vice
versa, for a period of three years prior to the initiation of the experimentation.
Their acclimation to laboratory growth conditions and growth rate constancy
was, therefore, assured. The composition of the liquid medium which was
used for the culture of the algae in the laboratory is described in Table 2-1.
This medium was designed to allow optimum growth of the algae for prolonged
periods of time, a feature which was of great benefit during the extended
periods of testing necessary for successful completion of the investigation.
Table 2-2 presents a breakdown of the elemental concentrations in the algal
growth medium shown in Table 2-1.
Throughout the laboratory phase of the investigation six species of
blue-green algae and four species of green algae were used as test orga-
nisms. These species are listed in Table 2-3 along with the bacteria
which were tested. Code numbers for the algae indicate their culture
number as cataloged by the culture collection group at Indiana University
from where they were obtained. Code numbers for the bacterial species
represent the American Type Culture Collection number or the culture
number from stock cultures at North Texas State University or both.
The algae were grown in culture and used in tests at a temperature
of 25 +. 1 C. Fluorescent lighting operating on a cycle of 14 hours on
and 10 hours off provided an intensity of 290-300 foot-candles .
All of the bacterial species were cultured in the laboratory with
trypticase soy broth or agar supplemented with 2 g/1 yeast extract.
Appropriate serial dilutions were made of the cultures followed by counting
9
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10
Table 2-1. Composition of Algal Culture Medium
Compound
Final Concentration (mg/1)
NaHCO,
MgSO,
3 ' 5H2°
KH2P04
NH4NO3
KNC>
200
75
20
50
20
75
40
Trace Element Solution (1 ml of the following mixture)
EDTA
ZnSO. ' 7H O
T Z
H3B°3
MnCl2 ' 4H2O
FeSO. ' 7H0O
4 2
CoCL ' 6H0Oq
2 2
CuSO. ' 5ELO
(NH4)6M°7°24
LiCl
4H20
18H20
SnCl
KI
KBr
10.0 g/1
1.0 g/1
1.0 g/1
0.5 g/1
0.5 g/1
0.15 g/1
0.15 g/1
0.10 g/1
0.0278 g/1
0.0556 g/1
0.0278 g/1
0.0278 g/1
0.0278 g/1
Distilled Water, to make 1 liter final volume
The pH of the above medium is adjusted to « 5.8 with
HCl before autoclaving, resulting in a final pH of « 7.4
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11
Table 2-2 . Element Concentrations in Algal Culture Medium
Element
N
K
P
Na
C
Cl
S
Mg
Ca
Si
Zn
B
Mn
Fe
Co
Cu
Mo
Li
Al
Sn
I
Br
Final Concentration (mg/1)
41.15
21.245
4.560
58.468
31.825
102.755
20.146
15.150
12.200
2.960
0.228
0.174
0.139
0.101
0.037
0.038
0.054
0.004
0.004
0.015
0.021
0.019
-------
12
Table 2-3. Algae and Bacteria Test Species
Organism
Code Number
Anabaena cylindrica
Anacystis nidulans
Gloeocapsa alpicQla
Oscillatoria chalybia
Oscillatoria formpsa
Phormidium faveolarum
Ankistrodesmus braunii
Chloreila pyrenoidosa
Chlorella vulgaris
Scenedesmus obliquus
Alcaligenes faecalis
Enterobacter aerogenes
Escherichia coli
Proteus viglgaris
Pseudomonas aeruginosa
Serratia marcescens
Salmonella joaratyphi
Salmonella typhosa
Shigella paradysenteriae
Shigella dysenter_ia_e
Vibrio comma
B 629
625
B 589
B 386
LB 390
B 427
245
26
29
72
ATCC 8748
ATCC 9621
ATCC 8677 NT 201
ATCC 8427
ATCC 7700 NT 99
ATCC 13880
NT 113
NT 118
NT 131
NT 127
NT 154
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13
and enumeration of the bacterial colonies on poured plates of the trypticase
soy agar. This mixture of nutrient sources was found to be superior to
nutrient broth or nutrient agar alone for growth and subsequent enumeration
of most of the test species. In tests involving laboratory cultures of
o
algae and bacteria, counts were made after 24 hours of incubation at 37 C.
The plates were then returned to the incubator and recounted at 48 and 72
hours because, in the majority of cases, the algal contaminants and some
of the test bacteria did not show adequate growth at 24 hours for accurate
enumeration. This recounting technique is time-consuming but necessary.
Two genera of bacteria were the primary contaminants of the filamentous
blue-green test algal species. These were Brevibacterium and Flavobacterium
and were identified through selective testing procedures by Dr. R. K. Guthrie.
Their presence was not detected in the test cultures of green algae.
Addition of test bacterial populations to algal cultures was uniformly
controlled throughout the investigation. A standard straight-wire inoculum
of the bacterial species in question was incubated for 24 hours in half
strength TS broth. The solution was mechanically agitated and 0.05 ml
was transferred to each 100 ml of algal culture. Before removing samples
from the algal cultures the volume of each 250 ml erlenmeyer flask was
adjusted with sterile distilled water to correct for evaporation losses.
After making appropriate serological dilutions the samples were plated
onto TS agar and counted.
Laboratory Investigation Series Identification
The first phase determined the effects that axenic cultures of blue-
green and green algae had on the dieoff of individual species of test bac-
teria. Additional tests involved studies of algal growth characteristics
when exposed to the bacteria and studies of the effects of the contaminating
bacteria on various enteric species. The tests are described below.
Series I. Viable bacteria were added to individual algal cultures
at controlled times (early to mid-log growth phase of
algae) and the'bactericidal or bacteristatic effects
noted. This series of tests was coded BG-I (blue-
greens) and G-I (greens).
-------
14
Series II. Viable bacteria were added to individual axenic cultures
of algae during the first twenty-four hour period of
the lag growth phase after inoculation of the algae
into sterile growth medium. Any inhibition of algal
growth was determined by this timing sequence.
The series was coded BG-II and G-II.
Series III. Control tests were conducted to evaluate normal
cyclic influences of contaminant bacteria in algal
cultures. This series was coded BG-III and G-III.
Series IV. Algal mass was determined by weighing procedures.
Comparison of these data from control cycles with
data obtained during Series I and II demonstrated
whether inhibition or enhancement of algal cultures
was the result of the presence of enteric bacteria.
This series was coded BG-IV and G-IV.
Series V. Dieoff rates of the contaminant bacterial species
were determined during series involving the addi-
tions of enteric bacteria in algal cultures. This
series was coded BG-V or "contaminants."
Series VI. Dieoff rates of the enteric bacteria alone in algal
growth medium were analyzed. This series provided
the basic control for the study described in Series V
above. This series was coded VI.
Series VII. Separation of the algal growth medium into a cell-
free filtrate during mid-log phase of algal growth
control with subsequent inoculation of enteric bac-
teria demonstrated the influences of algal metabolic
exudates on the enteric bacteria. This series was
coded BG-VII and G-VII.
Series VIII. Equal quantities of each of the six test species of
blue-green algae were mixed when in their mid-log
growth phase. Additions of suspensions of indivi-
dual species of enteric bacteria in each resulting
-------
15
heterogeneous algal culture demonstrated compara-
tive dieoff rates with Series I and II above. This
study was conducted with the blue-greens and the
greens separately and was coded BG-VIII and G-VIII.
Series IX. Duplication of series BG-VIII and G-VIII using
mixtures of all the enteric bacterial test species
provided bacterial dieoff rates which might be
expected from field conditions. This series was
coded BG-IX and G-1X.
Series X. Testing in the majority of the series was continued
for periods up to 90 days to establish patterns of
aftergrowth of each bacterial species. This series
was coded BG-X and G-X.
Laboratory Data Analyses Methods
Data for all of the series, I through X, and for the runs involving the
pathogenic bacteria, were analyzed using a method of least squares. The
program (BETA) is shown in Appendix C. It should be noted that when after-
growth occurred, the data were not subjected to statistical analysis. In
order to be acceptable, only decreases in numbers which extended over
periods of 90 days were programmed. Data for aftergrowth characteristics
are presented separately. Basically, the function of program BETA was the
calcultion of the constants for an equation similar to the following:
Y is the dependent variable, X is an independent variable, and C is con-
stant. Equation 2-2 was used tc calculate the log (base 10) death rates:
, /-. v 9-?
+ I /_ A. £ £
where
X,
log (Y) =
= the density of bacteria in number per ml, or mg/1 weight
= the Y-axis intercept
= the time in days corresponding to Y
= the death rate coefficient, log base 10
-------
16
Additional computer output provided: (a) the variance ratio from the hori-
zontal line; (b) variance about the regression line; and (c) the multiple
correlation coefficient. Data were analyzed at the 90 percent confidence
limit.
Laboratory and Field Waste Stabilization Pond Studies
Additional data were obtained from model waste stabilization ponds.
These laboratory units consisted of two serial connected aquaria similar
to those described by Malina and Yousef (60). The total capacity was
46 liters. A diagram of these units is shown in Figure 4-1. All of the
model pond experiments were conducted at ambient temperature on location
at the Govalle Wastewater Treatment Plant in Austin, Texas. Lighting was
provided by banks of fluorescent bulbs held at approximately 25 cm above
the water surface. The intensity was 325-350 foot-candles during the 12
hours they were cycled.
Three different design concepts were represented by the model ponds.
The volumes and detention times were calculated to correspond to the
series of full-scale ponds existing at the Govalle facility. The first set
of ponds consisted of an anaerobic pond followed by two 46-liter faculta-
tive ponds and a maturation pond. The second set was represented by
facultative ponds which contained an anaerobic "trench," followed by a
maturation pond. The third set contained facultative ponds followed by
a maturation pond.
Daily additions of 500 ml untreated domestic wastewater were added
to each model pond; a similar quantity was removed from the opposite end
to effect a balanced system, and evaporation losses were corrected by
the addition of tap water.
-------
CHAPTER 3
LABORATORY CULTURE DIEOFF EXPERIMENTS
Regardless of the degree of laboratory control, considerable variance
occurred in the bacterial population during some of the tests . These fluctua-
tions in numbers were mainly attributed to the growth phase of the algal
cultures and the inherent nature of bacteria to adhere to filaments or aggre-
gates of algae. The fluctuations also resulted in low multiple correlation
coefficients and high variances. All results of the analyses made during
the laboratory axenic culture series are presented in Tables A-l through
A-72 of Appendix A. Those data represent the analyses of laboratory data
as taken from program BETA printouts.
Column headings in Tables A-l through A-72 inclusive, Appendix A
are as follows:
N = number of data points used in computing that particular
regression line
S 2 = the variance of data points about the mean of all data
H
points
S 2 = the variation of data points about the regression line
r
S 2/S2= the variance ratio which if referenced against appropriate
standard "F" tables would indicate the statistical validity
of the data
b = calculated y - intercept
k = the dieoff (-) or growth (+) rate coefficient of the typical
kt
C =Cn 10 formulation
R = the multiple correlation coefficient
The numbers of data points indicated for each experiment do not necessarily
correspond to the total number obtained during the duration of the run.
Where significant aftergrowth of test bacterial populations occurred, the
test results were not included in the computer program and those data are
discussed separately.
17
-------
18
Enteric Bacteria Dieoff Studies
Although single species of bacteria do not exist in nature with axenic
cultures of algae, without these basic data it would be impossible to assess
the true value of each algal species with respect to its effect on the dieoff
rate of the bacteria in question. Dieoff rate coefficients for virtually all
of the laboratory series involving axenic algal cultures and enteric bacteria
are presented in Table 3-1. Considering the series involving the blue-green
algal test species, it is easily discernible that no two species exerted the
same dieoff effect on any two bacterial species. Additionally, the dieoff
rate coefficients vary considerably. One primarily important conclusion
which is derived after examination of the data in Table 3-1 is that the com-
paratively rapid dieoff rates of enteric bacteria which occur in nature are
apparently not due to the effects of individual algal species.
Differences in the dieoff rate coefficients (hereinafter called "coefficients")
between series BG-I and BG-II were minimal. There was little difference
in the effects which algae had on the dieoff rates of the test bacteria when
the algae were exposed to the bacteria during algal log growth phase (Series
BG-II) or algal log growth phase (Series BG-I). Several of the coefficients
seen in Table 3-1 appear with relatively high ranges, as for example, fil-
trate of Anabaena cylindrica and Alcaligenes faecalis , -. 0230 + . 0570 day
As in this example, some actually exceed the numerical values of the coef-
ficients themselves. It is believed that significant fluctuations occurred
in the bacterial populations throughout the experiment duration because of
aggregation and adhesive phenomena which are constantly occurring. For
these reasons the seemingly high coefficient ranges are not to be regarded
as errors.
On the other hand, comparatively low coefficients were due to the
abilities of the test bacteria to derive nutritional benefits from the cellular
materials of the blue-green algae. One such material was the gelatinous
matrix which is a characteristic of all the blue-green algae. Additional
evidence of these occurrences can be seen by the coefficients derived from
the tests using blue-green algal filtrate. On a comparative basis it appeared
that prolonged survival of enteric bacteria occurred when blue-green algae
were present as compared to green algae.
-------
Table 3-1. Dieoff Coefficients for Series Utilizing Axenic Algal Cultures and Enteric Bacteria.
Series
Alcaligenes
faecalis*
Enterobacter
aerogenes*
Escherichia Proteus
coli* vulgaris*
Pseudomonas Serratia
aeruginosa* marcescens*
Anabaena cyUndrica
Mid-log inoc. BG-I
Contaminant redn.
BG-I
Day-0 inoc. BG-II
Contaminant redn.
BG-II
Filtrate BG-VII
-.0774+.0192 -.0854+.0107 -.0397+.0125 -.1000+.0213 -.0512+.0241 -.0366+.0404
0157+.0526 -.0175 +.0147 -.0173 +.1320 -.0046+.0118 -.0154 +.0066
!0582+.0419 -.0816 + .0557 -.1132 + . 0534 -.0687 + .0307 -.0702 +.0104
.0187 + .0054 -.0159+.0128 -.0175 + .0258 -.0094+.0215 -.0198+.0163
!o230+.0570 -.0316 +.0832 -.0164 +.0575 -.0253 +.1009 -.0167 +.0548
-.0027 + .0072
-.0583 + .0249
-.0234 + .0157
-.0118 + .0494
Anacystis nidulans
Mid-log inoc. BG-I
Contaminant redn.
BG-I
Day-O inoc. BG-II
Contaminant redn.
BG-II
Filtrate BG-VII
-.1145 + .0260 -.1172+.0239 -.0796+.0137 -.0899+.0161 -.0614+.0158 -.0480+.0099
.0051 + .0275
.0640 + .0320
.0197 + .0074
.0375 + .0684
.0078 + .0099
.0448 + .0225
.0213 + .0114
.0469 + .0405
.0114 + ..0105
.0596 + .0149
.0161 + .0168
.0212 + .0660
-.0177 + .0453
-.0522 + .0470
-.0279 + .0140
-.0525 + .1060
.0589 + .1216
.0474 + .0136
.0068 + .0263
.0316 + .0873
,0147 + .0059
,0437 + .0306
.0323 + .0133
,0301 + .1084
Gloeocapsa alpicola
Mid-log inoc. BG-I
Contaminant redn.
BG-I
Day-O inoc. BG-II
Contaminant redn.
BG-II
Filtrate BG-VII
-.0688+.0212 -.1356+.0357 -.0849+.0246 -.1205+.0980 -.0484+.0157 -.0466+. 0301
.0194 + .0159
.0868 + .0328
.0110 + .0130
.0395 + .0437
.0606 + .0075
.0838 + .0278
.0198 + .0117
.0596 + .0118
.0802 + .0516
.0494 + .0176
.0271 + .0265
.0656 + .0082
-.0462 + .0217
-.0794 + .0438
-.0139 + .0104
-.0473 + .0309
,0484 + .0157
.0781 + .0272
.0141 + .0130
.0197 + .0460
,0466 + .0301
.0596 + .0179
.0213 + .0111
.0208 + .2174
Oscillatoria chalybia
Mid-log inoc. BG-I
Contaminant redn.
BG-I
Day-O inoc. BG-II
Contaminant redn.
BG-II
Filtrate BG-VII
-.0966 +.0386 -.0637 +.0518 -.1255 +.0478 -.1153 +.0199 -.1149 +.0288 -.0607 +.0108
.0704 + .0311
.0761 + .0327
.0110 + .0052
.0996 + .1473
,0341 + .0121
,0571 + .0154
,0138 + .0066
.0963 + .1387
,0393 + .1598
,1986 + .0631
,0143 + .0087
.1012 + .2162
-.0468 + .0353
-.1074 + .0450
-.0037 + .0165
-.0949 + .1623
.0234 + .0272
.0614 + .0181
,0006 + .0112
.0847 + .0721
,0285 + .0226
,0905 + .0272
,0047 + .0130
,0805 + .2214
-------
Table 3-1 Continued
Alcaligenes
Series
Oscillatoria formosa
Mid-log inoc. BG-I
Contaminant redn.
BG-I
Day-O inoc. BG-II
Contaminant redn.
BG-II
Filtrate BG-VII
Phormidium faveolarum
Mid-log inoc. BG-I
Contaminant redn.
•BG-I
Dau-O inoc. BG-II
Contaminant redn.
BG-II
Filtrate BG-VII
Ankistrodesmus braunii
Mid-log inoc. G-I
Day-O inoc. G-II
Filtrate G-VII
Chlorella pyrenoidosa
Mid-log inoc. G-I
Day-O inoc. G-II
Filtrate G-VII
Chlorella vulgaris
Mid-log inoc . G-I
Day-O inoc. G-II
Filtrate G-VII
faecalis*
-.1546 +
-.0410 +
-.2957 +
-.0018 +
-.0761 +
-.0880 +
.0029 +
-.0880 +
-.0195 +
-.0697 +
-.0701 +
-.0857 +
-.0769 +
-.1586 +
-.0743 +
-.0925 +
-.1255 +
-.1003 +
-.0852 +
Enterobacter
aerogenes*
.0224
.0139
.2187
.0139
.3122
.0431
.0142
.0275
.0630
.1664
.0193
.0435
.2780
.0219
.0942
.2509
.1253
.0137
.1884
-.0910 +
-.0278 +
-.2218 +
-.0051 +
-.0732 +
-.0640 +
-.0368 +
-.0594 +
-.0174 +
-.0841 +
-.0764 +
-.0801 +
-.0965 +
-.1013 +
-.1265 +
-.0851 +
-.0949 +
-.1138 +
-.0656 +
.0450
.0191
.5164
.0158
.1638
.0460
.0216
.0367
.0189
.1405
.0172
.1205
.1696
.0781
.0148
.2041
.0858
.0155
.1201
Escherichia
coli*
-.0864 -f
-.0182 +
-.2275 +
-.0120 +
.0136 +
-.0966 +
-.0196 +
-.0552 +
-.0262 +
-.0646 +
-.0365 +
-.0542 +
-.0520 +
-.0624 +
-.0763 +
-.0708 +
-.0464 +
-.0407 +
-.0553 +
.0393
.0194
.0640
.0132
.0965
.0599
.0137
.0313
.0235
.2584
.0290
.1598
.0090
.0305
.1384
.3320
.0480
.1286
.1392
Proteus
vulgaris*
-.0964 +
-.0250 +
-.2231 +
-.0124 +
-.0678 +
-.1782 +
-.0266 +
-.1567 +
-.0142 +
-.0643 +
-.0756 +
-.0745 +
-.0904 +
-.1800 +
-.0552 +
-.0830 +
-.1826 +
-.1352 +
-.0757 +
Pseudomonas
aeruginosa*
.0507
.0272
.1002
.0044
.1897
.0757
.0347
.1194
.0247
.0767
.0288
.3424
.0590
.1094
.0419
.2321
.4619
.0176
.1238
-.0795 +
-.0428 +
-.0751 +
-.0058 +
-.0568 +
-.1378 +
-.0188 +
-.2437 +
-.0047 +
-.0704 +
-.1129 +
-.1977 +
-.0890 +
-.0985 +
-.0574 +
-.0550 +
-.1092 +
-.1901 +
-.0683 +
.0379
.0219
.0260
.0101
.0728
.0348
.0084
.0778
.0085
.1549
.0504
.0297
.0751
.0767
.1100
.3776
.0581
.0171
.2253
Serratia
marcescens*
-.0679 +
-.0451 +
-.0709 +
-.0129 +
-.0671 +
-.0999 +
-.0515 +
-.0456 +
-.0542 +
-.0811 +
-.0620 +
-.1753 +
-.0558 +
-.0855 +
-.0552 +
-.0686 +
-.0651 +
-.0989 +
-.0644 +
.0159
.0309
.0165
.0352
.2056
.1055
.0308
.0254
.2044
.1332
.0438
.0254
.1196
.0481
.0787
.3630
.0547
.0148
.2491
-------
Table 3-1 Continued
Alcaligenes Enterobacter Escherichia Proteus Pseudomonas Serratia
Series faecalis* aerogenes* coli* vulgaris* aeruginosa* marcescens*
Scenedesmus obliquus
Mid-log inoc. G-I -.0458 +.0365 -.0574 +.0238 -.0583 +.0215 -.0891 + .0506 -. 0777 +_. 0357 -. 0974 +_. 0354
Day-O inoc. G-II -.0655 +.0142 -.0792 +_. 0065 -.0541 +_. 0631 -.0629 + .0093 -.0466 +.2023 -. 0492 +_. 1126
FiltrateG-VII -.0324 +.0214 -.0508 +.0307 -.0401 + .0352 -.0151 + .0809 -.0550 +.0852 -.0325 +.0908
Mixed blue-greens,
single bacteria
inoc. BG-VIII -.2463 + .3895 -.2368+.5988 -.1666 +.1590 -.2392 +.6491 -.1730 +.1538 -.1479 + .2096
Mixed blue-greens,
mixed bacteria
inoc. BG-IX -.2600 +.4379 -.2741 + .7115 -.1081+2.433 -.1632 +.4947 -.1536 +.1237 -.1397 +.0671
Mixed greens,
single bacteria
inoc. G-VIII -.1635 +.0689 -.1462 +.0353 -.1280 +.0773 -.1912 +.4282 -.1744 +.0601 -.1493 +.0567
Mixed greens,
mixed bacteria
inoc. G-IX -.1176 +.3440 -.2082 + .2771 -.1417 +.1001 -.1743 +.5893 -.1493 +.1280 -.1579 +.0885
Brevibacterium sp. ,
effect on dieoff of ... -.0513+.0300 -.0494+.0127 -.0755+.0123 -.0951 + .0178 -.1011 + .0407 -.0624+.0168
Flaveobacterium sg. ,
effect on dieoff of ... -.1437 +.0945 -.1616 +.0743 -.0947 +.0431 -.1520 +.0306 -.1042 +.0519 -.0666 +.0333
Bacteria alone, dieoff
in algal growth medium,
VI -.0228+.0279 -.0097+.0317 -.0214+.0263 -.0149+.0325 -.0133+.0222 -.0098+.0283
Bacteria alone, anaerobic
dieoff rates of ... -.0131+. 0253 -.0352 +.0175 -.0490 +.0072 -.0131 +.0307 -.0315 +.0259 -.0563 +.0108 to
-------
Table 3-1 Continued
Series
Alcaligenes
faecalis*
Enterobacter Escherichia Proteus
aerogenes:
coli*
vulgaris *
Pseudomonas Serratia
aeruginosa * rnarcescens *
Growth rates during Series I
Anabaena cylindrica
Control .0130 +
.0021 .0117 + .0099
Anacystis nidulans
Control .0171 +
.0111 .0116 + .0148
Gloeocapsa alpicola
Control .0041 +
.0071 .0083 + .0128
Oscillatoria chalybia
Control .0121 +
.0021 .0084 + .0059
Oscillatoria formosa
Control .0290 +_
.0077 .0285 + .0174
Phormidium faveolarum
Control .0025 +
.0018 .0013 + .0116
Ankistrodesmus braunii
Control .0085 +
.0021 .0074 + .0051
Chlorella pyrenoidosa
Control .0199 +
.0121 .0143 + .0091
Chlorella vulgaris
Control .0102 +
.0037
Scenedesmus obliquus
Control .0075 ±
.0065 .0071 + .0091
.0107+.0079 .0043+..0139 .0114 +.0143 .0155 +.0101 .0152 +.0093
.0112 + ,0101 .0131 + .0034 .0064 +.0083 .0102 +.0173 .0113 +.0095
.0070 +.0072 .0085 +.0076 .0039 +.0109 .0049 +.0050 .0119 +.0060
.0067 + .0048 .0049 + .0123 .0189 + .0078 .0155 + .0101 .0152 + .0093
.0256 + .0115 .0209 + .0146 .0160 +.0197 .0101 +.0176 .0159 +.0187
.0005 + .0094 .0027 + .0088 .0048 +.0148 .0019 +.0118 .0059 +.0119
.0042 + .0061 .0087 +.0071 .0027 +.0031 .0049 +.0008 .0111 +.0029
.0171 + .0061 .0171 + .0091 .0199 +.0210 .0100 +.0067 .0121 +.0077
.0177+.0041 .0120+.0141 .0200 +.0040 .0099 +.0061 .0154+.0071 .0136 +.0088
.0089 + .0072 .0101 + .0214 .0116 +.0100 .0171 +.0065 .0144 +.0061
tsj
_ i
-------
23
Additional data on dieoff effects of enteric bacteria were obtained
using a typical soil inhabitant blue-green algal species, Nostoc muscorum.
