A QUALITATIVE SURVEY OF THE AIRBORNE ALGAE, PROTOZOA, AND EACTERIA AT THE DENTON SEWAGE TREATMENT PLANT APPROVED; jor Professor * ~p" If Major Professor f -v . Z'—Vzi-~ <- 'X ^A.-t. -if Minor/Profe ssor Director of the Department of Bibl.og} V Dean of the Graduate School"
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A QUALITATIVE SURVEY OF THE AIRBORNE ALGAE, PROTOZOA,
AND EACTERIA AT THE DENTON SEWAGE TREATMENT PLANT
APPROVED;
jor Professor * ~p" If Major Professor
f -v . Z'—Vzi-~ <- 'X ̂A.-t. -if
Minor/Profe ssor
Director of the Department of Bibl.og} V
Dean of the Graduate School"
A QUALITATIVE SURVEY OF THE AIRBORNE ALGAE, PROTOZOA,
AND BACTERIA AT THE DENTON SEWAGE TREATMENT PLANT
THESIS
Presented to the Graduate Council of the
North Texas State University in Partial
Fulfillment of the Requirements
For the Degree of
MASTER OF ARTS
By
Joseph L. Mahoney, III, B. A.
Denton, Texas
May, 1968
TABLE OF CONTENTS
Page
LIST OF TABLES iv
LIST OF ILLUSTRATIONS V
Chapter
I. INTRODUCTION 1 Purpose History
II. MATERIALS AND METHODS 15
Air Sampling Location Air Sampling Procedure Microbiological Analysis Monitoring Meterological Conditions
III. RESULTS 30
IV. DISCUSSION . . . . . . 41
V. SUMMARY AND CONCLUSIONS 53
BIBLIOGRAPHY 55
iii
LIST OF TABLES
Table Page
I. Microorganisms Cultured from Air Samples 31
II. Collection Frequency Distribution of Viable Organisms 35
III. Heterological Conditions During Air Sampling . . . 37
iv
LIST OF ILLUSTRATIONS
Figure Page
1. Denton Sewage Treatment Plant . . . . . 17
2. Air Sampling Stations, Denton sewage treatment plant . . . . . . . . 19
3. Bubbler impinger used in air sampling 21
CHAPTER I
INTRODUCTION
Viable airborne microorganisms have been found by many
workers. A few have studied the addition of bacteria to the
air by the treatment of sewage. The algae and protozoa play
an important role in sewage treatment, but no one has
reported the emission of these organisms into the atmosphere
by wastewater treatment processes.
Purpose
This study had a three-fold purpose. First, it was
decided to determine if algae and protozoa were emitted to
the air at the Denton sewage treatment plant. The information
obtained could be of future importance in the fields of
algal and protozoan ecology and public health. Second, it
was decided to make a survey of the airborne bacteria at this
plant. Some researchers have described bacterial air contam-
ination at similar sewage treatment plants, but the one in
Denton has not been studied. Third, it was hoped that in
this research some relationships could be found between the
bacteria and the algae and protozoa in the air in the vicinity
of the sewage aeration basin. It was hypothesized that
pathogenic bacteria were carried in the air with these
other organisms.
History
There has been an interest in atmospheric organisms
since very ancient times. According to Gregory (14),
Hippocrates, the "Father of Medicine," who lived in the late
Fifth Century, B. C., felt that epidemic fevers were caused
by breathing air infected with hostile pollutants. Gregory
observed that Lucretius, in 55, B. C., watched dust particles
in a sunbeam and speculated that minute living particles in
the air accounted for the origin of plagues and other
phenomena. In more recent times, germ theories of disease
have been proposed, many of them founded on the proposition
that microbes are carried in the air.