Filtrate from an actively growing axenic culture of this organism was exposed
to the enteric bacteria in the same manner as the other filtrate series .
Since Nostoc is not ordinarily found as a phytoplankton member in waste
stabilization ponds, all of the series of combinations were not run on this
organism. The data for the filtrate run with Nostoc (Appendix A, Table A-50)
are included and intended for comparison with the data for the other enteric
bacterial-algal runs. The coefficients were: Alcaligenes, -.0862 + .2171
day" ; Enterobacter, -. 1021 +. 1180 day" ; Escherichia, -.0881+ .1177
day" ; Proteus, -. 102 1 + .0078 day" ; Pseudomonas, -.0639+ .1352 day" ;
Serratia, -.6020 +_ .0953 day . These are comparatively higher rates than
those obtained for many of the filtrate runs involving the other six species
of blue-greens. The most pronounced effect on any bacteria by Nostoc was
that exerted on Serratia. This bacteria appeared to be more resistant as
compared to the other species, yet the coefficient was-0.602 +_ .0953 day
Few genera were found to be persistent contaminants of the blue-green
algal cultures. Brevibacterium and Flaveobacterium were the two most
frequent contaminants, occurring primarily in filamentous blue-green species.
Coefficients for these two bacterial genera in control runs of the test blue-
green algae are shown in Table A-25, Appendix A. When enteric bacteria
were present, definite inhibitory effects were noted in the coefficients for
the culture contaminants (Table 3-1). At the same time, pronounced antag-
onistic effects of the enteric bacteria were noted, exerted by the contaminants
Coefficients were higher when the enterics were in the presence of Flaveo-
bacterium as compared to Brevibacterium. Coefficients for enteric dieoffs
were higher when in the presence of Flaveobacterium alone than when in
the presence of many of the axenic algal cultures , further evidence that
blue-green protoplasmic constituents were furnishing nutritional compounds
to the enteric bacteria.
A comparison of these coefficients with those obtained when the enteric
bacteria were placed in the sterile algal growth medium (Series VI control) is
noteworthy because of the differences which occurred as a result of any
-------
24
biological antagonism. Dieoff of enteric bacteria in algal growth medium under
anaerobic and aerobic conditions was similar, for the most part, to dieoff when
the enterics were present with the algae (Series BG-I, BG-II). Considering
the trace quantities of nutritive organics which were present in the algal
growth medium, those data demonstrate the persistent nature of the enteric
bacteria and their ability to survive in situations which would be considered
inadequate for life support of the bacteria.
Of the four species of green algae studied, Chlorella pyrenoidosa and
C. vulgaris exerted more antagonism than did Scenedesmus obliquus or
Ankistrodesmus braunii. Approximately similar dieoff rates for the enteric
bacteria occurred when in the presence of Scenedesmus obliquus and Ankistro-
desmus braunii and all the blue-green algae tested. Possibly Chlorella pro-
duced some substance such as chlorellin which was responsible for the
accelerated dieoff of the enterics. Numerically larger coefficients were
obtained for the series employing filtrate from the green algae (G-VTI). Most
of these coefficients were higher than those developed by the blue-green
algae under similar circumstances.
Perhaps the most significant runs were those incorporating mixed algae
and the additions of single bacterial and mixed bacterial species. The
resulting coefficients are shown in Table 3-1. Competition among algal
species for survival apparently accelerated the dieoff of the bacteria. Coef-
ficients calculated for the individual bacterial species were similar to the
rates for the same individual bacterial species when in mixed culture (com-
paring BG-VTII and BG-IX; G-VIII and G-IX). Oddly enough, in these series
a significant number of the coefficients were higher for the mixed blue-green
species than for the mixed green species. These data infer that the blue-
green algae secreted antibacterial substances when in the presence of other
blue-greens, whereas the green algae tested secreted their antibacterial
materials in heterogeneous populations or in axenic culture. No runs were
conducted with green algal contaminants due to their near total absence from
the cultures of green algae. For the mixed enterics with mixed blue-green
algae and separately with mixed green algae, coefficients were computed
for the total numbers of enterics present. The data are as follows: mixed
-------
25
blue-greens and enterics, -0.1536 + .0990 day~ ; mixed green algal species
and enterics, -.1487 + .0935 day" . These coefficients would correspond
to what is ordinarily considered to be a "total coliform" count dieoff coef-
ficient.
Only a small but significant part of the total research effort was devoted
to establishing the effects of the presence of enteric bacteria with algae.
Biomass of controls (axenic algal cultures) were compared with samples taken
during Series I runs. Coefficients representing these effects are presented
last in Table 3-1. In some instances, the presence of the enteric bacteria
effected a reduction in the total biomass productivity of the test algal species,
Pathogenic Bacteria Dieoff Studies
Five species of pathogenic bacteria were subjected to tests which were
similar to those involving the enteric bacteria. The dieoff coefficients for
those series are presented in Table 3-2 and Tables A-58 through A-72,
Appendix A. Considering the difficulty encountered in maintaining those pure
cultures of pathogens in the laboratory, their dieoff was slower when in the
presence of algae; however, no aftergrowth was found for any of those bac-
terial species. Considering the coefficients in Table 3-2, it would appear
that the blue-green and green algal test species had approximately the same
effect on those bacteria as they did on the enteric species. Surprisingly,
the mixed algal cultures did not exert as great an effect on the dieoff coef-
ficients of the pathogens as on those of the enteric bacteria. Coefficients
produced under anaerobic conditions were significantly lower than the rates
in the same medium under aerobic conditions. Therefore it may be concluded
that the algae had little effect on the pathogenic bacterial species.
Aftergrowth Potential Measurements
Extending the duration of the runs involving the test bacteria permitted
evaluation of one of .the original purposes for this investigation; namely,
identification of any aftergrowth potentials of each bacterial species tested.
Of the eleven species of bacteria tested, three demonstrated abilities to
regenerate their populations . These were Serratia marcescens , Pseudomonas
aeruginosa, and less frequently, Escherichia coli. The other enteric bac-
terial species as well as the pathogens apparently did not possess this
-------
Table 3-2. Dieoff Coefficients for Series Utilizing Pathogenic Bacterial Species
Salmonella
Series
Mid-log inoculation of ...
Anabaena cylindrica
Anacystis nidulans
Gloeocapsa alpicola
Oscillatoria chalybia
Oscillatoria formosa
Phormidium faveolarum
Ankistrodesmus braunii
Chlorella pyrenoidosa
Chlorella vulgaris
Scenedesmus obliquus
Mixed blue-green species
Mixed green species
Dieoff rates in algal
growth medium, Aerobic
Dieoff rates in algal
growth medium , Anaerobic
paratyphi*
-.0751
-.0840
-.0657
-.0613
-.0839
-.0790
-.0726
-.0950
-.0669
-.0833
-.0759
-.1553
-.0622
-.0207
+
+
+
+
+
+
+
+
+
+
+
+
+
+
.0098
.0174
.0401
.0309
.0199
.0106
.0193
.0495
.0334
.0276
.0259
.0617
.0454
.0293
Salmonella
typhosa*
-.0601 +
-.0758 +
-.0609 +
-.0523 +
-.0684 +
-.0602 +
-.0658 +
-.0986 +
-.0572 +
-.0700 +
-.0775 +
-.1156 +
-.0775 +
-.0268 +
Shigella
paradysenteriae*
.0351
.0157
.0156
.0303
.0093
.0195
.0181
.0386
.0317
.0381
.0244
.0402
.0174
.0262
-.0707 +
-.0981 +
-.0832 +
-.0673 +
-.0622 +
-.0791 +
-.0730 +
-.0996 +
-.0633 +
-.1061 +
-.0564 +
-.1702 +
-.0738 +
-.0492 +
.0141
.0317
.0087
.0330
.0071
.0181
.0250
.0261
.0322
.0411
.0187
.0586
.0276
.0146
Shigella
dysenteriae*
-.0742 +
-.1249 +
-.0745 +
-.0717 +
-.0688 +
-.0670 +
-.0707 +
-.0840 +
-.0700 +
-.0935 +
-.1124 +
-.1345 +
-.0728 +
-.0194 +
.0139
.0208
.0091
.0178
.0217
.0186
.0272
.0520
.0261
.0440
.0673
.0457
.0256
.0346
Vibrio
comma*
-.0511 +
-.0997 +
-.0755 +
-.0639 +
-.0706 +
-.0658 +
-.0593 +
-.0747 +
-.0465 +
-.0872 +
-.0933 +
-.1460 +
-.0625 +
-.0162 +
.0137
.0247
.0329
.0082
.0149
.0166
.0146
.0383
.0283
.0480
.0272
.0535
.0204
.0211
*log1(]day~
K>
CT)
-------
27
capability under the conditions of testing during these experiments. Data
for the aftergrowth, and times of occurrences in the test periods are presented
in Table 3-3. Aftergrowth was caused by the readily available protoplasmic
constituents of the algae as the algae reached their declining or log death
phase. The danger of recurrence of these bacterial species which showed
the aftergrowth potential is therefore present when sufficient organic nutrients
are present in the surrounding aquatic environment. And further, regardless
of the efficiencies of removal of any treatment process or design parameter
such as waste stabilization ponds, if absolutely 100 percent kill of these
bacteria is not accomplished, aftergrowth can indeed occur in the effluent
receiving-waters.
Organic Carbon Production by Algae
Little is known concerning the contribution by algae to the organic
carbon content of waters and the resulting effects of the organic carbon on
such parameters as bacterial survival or reproduction capacities. During
Series BG-I, G-I, BG-VIII, and G-VIII, measurements were made at the
90-day time period in an attempt to determine the maximum yield of total
carbon and total organic carbon by the algae, or biomass present in culture.
These data are presented in Table 3-4. Significant amounts of organic
carbon were present in the cultures after 90-days of testing. Comparison
with the controls reveals by yet another method that some inhibition by the
enteric bacteria on the overall productivity of the algae occurred. The con-
tribution by the bacteria to the organic carbon content was negligible in all
cases. This can be proven due to the fact that, on the average, it takes
12
10 bacterial cells to equal one milligram of biomass weight and the cells
are obviously not totally organic carbon. Consequently, the total organic
carbon values, as presented in Table 3-4, may be assumed to have been
derived from the algae themselves . These levels of organic carbon represent
adequate quantities for, at least, the survival of the enteric bacteria, if
not multiplication of same over a period of time.
-------
Table 3-3. Aftergrowth Characteristics of Enteric Bacterial Species with
Single Species of Algae (Series I).
28
Algal
Species
Anabaena
cylindrica
Anacystis
nidulans
Gloeocapsa
alpicola
Oscillatoria
chalybia
Phormidium
faveolarum
Ankistrodesmus
braunii
Chlorella
pyrenoidosa
Chlorella
vulgaris
Scenedesmus
obliquus
Bacterial
Genera
Pseudotnonas
Serratia
Pseudomonas
Serratia
Serratia
Pseudomonas
Escherichia
Pseudomonas
Serratia
Pseudomonas
Serratia
Pseudomonas
Serratia
Pseudomonas
Escherichia
Pseudomonas
Serratia
Min. No.
Bacteria
in run, No/ml
10,000
6,000
33,000
48,000
250
310
<100
<100
<100
<2,000
50,400
1,030
286
132
324
4,600
1,290
Day Min.
No.
Occurred
63
63
63
63
56
56
56
63
42
56
63
77
77
63
63
63
63
Aftergrowth ,
Max. No/ml
180,000
800,000
310,000
340,000
8,110
160,000
1,920
3,300
3,320
280,000
542,000
525,000
38,100
4,000
64,200
60,000
41,000
Day Max,
No.
Occurred
70
84
70
84
91
91
84
70
56
91
91
91
91
91
91
91
91
-------
Table 3-4. Total Carbon and Total Organic Carbon Content of Biomass After Ninety Days
(Series EG-I, G-l, BG-VIII, andG-VIII, in mg/1) .
Bacteria Added
Algal Alcaligenes
Species faecalis
T.C.
Anabaena
cylindrica 47
Anacystis
nidulans 36
Gloeocapsa
alpicola 64
Oscillatoria
chalybia 128
Oscillatoria
formosa 100
Phormidium
faveolarum 77
Ankistrodesmus
braunii 62
Chlorella
pyrenoidosa 50
Chlorella
vulgaris 53
Scenedesmus
obliquus 60
Mixed Blue-greens
BG-VIII 92
Mixed Greens
G-VIII 65
T.O.C.
41
34
56
124
88
73
41
37
41
41
58
61
Enterobacter
aerogenes
T.C. T.O.C.
60
49
67
79
198
81
57
61
59
62
83
67
58
45
58
69
154
73
43
50
43
40
49
65
Escherichia
coli
T.C.
47
36
71
64
206
69
67
47
60
61
118
137
T.O.C.
33
34
66
60
166
69
52
40
51
50
95
63
to Algal Culture
Proteus
vulgaris
T.C. T.O.C.
53
38
77
56
200
104
47
51
54
51
169
61
41
36
62
56
166
77
40
45
50
41
124
47
Pseudomonas
aeruginosa
T.C. T.O.C.
51
33
64
62
252
73
47
56
58
53
95
130
41
33
60
62
198
71
38
47
49
38
67
81
Serratia
marcescens Control
T.C.
49
41
58
86
120
69
59
56
55
57
77
94
T.O.C. T.C. T.O.C.
41 77 47
35 52 47
53 67 67
75 65 42
75 172 168
69 85 82
48 54 50
47 97 96
39 54 33
55 51 39
58
c-o
75 <-£>
-------
CHAPTER 4
LABORATORY AND FIELD WASTE STABILIZATION POND STUDIES
The objective of these experiments were to establish dieoff coefficients
for selected species of bacteria under conditions which would occur in
operational waste stabilization ponds. Two kinds of pond systems were
investigated; namely, laboratory scale units which were designed on a
volume detention time basis to closely correspond to the field units, and
field scale pilot units .
Laboratory Waste Stabilization Pond Studies
A diagram of the laboratory waste stabilization ponds depicting the
three different design concepts is shown in Figure 4-1. Throughout the
test period of approximately 60 days, supplementary data were taken on
phytoplankton populations to relate their concentrations to possible effects
on the bacterial populations. The procedure was as follows. A liter of
domestic wastewater was added to each of Series I, II, and III daily.
Series I differed from Series II and III in that Series I began the treatment
cycle with a six-liter anaerobic pretreatment chamber. Also, on a daily
basis, 0.5 liter of effluent from the anaerobic unit was added to each of
the two following facultative units. This was followed by the addition
of one liter, combined from each of the two facultative units, to the
maturation pond. In Series I the volume of the facultative units was 90
liters and that of the maturation pond was 18 liters. These volumes pro-
vided detention times of 6, 90, and 18 days respectively. The maturation
ponds in Series I, II, and III were similar.
Series II facultative units provided anaerobic treatment in an anaerobic
trench located at the influent end of each unit. The volume of these faculta-
tive units was 84 liters. The facultative units and the maturation pond
unit in Series III had the same volume as each of the similar units in
Series I. In Figure 4-1 the locations of bacterial inoculation points (i)
and sampling stations (numbers) are shown. Duplicated sampling station
30
-------
31
SERIES PRE-
TREATMENT
(AWSP)
INFLUENT
FACULTATIVE MATURATION
H
tfe
(FWSP) (MP)
^-BAFFLES
^T-
/
^
. _ .
2
*
3
v.
—
4
y
\
/
INOCULATION
STATIONS
INFLUENT
X
ANAEROBIC
TRENCH —
\
/
INFLUENT
X
1
_i
f
V.
8
X
9
v_
10
y
SAMPLE
STATIONS
/'
v^
^ — __t*
8
>
9
v
10
*
FIG. 4-1. SCHEMATIC OF LABORATORY PONDS
-------
32
numbers mean that equivalent volumes were sampled at those points and
mixed prior to analysis. Overall, the laboratory waste stabilization pond
units proved to be amenable to bacteriological analysis because of their
relatively small size, which permitted accurate bacterial inoculations.
Data for all bacteria counts were obtained by plating duplicates of
two dilutions from each sample. These data, representing the statistical
mean values of four counts per sample are presented in Appendix B as Tables
B-l through B-12. Blanks in these tables are the result of inconsistent
plating; or the types or species of bacteria in question did not appear on
the plates on that date; or the counts were too high to be statistically
valid. The gaps do not imply the absence of the bacteria. Incubation periods
were not consistent throughout the tests for the following reasons. In
many instances it was found that room temperature incubation, as opposed
to incubation at 35°C, enhanced some of the coliform species as well as
other bacterial species found on the plates used for the total counts.
Also, in several instances, periods of 72 hours of incubation were necessary
to obtain representative counts. This peculiar characteristic of many
bacterial species has been observed by the authors before. Therefore,
these data provide a more accurate account of the actual numbers of bac-
teria present at the sampling times than would have been recorded by
incubation for, say, only 24 hours at 35 C.
On July 7 and 29 cultures of Escherichia coli, Pseudomonas aeruqinosa,
and Serratia marcescens were inoculated into the selected locations in the
laboratory units (Figure 4-1). The total bacterial numbers for each inoculum
are presented in Table 4-1. By taking into account the daily additions of
wastewater, its complement of the bacteria in question, volumes of the
laboratory ponds and other pertinent quantitative physical factors, dieoff
coefficients for each of the three added species were calculated. These
coefficients are shown in Table 4-2 for the vicinities adjacent to the point
of inoculation. In the majority of cases the dieoff coefficients were much
higher than those found for the axenic culture experiments . The exceptions
to this can be seen in the data in Table 4-2 as for, by way of example,
Pseudomonas aeruginosa at station 2. Obviously, conditions prevailing
-------
33
Station*
Table 4-1. Bacteria Inoculated Into Selected Stations In
Laboratory Waste Stabilization Ponds
Species and Date
Escherichia
coli
Tuly 7 July 29
Pseudomonas
aeruginosa
July 7 July 29
Serratla
marcescens
Tuly 7 Tuly 29
1
2
5
6
7
8
11
13
12
12
11
13
12
11
.2406**
.2579
.4595
.6730
.1105
.5841
.2480
9
13
9
11
9
.3010
.8000
.0000
.3656
.3980
—
—
11
12
12
11
11
11
11
.4700
.1847
.3180
.7136
.8720
.6200
.7780
9
11
11
10
11
11
9
.9030
.6020
.6535
.0608
.9030
.4775
.7401
9.
11.
11.
12.
12.
13.
10.
9607
9240
1760
0415
6445
4345
2600
9.1760
11.3010
9.5440
—
—
9.6020
11.0000
*Locations indicated in Figure 4-1.
**Log _ total number inoculated.
-------
34
Table 4-2. Dieoff Coefficients of Inoculated Bacteria in Laboratory
Scale Waste Stabilization Ponds
Station Inoculated*
1
2
5
6
7
8
11
Coefficients
Escherichia
coli
-1.03
-0.44
-0.37
-0.31
-0.61
-0.37
-0.51
for Bacterial
Species (day"1)
Pseudomonas Serratia
aeruginosa
-0.34
-0.14
-0.93
-1.00
-1.10
-0.63
-1.26
marcescens
-0.43
-0.41
-1.38
-1.78
-1.53
-1.16
-1.15
*Inoculation point at or adjacent to indicated station; Figure 4-1.
-------
35
in the early stages of the treatment units caused higher dieoff coefficients
to occur than did those in the later stages. Anaerobic pretreatment did not
cause the accelerated dieoff for the three inocula bacteria species, expecially
Pseudomonas and Serratia, as was expected. It might be concluded that
the corresponding decreases in nutrient materials aided in acceleration of
that dieoff which did occur. It should be noted, however, that the addi-
tions of the inocula bacteria at station 8 resulted in reduced dieoff coeffi-
cients for two of the three test species. Fewer algae were present at that
station during part of the test period than were present at other stations
except station 11 (Table 4-4).
The reduced phytoplanktonic concentrations throughout all runs involving
the laboratory ponds in Series III appeared to be a characteristic of that
series throughout the test period. Of the three test bacteria, E. coli
appeared to possess the greatest capacity for survival through all three
types of pond combinations. Many of the enteric bacteria remained in
the final effluents. Examination of all three effluents revealed that the
quality of the effluent permitted some aftergrowth of all three test bac-
teria species. The increases in numbers of bacteria did not exceed two
orders of magnitude. However, the mere fact that aftergrowth did occur
is in itself additional evidence that a much greater understanding of the
behavior of these bacteria in ponds is needed.
Relationships between the bacteriological concentrations and the
corresponding phytoplankton concentrations may be observed by referring
to Tables 4-3 and 4-4 and Figures 4-2 through 4-5. Surprisingly few
diatoms were present in any of the treatment units during the course of the
study. Euglena sp. did not appear until on or slightly prior to August 20.
For the purposes of this investigation the intermittent appearances of
representative species of these divisions (Euglenophyta and Chrysophyta)
permitted evaluation of the two divisions which were of primary concern,
the blue-green algae (Cvanophyta) and the green algae (Chlorophyta).
Considering the behavior of the test species in the laboratory ponds ,
the following observations were made during the duration of the experiments.
Periodic increases in concentrations of total bacteria, as noted for day 36
-------
Table 4-3. Phytoplankton Found in Laboratory Waste Stabilization Ponds.
Algal
Division
Stat
Cyanophyta
Euglenophyta
Chrysophyta
Chlorophyta
Total
ion; 3
30,000*
(23,000)**
-0-
1,000
( 1,000)
1,400
(17,000)
32,400
(41,000)
Table 4-3 Continued
Algal
Division
Stat
Cyanophyta
Euglenophyta
Chrysophyta
Chlorophyta
Total
ion: 3
17,000
( 5,000)
1,000
( 1,000)
-0-
10,000
(34,000)
28,000
(40,000)
July 9
6
37,000
( 36,000)
-0-
-0-
1,500
( 2,000)
38,500
( 38,000)
August 20
6
13,000
(144,100)
500
( 400)
-0-
19,500
( 60,500)
33,000
(205,000)
9
-0-
-0-
-0-
6,700
( 8,000)
6,700
( 8,000)
9
4,000
(21,500)
100
( 100)
-0-
6,900
(38,400)
11,000
(60,000)
Date and Station Number
July 23 August 6
36936
17,000 52,000 4,000 10,700 37,200
(31,500) ( 47,000) ( 7,500) ( 7,000) ( 60,000)
— r\ A n — n~ — 0—
-°- , !:Z -°- -°- -«-
3,000 43,000 10,000 2,700 6,000
( 6,000) (187,000) (20,000) (28,000) ( 23,000)
20,000 96,000 14,000 13,400 43,200
(37,500) (235,000) (27,500) (35,000) (290.000)
Date and Station Number
9
-0-
-0-
-0-
4,000
(36,000)
4,000
(36,000)
*Areal StandardeUnits of phytoplankton per ml.
**No. of phytoplankton per ml.
OJ
-------
37
Table 4-4. Total Phytoplankton Concentrations Found In
Laboratory Waste Stabilization Ponds
Date
July 9
July 23
Aug. 6
Aug. 20
3
32,400*
(41,000)**
20,000
(37,500)
13,400
(35,000)
28,000
(40,000)
5
4,100
(12,000)
6,100
(11,000)
6,300
(16,000)
13,000
(27,500)
Station Number
6 7
38,500
( 38,000)
96,000
(235,000)
43,200
(290,000)
33,000
(205,000)
100
( 270)
2,500
( 3,500)
41,900
(52,500)
20,000
(31,500)
9
6,700
( 8,000)
14,000
(27,500)
4,000
(36,0001
11,000
(60,000)
11
3,000
( 6,000)
3,750
(13,000)
1,670
( 4,800)
9,600
(27,000)
*Areal Standard Units of phytoplankton per ml.
**( ) No. phytoplankton per ml.
-------
7
3 6
INFLUENT
STATION 7
(EFFL. H)
A/
STATION
(EFFL.m)
STATION 5
(EFFL.I)
0 Days 10 30 40
(Sampling Period July 3-Aug. 26)
50
FIG. 4-2. TOTAL BACTERIA, LABORATORY PONDS
-------
•
o
o.
0 Days
10 20 30 40
(Sampling Period July 3-Aug. 26)
FIG. 4-3. TOTAL COLIFORM, LABORATORY PONDS
-------
INFLUENT
STATION 5
(EFFL.I)
STATION I
(EFFL.IK
STATION
(EFFL
Days
10 20 30 40
( Sampling Period July 3- Aug. 26 )
FIG.4-4. E.coh, LABORATORY PONDS
-------
41
CO
10
9
8
£" 7 -
o
"2 6
o x
= r 5
2 e
en 4
3
2
I
o
X
O.
o i
o
0
STATION 6
STATION 3
STATION 9
STATION
STATION 9
STATION 3
7-9
7-23 8-6
SAMPLE DATE
8-20
FIG. 4-5. PHYTOPLANKTON DENSITIES
IN LABORATORY PONDS
-------
42
(Figures 4-2), were followed closely by a sudden increase in the total
coliform count (day 44, Figure 4-3). Only a small fraction of the total
coliform count was attributed to E. coli, per se. Examination of the plates
for the samples in question revealed colonial morphology similar to that for
Enterobacter, Alcaligenes , or Proteus , rather than Pseudomonas and Serratia .
The total coliform count as well as the populations of E. coli decreased
significantly on or about July 23 to about August 6 when the phytoplankton
populations were relatively high. During these times the blue-green popula-
tion was in the majority. Figures 4-2 through 4-4 relate other pertinent
points. Acclimitization of the pond systems was occurring for approximately
thirty days prior to any stabilization of the effluent quality so far as total
coliform or E. coli concentrations were concerned. Statistical means were
calculated for all values of effluent concentrations. These indicate the
systems' overall capability in reduction of the group or species in question.
Data for these follow:
Total Bacteria
Total Coliform
E. coli
Pseudomonas sp.
Influent
6.92
5.87
4.73
Effluent
Series I
(Station 5)
4.95
2.36
2.45
3.83
Effluent
Series II
(Station 7)
5.37
2.34
2.64
3.50
Effluent
Series III
(Station 11)
5.13
2.02
2.00
3.21
The values are all log-^Q of the mean number per milliliter. Values were
not calculated for Serratia because of its erratic occurrence and detecta-
bility. By the same token the presence of Pseudomonas in the influent
wastewater was known and detected; however, detection by standard plate
techniques was hampered due to the difficulty encountered in culturing
this genus at standard laboratory incubation temperatures.
The data presented above demonstrate that pond Series I were the
most efficient in reducing the total bacterial populations. The slightly
increased detention time may have been a contributing factor in this
reduction. For total coliform bacteria all three series exhibited similar
capabilities; however, Series III exhibited slightly more efficiency in this
-------
43
regard than did I or II. Similarly, Series III performed slightly more
efficiently than did Series I or II in reducing E. coli numbers as well as
Pseudomonas.
Escherichia coli, Pseudomonas aeruginosa, and Serratia marcescens
all exhibited some survival at the three effluent sample stations 5,7, and
11. Some correlation between the phytoplankton concentrations and the
bacterial densities were observed. Pseudomonas appeared in higher nub-
bers when reduced phytoplankton concentrations were present. E. coli
apparently was capable of survival in consistently higher numbers regard-
less of the phytoplankton concentrations. Serratia was rarely present in
consistently large concentrations in the effluents; however, the species
inoculated was apparently different from that (or those) present in the
wastewater. The periodic increases in Serratia (Tables B-9 and B-10,
Appendix B) were observed to be associated with clumps of blue-green
algae, a phenomenon which is common to all waste stabilization ponds
during the summer and early fall seasons .
The group of organisms which are reported in Tables B-ll and B-12
of Appendix B, the chromogens, includes such genera as Flaveobacterium
and Brevibacterium. These organisms were found to be present in waste-
water in high concentrations. Very little is knownabout their physiology,
pathogenicity (if any), or their contribution to the overall dieoff of the
coliform group. From the data presented in Tables B-ll and B-12 it can be
seen that aftergrowth of these bacterial genera did occur in the effluent
sampling station zones 5, 7 and 11. The comparatively high numbers of
these bacteria may have been responsible, at least in part, for a certain
amount of the dieoff of the coliforms and/or test species . This may have
occurred by either antibiosis or nutrient competition. At any rate, Flaveo-
bacterium and Brevibacterium will require considerably more research
effort before their exact contribution to waste treatment processes can be
effectively evaluated.
-------
44
Field Waste Stabilization Pond Studies
During and preceding the period of investigation with the laboratory
waste stabilization ponds, data were obtained on three series of field
waste stabilization ponds. These data were taken for a three-month period
beginning on June 4 and extending through August 26. A diagram of the
field ponds is shown in Figure 4-6. The laboratory waste stabilization
ponds described earlier in the text were designed to approximate the
detention times and types of systems of the field ponds. Series I was
preceded by an anaerobic pretreatment unit which had a volume of 8,900
cubic feet and a detention time of about 4 days. The facultative pond in
Series I had a volume of 117,500 cubic feet and a detention time of about
55 days. The small maturation ponds all had volumes of 18,000 cubic feet
and detention times of about 8 days . Series II and III facultative ponds
had volumes of 126,400 cubic feet and 126,300 cubic feet respectively with
detention times of about 51 days and 59 days each. Sample stations were
in the vicinities of the numbered areas of the ponds in Figure 4-6. Surface
samples were taken one foot below the surface and bottom samples were
taken about one foot from the bottom. This was done to avoid excessive
concentrations of algae at the surface and excessive amounts of settled
sludge at the bottom. Data obtained from these 12 sampling points are
presented in detail in Tables B-13 through B-28, Appendix B. Objectives
of this phase of the investigation were to compare the efficiencies of the
three different types of waste stabilization ponds as to coliform reduction,
to compare the efficiencies of the field and laboratory scale ponds, and to
attempt massive inocula of selected bacteria for dieoff studies.
Inoculations with laboratory-cultured bacteria occurred on the dates
indicated in Table 4-5. The numbers of bacteria which were inoculated
into those ponds in certain cases proved to be ineffective as tracer method-
ology for dieoff studies. Dieoff of the test species was rapid in the field
ponds. Calculating by volume of the ponds and daily flow rates from the
influent to the sample stations indicated the following dieoff coefficients
(as log1Q day ).
-------
45
ANAEROBIC
MATURATION
4(S)
5(B)
FACULTATIVE
INFLUENT
FACULTATIVE
7(S)
8(B)
C AMAPRORir
MATURATION
—I/
ANAEROBIC TRENCH
9(S)
nr
IO(S)
1KB)
FACULTATIVE
MATURATION
SAMPLE STATIONS
(S)-SURFACE (B)- BOTTOM
FIG. 4-6. SCHEMATIC OF WASTE STABILIZATION PONDS
-------
46
Table 4-5. Bacteria Inoculated Into Selected Stations
of Waste Stabilization Ponds
Station
2
4
4
7
7
10
10
Date
June 5
June 5
June 16
June 19
July 29
June 23
July 2 1
Escherichia
coli
13.2265*
12.9350
13.7164
13.9235
15.0281
13.4510
14.0720
Pseudomonas
aeruginosa
14.0592
12.9351
14.2812
14.0214
15.1146
14.1565
14.5761
Serratia
marcescens
—
—
14.1763
13.9855
14.4340
14.5513
14.2140
*log. n numbers per ml.
-------
47
Sample Stations
Escherichia coli
Pseudomonas aeruginosa
Serratia marcescens
#4
-1.42
-0.89
-1.81
#7
-1.67
-1.10
-2.02
#10
-1.21
-0.91
-.199
These coefficients were, without exception, higher than those found for
the laboratory scale ponds, indicating accelerated antibiotic activites.
Solar radiation contributed to some extent to those accelerated rates.
Data for total bacteria, total coliform bacteria, and phytoplankton popula-
tions are shown in Figures 4-7 through 4-9, and Tables 4-6 and 4-7.
Those data indicate, as in the case of the laboratory waste stabilization
pond studies, that a period of acclimatization was occurring for approxi-
mately half the test period of three months. Means of total bacteria,
total coliform bacteria and £_._ coli concentrations were calculated for the
raw influent (station 1) and the three effluents for the test period. They
were as follows (as log1Q No./ml).
Series I Series II Series III
Influent Station 6 Station 9 Station 12
Total Bacteria
Total Coliform
E_. coli
These values indicate that reduction of the total bacteria was not as effi-
cient as could be hoped for. Total coliform bacteria were reduced signifi-
cantly, with the majority of reduction being due to the dieoff of E._ coli,
per se.
Algae which were present in the ponds throughout the test period were
predominantly blue-green and green algae, as was the case for the labo-
ratory waste stabilization ponds. Surprisingly high concentrations of
algae were found at the surface of the anaerobic pretreatment pond of
Series I, contributing, no doubt, to some aerobic activity. There appeared
to be a direct relationship between the high phytoplankton concentrations
in August and consistently lower coliform counts. Increases in total coliform
7.14
5.83
5.26
5.04
1.35
0.34
4.91
1.31
0.78
5.25
1.37
0.57
-------
'. r
•
•
-
STATION
(INFLUENT)
STATION 12
EFFL JH
STATION 9
(EFFL.n
STATION 6
(EFFL.I)
0 Days 5
15
20
25 30 35 40 45 50 55 60 65 70
( Sampling Period June 4 - Aug. 26 )
FIG. 4-7. TOTAL BACTERIA IN WASTE STABILIZATION PONDS
75 80 85
-------
~ 5
o
C7>
O
- 4
-,
>\
STATION 6
/;
/ <
/ i
STATION 9
(EFFL. H)
(EFFL. I) ' ',
STATION 12
(EFFL. HI)
0 Days 5 10 15 20 25 30 35 40 45 50 55 60 65 70
( Sampling Period June 4 -Aug. 26)
FIG. 4-8. TOTAL COLIFORM IN WASTE STABILIZATION PONDS
75 80
85
-------
50
en
10
9
8
-------
Table 4-6. Phytoplankton Found in Waste Stabilization Ponds
Algal
Division
Station: 4
C y_a ripphyta.
Euglenophyta
Chrysophyta
Chlorophyta
Total
5,000*
( 7,500)**
-0-
-0-
19,100
( 42,500)
24,100
( 45,000)
June 1 1
7
5,500
( 4,800)
-0-
-0-
15,000
(3_1,200J
20,500
(36,000)
10
4
( 2
1
( 1
,100
,500)(
,500
,750)(
-0-
37
(612
43
(617
,500
,750)(
,100
,000)(
Date
4
4,200
8,000)
600
650)
-0-
40,150
51,350)
45,000
60,000)
and
Station
June 25
7
5
( 5
,000
,000)
-0-
-
20
(22
2b
(27
0-
,000
,500)
,000
,500)
Number
10
1,500
( 4,700)
-0-
-0-
47,500
(445,300)
49,000
(450,000)
4
7,000
( 22,000)
-0-
-0-
24,500
L27,OOOJ_
31,500
( 49,000)
July 2
7
3,400
( 3,000)
4,000
( 3,000)
-0-
6,100
( 8,0001
13,500
(14,000)
10
21,000
( 24,500)
6,000)
( 4,500)
-0-
54,000
(488,000)
81,000
(517,000)
Table 4-6 Continued
Algal
Division
Station: 4
Cyanophyta
Euglenophyta
Chrysophyta
Chlorophyta
Total
72,000
(145,000)
-0-
-0-
9,000
( 20,000)
81,000
(165,000)
July 23
7
9,000
(17,000)
-0-
900
( 1,000)
16,000
(57,000)
25,400
(75,000)
1
1
(
-1
.0
,000
50 OX
3- ,
-0-
3
( 19
4
( 20
,900
,500)(
,900(
iOOO)(
Date
4
20,000
8,000)
1,000
1,000)
-0-
4,000
123,000)
25,000
132,000)
and
Station
August 6
7
21
(10
1
( 1
44
(50
67
(62
,200
,000)
,500
,200)
0-
,300
,800)
,000
,000)
Number
10
15,500
( 12,000)
250
( 100)
-0-
5,250
( 13,400)
21,000
( 25,500)
4
3,000
( 2^000]
2,000
( 1,000)
-0-
13,000
(207,000i_
17,000
(210,000)
August 20
7
82,000
(17,000)
-0-
-0-
11,000
(22,000)
93,000
(39,000)
10
-0-
-0-
-0-
40,000
( 27,000)
40,000
( 27,000)
*Areal Standard Units of phytoplankton per ml.
**( ) No. of phytoplankton per ml.
en
-------
Table 4-7. Phytoplankton Concentrations Found In Waste Stabilization Ponds
Station Number
Date
June 11
June 25
July 2
July 23
Aug. 6
Aug. 20
2
1,100*
( 900)**(
2,100
( 1,300)(
96,900
(2,001,000)(1
16,670
( 27,200)(
13,700
( 20, 000) (
17,500
( 25,500)(
3
3,
,009
60
80
7
13
6
11
4
500 24,
400) ( 45,
250 45,
100) ( 60,
600 31,
,000)( 49,
,000 81,
,000) (165,
,500 25,
,500) (132,
,500 17,
,500) (210,
5
100 22,500
OOOX 30,000)
000 30,000
OOOX 27,000)
500 28,700
000)( 55,000)
000 88,000
000)(175,000)
000 22,500
OOOX 35,750)
000 19,500
000)(218,000)
6
7,000
( 13,000)
6,500
( 12,500)
6,400
( 17,000)
66,000
(285,000)
25,500
( 38,500)
40,000
( 67,500)
7
20,500
(36,000)
25,000
(27,500)
13,500
(14,000)
25,900
(75,000)
67,000
(62,000)
93,000
(39,000)
8
17,000
(22,500)
7,900
(15,000)
4,600
( 6,000)
2,000
( 3,000)
11,500
(13,000)
21,000
(19,000)
9
5,500
(11,000)
4,900
( 7,200)
9,800
(23,000)
19,700
(21,000)
21,500
(30,000)
8,100
(13,400)
10
43,100
(617,000)
49,000
(450,000)
81,000
(517,000)
4,900
( 20,000)
21,000
( 25,500)
40,000
( 27,000)
11
11,000
( 13,500)
6,200
( 12,500)
31,900
(555,000)
5,000
( 38,000)
13,700
( 33,000)
1,000
( 1,100)
12
2,700
( 3,500)
3,850
( 14,000)
26,000
(380,000)
7,850
( 43,000)
7,000
( 11,000)
14,000
( 17,500)
*Areal Standard Units of phytoplankton per ml.
**( ) No. of phytoplankton per ml.
en
-------
53
counts in late August (day 70 on) corresponded closely with the compara-
tively lower algae counts for the period.
Survival of Pseudomonas sp. in these ponds was apparently very
difficult as can be seen by the data in Tables B-22 through B-24. Some
carryover and aftergrowth was evident in the maturation pond of Series III
(station 12). That particular station had a bloom of Brachionus around
July 17 in concentrations of up to 200 per milliliter in the surface waters.
Those large numbers should have significantly reduced the bacterial popula-
tion in that time period; however, no significant reduction was noted.
On many occasions the concentration of coliform bacteria were lower in
the deeper waters than in the surface waters. It is entirely possible
that a greater amount of antibiotic activity was occurring in the deeper
waters. Apparently few Serratia sp. were present in the influent waste-
water. Those individuals which were present appeared to survive until
reaching the facultative or maturation ponds. Some aftergrowth of this
genus was observed in the maturation ponds. The chromagens exhibited
very low, if any, dieoff in most instances. Their higher numbers may
have contributed to the overall comparatively efficient reduction of the
coliforms . It is clear that the field waste stabilization ponds were
more effective in bacterial reduction than were the laboratory units, so
far as the coliform group were concerned.
These investigations on the field pilot waste stabilization ponds
and the laboratory ponds were accomplished in conjunction with investi-
gations in progress under grant WTRD 178-01-68, Federal Water Pollu-
tion Control Administration, "Design Guides for Selected Wastewater
Treatment Processes."
With the information presented by this investigation, remaining
efforts in this important area of sanitary engineering should be directed
toward establishing: (a) guidelines on amounts of disinfectant necessary
to eliminate proportions of enteric bacterial populations to meet effluent
specifications; and (b) cost-benefit specifications based on the as yet
unknown pathogenicity of other bacterial species found in waste stabiliza-
tion ponds and related wastewater treatment systems. An undeniable
need exists at the present time for this information.
-------
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pyrenoidosa TX7110-5 and Various Bacteria," Jour. Gen. Microbiol.,
42., 123-131 (1965).
53. Vladimirava, M. G. "Dynamics of the Bacterial Microflora Growth in
Chlorella Cultures," Mikrobiologiya, 30., 374 (1960).
54. David, E. M., Wilcomb, M. J., Reid, G. W. "Algal Succession and
Bacterial Reduction in Bio-oxidation Ponds," Proc. Okla. Acad.
Sci., 45_, 220-227 (1965).
55. Gloyna, E. F. "Design Considerations as Based on Disease Transmission,"
Chapter 6, Monograph on Waste Stabilization Ponds, World Health
Organization (1966).
56. Gloyna, E. F. "Low Cost Waste Treatment-Waste Stabilization Ponds,"
Proceedings, United Nations Conf. on the Application of Science
and Technology for the Benefit of Less Developed Areas, Geneva,
Switzerland (1963).
57. Gloyna, E. F., Suwannakarn, V. "Efecto de la temperature en el tratamiento
de aguas residuales mediante estangues de establization," Boletin
do la Oficina Sanitaria Panamericana, _56_ (2), 128-139 (1964).
58. Gloyna, E. F., Espino de la O., E. "Formal Discussion of Paper 1-6,"
Presented at the International Conference on Water Pollution Research,
Munich, Germany, ^September 5-9, 1966). (Paper deals with work
accomplished under Grant WP00688-02.)
59. Hermann, E. R., Gloyna, E. F. "Waste Stabilization Ponds, Part III,
Formulation of Design Equations," Sewage and Industrial Wastes,
30, 963-975 (August, 1958).
60. Malina, J. F., Jr., Yousef, Y. A. "The Fate of Coliform Organisms in Waste
Stabilization Ponds," Jour. Water Poll. Cont. Fed., 36. (11), 1432-
1442 (1964).
-------
APPENDIX A
STATISTICS OF ALL LABORATORY AXENIC CULTURE STUDIES
Key to Appendix A tabulated columns:
N = number of data points used in computing that
particular regression line
2
S = the variance of data points about the mean of
H
all data points
2
S = the variation of data points about the regression
line
2 2
S /S = the variance ratio which if referenced against
H r
appropriate standard "F" tables would indicate
the statistical validity of the data.
b = calculated y-intercept
k = the dieoff (-) or growth (+) rate coefficient of
kt
the typical C = Cn 10 formulation
R = the multiple correlation coefficient
59
-------
60
Table. A-1
Reduction statistics of enteric bacteria species with Anabaena
cylindrica. Series BG-I. Bacteria added to algae when in mid-log growth phase,
N
Alcaligenes faecalis
12 57.966 0.6783
Enterobacter aerogenes
12 227^04 0.2110
Escherichia coli
12 35.917 0,2882
Proteus vulgaris
12 79.048 0,8310
Pseudomonas aeruginosa
13 17,007 0.6349
Serratia marcescens
14 3.1013 1,7802
R
(8.7213*.8873) (-.0774-.0192) 0.892
(8, 3621* ,4949) (-.0854*^0107) 0.970
(8.0230-.5784) (-.0397-.0125) 0,837
(8.3912* ,9822) (-. 1000*.0213) 0,919
(8.2665*.9368) (-.0512*.0241) 0.739
(6,8402*1.5687) (-.0366*. 0404) 0,341
Table A-2
Reduction statistics of enteric bacteria species with Anacystis nidulans,
Series BG-I. Bacteria added to algae when in mid-log growth phase.