Fracastorius described various means of disease trans-
mission and, in 1546, speculated that some diseases were
transmitted "ad distans"--or through the air (41). With the
aid of some of the first crude microscopes, Antony van
Leeuwenhoek (41) in 1676 described tiny "animalcules"-"
bacteria and protozoa--and concluded that the air was one
source of these creatures. Leeuwenhoek did not consider
these organisms to be of any consequence in regard to
diseases. By 1650 the first theories of spontaneous gen-
eration of living from non-living matter were advanced (14).
Throughout the ensuing abiogenesis controversy, evidence for
microscopic contamination through the air was found. The
use of cotton stoppers in flasks and test tubes was known as
an effective means of filtering these microbes out of the
air (41). In 1860 and 1861, Louis Pasteur (14, 41) performed
several experiments to demonstrate that bacteria were found
in dust. He also took air samples (14) and found bacteria
at different elevations and at several locations which
demonstrated the variety and ubiquity of these microorganisms.
In 1844 Ehrenberg (9) reported diatoms in dust from air
obtained from Charles Darwin (5,6) who speculated that one
explanation for the distribution of species might be found
in dust clouds, as cited by Gregory (14) and Schlichting (40).
Although interest in airborne microbes began to wane toward
the end of the Nineteenth Century, the French microbiologist
Pierre Hiquel continued to make extensive studies of the
air flora (14). Miquel was especially interested in the
number of bacteria and fungal spores to be found in the
atmosphere.
New interest in aerobiology has developed in more recent
years. Extensive air sampling for bacteria began anew in
the 1930's. Airplanes became a popular means of obtaining
atmospheric samples and Proctor (32,33,34,35) was one of the
first to describe species of bacteria collected in the upper
air. Meier and Lindberg (26) in 1935 reported the collection
of a number of bacteria in flights over the arctic regions of
Greenland. In the late 1930's, ZoBell (46) was studying
populations of airborne bacteria over the Pacific Ocean.
By the early 1950's, the list of atmospheric bacteria was
again lengthened by reports of microbes found over Canada
by Kelly and Paddy (17,31). Few of thes e workers in the
field of aerobiology have been interested in organisms other
than bacteria, fungi, and the pollen of higher plants.
Reports by persons attempting to collect, identify, and
culture airborne algae or protozoa are relatively few.
According to Schlichting (38), the Italian priest and
scientist Lazaro Spallanzani probably isolated and studied
the first protozoa from the atmosphere by exposing sterile
media to the air in 1777. According to Gregory (14),
Ehrenberg reported finding great quantities of protozoa in
the air between 1849 and 1872 (10). In 1883, Miquel (14)
found protozoan cysts in his aerial samples. Gregory (14)
has also reported that in 1913 Puschkarew found amoebae,
flagellates, and a ciliate in air sampled in extensive
studies along the Neckar River in Germany. Lackey, greatly
interested in the microbiology of water found standing in
tree-holes, reported great numbers of protozoa and algae
present in these habitats in 1939 (19). Between 1955 and
1964, Schlichting (38,39,40) cultured many species of algae
and protozoa from air samples collected in Michigan and Texas.
Stevenson and Collier (43) reported airborne flagellates and
diatoms in the air along the Texas Gulf coast in 1962.
Brown, Larson, and Bold (4) reported a few airborne flagellates
along with numerous algae in various air samples obtained in
1963.
It has been well documented that algae are often air-
borne. According to Schlichting (38), Ehrenberg (9) had
identified about thirteen genera of algae in dust samples
collected at sea in 1844 and Salisbury (37) in 1866 sampled
air in the United States to collect "disease producing algoid"
organisms which he called "Palmellae." H. Molish (27), as
cited by van Overeem (30), described many airborne diatoms
in 1920 and coined the term "aeroplankton." A number of
unclassified algae were collected from airplane flights over
Greenland in 1935 by Meier and Lindberg (26). Marie van Overeem
made a number of studies on airborne algae in 1936 and
1937 (29,30). Over forty genera were described from air
samples taken by stationary sampling devices at Leiden and
samplers on.airplane flights. Gregory, Hamilton, and
Sreeramula (15) reported the blue-green alga Gleocapsa and
other members of the Chrococcaceae in air samples obtained
during the summer of 1954 in England.