SH2/Sr2 Sr2 b k
N
Alcaligenes faecalis
12 78.809 0,4033
Enterobacter aerogenes
12 97.721 0.3406
Escherichia coli
12 116,24 0;3905
Proteus vulgaris
12 111.76 0.3614
Pseudomonas aeruginosa
13 57*109 0.2387
Serratia marcescens
14 83.845 0.1375
R
(7.9753*.8313) (-. 1145*. 0260) 0.940
(7U7923*.7639) K1172-.0239) 0.951
(8.1602-.6598) (-.0796-.0137) 0.936
(7,618li.6798) (-.0899-.0161) 0,941
(7.8915-.5883) (-.0614-. 0158) 0.-904
(8c3103-u4194) (-.0480-.0099) 0,923
-------
61
Table A-3
Reduction statistics of enteric bacteria species with Gloeocapsa alpicola.
Series BG-I. Bacteria added to algae when in mid-log growth phase.
N SH2/Sr2 Sr2 b k R_
Alcaligenes faecal is
12 36.494 0.9304 (6.5049*1.0184) (-.0688*.0212) 0,820
Enterobacter aeroqenes
12 79.796 0.1942 (8.1088 + . 7585) (-.1356^.0357) 0.964
Escherichia coll
12 44.764 0.5826 (7.7978±.9192) (-.0849*.0246) 0.882
Proteus vulgaris
12 8.3744 1.4614 (8.3445*2,0807) (-. 1205*.0980) 0.736
Pseudomonas aeruginosa
13 52.261 0.1064 (7.7185*,5751) (-. 0484*. 0157) 0.946
Serratia marcescens
14 20.3386 0.1045 (8.0011*. 7897) (-.0466*.0301) 0.910
Table A-4
Reduction statistics of enteric bacteria species with Oscillatoria chalybia.
Series BGI. Bacteria added to algae when in mid-log growth phase.
N SH2/Sr2 Sr2 b k R
Alcaligenes faecalis
12 22.511 2.0704 (6.5580*1.6270) (-.0966-.0386) 0.763
Enterobacter aerogenes
12 6.1434 1.5995 (6.1868-1.6554) (-.0637-.0518) 0.551
Escherichia coli
12 31.303 0.6905 (6.4209-1.2376) (-. 1255*. 0478) 0.887
Proteus vulgaris
12 136.60 0.2356 (6.9627-.6353) (-. 1153*.0199) 0.965
Pseudomonas aeruginosa
13 64.500 0.4957 (8.4764*.9215) (-.1149*00288) 0.928
Serratia marcescens
14 109,20 0.2419 (7.4993*.5193) (-.0607*.0108) 0.932
-------
62
Table A-5
Reduction statistics of enteric bacteria species with Oscillatoria formosa
Series BGI. Bacteria added to algae when in mid-log growth phase.
> n 2 1_ L. D
N
Alcaligenes faecalis
12 216.93 0.1511
Enterobacter aeroqenes
12 14.656 3.7099
Escherichia coli
12 17.347 2.8252
Proteus vulgaris
12 12.962 4.7076
Pseudomonas aeruginosa
13 15.716 2.6409
Serratia marcescens
14 65.150 0.4653
(8o6823*.5790) (-. 1546*.0224) 0.982
(7.3945*2.0752) (-.0910±. 0450) 0.677
(7.1810-1.8109) (-.0864-.0393) 0.712
(6.5127-2.3376) (-.0964*. 0507) 0.649
(7.1070*1.7509) (-.0795*.0379) 0.692
(7.6482^.7349) (-.0679-.0159) 0.903
Table A-6
Reduction statistics of enteric bacteria species with Phormidium faveolarum.
Series BGI. Bacteria added to algae when in mid-log growth phase.
SH2/Sr2 Sr2 b k
N
R
(6.9144*1.9852) (-.0880*. 0431) 0.681
Alcaligenes faecalis
12 14.979 3.3952
Enterobacter aerogenes
12 6.9355 3.8784 (5.0351*2.1218) (-.0640*. 0460) 0.498
Escherichia coli ,
12 9.8227 3.9133 (5.0539*2.3258) (-.0966-.0599) 0.621
Proteus vulgaris ,
12 25.151 1.7319 (6.2764-1.9600) (-. 1782-. 0757) 0.863
Pseudomonas aeruginosa
13 71.313 0.3654 (7.1779-.9003.) (-. 1378*. 0348) 0.947
Serratia marcescens
14 4.9669 1.6954 (6,8908*2.2411) (-.0999*.1055) 0.623
-------
63
Table A-7
Reduction statistics of bacterial contaminants of Anabaena cylindrica
during series BG-I with enteric bacteria .
I Q 2 u v
N
Escherichia coli
0.1714
45.860 0.0051
Serratia marcescens
12
0.7622 0.0224
R
Alcaligenes faecalis
12 0.7611 0.3184 (9.3911-1.3784) (-.0157±.0526) 0.276
Enterobacter aerogene_s
12 12.004 0.0249 (9,7528^.3856) (- .0175*.0147) 0.857
(9.6374-2.3863) (-.0173-. 1320) 0.408
12 0.6891
Proteus vulqaris
11 0.8337 0.0605 (9.7131-.4338) (-.0046-.0118) 0.217
Pseudomonas aeruginosa
12
(9.3210^.1736) (-.0154-o0066) 0.958
(9.6187-. 2641) (-. 0027-. 0072) 0.203
Table A-8
Reduction statistics of bacterial contaminants of Anacystis nidulans
during series BG-I with enteric bacteria.
SH2/Sr2 Sr2 b k
N
R
Alcaligenes faecalis
12 0.1918 0.3267 (8.4975-1.0079) (.0051*.0275) 0.060
Enterobacter aerogenes
12 5.2952 0.0113 (9.7894-.2597) (-. 0078*.0099) 0.726
Escherichia coli
12 6.5719 0.0472
Proteus vulgaris
12 0.8462
(9.4214*.3833) (- ,0114±. 0105) 0.686
0.8866 (8.9210-1,6604) (-.0177-.0453) 00220
Pseudomonas aeruginosa
12 2.0035 1,6987 (9.9329-3,1842) (-,0589-.1216) 0,500
Serratia marcescens
12 34.179 0.0150
(9.2648-.2163) (-.0147^.0059) 0.919
-------
64
Table A-9
Reduction statistics of bacterial contaminants of Gloeocapsa alpicola
during series BG-I with enteric bacteria.
" " Sr2 b k
N
R
(8. 9674-. 2769) (- .0606- .0075) 0.992
Alcaligenes faecalis
12 12.7660 0.0290 (7.6995*.4162) (-.0194^.0159) 0.864
Enterobacter aerogenes
12 355.73 0,0246
Escherichia coli
12 20.605 0,3061 (8.2531*1.3517) (-.0802*. 0516) 0.911
Proteus yulgaris
12 25.218 0.2025 (8.5118-. 7937) (-.0462*.0217) 0.894
Pseudomonas aeruginosa
12 52,609 0.1064
Serratia marcescens
12 20.338 0.1050 (8.0011*.7897) K0466*.0301) 0.910
(7.7185-.5751) (- = 0484- .0157) 0,946
Table A-10
Reduction statistics of bacterial contaminants of Oscillatoria chalybia
during series BG-I with enteric bacteria.
N SH2/Sr2 Sr2
Alcaligenes faecalis
12 43.578 0.1113
Enterobacter aerogenes
12 44.1414 0,0632
Escherichia coli
13 Oo5146 2,9367
Proteus yulgaris
11 9.7113 0,5394
Pseudomonas aeruginosa
12 6.3348 0,0851
Serratia marcescens
12 13,609 0.0587
R
(5.1798-.8152) (-.0704-.0311) 0.956
(5.9133*04432) (-.0341-.0121) 0.936
(5,6901-4,1866) (-.0393-. 1598) 0.205
(6,9309*1.2951) (-,0468±.0353) 0.764
(7n8599±.7l27) (-.0234-.0272) 0.760
(8.8888*05918) (-.0285-.0226) 0.872
-------
65
Table A-11
Reduction statistics of bacterial contaminants of Oscillatoria formosa
during series BG-I with enteric bacteria 0
SH2/Sr2 Sr2 b k _
N
R
(9,6125±.5447) (-.0410*.0139) 0.941
(9,7453-.7484) (-.0278-.0191) 0.796
Alcaliqenes faecalis
12 48.049 0,1016
Enterobacter aeroqenes
12 11.688 0.1919
Escherichia coli ,
9478621 0.1967 (9,8661^.7578) (-«0182±00194) 0.618
Proteus vulgaris , ,
12 7.222 0.0850 (9,6902-.7123) (-.0250^,0272) 0.783
Pseudomonas aeruqinosa +
H2l7l77 0.2511 (10.0314^.8560) (-.0428-.0219) 0.876
Serratia marcescens
1118.190 0.1096 (9.7702^.8088) (-.0451^.0309) 0.901
Table A-12
Reduction statistics of bacterial contaminants of Phormidium faveolarum
during series BG-I with enteric bacteria.
>. o 2 K k
N
R
Alcaliqenes faecalis ,
12 0.2333 0.1059 (7,2847-.5559) (.0029^.0142) 0.072
Enterobacter aeroqenes
12 ^099 0.2447 (9.0394-.8451) (-.0368±.0216) 0.843
Escherichia coli , , x „„,
12 il."352 0.0985 (8.7132-.5361) (-.0196^.0137) 0.791
Proteus vulqaris ,
IT" 3.2495 0.630.8 (9.0526-1.3569) (-.0266^.0347) 0.520
Pseudomonas aeruqinosa
12 27.377 0.0374
Serratia marcescens
12 23.895 0.1088
(8.7549^.3303) (-.0188^.0084) 0.901
(8,7565^.8059) (-n0515^.0308) 0.923
-------
66
Table A-13
Reduction statistics of enteric bacteria species with Anabaena cylindrica .
Series BG-II, Bacteria and algae inoculated within
twenty-four hours of one another.
N SH2/Sr2 Sr2 b k R
Alcaligenes faecalis
11 6.459 4,5436 (5,4092*2.0979) (-.0582-.0419) 0.418
Enterobacter aerogenes
9 7.704 4.2371 (5.6137*2.2365) (-.0816-.0557) 0.524
Escherichia coli
9 16.612 3.8936 (6.9423*2,1439) (-. 1132-.0534) 0.697
Proteus vulgaris
12 16.439 3.1431 (5,6887-1,6783) (-.0687-.0307) 0.622
Pseudomonas aeruginosa
12 14.83 0.3628 (7.3219-.5702) (-.0702-.0104) 0.937
Serratia marcescens
12 18.062 2.0567 (5.8236-1,3576) (-.0583*.0249) 0.644
Table A-14
Reduction statistics of enteric bacteria species with Anacystis nidulans.
Series BG-II. Bacteria and algae inoculated within
twenty-four hours of one another.
N SH2/Sr2 Sr2 b k R
Alcaligenes faecalis
12 15.144 0,9024 (6.0191*1.1080) (-.0640*.0320) 0.716
Enterobacter aerogenes
12 13.014 1.6865 (5,3492*1,2294) (-,0448^,0225) 0.565
Escherichia coli
12 52.405 0.7412 (5.6975*.8150) (-,0596*. 0149) 0.840
Proteus vulgaris
10 4.2597 5.0709 (4.4056*2.2207) (-. 0522*. 0470) 0.347
Pseudomonas aeruginosa
11 40.969 0,5410 (7.0880*.7078) (-.0474*.0136) 0.820
Serratia marcescens
12 6.6982 3.1234 (5,7883*1,6731) (-.0437-.0306) 0.401
-------
67
Table A-15
Reduction statistics of enteric bacteria species with Gloeocapsa alpicola.
Series BG-II, Bacteria and algae inoculated within
twenty-four hours of one another.
N
Sr<
Alcaligenes faecalis
12 23.541 2.7722
Enterbbacter aerogenes
12 30.543 1.9952
Escherichia coli
12 27.325 0,7285
Proteus yulgaris
12 11.356 3.6999
Pseudomonas aeruqinosa
12 27.164 2.4549
Serratia marcescens
12 36.312 1.0680
R
(6.0652-1.6387) (- .0868-.0328) 0,723
(5,5931*1.3902) (-.0838*. 0278) 0.772
(5.0102^.8524) (-.0494*.0176) 0.773
(5.1014-1.9798) (-.0794*00438) 0.587
(6.4011-1.4833) (-.0781^.0272) 0.731
(5.4419-.9783) (-.0596^.0179) 0.784
Table A-16
Reduction statistics of enteric bacteria species with Oscillatoria chalybia
Series BG-II. Bacteria and algae inoculated within
twenty-four hours of one another.
N
SH2/Si
Alcaligenes faecalis
12 18o249 2.7641
Enterobacter aeroqenes
12 46,006 0.6161
Escherichia coli
12 44.993 0.751
Proteus yulgaris
12 21.521 1.7036
Pseudomonas jaeruginosa
12 41.208 0.4405
Serratia marcescens
12 38.386 1.4187
R
(5,4482*1.6169) (-.0761^.0327) 0.670
(5.4358*.7633) (-.0571^.0154) 0.836
(8.2274-1.3374) (-.1986^.0631) 0.918
(5,5990*1.5143) (-.1074^.0450) 0.782
(6.4460^.7140) (- ,0614-.0181) 0.855
(6.7981-1.2119) (-.0905-.0272) 0.827
-------
68
Table A-17
Reduction statistics of enteric bacteria species with Osciliatoria formosa
Series BG-II. Bacteria and algae inoculated within
twenty-four hours of one another.
N SH2/Sr
2 o_2 h k R
Alcaligenes faecalis
12 15.581 1.3746 (6.8638-2.8644) (-.2957-.2187) 0,886
Enterobacter aerogenes
12 7.3510 0,6556 (6.8156*4.6670) (-.2218*.5164) 0.880
Escherichia coli
12 69.975 0=3625 (8,0650-1.0976) (-.2275-.0640) 0.959
Proteus vulgaris
12 27.448 0.8883 (7,9273-1.781) (-.2231*.1002) 0.901
Pseudomonas aeruginosa
12 27,483 2,2444 (6,7767*1.4182) (-.0751-.0260) 0.733
Serratia marcescens
12 60.798 0,9046 (6.5623-.9004) (-,0709-.0165) 0,859
Table A-18
Reduction statistics of enteric bacteria species with Phormidium faveolarum.
Series BG-II. Bacteria and algae inoculated within
twenty-four hours of one another.
N SH2/Sr2 Sr2 b k R
Alcaligenes faecalis
12 35.483 1,4550 (5.5216-1,2415) (-,0880*.0275) 0.816
Enterobacter aerogenes
12 9.3918 1,8409 (4.7479*1.4742) (-.0594*.0367) 0.573
Escherichia coli
12 10.2230 3.2589 (5.1780*1,7090) (-.0552*.0313) 0.505
Proteus vulgaris
12 9.5460 1.2614 (5.7131-2.0473) (-.1567*.1194) 0.761
Pseudomonas aeruginosa
12 54.309 0.5361 (6.9504-1.3347) (-.2437*.0778) 0.948
Serratia marcescens
12 10.628 2.1393 (4.4701*1.3846) (-.0456-. 0254) 0.515
-------
69
Table A-19
Reduction statistics of bacterial contaminants of Anabaena cylindrica.
during series BG-II with enteric bacteria.
N
2/c 2
H /QT
R
(8.9466±,2816) (-.0187^.0054) 0.881
Alcaliqenes faecalis^
13 44.545 Oo0424
Enterobacter aeroqenes , +
12 578129 2.3427 (8,2424- . 6616) (-.0159-.0128) 0.492
Escherichia coli , + . _ . ._
12 2To"875~ 0.3648 (8.6235^1.0190) (-.0175-.0258) 0.343
Proteus vulWs ^^ (g_1277±J-1123) (..0094±.021S) 0.106
Pseudomonas aeruqinosa . n^n.
13 T?898 0.2366 (8.6531^,7359) (-.0198^.0163) 0.545
Serratia marcescens noo,+ nic-7\ n 717
13 ToTill 0.1349 (8,8000^.6195) (-.0234-.0157) 0.717
Table A-20
Reduction statistics of bacterial contaminants of Anacystis nidulans
during series BG-II with enteric bacteria.
SH2/Sr2 Sr2 b k _
N
R
(8.5258±.2666) (-.0197-+.0074) 0.890
(8.8843±.5258) (-.0213±.0114) 0.738
(8,3519-.7735) (-.0161^.0168) 0.427
Escherichia coli
12 3.7262 Oo3657
Proteus vulgaris + . „,... _ „,.,.
12 16.150 0.2533 (8.5266-.6437) (-.0279^.0140) 0.763
Pseudomonas aeruqinosa , n
U o7?013 0,4104 (7.6799^.9494) (-.0068^.0263) 0.070
Serratia marcescens^ + ..... n OCQ
12 267612 0.1054 (8,4857^.4812) (-.0323-.0133) 0.869
-------
70
Table A-21
Reduction statistics of bacterial contaminants of Gloeocapsa alpicola
during Series BG-II with enteric bacteria.
21 9
/c Z c " U 1^ 1
N
Alcaligenes faecalis
13 3.2359 0.2076
Enterobacter aeroqenes
12 13.108 0.1672
Escherichia coli
12 5.7870 0.3437
Proteus vulgaris
12 8.1439 0.1337
Pseudomonas aeruginosa
12 6.4973 0.0826
Serratia marcescens
12 16.613 0.1527
(7.96S8-.6747) (-.0110-.0130) 0.447
(8.1383^.6055) (-.0198-.0117) 0.766
(8.8736*1.0537) (-.0271-.0265) 0.658
(8,5076-.5415) (-.0139-.0104) 0.671
(7.9595-.5167) (-.0141-.0130) 0.684
(8.2359-.5786) (-.0213-. 0111) 0.806
Table A-22
Reduction statistics of bacterial contaminants of Oscillatoria chalybia
during series BG-II with enteric bacteria .
N SrT2/S 2 S,2 b k R
Alcaligenes faecalis
12 24.990 0.0192
Enterobacter aerogenes
12 24.106 0.0310
Escherichia coli
12 12.224 0.0996
Proteus vulgaris
12 0.2826 Ool938
Pseudomonas aeruginosa
12 0.0174 0.0899
Serratia marcescens
(7.6573-.2627) (-.0110-. 0052) 0.893
(707886*. 3343) (-.0138^.0066) 0.889
12 0.7189
0.1208
(8.3854^.5177) (-.0143-. 0087) 0.753
(7.4938-.83S3) (-.0037^.0165) 0.086
(7.1715-.5690) (.0006 -.0112) 0.006
(7.3817-.6596) (-.0047^.0130) 0.193
-------
71
Table A-23
Reduction statistics of bacterial contaminants of Oscillatoria formosa
during series BG-II with enteric bacteria „
N STT2/S .2 S.2 b k R
Alcaligenes faecalis .
12 0,0758 Oo2549 (7.7277*,7207) (-.0018-.0139) 0.019
Enterobacter aerogenes +
12 0.4654 0.3258 (7.7450±.8148) (-.0051-.0158) 0.104
Escherichia coli
12 3.7691 0.2270 (8.0401-. 6801) (-.0120-.0132) 0.485
Proteus vulqaris
12 36.3346 0.0251
Pseudomonas aeruginosa
(8.0503-.2259) (-.0124-.0044) 0.901
12 1.5084 0.1332 (7.7167-.5209) (- ,0058- ,0101) 0.274
Serratia marcescens
12 1.1490 1.4257 (7.8396-.9224) (-.0129±,0352) 0.365
Table A-24
Reduction statistics of bacterial contaminants of Phormidium faveblarum
during series BG-II with enteric bacteria.
N
Sr'
R
Alcaligenes faecalis
12 3.8100 0.1202 (7.7007-2.0520) (-,0195±,0630) 0.792
Enterobacter aerogenes
12 7,2137 0,1341 (7,8364-.9043) (-.0174-.0189) 0.783
Escherichia coli +
12 10.6154 0.2067 (8.1078-1.1228) (-.0262-.0235) 0.841
Proteus vulgaris ,
12 2.8200 0,2284 (7.7159*1.1805) (- .0142-. 0247) 0.585
Pseudomonas aeruginosa
12 2.6126 0.0269 (6,2282-. 4053) (.0047^,0085) 0.566
Serratia marcescens ,
12 2.8029 1.2669 (7.4335-6.6603) (-.0542^.2044) 0.737
-------
72
Table A-25
Growth statistics of bacteria found in axenic algae cultures
Series BG-III. Controls „
N SH2/Sr2 Sr2
Anabaena cyllndrica
b
k R
i nccn+ r\coi\ n 47R
5.8969 (3.5555±3.5177) (.0495^0669) 0.383
(1.8642±1.7482) (.0592^.0333) 0.783
Oscillatoria chalybia +
12 670545 2,3982 (3 = 9318*2.2433) (. 0493-. 0427) 0.602
Oscillatoria formosa . .
12 37o"423 3.4652 (4 ,4801-2 .6966) (. 0420-.0513) 0.432
Phormidium faveolarum ,
475869 2.7219 (4.1262-2.3899) (.0457^.0455) 0.534
12
Table A-26
Growth statistics of axenic culture of Anabaena cylindrica with enteric
bacteria during series BG-Irun, Series EG-IV,
Results based on mg/1 dry weight 0
N
(.0117±.0099) 0.61!
(1.7229^.4403) (.0107±.0079) 0.683
0.1098 (1.8928-+.7893) (.0043-^0139) 0.098
0.1148 (1.4919-+.8073) (.0114-^0143) 0.421
(1.2489-^5738) (.0155±%0101) 0.725
Serratia marcescens
--
ratia marcescens. , + nnnn. . „...
U -- IO66 0.0493 (1.2862-.5288) (.0152-. 0093) 0.751
-------
73
Table A-27
Growth statistics of axenic culture of Anacystis nidulans with enteric
bacteria during series BG-Irun, Series BG-IV,
Results based on mg/1 dry weight„
N
O 2/0
bH /br
R
Alcaligenes faecalis
11 207980 0.1230
Enterobacter aerogene.s
11 5.5746 0.0575
Escherichia coli
11 67,451 0.0065
Proteus vulgaris
11 2.7309 0.0389
Pseudomonas aeruginosa
11 1.5814 Ool693
Serratia marcescens +
11~~~ 6.3484 0.0513 (1.3299^.5398) (.0113-.0095) 0.613
(1.4449-.8355) (.0116^.0148) 0.411
(1.1917-.5714) (,0112*.0101) 0,582
(1.602-U1919) (.0131-.0034) 0.944
(1.5815-.4699) (.0064-^0083) 0,406
(1.3273-.9802) (. 0102-, 0173) 0,283
Table A-28
Growth statistics of axenic culture of Gloeocapsa alpicola with enteric
bacteria during series BG-Irun, Series BG-IV*
Results based on mg/1 dry weight.
c 2 K V
N
« 2/0 2
SH /sr
Alcaligenes faecalis
11 1.9229 0,0928
Enterobacter aerogenes
11 4o2808 0.0297
Escherichia coli
11 5.7088 0,0326
Proteus vulgaris
11 0.6004 Oc0673
Pseudomona s aeruginosa
11 4.3449 0<0140
Serratia marcescens
11 17o924 0.0203
(1U6702-.72S7) (00083-0 0128)
(1.9153-.4106)
(1.7957-.4299)
(.0070±,0072)
(00085-o0076)
(2.1234-.6180) (.0039-.0109)
(2,0407-U2821) (.0049*^0050)
(1.5473-.3399 (.0119^,0060)
R
Oo324
0.517
0«588
0,130
0,521
0.817
-------
74
Table A-29
Growth statistics of axenic culture of Oscillatoria chalybia with enteric
bacteria during series BG-Irun. Series BG-IY.
Results based on mg/1 dry weight.
N SH2/Sr2 Sr2 b k R
Alcaligenes faecalis
11 8.9103 0.0203 (1,5632-.3391) (,0084-.0059) 0.690
Enterobacter aeroqenes
11 8.5231 0.0134 (1,6601-.2759) (.0067^.0048) 0.681
Escherichia coli
11 0.7271 0.0860 (1.9015-.6988) (.0049-.0123) 0.154
Proteus vulgaris
11 26.7943 0.0343 (1.7519-.4414) (.0189-.0078) 0,870
Pseudomonas aeruqinosa
11 10.575 0.0580 (102489-.5738) (.0155^.0101) 0.725
Serratia marcescens
11 12.066 0.0493 (1.2862-.5288) (.0152-.0093) 0.751
Table A-30
Growth statistics of axenic culture of Oscillatoria formosa with enteric
bacteria during series BG-Irun. Series BG-IV.
Results based on mg/1 dry weight.
Nq 2 /q 2 o " K V R
OTT / O_ O_ U JS. I\
Alcaligenes faecalis
12 12.215 0.1702 (.6819^.9829) (,0285±.0174) 0.753
Enterobacter aerogenes
11 22.734 0.0742 (1.0015-.6489) (.0256-.0115) 0.850
Escherichia coli +
13 9.3189 0.1202 (1.1998±.8261) (.0209-.0146) 0.699
Proteus vulgaris
12 2.9976 0.2196 (1.3486-1.1164) (.0160-.0197) 0.428
Pseudomonas aeruginosa
11 1.4971 0.1744 (1.5118-.9948) (.0101*.0176) 0.272
Serratia marcescens
11 3.3157 0.1967 (1,3871*1.0566) (.0159-.0187) 0.453
-------
75
Table A-31
Growth statistics of axenic culture of Phormidium faveolarum with enteric
bacteria during series BG-Irun, Series BG-IV,
Results based on mg/1 dry weight ,
M q 2/0 2 o 2 , k
JM OTT /b_ Of D Jv
Alcaligenes faecalis
11 0.0620 0.0763
Enterobacter aerogenes
12 0.0144 0.0504
Escherichia coli
12 0.4124 0=0444
Proteus vulgaris
12 0.4748 0.1242
Pseudomonas aeruginosa
12 0.1167 0.0785
Serratia marcescens
12 1.1185 0.0804
Reduction statistics
contaminant
N SH2/Sr2 Sr2
Alcaligenes faecalis
12 11.847 1.3542
Enterobacter aerogenes
12 61.275 2.4412
Escherichia coli
12 15.235 0.2289
Proteus vulgaris
12 115.08 0.4806
Pseudomonas aeruginosa
12 28.100 1,2490
Serratia marcescens
12 55.697 0.427
(2.1178-.6583) (.00131.0116)
(2.0452-.5349) (. 0005- , 0094)
(2.16461.5023) (-.00271.0088)
(1.64431.8396) (.0048-. 0148)
(2.00891.6676) (- .0019*. 0118)
(2.3023^6755) (-.0059-.0119)
Table A- 3 2
of enteric bacteria species with algal
Brevibacterium, Series BG-V.
b k
(6.7687-1.5672) (- .0513- .0300)
(8.30111.6642) (-.04941=0127)
(8.43661.6432) (-.07551.0123)
(8.46801.9319) (-.0951±.0178)
(7.6973*1.7243) (-. 1011-.0407)
(8. 6455-. 8787) (-.06241.0168)
R
0.015
0.004
0,093
0.106
0.028
0.218
R
0.703
0.924
0.968
0.958
0.875
0.918
-------
76
Table A-33
Reduction statistics of enteric bacteria species with algal
contaminant, Flaveobacterium , Series BG-V.
N
H
2/q 2
./sr
R
(8.1993-2,4757) (-. 1437-.0945) 0,908
Alcaligenes faecalis
12 19.699 1.0269
Enterobacter aerogenes
12 188.33 0.0543 (8.0086*1.3435) (-.1616*.0743) 0.995
Escherichia coli
12 21.881 1.4051 (8.1903*1.8289) (-.0947*.0431) 0.845
Proteus vulgaris
12 136.43 0.3321 (10.0665*1,0505) (-. 1520*.0306) 0.978
Pseudomonas aeruginosa
12 22.289 0.9558
Serratla marcescens
12 16.282 1.6677 (6.5629*1.7360) (-.0666*.0333) 0.765
(7.6614*1,7822) (-. 1042-.0519) 0.881
Table A-34
Reduction statistics of single species of enteric bacteria in presence
of mixed cultures of six species of blue-green algae. Series BG-VIII,
O 2 1- \r
N
q 2/q 2
SH /sr
R
(707778±7.0397) (-.2463-.3S95) 0.941
Alcaligenes faecalis
13 15.939 1.4918
Enterobacter aerogenes
12 6.2346 3.5258 (8,1472- 10.8224) (-,2368-.5988) 0.862
Escherichia coli
13 0.3556 2.9060 (7.0455-7.1646) (-.1666-,1590) 0U824
Proteus vulgaris
13 5.4117 4.1432
Pseudomonas aeruginosa
12 10.783 2.7206 (7.0364-4.0296) (-.1730-.1538) 0.843
Serratia marcescens
12 4.2473 5.0527 (6.4926-5.4915) (-»1479*.2096) 0.679
(8,1208-11.7319) (-.2392^.6491) 0,844
-------
77
Table A-35
Reduction statistics of mixed enteric bacteria in presence of mixed
cultures of six species of blue-green algae. Series BG-IX.
N SH2/Sr2 Sr2 b k R_
Alcaliqenes faecalis
11 14.056 1,8856 (6.7616^.9146) (-.2600-.4379) 0.933
Enterobacter aeroqenes ,
11 5.9160 4.9779 (6.8051*12.8595) (-.2741-.7115) 0.855
Escherichia coli ,
11 0,0786 19.409 (4.1152-19.6689) (-.1081*2.4333) 0.073
Proteus vulgaris ,
11 4.3410 5.6157 (6 .2648±12 .6453) (-. 1632-.4947) 0.813
Pseudomonas aeruqinosa
11 13.145 1.7591 (6.9496±3.2402) (-. 1536*. 1237) 0.868
Serratia marcescens
11 36.931 0.5183 (7.0223^1.7588) (-.1397^.0671) 0.948
Total* of all 6 enterics
11 20.528 1.1271 (7.5912*2.5936) (-.1536^.0990) 0.911
* Data for the total number of enteric bacteria, not sum of individual
statistical results for each species.
-------
78
Table A-36
Reduction statistics of enteric bacteria species with Ankistrodesmus braunii,
Series G-I. Bacteria added to algae when in mid-log phase,
N SH2/Sr2 Sr2 b k R
Alcaligenes faecalis
13 59.850 0,45717 (7.0994^9669) (-.0701^.0193) 0.937
Enterobacter aerogenes
14 89.272 0.36307 (8.2215-.8617) (-.0764^0172) 0.957
Escherichia coli
14 7.1998 1.0295 (6.7241-1.4509) (-.0365±.0290) 0.643
Proteus vulgaris
11 38.053 0.35906 (6.9891-1.0566) (- .0756^.0288) 0.927
Pseudomonas aeruginosa
12 42.717 0.29243 (7.9607-1.3211) (-.1129-.0504) 0U955
Serratia marcescens
12 11.093 0.82891 (9.1086-1.6055) (-.0620^0438) 0,787
Table A-37
Reduction statistics of enteric bacteria species with Chlorella pyrenoidosa
Series G-I. Bacteria added to algae when in mid-log phase.
N SH2/Sr2 Sr2
R
Alcaligenes faecalis
13 447.65 ,05507 (8,1764^.5733) (-.1586-.0219) 0.995
Enterobacter aerogenes
14 9,3163 2.6364 (7.7223-2.8633) (-.1013-.0781) 0.756
Escherichia coli
14 19.113 1.1338 (6.6677*1.5227) (-.0624t.0305) 0.827
Proteus vulgaris
11 23.078 1.3761 (8,2918-2.8659) (-.1800-.1094) 0.920
Pseudomonas aeruginosa
12 9.1331 2.5412 (8,0134-2.8111) (-. 0985±.'O767) 0,753
Serratia marcescens
12 17.501 0.9995 (7.0495-1,7629) (-.0855±.0481) 0.854
-------
79
Table A-38
Reduction statistics of enteric bacteria species with Chlorella vulgaris.
Series G-I. Bacteria added to algae when in mid-log phase.
N
Alcaligenes faecali_s
12 8.5477 1.8059
Enterobacter aeroqenes
12 6.7731 3ol823
Escherichia coli
12 4,2474 2,8177
Proteus vulgaris
12 6.2347 2.0976
Pseudomona_s aeruginosa
12 19.549 1.4598
Serratia marcescens
12 I2o080 1.8887
R
(7.1179*3.2831) (-. 1255*. 1253) 0.810
(7,1567-3.1458) (-.0949*0.0858) 0 = 693
(6.5618*2.4005) (-.0464*0.0480) 0.515
(8.1411*8.3476) (-. 1826*. 4619) 0.862
(8,1344-2.1307) (-. 1092*. 0581) 0.867
(7.9809-3.0490) (-.0651*. 0547) 0.858
Table A-39
Reduction statistics of enteric bacteria species with Scenedesmus^bliguus..
Series G-I. Bacteria added to algae when in mid-log phase,
> o 2 K k
N
Alcaligenes faecalis
12 7.1587 1.6250
Enterobacter aeroqenes
12 26,533 0.6903
Escherichia^ coli
12 40.527 0.2004
Proteus vulqarls
Serratia marcescens
12 41.964 0.5409
R
(6.1286-1,8230) (-.0458*. 0365) 0.642
(6,9105*1.1881) (-.0574*.0238) 0.869
(7,4914*0.7894) (-.0583*.0215) 0.931
(6,9057*1.8545) (-. 0891*. 0506) 0.851
(7.9135*1,3072) (-.0777-;0357) 0.898
(8,8431*1.2969) (-,0974*.0354) 0.933
-------
80
Table A-40
Reduction statistics of enteric bacteria species with Ankistrodesmus bra unit.
Series G-II. Bacteria and algae inoculated within twenty-four hours of one another
N SH2/Sr2 Sr2 b k
R
(7.8609*8.9976) (-.0542*. 1598) 0.821
Alcaligenes faecalis
12 154.98 0.19994 (8.7434-2.4481) (-.0857^.0435) 0,994
Enterobacter aeroqenes
12171603 1.5360 (8.5471±6.7856) (-.0801*.1205) 0=946
Escherichia coli
12 4.5853 2,7007
Proteus vulgaris
12~ 1.8897 12.398 (7.2077*19,278) (-.0745*.3424) 0.654
Pseudomonas aeruqinosa
12 1765.9 0,01860 (8.9704*.8609) (-.1977^.0297) 0.999
Serratia marcescens
12~ 1891.1 0.01366 (9.7349-.7378) (-. 1753*.0254) 0.999
Table A-41
Reduction statistics of enteric bacteria species with Chlorella pyrenoidosa.
Series G-II. Bacteria and algae inoculated within twenty-four hours of one another.
N
o 2/q 2 g 2
SH /Sr br
Alcaligenes faecalis
12 24.159 0.9386
Enterobacter aeroqenes
12 2899,2 0.0046
Escherichia coli
12 12.115 2.0249
Proteus vulgaris
12 69.343 0.1855
Pseudomonas aeruginosa
12 10.876 1,2791
R
(7.9536*5.3042) (-.0743*.0942) 00960
(9,2054^.4299) (-.1265^.0148) 0.999
(7.7306-7.7908) (-. 0763*. 1384) 0,924
(7.0642*2.3582) (-,0552-.0419) 0.986
(8.0599*6.1921) (-.0574*. 1100) 0.916
Serratia marcescens ,
12 19.603 0.6550 (8.2736*4.4309) (-.0552-.0787) 0.951
-------
81
Table A-42
Reduction statistics of enteric bacteria species with Chlorella vulgaris. Series
G-II. Bacteria and algae inoculated within twenty-four hours of one anotter,
_N_ SH2/Sr2 Sr2 b k _R
Alcaligenes faecalis
12 2125,2 0,00398 (5,9987^.3983) (-. 1003^.0137) 0,999
Enterobacter aerogenes ,
1221?6.9 0.00505 (7,5548^.4487) (-.1138-.0155) 0,999
Escherichia cpli +
12 470005 1,7486 (5.6424^7.2399) (-. 0407-. 1286) 0.800
Proteus vulgaris . ,
12~ 2362.9 0.0065 (8.1123^.5091) (-.1352^.0176) 0,999
Pseudomonas aeruginosa +
124933,7 Oo0062 (7,4898-.4954) (-.1901-.0171) 0,999
Serratia marcescens , nnn
12 1781,4 0,0046 (6.6976^,4292) (-,0989±a0148) 0,999
Table A-43
Reduction statistics of enteric bacteria species with Scenedesmus obliguus .
Series G-II. Bacteria and algae inoculated within twenty-four hours of one
another.
N SH2/Sr2 S2 b __ k _R _
Alcaligenes, faecalis
- 12 847.23 0.02134 (7 ,4529- . 7997) (- ,0655±. 0142) 0.998
Enterobacter aerogene^ ,
- 12 - sTil.5 0.00448 (8,3051- ,3663) (-.0792^,0065) 0,999
Escherichia. coli , +.„,.„
- 12 2?,286 0.42066 (6.7184^3,5509) (-.054^.0631) 0,967
Proteus vulgaris +
- 12 -- 182T75 0.00183 (6 n4763^.2699) (-.0629^,0093) 0,999
Pseudomonas aeruginosa ,
- 12 2TIl69 4,3286 (6.6961-11,3908) (- ,0466-, 2023) 0,679
Serratia marcescens , ,
- U -- T6209 1.3397 (7,5169-6,3371) (-,0492^.1126) 0.884
-------
82
Table A-44
Reduction statistics of single species of enteric bacteria in presence
of mixed cultures of four species of green algae. Series G-VIII.
R 2 h k R
N
SH2/S_2
(6.8582-2,0250) (-.1280^.0773) 0.921
Alcaligenes faecalis ,
12 47.994 0.5460 (7.5188-1.8052) (-.1635-.0689) 0.959
Enterobacter aerogenes
12 145.79 0.1437 (6.6479*.9259) (-.1462*.0353) 0.986
Escherichia coli
12 23.382 0.6871
Proteus vulgaris
12 7.9498 1.8035 (7.3760-7.7403) (-.1912*.4282) 0.888
Pseudomonas aeruginosa
12 71.674 0.4158 (8.1735*1.5754) (-.1744^.0601) 0.973
Serratia marcescens
12 59.165 0.3693 (7,2299*1.4847) (-.1493*.0567) 0.967
Table A-45
Reduction statistics of mixed enteric bacteria in presence of mixed cultures
of four species of green algae. Series G-K.
N SH2/Sr2 Sr2 b k
R
(6.6735*6.2179) (-. 1176*.3440) 0.823
Alcaligenes faecalis
11 4.6588 1.1638
Enterobacter aerogenes
13 2.2508 0.7552 (6.9674-5.0089) (-.2082^.2771) 0.957
Escherichia coli
13 17.089 1.1520 (7.3603-2.6222) (-.1417^.1001) 0.895
Proteus vulgaris
12 3.4883 3.4136
Pseudomonas aeruginosa
12 11.588 1.8844 (7.4373-3.3536) (-.1493*.1280) 0.853
Serratia marcescens
12 27.099 0.9017 (7.6250*2.3198) (-. 1579-.0885) 0.931
Total enteric count (all six above)
12 21.552 1.0055 (8.1321*2.4497) (-. 1487-.0935) 0.915
(6,8254*10.650) (-. 1743*. 5893) 0,777
-------
83
Table A-46
Reduction statistics of enteric bacteria species in algal growth medium.
Series VI.
N
sH2/sr2
R
Alcaligenes faecalis
12 2.2010 2o7828
Enterobacter aeroqenes
12 0.3045 3.6089
Escherichia coli
12 2.1764 2.4862
Proteus vulqaris +
12~ 0.6976 3.7817 (6,1353-1.4299) (-.0149-.0325) 0,065
Pseudomonas aeruginosa
12 1.1722 1.7682
Serratia marcescens
120.3976 2.880 (7.1737-1.2479) (-.0098^,0283) 00038
(6.9431-1,2267) (-00228^.0279) 0.180
(6.0233-1.3969) (-.0097^.0317) 0.029
(6.3741*1.1595) (-.0214^.0263) 0.179
(6.9922^.9778) (-.0133^.0222) 0.105
-------
84
Table A-47
Reduction statistics of enteric bacteria species in filtrate from Anabaena
cvlindrica at mid-log growth phase , Series VII.
N SH2/Sr2
R
Alcaliqenes faecalis . + nr.^n\ „ ncc
11 6.4853 0,2025 (7.7104-2.4823) (-.0230-.0570) 0.866
Enterobacter aerogenes +
12 5~,7437 0.4313 (7,4657-3.6224) (-.0316-. 0832) 0.852
EscherichiacglL ^^ (8,0062±2,5035) (-.0164^.0575) 0.765
0.6346 (7.4696±4.3939) (-.0253^.1009) 0.715
(7. 7903±2 .3862) <-.0167±.0548) 0.788
Serratia marcescens . + nAn.. . ....
12 2.2672 0.1520 (7.8025-2.1506) (-.0118-,0494) 00694
Table A-48
Reduction statistics of enteric bacteria species in filtrate from Anacystis
nidulans at mid-log growth phase. Series VII.
N SR2/Sr2
R
Alcaligenes faecalis . + n/.n^ n AOO
- 12 - H^72 0,2918 (7.8427-2.9796) (-.0375-. 0684) 0.923
Enterobacter aerogenes + + rt.nrx n Ooo
- 12 - 5T.426 0.1023 (7.8034-1.7639) (-.0469-. 0405) 0.982
(7,5976-2.8743) (-.0212-. 0660) 0.805
(7.3661-4.6137) (-.0525-. 1060) 0.907
Pseudomonsa aeruginosa + „„,,.+ oo-,o^ n Q-JQ
- 12 - 5T2270 0.4748 (8,0166-3,8006) (-.0316-. 0873) 0.839
Serratia marcescens , + , -„.. n _cyl
- 12 -- 3,0764 0.7320 (7,4349^4.7190) (-.0301-. 1084) 0.754
ro eus vu aaris
-------
85
Table A-49
Reduction statistics of enteric bacteria species in filtrate from Gloeocapsa
alpicola at mid-log growth phase. Series VII.
N
Alcaliqenes faecalis
12 3.2476 0.1193
Escherichia coli
12 253772 0.0042
R
(8.5132-1.9048) (-, 0395-. 0437) 0.970
Enterobacter aeroqenes , +
12 fOie.S 0,0087 (9.2218- .5138) (-.0596-. 0118) 0.999
, +
(8*6674- .3579) (-.0656-. 0082) 0,999
Proteus vulgaris . +
12 93.455 0.0594 (9.1244-1.3448) (-.0473-, 0309) 0.989
Pseudomonsa aeruqinosa , +
14 773096 0.1320 (8. 6017-2 .0040) (-.0197-. 0460) 0.879
Serratia marcescens , +
12 0.3661 2.9428 (7.7528-9.4620) (-.0208-. 2174) 0.268
Table A-50
Reduction statistics of enteric bacteria species in filtrate from Nostoc
muscorum at mid-log growth phase. Series VII.
N
SH2/Sr2
R
Alcaliqenes faecalis , +
14 672929 2.9351 (7,5992-9.4495) (-.0862-. 2171) 0.863
Enterobacter aeroqenes . +
TI 2T.8502 0..8679 (8.2287-5 = 1385) (-. 1021-. 1180) 0.967
(8.0406-5.1242) (-.0881-. 1177) 0.957
(8.2417- .3411) (-.1021-.0078) 0,999
Escherichia coli
12 22.339 0,8631
Proteus vulgaris
12 6768.7 0,0038
Pseudomonsa aeruqinosa , +
12 879025 1.1393 (7.5259-5,8874) (-.0639-. 1352) 0,899
Serratia marcescens , +
IF 15.896 Oc5653 (7.6208-4.1472) (-.602 -,0953) 0.941
-------
86
Table A-51
Reduction statistics of enteric bacteria species in filtrate from Oscillatoria
chalybia at mid-log growth phase» Series VII.
N SH2/Sr2 Sr2 b k R
Alcaliqenes faecalis , +
- 12 18.221 1.2523 (8.0115-6.4142) (-.0996-. 1473) 0.948
Enterobacter aerogenes , +
12 19,231 1.1985 (7.4310-6.0384) (-.0963-. 1387) 0.951
Escherichia coli . + 4 nnn
- 12 - '8.7375 2.9108 (7.7619^9.4103) (-.1012-. 2162) 0.897
Proteus vulgaris . +
- 12 13.628 1.6409 (7.2278*7.0653) (-.0949-. 1623) 0.932
Pseudomonsa aeruginosa , +
- 12 - 54.986 0.3240 (7.3250*3.1396) (0.0847-.0721) 0.982
Serratia marcescens . +
- 12 -- 5.2723 3c0540 (7. 7516±9.6390) (-.0805-. 2214) 0.840
Table A-52
Reduction statistics of enteric bacteria species in filtrate from Oscillatoria
formosa at mid-log growth phase. Series VII.
22 2 k R
N S /Sr Sr
ji r r
Alcaligenes faecalis . +
12 2.3677 6.0714 (7.1292-13.5907) (-.0761-.3122) 0.703
Enterobacter aeroqenes , +
12 7.9584 1.6716 (8.1111- 7.1312) (-.0732-. 1638) 0.888
Escherichia coli . +
12 0^880 0.5797 (3.1487* 4.1997) ( .0136-.0965) 0.441
Proteus vulqaris . +
12" 5.0901 2.2407 (7.5433- 8.2564) (-.0678-. 1897) 0.836
Pseudomonsa aeruginosa , +
12 247278 0.3300 (8.4717- 3.1687) (-.0568-.0728) 0.960
Serratia marcescens . +
12 4.2481 2.6330 (8.1498* 8.9501) (-.0671-.2056) 0.809
-------
87
Table A-53
Reduction statistics of enteric bacteria species in filtrate from Phormidium
fayeolanum at mid-log growth phase. Series VII.
N STT2/S_2 S_2 b k R
Alcaliqenes faecalis + +
12 679894 1.7256 (7.4942-7.2456) (-. 0697-. 1664) 0.875
Enterobacter aerogenes , + ,, . _ ....
14 rT.285 1.2290 (8.1289-6.1147) (-.0841-. 1405) 0.934
Escherichia^c|_i^ 4ol571 (7.7636-11.2459) (-.0646-. 2584) 0.714
0.3666 (7.7698-2.2297) (-.0643^.0767) 0.965
roteus
11 2£
Pseudomonsa aeruqinosa + „„„„+ , r/ir,\ n ooo
13 872261 1.4944 (7.6431-6.7427) (-.0704-. 1549) 0.892
(..08ll±.1332) 0.936
Table A-5 4
Reduction statistics of enteric bacteria species in filtrate from Ankistrodesmus
braunii at mid-log growth phase. Series VII.
N SH2/Sr2 Sr2 b k R
(7.7835-12.1020) (-.0769-.2780) 0.753
(-.0965-.1696) 0.928
Escherichia ss&^ ^^ (?^^t ^gm} (._0520i_0090) 0.999
Proteus vulqaris s ^^ (7.3531±2.5691) (-.0904±.0590) 0.989
(..OB90±.0751) 0.982
,..0558±. 1196) 0.897
-------
88
Table A-55
Reduction statistics of enteric bacteria species in filtrate from Chlorella
pyrenoidosa at mid-log growth phase. Series VII.
^J SH2/Sr2 Sr2 b k __R_
Alcaligenes faecalis
12 5.4223 3.9217 (7.4986-10,9228) (-.0925^.2509) 0.844
Enterobacter aeroqenes
12 6.9282 2,5946 (7,5341*8.8844) (-.0851-.2041) 0.874
Escherichia coli ,
12 1.8148 6.8664 (7.2231*14,4531) (-. 0708-.3320) 0.645
Proteus vulgaris
12 5,1004 3.3545 (7,3782*10.1021) (-.0830-.2321) 0.836
Pseudomonas aeruqinosa ,
12 0.8458 8,8804 (7,3886-16.4367) (-.0550-.3776) 0.458
Serratia marcescens
12 1.4244 8.2050 (7.5455*15.7993) (-.0686-.3630) 0.587
Table A-56
Reduction statistics of enteric bacteria species in filtrate from Chlorella
yulgaris at mid-log growth phase. Series VII.
N S 2/S 2 S 2 b k R
rl r i , _— __
Alcaligenes faecalis
14 8.1485 2.2109 (7,0826*8.2012) (-.0852*.1884) 0.891
Enterobacter aeroqenes
14 11.8899 0,8985 (5.9278*5.2283) (-.0656-. 1201) 0.922
Escherichia coli ,
14 6,2986 1.2075 (7,0093-6.0610) (r .0553-. 1392) 0.863
Proteus vulgaris ,
14 14.888 0.9550 (6.5244*5.3902) (-.0757-. 1238) 0.937
Pseudomonas aeruqinosa
14 3.6679 3,1607 (7,2067*9.8060) (-.0683*.2253) 0.786
Serratia marcescens
14 2.6636 3.8659 (6.5441*10.8448) (-.0644^.2491) 0.727
-------
89
Table A-57
Reduction statistics of enteric bacteria species in filtrate from Scenedesmus
obliquus at mid-log growth phase. Series VII.
! c 2 v, k
N
R
(8.8573-.9313) K0324±.0214) 0.989
Alcaliqenes faecalis
14 91.4609 0.0285
Enterobacter aerogenes ,
14F09.62 0.0585 (8.9597-1.3346) (-.0508^.0307) 0.991
Escherichia coli ,
1457.6465 0.0773 (8.4025^1.5335) (-.0401^.0352) 0.981
Proteus vulgaris ,
14~ 1.3987 0.4076 (8.2736*3.5216) (-.0151^.0809) 0.583
Pseudomonas aeruginosa
14 16.5995 0.4526
Serratia marcescens
14 5.1187 5.1345
(8.0305-3.7108) (- .0550- . 0852) 0.943
(8.3673*3.9523) (-.0325^.0908) 0.836
Table A-5 8
Reduction statistics of pathogenic bacteria species with Anabaena cylindrica.
Bacteria added to algae in mid-log growth phase.
N
c 2
br
Salmonella paratyphi
8 222.0690 .1189
Salmonella typhosa
8 11.0652 1.5269
Shigella paradysenteriae
8 95.2742 .2458
Shigella dvsenteriae
8 106.4579 .2418
Vibrio comma
8 52.2653 .2336
b
R
(6.0783*.3755) (-.0751^.0098) .9737
(6.6067*1.3458) (-.0601-.0351) .6484
(6.8035*.5399) (-.0707^.0141) .9408
(6.7365*.5356) (-.0742^.0139) .9466
(5.1157*.5265) (-. 0511*. 0137) ,8970
-------
90
Table A-59
Reduction statistics of pathogenic bacteria species with Anjc^stis nidul§ns_.
Bacteria added to algae in mid-log growth phase.
,.T o 2/q 2 Q 2 b k R
N SH /br or -
<7.3962±.66S8) (-.0840±.0174) .9364
(6.3929±.6028) (-.0758±.01S7) -9359
(6.6402,1.2 HO) <-.0981±.0317> .8579
(6.8716i.5993) (-.1249^0208) .9671
.3186 (5.2S23±.7I16) (-.0997±.0247) .9299
Table A-60
Reduction statistics of pathogenic bacteria species with Gloeocapsa aMgola
Bacteria added to algae in mid-log growth phase.
N SH2/Sr2 V2 b k —
(7.5863-1.5407) (-.0657^.0401) .6270
(5.6856^.5965) (-.0609-.0156) .9062
Shiaella paradysenteriae rtn^0+ nno^
~~8 341.4663 .0948 (6.5899^.3353) (-.0832^.0087)
Shigella dysenteriae , nr,AC+ nnm\
8 250.8821 .1036 (5.8648-.3505) (-.0745-.0091)
Vibrio comma . »^r+ n»r,n\
8 191350 1.3396 (6.2735*1.2606) (-.0755±.0329)
-------
91
Table A-61
Reduction statistics of pathogenic bacteria species with Oscillatoria chalybia
Bacteria added to algae in mid-log growth phase.
O 2 U T,
N
sH2/sr2
Salmonella paratyphi
8 14.8942 1.1813
Salmonella typhosa
8 11.2542 1.1372
Shigella paradysenteriae
8 15.7010 1.3518
Shigella dysenteriae
8 61.3114 .3928
Vibrio comma
8 228.8732 .0834
R
(6.6241-1.1837) (-.0613-.0309) .7128
(5.5874-1.1614) (-.0523-.0303) .6523
(6.3148-1.2663) (-.0673-.0330) .7235
(6.2078-.6826) (-.0717-. 0178) .9109
(5.1152^.3146) (-.0639^.0082) .9745
Table A-62
Reduction statistics of pathogenic bacteria species with Oscillatoria formosa.
Bacteria added to algae in mid-log growth phase.
S 2/Sr2 S 2 b k
N
Salmonella paratyphi
8 67.3622 .4893
Salmonella typhosa
8 206.1572 .1061
Shigella paradysenteriae
8 291.2393 .0623
Shiqella dysenteriae
8 37.9589 .5839
Vibrio comma
8 84.3495 .2766
R
(6.2018^.7618) (-.0839-.0199) .9182
(5,2634-. 3547) (-.0684±.0093) .9717
(5.3232*.2718) (-.0622*. 0071) .9798
(5.0350^.8323) (-.0688*. 0217) .8635
(5.0534-.5728) (-,0706-.0149) .9336
-------
92
Table A- 63
Reduction statistics of pathogenic bacteria species wit
faveolarum. Bacteria added to algae in mid-log growth phase
N
b k
(6.364li.4069) (-.0790*.0106) .9721
(4.8470±.7478) (-.0602-+.0195) .8571
(5.589^.6926) (-.0791±.0181) .9235
(5.1532±.7147) (-.0670±.0186> .8903
.3413 (4.6406±.6363) (-.0658^.0166) .9081
Table A-64
Reduction statistics of enteric bacteria in algal growth medium under
anaerobic conditions.
2 k
Sr
(4.2661i1.0626) (-.0131±.0253) .179
(6.0776±.7381) (-.0352±.0175) .7657
Escherichia coli + . _.Qn+ ^771 q?4l
- 12 - 189.839 .0604 (6.8443i.3016) (-.0490-. 0072) .9743
1.1097 (4,7693±1.2928) (-.0131±.0307) .1287
(5.7946*1.0879) f-.03i5t.0259) .5459
(7.5824t.4524) (-.0563*.0108, .9571
-------
93
Table A-65
Reduction statistics of pathogenic bacteria species in algal growth medium
under anaerobic conditions.
N
Salmonella paratyphi
12 2.0283 1.0080
Salmonella typhosa
12 4.2477 .8056
Shigella dysenteriae
12 46,3827 .2490
Shigella paradysenteriae
12 1.2762 1.4087
Vibrio comma
12 2.4089 .5228
R
(5.2320*1.2322) (-.0207-.0293) .2886
(4.6221*1.1016) (-.0268*.0262) .4593
(6.5081*.6124) (-.0492*.0146) .9027
(4.2391*1,4567) (-. 0194-. 0346) .2033
(4.1682-.8874) (-.0162-. 0211) .3251
Table A-66
Reduction statistics of pathogenic bacteria in presence of culture of four
green algae species. Bacteria added to algae when in their mid-log growth
pha se.
N
Salmonella paratyphi
12 35.0416 .3373
Salmonella typhosa
12 37.5767 .3051
Shigella paradysenteriae
12 46.6440 .3042
Shigella dysenteriae
12 48,0406 .1846
Vibrio comma
12 41.3008 .2528
R
(5.0683*1.0587) (-. 1553-. 0617) .9211
(5.8014-.8522) (-. 1156*. 0402) .9038
(5.7432*1.0055) (-. 1702*.0586) .9396
(5.4882-.7831) (-. 1345*.0457) ,9412
(5.0728*.9165) (-. 1460*.0535) .9323
-------
94
Table A-67
Reduction statistics of pathogenic bacteria in presence of culture of six
blue-green algae species. Bacteria added to algae when inttheir mid-log
growth phase.
N SH2/Sr2 Sr2 b k R
Salmonella paratyphi «
1234.9980 .3504 (5.6754^.7463) (-.0759*.0259) .8750
Salmonella typhosa
1240.8310 .3134 (4.9758*.7058) (-.0775^.0244) .8909
Shiqella paradysenteriae
12 36.7898 .1841 (4.2882^.5409) (-.0564^.0187) .8804
Shiqella dysenteriae
12 12.6987 .6502 (5.8225^1.4382) (-.1124*.0673) .7605
Vibrio comma
IF 53.4203 .1396 (4.9777^.5765) (-.0933^.0272) .9303
Table A-68
Reduction statistics of pathogenic bacteria species in algal growth medium
Controls.
N SH2/Sr2 Sr2 b ^ R
Salmonella paratyphi
8 7.084 2.936 (4.7225*1.6467) (-.0622*.0454) .5414
Salmonella typhosa ,
8 75.1391 .3737 (S.3975-.6658) (-.0775-.0174) .92605
Shigella paradysenteriae
8 26.9832 .9442 (5.0349*1.0583) (-.0738*.0276) .8181
Shigella dysenteriae
8 30.5534 .8124 (5.0675*.9817) (-.0728*.0256) .8359
Vibrio comma ,
8 35.4679 .5158 (4.3030*.7822) (-.0625*.0204) .8553
-------
95
Table A-69
Reduction statistics of pathogenic bacteria species with Ankistrodesmus
braunii. Bacteria added to algae in mid-log growth phase.
N SH2/Sr2 Sr2 b k R
Salmonella paratyphi
8 53.4130 .4618 (5 .2557^.7401) (-.0726-. 0193) .8990
Salmonella typhosa
8 50.0659 .4051 (4.5382-.6932) (-.0658*.0181) .8930
Shigella paradysenteriae
8 32.6928 .7621 (4 .9435*.9508) (-.0730*.0250) .8449
Shigella dysenteriae
8 25.4007 .9202 (4.7347-1.0448) (-.0707*.0272) .8089
Vibrio comma
8 62.7151 .2627 (4.1532*.5582) (-.0593*.0146) .9127
Table A-70
Reduction statistics of pathogenic bacteria species with Chlorella
pyrenoidosa. Bacteria added to algae in mid-log growth phase.
N SH2/Sr2 Sr2 b k R
6 16.7589 1.0565 (5.0966-1.3713) (-.0950-.0495) .8073
Salmonella paratyphi
6 16.7589
Salmonella jyphosa
6 29.6214 .6437 (5.4513-1.0704) (-.0986±.0386) .8810
Shigella paradysenteriae
6 66.4033 .2931 (5.1800-. 7223) (-.0996*.0261) .9432
Shigella dysenteriae
6 11.8522 1.1679 (4.4327*1.4418) (-.0840-.0520) .7477
Vibrio comma
17.2831 .6330 (4.3459-1.0614) (-.0747*.0383) .8121
-------
96
Table A-71
Reduction statistics of pathogenic bacteria species with Chlorella vulgaris.
Bacteria added to algae in mid-log growth phase.
o 2 /o 2 a 2 u V
N
Salmonella paratyphi
7 16.3117 1.2757
Salmonella typhosa
7 13.2037 1.1533
Shiqella paradysenteriae
7 15.7464 1.1842
Shiqella dysenteriae
7 29.2807 .7786
Vibrio comma
7 10.9643 .9153
R
(4.6525*1.2950) (-.0669*.0334) .7654
(4.5957*1.2314) (-.0572*.0317) .7253
(4.1637*1.2477) (-.0633*.0322) .7590
(4.7370*1.0117) (-.0700*.0261) .8541
(3.5452*1.0970) (-.0465*.0283) .6868
Table A-72
Reduction statistics of pathogenic bacteria species with Scenedesmus
obliquus. Bacteria added to algae in mid-log growth phase.
•vr o 2 /o i* o £•
Salmonella paratyphi
8 34.3155 .9453
Salmonella typhosa
7 13.7076 .7617
Shigella paradysenteriae
7 27.1390 .8834
Shigella dysenteriae
7 18.2956 1.0167
Vibrio comma
,+
R
(6.2250-1.0590) (-.0833-.0276) .8512
(4.5996*1.1003) (-.0700-.0381) .7327
(6.1021*1.1849) (-.1061^.0411) .8444
(5.3369*1.2712) (-.0935-.0440) .7854
13.4125 1.2051 (4.9428*1.3843) (-.0872*.0480) .7285
-------
APPENDIX B
BACTERIOLOGICAL DATA FROM LABORATORY AND
FIELD WASTE STABILIZATION POND STUDIES
97
-------
Table B-l. Total Bacteria Densities In Laboratory Scale Waste Stabilization Ponds, As Log10/ml.
Date
7- 3-69
7- 7-69
7- 9-69
7-11-69
7-14-69
7-16-69
7-18-69
7-23-69
7-25-69
7-29-69
Sample Station —
Raw #1
6.72673
6.46613
7.05757
7.34782
7.19576
7.49066
8.52022
7.92505
7.79449
7.12385
6.66346
5.97772*
6.99717
7.07372
6.10380
6.79239
\
6.79379
7.47276
6.26834
6.28948*
#2
5.97081
5.50106*
6.72148
6.41330
4.65369
5.01072
4.95425
6.50827
5.44871
5.01589*
#3
5.54407
5.08991
6.90227
5.78426
4.79727
5.09777
4.91803
5.19728
4.75587
#4
5.10380
6.12222
5.96190
5.98520
5.50718
5.44248
5.48572
6.20352
5.04922
5.13672
#5
3.32593
4.92942*
6.81023
6.60152
5.04115
4.59660
4.97405
5.34193
4.34133
4.33445*
#6
5.65992
6.11227*
6.58743
5.69174
5.47857
5.68679
6.04139
5.79344
5.94374
5.22272*
#7
4.88081
5.68574*
5.99388
6.23553
4.64836
4.97058
4.53782
5.07555
5.98989
5.48180*
#8
6.41119
4.00000*
5.48714
5.43377
5.32919
6.02794
5.62014
5.67486
5.42488
5.51455*
#9
5.53593
5.23045
5.64444
5.58546
5.49406
5.08955
6.38382
6.44739
5.31597
5.79623
#10
5.22011
6.65002
6.13928
5.62273
5.24613
5.21617
5.71012
5.77815
5.43933
#11
4.69940
6.07188*
6.28780
5.94052
5.20276
3.87506
4.58433
4.38075
4.09377
4.98453*
*lnoculation with laboratory cultures: 7-7 and 7-29 with E. c. , Pseud. , and Serr.
CO
-------
Table B-2. Total Bacteria Densities In Laboratory Scale Waste Stabilization Ponds, As Log1()/ml.
Date
8- 4-69
8- 6-69
8- 8-69
B-ll-69
8-13-69
8-15-69
8-18-69
8-20-69
8-22-69
8-26-69
Sample Station —
Raw #1 #2
7.32531
7.66229
6.78013
6.74036
7.04630
6.96379
7.42243
7.12710
7.51851
6.87216
6.26411 5.12988
5.62685 4.32736
4.74819 6.95624
5.67669 4.19033
6.17099 5.29667
6.24304 5.36549
5.47712 4.75397
6.02531 3.98677
6.36680 5.20412
5.30103 4.16137
#3
4.69152
4.05115
6.81471
4.31175
4.63347
5.23553
4.45864
4.81624
5.94694
4.52504
#4 #5 #6
5.30211 6.19209 5.13751
3.97428 4.98080 5.56732
7.21885 7.60590 8.55781
4.61805 4.07278 5.67669
5.40184 4.15381 5.88804
5.02531 4.48714 6.29831
5.12711 4.48572 6.13513
4.49136 4.29994 6.30428
4.61278 6.69329
4.43537 4.11227 4.79588
#7 #8
5.63220 5.45255
5.33244 5.08279
8.46310 7.60487
5.04139 5.29003
5.09552 5.08458
5.16732 5.53782
4.92428 5.39226
4.07188 5.31175
5.49136 5.70372
4.70757 4.57113
#9
5.85643
4.96497
8.38462
5.37107
5.13988
5.73139
5.46613
5.41497
5.72937
4.68350
#10 #11
4.94349 4.96848
4.75397 4.51455
7.65715 7.34922
5.35218 4.75967
5.26717 5.09691
4.93197 5.70948
4.85126 5.13830
5.86332 5.09342
5.93952 5.54064
5.02119 4. 59934
to
CO
-------
Table B-3. Total Coliform Bacferia Densities In Laboratory Scale Waste
Stabilization Ponds, As Log^Vml.
Date
7- 3-69
7- 7-69
7- 9-69
7-11-69
7-14-69
7-16-69
7-18-69
7-23-69
7-25-69
7-29-69
Sample Station —
Raw #1 #2 #3 #4
5
4
5
6
5
6
5
6
.14613
.69907
.60746
.81258
.87216
.66039
.62839
.01807
4,
3
5
5
5
6
6
4
4
4
.88930 4.39794 2.08279
.92942* *
.07918 4.55630 5.59106 4.30103
.00000 5.36173 4.50515 5.06070
.07918 3.13830 3.62839 3.90309
.32222 4.13033
.43537 3.79588 2.81291 3.79588
.70329 0.97772 0.17609 0.17609
.73679
.28443* *
#5
0.74036 3.
* 4.
4.90714 3.
4.74036 4.
3.00000 3.
4.
4.31175 3.
3.
* 2,
#6 #7 #8 #9
71642 0.39794 3.87506 3.S4407
39902* * 2.00000* 3.97772
96614 4.67482
30103 4.60206 4.14613 4.38021
00000 3.77815 3.15381 3.12222
63599 3.75967 4.76343 4.76080
72632 4.25539 3.27875 2.87040
,87535 2.39750 2.95904
,95036* * 2.72815* 2.79571
2
2
4
2
3
4
4
2
2
#10
.00000
.30103
.00000
.14922
.56526
.17609
.49693
.51587
.63246
#11
1
3.26682
4.23045
1.57978
2.13033
inoculation with laboratory cultures: 7-7 and 7-29 with E. c., Pseud., and Serr.
o
o
-------
Table B-4. Total Coliform Bacteria Densities In Laboratory Scale Wastes
Stabilization Ponds, As Log10/ml.
Date
8- 4-69
8- 6-69
8- 8-69
8-11-65
8-13-69
8-15-69
8-18-69
8-20-69
8-22-69
8-26-69
Sample Station —
Raw #1 #2
5.19866
5.85126
5.75587
5.71600
5.78176
5.57978
5.08279
6.10551
5.86332
5,72222
3.40449 1.72222
1.07918
4.06633 0.30103
3.81291 1.49136
4.39794
4.65321 0.77815
4.37566
4.51521
3.84510 0.17609
3.84510
0
0
0
2
2
0
0
0
0
#3 #4
.54407
.84510 0.30103
.30103
.32222
.04139
.00000 0.00000
.00000
.00000
.00000
#5
3.00432
1.97543
1.02110
0.81291
1.25527
3.30103
0.90309
1.71391
1.71181
2.04238
2
2
3
1
4
3
2
2
2
#5
.02632
.95424
.17609
.99123
.65321
.29003
.95424
.52114
.49554
#7
2.20140
1.23045
1.06446
0.30103
1.51188
3.17609
1.72428
0.00000
0.17609
#8
2.05881
3.61278
3.06446
3.51851
3.69897
2.38021
3.14613
3.17609
2.77815
#9
1.80787
1.79934
0.95424
3.27875
3.50515
4.17609
3.34242
3.27875
3.14613
2.51055
#10
2.09078
0.69897
1 .92428
3.23045
3.14613
4.53148
3.2787S
3.11394
3.32736
2 .13672
#11
1.74624
0.90309
2.30103
1.76343
4.43136
2.40867
0.17609
0.30103
1.07918
-------
Table B-5. Escherichla coli Densities In Laboratory Scale Waste
Stabilization Ponds, As Log.-/ml.
Date
Sample Station—
Raw #1
#3
#5
t8
tt9
#10
#11
7- 3-69
7- 7-69 4.74036
4.54407
3.74036*
7- 9-69 4.69954 5.07918 4.55630
7-11-69
7-14-69
7-16-69 5.49381
7-18-69 5.27875
4.69897 5.36173
4.95424 3.13033
6.29447 3.00000
5.06004
7-23-69 6.49206 4.86332 0.00000
7-25-69 5.06070 4.11394
7-29-69 5.41497 3.62325* *
* 3.00496* *
5.59660 4.30103 4.90687 3.96614 3.67685
4.38021 4.84510 5.74036 4.30103 4.39794
3.62839 3.90309 3.00000 3.00000 3.77815
3.17609 4.30103 4.02119 3.47712
4.11394
3.60206 3.21748
* 2.41664*
3.15381
3.17609
2.74036 3.84510
2.92942 2.03443
3.12222
3.60206
4.04115
0.00000
1.14613 2.81291
3.17609* 1.89487
3.00000
4.00000
3.56526
3.90309
3.77815
4.44560
2.60206
2.02531
3.03080
1.68124
4.07918
3.00000
1.54407
1.11394
*Inoculation with laboratory cultures: 7-7 and 7-29 with B.C., Pseud., and Serr.
O
oo
-------
Table B-6. Escherichia coli Densities In Laboratory Scale Waste
Stabilization Ponds, As Log.g/ml.
Date
8- 4-69
8- 6-69
8- 8-69
8-11-69
8-13-69
8-15-69
8-18-69
8-20-69
8-22-69
8-26-69
Sample Station —
Raw #1 f2 #3 f4 #5
4.95785 2.20276 1.72222 0.54407
4.37107 0.90309 0.60206
4.14613 3.27875 0.00000
3.65321
5.21748 3.81291
4.92942 4.00000
5.55023 3.54407 0.00000
5.27875 4.14613
5.26717 3.54407 0.00000
5.00432 3.39794
3.00453
1.97543
0.69897
0.30103
0.54407
0.84510
1.70969
1.71181
2.03941
#6
1.90309
2.07918
3.07918
1.30103
0.47712
2.69897
2.54407
1.73640
0.39794
#7 f8
2.12385 2.02531
0.87506
0.84510 3.00000
0.00000 3.84510
0.00000 2.60206
1.70969 2.92942
2.47712
2.30103
2.30103
#9
1.14613
1.30103
3.92942
2.54407
2.00000
2.84510
2.65321
2.79588
1.73838
#10
2.09078
1.11394
2.69897
2.30103
2.77815
2.95424
2.47712
2.81291
1.71181
#11
1.74624
0.90309
2.30103
0.69897
0.00000
0.00000
o
co
-------
Table B-7. Pseudomonas aeruginosa Densities In Laboratory Scale
Waste Stabilization Ponds, As Log /ml.
Date
Sample Station—
Raw #1 #2
#3
#5
#8
#9
#10
#11
7- 3-69
7- 7-69
7- 9-69
7-11-69
7-14-69
7-16-69
7-18-69
7-23-69
7-25-69
7-29-69
4.30103
* *
5.97772 5.97772
4.00000 5.30103
5.17609
3.87506
0.00000
* - * *
5.34242 3.00000
4.92942 3.00000 3.81291
4.00000
4.00000 3.77815
3.74036
4.00000
3.00000
2.00000
*Inoculation with laboratory cultures: 7-7 and 7-29 with E. c.. Pseud. , and Serr.
-------
Table B-8. Pseudomonas aeruqlnosa Densities In Laboratory Scale
Waste Stabilization Ponds, As Log10/ml.
Sample Station —
Date Raw #1
8- 4-69
8- 6-69
8- 8-69
8-11-69
8-13-69
8-15-69
8-18-69
8-20-69
8-22-69
8-26-69 5.30103
#2 #3 #4 #5 #6 #7 #8 #9 #10 #11
4.17609 3.30103 3.69897 3.00000 3.47712
3.00000 3.00000
3.00000 3.00000 3.84510
3.00000 3.00000
3.00000
3.00000 3.77815 2.30103 4.74036
3.00000 3.74036 2.90309 2.79588 3.00000 3.00000 3.39794
o
en
-------
Table B-9. Serratia marcescens^ Densities In Laboratory Scale
Waste Stabilization Ponds. As Log /ml.
Date
Sample Station—
Raw #1
#2
#3
#5
#7
#8
#9
#10
#11
7- 3-69
7- 7-69
7- 9-69
7-11-69
7-14-69
7-16-69
7-18-69
7-23-69
7-25-69
7-29-69
5.64836 5.53782
4.75967 3.47712
2.81291 2.92942
2.00000
4.73038 4.74036
3.77815
3.90309
3.77815
4.90309
* 4.12222* 4.24304 3.95424
* 3.00000* 4.30103*
4.00000
4.30103
4.00000
*Inoculation with laboratory cultures: 7-7 and 7-29 with E. c., Pseud., and Serr.
O
CT)
-------
Table B-10. Serratia marcescens Densities In Laboratory Scale
Waste Stabilization Ponds, As Log1()/ml.
Sample Station—
Date Raw #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11
8- 4-69 4.30103 4.16137 3.77815 4.19033 3.00000 3.39794
8- 6-69 4.19033 3.30103
8- 8-69 4.74036
8-11-69
8-13-69
8-15-69 3.00000
8-18-69 5.00000
8-20-69
8-22-69
8-26-69
O
•si
-------
Table B-ll. Chromagen Densities In Laboratory Scale Waste Stabilization Ponds, As Log /ml.
Sample Station —
Date Raw #1 #2 #3 #4 #5
7_ 3-69 5.36642 5.29003 4.74036 4.38021
7_ 7-69 4.00000
7- 9-69 5.07004 5.45484
7-11-69 5.84510 5.32222 5.54407 4.64836 5.37475 6.26245
7_14_69 3.87506 3.74036 4.06070
7_16-69 3.84510 3.77815 4.27875
7-18-69
7-23-69 6.06070 5.47712 4.52114 3.77815 5.35781 4.35784
7-25-69 6.63849 3.47712 3.00000
7 29 6g 3.47712 4.27875 2.69897
t6
4.70757
6.02531
5.01599
3,74036
4.77761
5.09342
4.41497
3.87506
#7
4.09691
5.74036
5.91566
4.00000
3.79588
4.07918
4.34242
4.46613
#8
5.62325
4.86332
4.74429
2.81291
3.19033
4.51851
4.60206
4.63347
#9
3.95424
5.07555
5.03743
3.66978
3.30125
6.00065
3.47712
4.77452
#10 #11
4.52504 4.13033
5.65992
5.74710
5.07918 5.63548
4.10551 4,30103
4.91116 2.55023
4.77815 0.81291
3.19033
o
00
-------
Table B-12. Chromagen Densities In Laboratory Scale Waste Stabilization Ponds, As Log1()/ml.
Date
Sample Station—
Raw #1
#3
#4
#5
#6
#8
#9
#10
#11
8- 4-69
8- 6-69
8- 8-69
8-llr69
8-13-69
8-15-69
8-18-69
8-20-69
8-22-69
8-26-69
6.47712
5.00000
5.69897
6.17609
4.94448 4.07004 3.86923
6.95424
3.47712
2.47712
4.81291
4.04139
6.81291
4.03141
3.74036
4.61805 5.19033
5.11394
3.07004
7.21748
3.00000
5.10037
4.30103
4.04139
4.7493
7.60206
2.91645
3.94511
3.85278
4.61542
3.74036
8.55630
2.69897
5.67210
5.43933
5.17245
4.00000
8.46240
4.95424
5.06070
4.62325
5.37566
7.60206
4.30103
4.00000
5.09691
4.72428
5.85431
3.00000
8.38021
4.30103
3.47712
5.14998
3.55023
7.65321
4.47712
4.41498
4.73038
3.84510
7.34242
4.09691
5.01284
5.30103
3.75967 5.04139 4.54407 5.08991 5.24304 5.46240 4.93450
o
CD
-------
Table B-13. Total Bacteria Densities In Waste Stabilization Ponds, As Log /ml.
Date
6- 4-69
6- 5-69
6- 6-69
6- 9-69
6-11-69
6-13-69
6-16-69
6-18-69
6-20-69
6-23-69
6-25-69
6-27-69
6-30-69
Sample Station —
#1 #2
6.34242
6.44716
6.73560
6.70842
6.70415
6.69984
6.59550
6.96100
6.69108
7.03262
6.35218
6.57978*
6.69897
6.02119
6.38202
6.58659
5.98677
5.72673
6.46240
6.82217
6.65706
6.35458
#3
6.30103
7.00000
6.67394
6.35025
6.28556
5.95904
5.90472
6.29885
6.48430
6.64246
6.34044
6
5
6
6
4
3
4
5
4
3
6
t4
.51455
.92428*
.14922
.20140
.81954
.00000
.74819*
.06819
.60206
.14613
.96656
#5
5.32222
5.43136
6.58883
5.55630
5.29667
5.67025
5.67302
4.47712
4.46850
4.38758
#6
4.98677
5.07918
5.69020
5.74036
4.56820
4.79934
4.74036
5.24304
4.27875
4.57978
3.95424
3.20412
3.25696
6
5
6
6
5
5
5
5
3
3
4
#7 f8
.15534
.66276 5.60206
.28780 5.69897
.65610 5.19866
.42975 5.39794
.45025 5.41497
.28330 6.34830
.17319 5.29885
*
.00000
.57978 3.50515
.57980 5.70672
#9
5.66276
S. 56820
4.84510
4.77815
4.94448
4.44716
3.60206
3.00000
4.85126
3.04139
3.20412
3.66783
#10 #11 #12
5.80482 5.79029
6.49136 6.25285
4.44716 5.29003 5.34242
5.17898 4.79239 3.30103
3.77815
* 3.60206
3.87506
5.46538 5.65992 3.89763
5.65300 5.78247 4.48195
*Inoculation with laboratory cultures: (6-5; E. c.. Pseud.) (6-16; E. c.. Pseud., Serr.) (6-19; shown as 6-20; E. C., Pseud., Serr.) (6-23; E. c..
Pseud., Serr.)
-------
Table B-14. Total Bacteria Densities In Waste Stabilization Ponds, As Log10/ml.
Date
7- 2-69
7- 7-69
7- 9-69
7-11-69
7-16-69
7-18-69
7-21-69
7-23-69
7-25-69
7-29-69
7-31-69
Sample Station —
#1 #2
6.73878
7.29612
6.86629
7.34587
7.19576
7.49066
8.52088
7.92505
7.79449
7.12385
7.22272
6.98644
7.35362
6.36605
7.27646
7.37767
6.60016
7.96497
7.19033
6.82445
7.27646
6.92298
#3
6.93865
7.19590
7.03993
7.53656
7.10806
6.03523
8.25600
7.34635
7.19451
6.56820
7.14301
#4
5.37220
5.43616
3.87344
5.36577
5.59106
6.63829
5.77706
5.53013
7.28319
5.20548
5.77379
#5
5.48853
5.58092
5.42922
6.09412
5.78319
6.48053
6.19089
6.73632
7.17713
5.61262
5.52504
#6
4.10072
3.58192
5.47787
5.28780
4.16443
6.18064
4.86540
6.02735
6.26174
4.15987
4.45102
#7
5.14426
5.21484
5.17342
6.64147
5.51521
6.59555
6.33163
6.50853
6.28171
5.29115*
5.33746
#8
5.23259
4.98227
5.40747
6.22154
5.38471
6.35005
6.18227
6.57119
6.50127
5.36949
5.26600
#9
5.18611
4.74819
4.87484
5.88053
4.52827
5.33011
5.25139
5.79955
5.44754
4.24055
2.94201
#10
5.72835
5.22660
6.27140
6.21163
6.44091
6.82086
6.38739*
7.12548
6.72016
6.08814
6.02325
#11
5.76530
5.61013
6.63624
6.37658
6.38292
6.62428
7.22789
6.93717
7.13815
6.14768
5.56229
#12
S. 30604
5.07004
5.74321
5.85187
5.96308
6.62926
6.43553
6.86608
5.39094
5.24920
4.92169
*Inoculation with laboratory cultures: 7-21 and 7-29 with E. c., Pseud., and Serr.
-------
Table B-15. Total Bacteria Densities In Waste Stabilization Ponds, As Log ./ml.
Date
8- 4-69
8- 6-69
8- 8-69
8-13-69
8-15-69
8-18-69
8-20-69
8-22-69
8-26-69
Sample Station —
#1 #2
7.33244
7.66229
7.05207
7.04630
6.96379
7.42243
7.12710
7.51851
6.87216
7.24981
6.93069
7.20880
7.08636
7.22789
6.91116
7.22531
7.29115
6.84819
#3
6.98000
6.99344
7.29170
7.08189
7.48996
7.29447
7.26186
7.29336
6.84973
4
6
6
6
7
6
6
7
6
#4
.67210
.12215
.40140
.88550
.28499
.80702
.46165
.05018
.67440
#5
6.33496
5.99100
6.63849
6.82102
7.14613
6.87938
6.71391
6.87520
6.54777
5.
5.
5.
4.
6.
6.
6.
5.
#6
63246
55991
49136
96379
13909
37475
89708
72428
#7
5.62480
5.58574
7.20412
7.05300
7.38828
7.15503
6.81067
7.42854
6.16584
5
5
6
6
6
6
5
7
5
#8
.56526
.79934
.44770
.61316
.84510
.73739
.79831
.25768
.72222
#9
5.04336
5.37794
5.93197
5.78645
5.93827
6.24748
5.64738
6.47276
5.59988
#10
6.62660
5.55961
6.46117
6.34586
6.71904
6.55023
5.93827
6.89070
5.54407
#11
5.63849
5.92634
6.35338
6.25467
6.66745
6.87795
6.02531
6.30049
5.55328
#12
4.97313
4.98453
5.66229
5.15076
6.12548
5.83727
5.60959
6.56926
5.31755
-------
Table B-16. Total Coliform Bacteria Densities In Waste Stabilization Ponds, As Log n/rnl.
Date
6- 4-69
6- 5-69
6- 6-69
6- 9-69
6-11-69
6-13-69
6-16-69
6-18-69
6-20-69
6-23-69
6-25-69
6-27-69
6-30-69
Sample Station —
#1 #2
5.47712
5.77815
5.77670
5.98137
5.77815
5.90309
5.02938
6.23045
5.75587
5.80618
6.51983
5.85733
5.69897
5.30103*
5.60206
5.70969
5.00000
6.21484
4.77085
5.17319
5.04139
4.92942
4.65321
4.77815
4.60206
#3
5.00000
5.47712
5.83569
4.81954
4.93450
4.66276
5.17609
5.04139
5.43136
4.96848
4.84510
4.77815
5
4
4
5
2
0
2
4
2
0
0
#4
.60206
.20412*
.17609
.15836
.69897*
.69897
.20140
.90309
.40483
.69897
.00000
4
4
3
3
3
4
1
1
0
1
0
#5
.11394
.39794
.69897
.00000
.17609
.39794
.86923
.64345
.69897
.25527
.54407
#6
3.90811
2.80625
1.74115
2.93450
1.63347
1.59106
1.72428
1.86332
1.87216
1.92942
5
4
5
4
3
3
2
3
1
2
3
1
#7
.47712
.54407
.81889
.88081
.92942
.84510
.71349
.00000*
.13033
.88053
.04532
.44716
#8
4.34242
3.90309
3.47712
4.14613
4.25527
3.69897
3.19285
3.84510
0.60206
1.32222
3.18752
0.45788
#9 #10 #11 #12
3.77815
4.74819 4.57403
4.83885 4.43136
3.74036 3.87506 2.69897
0.00000 3.37840 2.00043 1.04139
3.84510 3.60206 0.97772
4.17609 * 3.00000
1.39794 3.03342 2.53020 0.77815
2.94349 3.48430 0.77815
0.54407 2.95665 0.30103
*Inoculation with laboratory cultures: (6-5; E.G., Pseud.) (6-16; E.G., Pseud., Serr.) (6-19; shown as 6-20; E. c., Pseud., Serr.) (6-23; E.G.,
Pseud., Serr.)
-------
Table B-17. Total Coliform Bacteria Densities In Waste Stabilization Ponds, As Log /ml.
Date
7- 2-69
7- 7-69
7- 9-69
7-11-69
7-16-69
7-18-69
7-21-69
7-23-69
7-25-69
7-29-69
7-31-69
Sample Station —
#1 #2
5.62325
6.12304
5.59934
5.65321
6.81258
5.87216
6.17464
6.66039
5.62839
6.01807
4.89209
4.90300
5.19728
5.77815
5.27875
5.24304
5.14922
4.96848
5.27068
4.59660
4.29003
5.05500
#3
5.07004
4.74036
5.17609
4.64098
4.89900
4.89209
4.65801
5.51117
4.94448
4.73838
4.06633
#4
2.70136
0.69897
0.60206
2.94201
2.84261
2.02735
0.00000
0.00000
2.00304
#5
0.02119
2.70265
2.70243
1.21748
3.20412
3.92686
0.00000
2.13354
2.84510
2.00000
#6
2
0.30103 3
0.54407 1
0.74036
4
3
3
0.90309 4
3
0.47712 2
0.14613 3
#7
.75797
.49534
.31175
.02119
.46240
.83727
.01912
.66745
.74036*
.31175
2
3
3
3
3
3
4
3
2
2
#8
.71204
.19770
.84510
.94939
.55630
.47349
.66978
.74036
.97772
.90309
#9
0.47712
0.60206
1.81291
1.00000
1.13033
1.29003
0.30103
0.30103
0.90309
3
3
3
2
4
4
3
1
5
#10
.42704
.49631
.69897
.03141
.48359
*
.09342
.37107
.90445
.13354
#11
2.77670
2.80058
0.84510
2.81291
3.86332
3.90982
3.11394
3.89487
3.50174
3.19033
#12
2.70286
0.47712
1.17609
3.90300
0.54407
0.77815
1.24304
0.47712
1.49831
*Inoculation with laboratory cultures: 7-21 and 7-29 with E. c. , Pseud., and Serr.
-------
Table B-18. Total Coliform Densities In Waste Stabilization Ponds, As Log., ./ml.
Date
8- 4-69
8- 6-69
8- 8-69
8-13-69
8-15-69
8-18-69
8-20-69
8-22-69
8-26-69
Sample Station —
tl #2
5.19866
5.85126
5.75587
5.78176
5.57978
6.08279
6.10551
5.86332
5.72222
4.99782
4.71809
4.54407
4.96142
4.81790
4.97081
4.92686
4.90037
5.07225
#3
4.86332
4.53148
4.92942
4.69461
4.88081
3.72428
4.96142
4.65801
4.84819
#4
0.30103
1.78176
1.17609
1.97658
3.92169
3.03141
2.15381
2.00432
3.13409
#5
2.00000
2.87852
2.88944
2.55509
3.47712
3.09691
3.71809
1.91116
3.50379
#6
1.30103
0.00000
1.00000
0.47712
1.55630
0.60206
2.47712
0.00000
#7
2.47712
3.51188
3.99123
4.11561
3.82607
3.96379
3.84354
3.84819
3.29003
#8
2.30103
3.27875
3.52504
3.46982
3.67210
3.72222
3.89625
3.34242
3.68124
#9
0.77815
1.00000
0.90309
1.72016
0.65321
1.72016
2.41664
#10
3.13033
2.81291
2.00647
3.65321
3.95424
3.16137
3.63849
3.61013
3.24304
3
3
4
3
4
3
3
3
#11
.00000
.75967
.59106
.60314
.14301
.47712
.55630
.74233
#12
0.77815
0.30103
1.17609
2.15076
1.71181
2.01072
1.78355
2.50651
Cn
-------
Table B-19. Escherichla coll Densities In Waste Stabilization Ponds, As Log. /ml.
iu
Date
6- 4-69
6- 5-69
6- 6-69
6- 9-69
6-11-69
6-13-69
6-16-69
6-18-69
6-20-69
6-23-69
6-25-69
6-27-69
6-30-69
Sample Station —
#1 #2
5
5
5
4
5
5
4
5
5
5
5
.00000
.00000
.23553
.81954
.00000
.39794
.19033
.97772
.34242
.67210
.58546
5
5
3
4
4
3
5
4
4
4
4
4
.30103
*
.00000
.92942
.11394
.88195
.69897
.03743
.84510
.54407
.00000
.74036
.47712
5
4
4
4
3
3
4
4
4
4
4
#3 #4 #5 #6 #7
3.84510* 3.30103
.00000
.06070 3.97772 3.30103
.46240
.34242 2.69897
.39794 *
.17609 0.30103
.72428 1.89763 2.43457
.60206 0.69897
.39794 0.60206 0.17609
.77815 0.00000 0.47712
.65321 0.00000 0.30103
4.04139 3
4.75587 3
4.42813 2
3.00000 3
3
3.17609 3
* 3
0.00000 0
2.40140 0
2.77085 3
1.30103 1
#8
.00000
.75587
.69897
.39794
.81291
.17609
.60206
.39794
.00000
.02531
.54407
#9 #10 #11 #12
3.00000
3.54407 3.47712
3.39794 3.00000
3.00000
0.77815 1.46240 0.87506 0.54407
0.17609 1.69897* 3.47712 0.47712
3.39794
0.17609 2.94448 1.54407 0.17609
3.35218 2.77815 0.00000
0.17609 1.99123
*Inoculation with laboratory cultures: (6-5; E. c.. Pseud.) (6-16; E. c., Pseud., Serr.) (6-19; shown as 6-20; E. c. , Pseud., Serr.) (6-23; E. c. ,
Pseud., Serr.)
-------
Table B-20. Escherichia coll Densities In Waste Stabilization Ponds, As Log /ml.
Date
7- 2-69
7- 7-69
7- 9-69
7-11-69
7-16169
7-18-69
7-21-69
7-23-69
7-25-69
7-29-69
7-31-69
Sample Station —
#1 #2
5.04139
5.46613
5.44248
5.49381
5.27875
5.54407
6.49206
5.06070
5.41497
4.67210
4.17609
5.06070
4.68970
4.69897
4.45102
4.75587
4.97772
3.92942
4.13830
4.78533
4
4
S
3
4
4
4
4
4
4
3
#3 #4 #5
.82930 0.30103 0.00000
.30103 0.39794 0.47712
.00000
.30103
.57403 2.00967 2.00000
.41497 2.57692 2.00000
.10551 0.00000
.58546 2.01599 1.90309
.39794 2.00000
.48430
.66976
#6
1.
0.30103 3.
0.54407
3.
2.
3.
0.00000 3.
3.
3.
3.
#7
46240
24920
63347
90309
49136
78176
14613
47712*
11394
#8
0.77815
3.24834
3.30103
3.41078
3.04139
3.50515
2.90309
2.54407
2.77815
#9
2
0.00000 3
2
2
0.30103 3
0.69897 3
0.00000
0.00000 3
0.00000 2
2
4
#10
.89154
.00000
.00647
.80647
.59660
.43933
*
.65801
.81291
.30103
.66276
#11
2.70906
1.60746
3.67669
3.24304
3.03141
3.69897
3.14613
2.84510
2.97772
#12
0.60206
0.00000
0.81291
0.30103
0.30103
*Inoculation with laboratory cultures: 7-21 and 7-29 with E. c. , Pseud., and Serr.
-------
Table B-21. Escherlchia coll Densities In Waste Stabilization Ponds, As Log /ml.
Date
8- 4-69
8- 6-69
8- 8-69
8-13-69.
8-15-69
8-18-69
8-20-69
8-22-69
8-26-69
Sample Station —
#1 #2
4.95785
5.37107
5.14613
5.21748
4.92942
5.55023
5.27875
5.26717
5.00432
4.66745
4.27875
3.90309
4.51188
4.32736
4.62066
4.14613
4.37107
4.30643
#3
4.11394
4.14613
4.25527
4.06691
4.16137
3.43933
4.20412
4.14613
4.00432
#4
0.84510
1.75967
3.86332
2.84510
1.73640
0.17609
2.14535
#5
0.47712
1.72428
2.18184
2.84510
2.82930
2.00000
1.72428
2.49693
#6
0.00000 3
0.30103 3
2
0.60206 3
3
3
3
3
#7
.20412
.23045
.92942
.20412
.56229
.30103
.26717
.07918
#8
3.00000
3.84510
2.90309
3.17609
3.33244
2.81291
2.94201
3.04139
#9
0.30103
0.30103
0.00000
1.25527
0.30103
0.30103
1.70329
2.35218
#10
2.65321
2.69897
2.54407
2.77815
2.90309
2.95424
3.06070
2.90309
2.91645
#11 #12
2.84510
3.. 2 78 75
2.65321 0.30103
2.92942 0.65321
2.00000
3.00000 0.00000
2.88930 1.71809
3.13830 0.77815
oo
-------
Table B-22. Pseudomonas aeruginosa Densities In Waste Stabilization Ponds, As Log1Q/ml.
Date
Sample Station—
#1 #2 #3
#4
#6
#8
#9
#10
#11
#12
6- 4-69
6- 5-69
6- 6-69 5.00000
6-11-69
6-13-69
6-16-69
6-18-69
6-20-69
6-23-69 4.81291
6-25-69
6-27-69
6-30-69 5.00000
3.00000
3.00000
3.17609
5.00000 4.00000
* 3.00000
0.00000
3.30103
3.00000
0.00000
0.60206 0.30103 0.00000
3.77815
3.00000
2.69906 3.00000 3.00000* 5.87506
4.60206
*Inoculation with laboratory cultures: (6-5; E. c., Pseud.) (6-16; E. c., Pseud., Serr.) (6-19; shown as 6-20; E. c.. Pseud., Serr.) (6-23; E. c.,
Pseud., Serr.)
CO
-------
Table B-23. Pseudomonas aeruainosa E)ensities In Waste Stabilization Ponds, A* Log
Sample Station —
Date #1 #2 #3 #4 #5 #6 #7 #8 #9
7- 2-69 5.30103
7- 7-69
7- 9-69 0.69897 0.97772 0.00000
7-11-69 3.00000
7-16-69
7-18-69
7-21-69
7-23-69
7-25-69
7-27-69
7-29-69
#10 #11 #12
3.00000
1.04139
Mnoculation with laboratory cultures: 7-21 and 7-29 with E. c., Pseud., and Serr.
-------
Table B-24. Pseudomonas aeruginosa Densities In Waste Stabilization Ponds, As Log n/ml.
10'
Sample Station —
Date #1 #2 #3 #4 #5 #6
8- 4-69
8- 6-69 6.30103 3.47712 3.00000
8- 8-69 5.00000
8-13-69 6.00000
8-15-69 5.00000
8-18-69
8-20-69
8-22-69
8-26-69 5.30103 4.00000
#7 #8 #9 #10 #11 #12
3.74036
3.09691 4.30103 3.17609
4.00000
3.30103 3.74036 3.00000
tsj
-------
Table B-25. Serratia marcescens Densities In Waste Stabilization Ponds, As Log1Q/ml.
Sample Station—
Date #1 #2
#3
#4
#5
#6
#7
#8
#9
#10
#11
#12
6- 4-69
6- 5-69
6- 6-69
6- 9-69
6-11-69
6-13-69
6-16-69
6-18-69
6-20-69
6-23-69
6-25-69
6-27-69 5.00000
6-30-69
5.00000
3.00000
3.60206 6.76716
4.30103
3.69897 0.30103 0.30103
2.69914 * 3.00000
*Inoculation with laboratory cultures: 7-21 and 7-29 with E. c. , Pseud. , and Serr.
-------
Table B-26. Serratia marcescens Densities In Waste Stabilization Ponds, As Log /ml.
Sample Station —
Date #1 #2 #3
8- 4-69
8- 6-69
8-< 8-69
8-13-69
8-15-69
8-18-69
8-20-69
8-22-69
8-26-69
#4 #5 #6 #7 #8 #9 #10 #11 #12
3.00000 3.77815 3.00000 4-.17609 3.74036
3.00000 4.04139 4.00000
4.00000 4.77815
4.00000 4.00000
5.17609 4.00000
CO
-------
Table B-27. Chromagen Densities In Waste Stabilization Ponds, As Log1Q/ml.
Date
6-30-69
7- 2-69
7- 9-69
7-11-69
7-18-69
7-21-69
7-23-69
7-25-69
7-29-69
7-31-69
Sample Station —
#1 #2
S
5
5
5
5
6
6
5
.17609
.54407
.07004
.84510
.69897
.06070
.63849
.00000
5
5
6
6
6
5
5
6
. 15381*
.39794
.11644
.96848
.50515
.60206
.69897
.32222
5
5
6
7
5
6
5
6
0
#3
,60206
.39794
.19451
.28103
.77815
.00000
.90309
.63347
.00000
3
4
2
5
6
3
6
6
3
#4 #5 #6
.35392 3.00475 3.00303
.38021
.70372 4.77452 5.01912
.17026 5.53593 4.87338
.23465 5.65562
,00000
.19866 6.74135 5.69858
.41747 6.86004 5.93044
,74086 2.54407
3.00000 2.47712
#7
3.00260 3
5.04532 4
4.33415 4
6.27875 4
6.17826 5
5
6.08027 5
5.91619 6
* 2
#8
.60206
.93952
.51382
.38021
.92763
.39794
.96755
,04001
.17609
#9 #10 #11 #12
1.73239 3.44404 3.47712 3.01452
3.47712 3.97772
4.37493 5.92428 6.27300 4.20656
5.92763 5.35698 5.69020 5.26007
4.99454 6.30750 5.72835 5.63949
4.40824 * 6.05500
5.40697 6.71809 6.59638 6.50583
4.83569 6.34044 6.71684
4.65321
*Inoculation with laboratory cultures: 7-21 and 7-29 with E.G., Pseud., and Serr.
to
-------
Table B-28. Chromagen Densities In Waste Stabilization Ponds, As Log10/ml.
Sample Station —
Date #1 #2 #3
8- 4-69 6.47712 6.53148 6.00000
8- 6-69 6.00000 6.27875 6.13830
8- 8-69 5.69897 5.30103
8-13-69 6.35218 6.47712
8_15_69 6.35218 6.477:2
8-18-69
8_20_69 6.69897 6.37107
8-22-69 6.17609 6.09691 6.27875
8-26-69
#4
4.91381
5.89625
5.96731
6.84042
6.84042
6.07004
5.69897
6.45102
6,
5
6
6
6
6
6
6
#5
.26717
.82930
.19728
.76343
.76343
.60206
.57978
.22337
#6 #7
5.60206 4.21748
5.46761 4.67897
5.17934
7.02016
7.02016
5.57403 7.11394
5.57978 6.70969
6.69461 7.07555
5,
5
5
6
6
6
5
6
#8
,24428
.33746
.39138
.54407
.54407
.69897
.34242
.86629
#9
4.95454
5.29885
5.69897
5.69897
6.19728
4.23045
6.13033
6,
5
5
6
6
6
5
6
#10
.61013
.29003
.37143
.18255
.18255
.51188
.69020
.51851
#11
5.49136
5.25888
5.29281
6.10551
6.10551
6.84510
S. 70757
5.84819
#12
4.62839
4.66511
4.81624
5.54095
5.60206
5.31702
6.24981
ts?
en
-------
APPENDIX C
PROGRAM BETA FORMAT
126
-------
127
000002
000002
000002
000002
00000?
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
000002
OOOOC2
000002
000002
000002
000002
000002
000002
000002
BFTA(INPUT,OUTPUT)
DIMFNSION IKOEXO*) »Y<*00> »X (20,400) ,«UOO) ,TABUF.13*»3) »A\20»20> .
JYX(20).8 ,CfoT(20,20)»RDrT(20),CINV(20»20)«NSU8(20)»0p(20)»
2CCVA«'20,?0)«SU1(2n),PSTAR(?0)«FORM2(5>.
3FCRM(7> ,CM20,2A) ,C (20,20 )»v/ARI (400) ,YHnT (400)
DIMENSION YHATT(*nn>» YUP(4nO), YLO»(40n), DELTY(400)
DIMENSION ,ICFLL
CCMMON/6/TABLF
CCMMON/C/\
KTA6LFB1.,2.»3.»4..5..6..7..fl..9.»lo..n..l2..13.»»*.,l5,»l6.
5ft.3138, 2.<9?00,2. 353*,?. 1318,?.0150 0.9*32, 1.89*6, 1.6595, 1.8331,
61.«125,1.7959, l.r«?-3, 1.7709. 1.7613 ,1,7530, 1.7459. 1.7396, 1.7341,
71.7291,1.7?47,1.7207,1.7171,1.7139.1.7lo^.l.7081,1.70S60.7033,
81.70U,1.A991.1.6973,J.6839,1.6707.1.6577,1.64*9.
1 I?. 706,«.3027,3.1625,2.7764,2.5706,2.4469,2.3646.2.3060,
22.2622,2.2281,2.2010,2.1788,2.1604,2.1448,2.1315,2.1199.?.1098,2.1
4,2.0*8*.2.0*52,2.0*23,?.0211,2.0003, 1.9799,1.9600)
1 FORMAT (I?,A3,5X,A7)
38 FCRMAT(29U<.?H?X(I2,17H) AFTFP REMOVING 12, 1*H (2HX (12,2H) )))
65 FORMAT(5F?0.In)
66 FCRMAt(SH A-M.ftTRlX /)
f,7 FCRMAT(//50H INVERTED A-MATBTX (C-MATRIX) /)
f 0 r v. T^ >" •'•* ' \ r f I i r i.. " M '" •"• ' ' ' ""-' "
75 FORMAT (X/Tfth RFGWESStrN COEFFICIENTS /) -...,,.
,«=60FCRMAT (//«CH TnTAL CALCULATED FROM DATA OI6FERS FROM TOTAL CALCUL
BY SUMMING COMPONENTS BY FeQ.10/YI
».05
mt)6
FORMAT (3H P(I?,"=H)
?r>). pUF TO
= F20.1f)
I502X.-5E20.10//15H
19, 12X.E20.10)
FROM
f,\*
A15
H61
<>17
FORMAT (1?..?*4)
FcKl'HTIn?,«,m - F2a.in,ioH */. F2ouo>
FCRMATU5I- VAPItNCE OK YII3.3H) *F2n.lO)
FCRfAT13HOV(I3,RH) sF20.1n,10H +/-
FORMAT ( -»HRY(13,?H) = F?0 . 1 0 ,5X ,F?Q . I 0 .5XF20 . 1 0.5XF20 . 1 0
FCRM6l(/4nM'"ML'LTlPl.F. CORRELATION COFFFIrlFNT (H) « E20.1"//
17H VAHI«\CF RATIO * E20.1"//)
F20.10,5X,F20.lO,Fl0
4)
I
FCRMAT(SF?n,10)
FCPMAT!//?4K CONFIDENCE LIMTTS CF 8 )
' ON •'
^eg FCRMATC3H ... ,._
fcqj FORMAT (12e,i2X,-»E2n. In)
^5 FCRMATOH x',I?.)OH) REMOVED 11 0 , l2X,3E2n . 10J
ft40 FORMAT{//Ofth SOURCE DEGREES OF FRE5DOM
IRE5 VARIANCE VARIANCE RATTO //)
*,61 FORMAT (1H(T2,15M(3H X(I2,2H) )))
700 FCRMAT(7H PSS = F20.1")
701 FCRMAT(13H F-KEto MATRIX//4(*F?0.1O/))
OF
SC.UA
000
001
002
003
004
005
006
007
008
009
010
t)U
012
013
014
015
016
017
018
020
021
022
023
024
025
026
027
02fl
029
030
031
032
033
03*
035
036
037
038
039
040
041
042
043
044
045
0*6
047
048
0*9
050
051
052
053
-------
128
000002
000002
000002
000002
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000002
000002
000002
000002
000002
000010
000022
000030
000040
000043
000052
000056
0000«4
000071
000076
000100
000102
000111
000111
000115
OOOIZ4
0001J5
0001)21
000132
000134
000135
000136
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000154
0001*4
0001^7
000171
000172
000202
000206
000212
000214
000215
000225
000330
000334
000341
000246
000352
000354
000273
000300
702 FORMAT <26w LEAVING CUT VARInPLE NC. I2//13H NEW C-MATRIX//) 054
705 FCRMATI///) 055
720 FCRMATllHl ) 056
721 FCRMAT(67x,27H DEVIATION 0/0 ) 057
7?2 FORMAT <35(- THE AVERAGE ABSOLUTE DEVIATION IS Fln.4,9H PERCENT.) Q58
7?21 FORMAT (3nH ABSOLUTE AVERAGE DEVIATION = ,F20. 10 .20HCONFTDENCE FAC
1TCR = .F?n.lO)
7?3 FORMAT < 10< , 1 OH INPUT 0«TAt//tSX» IHYi 15X t ?*M VALUE (S) -LEFT TO RIGHT 059
1) 060
72* FORMAT
7?41 FORMAT
726 FORMAT!//) 061
13?7 FCRMAT(//Pl|- ANALYSIS OF VAojANCE ) 062
16?8 FCRMAT(//?4H COfFJDENrE LIMTTS OF Y ) 063
4040 FORMAT (19M26H wlTH LIMITS rFtlS.llH PERCENT//)) 064
99 READ 600.TMCFX 065
RtAO l.NGl FSS.Mf/Q.TYPp
READ 724, ( ICELL ( I ) • 1 = 1 18)
READ 60). ".K 066
KKON = PO * 5 • NGUESS OAS
ENCODE (33. 4<>4ntFORM2)KKON 069
PRINT 720
PRINT 7241 .(ICELL(T) »T"1«8)
PRINT 723*NCTff = n
IF (MMG.fcO.ii-NCNlS.SSg 071
5 MG=] $vMC=lfNOTE=] 072
DC 100 „=! ,M 073
100 C*LL IOATA (Y(J) ,X(ltJ> .NOTE) 074
00 TO 863 075
t; 111.4930 098
4038 TF ( INDEX (1 3) .F0.3HYFS) 1000. 1001 099
iftOO Pf-TNT 66 100
DC 61 1=1. M 101
61 PKJMT 65. M (I,J) »J=1,M 102
infll IF ( INDEX ni) .F0.3HYES) 1002.1003 103
1002 PRINT 67 104
-------
129
0003C*
0003C6
0003?5
000332
00033*
000335
000336
000350
0003?*
00036*
000367
000371
000*10
000*15
000*21
000*30
000*3*
000*41
000443
000444
0004=7
000461
000*62
00047?
000500
000501
000512
000513
000515
000515
000516
000517
000527
000532
00053*
000540
000545
000547
000550
000552
000554
000555
000560
000562
000563
000570
000574
000600
000626
000630
000631
000635
000643
000647
000654
000655
000657
000660
DC fcO 1 = ] ,N
*0 PRINT 65, If(I,J) ,J = 1»N )
1003 IF ( INDF.X (1 0) .F0.3HYFS)42,43
42 DC 44 1=1,N
DC 44 J=l,N
9lM=0»
DC 45 K=] ,N
45 9lM=SUM»C(T,K)««(K,J)
44 CCnT(I,vJ)=SLM
PRINT 70
DC 71 I=1,M
71 PRINT 65,(TOOT(I•J)»J=l«N)
43 IF(INDFX(14).E0.3HYES) 1004,1005
100* PRINT 68
PMNT 65, (YX (I) , 1 = ] ,N)
1005 PRINT 75
111 IF ( INOF.X (i ) .EC.3HYFS) 7,150
7 DC « I = 1 . K
a
150 DC
605.K|R(T>
152 W' = 1.M
1*3
15*
DC 152 1 = 1,N
Yt-AT(J)»YMM(j)*X(t»J)»P(I)
SlMl=0.0
DC 153 jsl.V
9LM1=SUM*Y(J)
R = SUfl/F[ OAT (M)
SSP=0.0
DIUFR=0.0
DIFFsO.O
DC 15* J=l,C
SSR=SSR+(YHAT(J)-YBAR)«»2*W(J)
DIVFR=OIVFP«(Y(J)-YHAT(J))»«2«W(J)
DIFF =OIFF «rY(J)-YHAC)«»2»W(J)
IF (MMQ.fcQ.^HNON;) 1006,1007
1006 NCR=N-1
RC TO lOOfl
1007 NCR=N
100B NCFR=f-N
NTOT = K -1
SCDf = SSW/FI.CAT
S2=DIVFfi/FI OAT
FsSMflR/S?
IF(INDEX (?).F0.3HYFS)1S1»ZOO
PRINT 1327
PRINT 660
PR TNT 60A,NDR,SSR,S^DR,F,NOFP,OIVFR,S2»KTCTfOIFF
TCTAL a SSP+DTVFR
DEV=OIFF-TOTAL
PRINT 156. OEV
PRINT 660
DC 175 1*1 ,A
NCFR=1
151
155
105
106
107
10«
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
1?7
12S
129
130
131
132
133
13*
135
136
137
138
139
A
B
C
0
141
142
1*3
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
-------
130
0006*1
0006*3
00070*
000711
000715
OOOT27
0007.36
0007*2
000752
0007=3
000756
000757
0007(1
000773
00077*
00100*
001010
001012
001013
001015
001032
00103*
001037
001041
001042
0010=2
001065
0010(2
001063
001070
001072
00107*
001075
001076
001101
001110
001171
0011*0
00114*
001146
001147
OOH57
0011*2
oom?
001170
001175
001177
001201
001203
001213
001232
001336
001837
00124*
001246
001356
0013(3
0012(6
175
167
IPO
157
15H
1*
17
16
18
15
13
1f3
If*
165
?00
?01
203
K=I-JKQ
PRINT 655,K«NnFP»SSZ««MStF
IF (INDEX (11 ) ,FQ.3HYfcSi 1 BO. 210
Pfi|NT ftfcfl
READ 607,KO, JKDt'X (B> « INDEX (11)
READ 608. (MStJpU) ,7=1. N0>
PRINT 65?
DC 157 o»l .NO
KSMSUB („•) *JfQ
RtnT ( J)=H(K)
OC 158 „ = ! ,NO
DC 158 K=i ,NO
KK=NSUB(K| *.,MC
CUnT ( J,K)=C I JJt«K)
IF (INDEX (0) ,FO.->HYFS) 14tl3
i/ronrcn
F=S*«AS/S?
PHI^T 37.f«:lw(
DC 15 I»2.MC
CALL 6ALS«1 ( I .
DC 16 JK = i ,1
QF(JK)=0.
DC 17 KLal .1
QF(JK)=CP< JK)+CtNvC.fC.3HYES)20l«25fl
DC 203 1»1 ,N
DC 203 ,at,N
203 CCVAR(I,J)=C(I.J)«S2
PRINT 651
DC 205 IBI.-N
160
161
162
163
164
165
166
167
16«
169
170
171
172
173
17*
175
176
177
17P
179
ISO
1S1
IS?
183
1S4
1«5
186
Ifl7
iae
1R9
190
191
192
193
19*
195
196
197
198
199
POO
201
202
203
20*
?05
206
207
20fl
209
210
211
212
213
?1*
215
216
217
-------
131
001270
001307
001312
00131ft
001323
001325
001327
OOJ332
001334
001343
001344
001345
001345
001347
001351
0013?7
001364
001370
001172
001400
0014T3
001417
001424
001430
001432
001433
001435
00143ft
001451
0014=4
0014(3
001470
001473
001475
001510
001513
001520
001525
001927
001530
001532
001533
001546
0015=1
0015*0
001545
001572
001577
001603
001607
001610
001611
001614
001617
001621
001622
001625
001630
?05 PRINT 627. (COVAC (T. J) . J=l »Ni
PRINT h2R
PRINT FCRw?
??0 IF (INDF* (4) .FR.1HYFSlp51.300
?* 1 OF=FLOAI (M.N)
1 = 1
7=2 IF (TABLE < T , 1 ) -DF ) ?53 • ?S5 «254
?53 1=1+1
GC TO 25?
P = 4 IL=I-1
Xl=TAbL6 (TI..NPUP S9)
X? = TftBLt ( I .NR.'FCS)
Yl = TAtHL£ ( Tl.» 1 )
Y?=TABLE (T.I)
TE=Xl-(Yl-nF)«(vl_y2)/(Yl-Y?)
?*0 IF ( INDFIX (4 ) .FC. 1HYFS) ?S6»36n
?55 Tt = TABLE ( T ,Nfi|.F «:S)
?Sft DC 257 1 = 1 .A
Tl. IMssTF^SCPT (S'*C(l«T))
K= T - J^O
?=7 PRTN'T 614.^tH(TitTLT^
TOO TF(INUFX(c),f£--aHYFS)'aol» 35 n
101 PR TN T FO^
P»C 30b I J= 1 ,M
VARI (!J)=0.0
DC 303 WK=1 ,N
91 1 { JK) in.n
OC 302 *l = i ,^'
102 9L) ( JK) =» (K| « I Jl »r fKl. «.IK) «S> 1 ( JK)
103 91 ) ( JK)=?i 1 ( JK) «X ( JK . IJ)
DC 30 I ( T J ) +"5l- 1 ( JK )
1=7 IF (INUEX (4 ) .FC.iHYFS) 1*<0«25l
1*0 PRINT lft?H
PR INT FCHw?
SFOY=0.
5DFLY=0.
PRINT 72]
DC ISP 1=1. V
ntl. Y = Y ( I )-YhAT (T )
PCY=100.«nFl_V/Y ( I)
SCFLY = SCFLY + At»S (OFI y)
SFHYsSPCY* APS (COY)
TLTM=TE«SCOT (S?«V&-'T ( I) )
210
21S
220
2?1
222
2?3
2?*
225
2?ft
2?7
22H
2?9
230
231
232
233
234
235
23*
237
243
247
?4*
249
2^0
251
252
253
254
255
256
257
258
259
260
2ftl
262
263
264
265
266
267
268
2ft9
270
271
272
273
-------
132
001636
001656
0016*2
001662
0016*6
001670
001674
001676
001705
001711
001716
0017?3
001T25
0017*6
0017=1
0017*1
0017.fl
0017*7
OOlTTi
002002
002004
OOP010
OOJ017
0020Z4
002047
002053
0020*3
002072
002074
002101
002103
0021J2
002125
0021,34
002140
002146
002155
002163
002170
002171
61*,T.YHAT (I) tT|.IM,DEl Y«POY
If (TYPE.FC^HSFk-ILOG) 3532. *s«3
CCMTliMlJE
PRTM 72»10.«*(YHAT-YLO« .YUP(I) .YLOW(I) tDELTY 'I)
PRINT 726
PRt*T 7221 ,AVr>EI. YfCCNFAC
CCMTINUF
SPnY=SPDY/FLOAT (M)
PRTNT 72?.
------- |