In June, 1956, Schlichting (39) exposed sterile media
to the air and obtained a number of algae and protozoa.
In 1961 he reported seven viable species cultured from air
samples collected weekly over a one year period in Michigan (40)
In 1962, Stevenson and Collier (43) reported airborne marine
phytoplankton by taking air samples on the Texas Gulf Coast.
During 1964, Brown and others (4) in Texas reported over
sixty genera of viable algae collected by exposing agar plates
at stationary locations and holding plates out of moving
automobile and airplane windows, in addition to obtaining
samples from twenty-one states taken by air pollution
monitoring stations. In March, 1964, Schlichting (38) presented
the results of his extensive airborne algae research in
Michigan and Texas; thirty-three species of algae and seven
species of protozoa were cultured from the Texas air samples.
In 19o5 Brown (2,3) reported collections of airborne algae
in Hawaii and suggested that biology classes might perform
laboratory experiments in airborne algae collection.
In 1967, Mahoney (23) reported finding the algae Phormidium
and Anklstrodesmus in short air sampling periods at a
trickling filter sewage treatment plant in Denton, Texas.
Several studies of air pollution and bacterial air
contamination by waste and sewage treatment have been made.
As early as 1907, Horrocks (16) had experimented with
conditions that would permit infectious bacteria to remain
viable in the air in London sewers. Fair and Wells (12)
measured bacterial air contamination at a variety of sewage
treatment plants in 1934. After a 1957 outbreak of psitta-
cosis in Oregon, Spendlove (42) used certain marker bacteria
to trace the emission of bacteria to the air by a rendering
plant. Woodcock (44) reported research into the release of
fine droplets and bacteria into the air by bubbling sewage
in 1955. Albrecht (1) made detailed studies of bacterial air
contamination in 1958 at several Florida trickling filter
sewage treatment plants. Reports of odor control (8) and
air pollution resulting from the treatment of sewage were
common by 1960. In 1964 Ledbetter (20) published his
observations on the addition of fine aerosols and odors to
the atmosphere. He speculated that this problem would
8
increase in importance with "the growth of the population,
advance of industrial technology, and the public awareness
of air pollution" (20,p. 62).
Ledbetter and Randall (21,36) in 1965 and 1966 reported
sampling the air upwind and downwind of an activated sludge
aeration unit for coliform bacteria. They found large numbers
of coliforms in the air and made proposals as to the effect
of certain environmental conditions on bacterial air contam-
ination. Air contamination was compared at different types
of sewage treatment plants in 1966 by Napolitano and Rowe (28).
They found that the aeration method emitted three times as
many bacteria as did the trickling filter method. Glaser
and Ledbetter (13) have made a detailed study of the mechanical
and physical factors in the production of aerosols in sewage
aeration, quantifying the particles by size, weight, and
other characteristics. None of these workers have searched
for or reported the finding of algae or protozoa in their
aerial sampling, although there have been many studies of
the organisms usually found in sewage (7,8,18,25)--algae,
protozoa, virtises, bacteria, and fungi as well as many of
the higher plants and animals.
Some evidence has been presented that airborne algae or
protozoa might cause allergies and fevers. In 1866,
9
Salisbury (37) attempted to show that a Palraella-like alga
was the cause of fevers. In 1948, A. H. Woodcock (45)
showed that some respiratory irritations occuring in coastal
communities were caused by inhaling mist containing high
concentrations of marine plankton or their waste products.
McElhenny and others (24) have demonstrated in 1962 laboratory
tests that green algae, as common air contaminants, are a
possible cause of some inhalant allergic sensitivities.
Mackenthum and Ingram (22) have cited fourteen studies of
human respiratory disorders associated with algae.
CHAPTER BIBLIOGRAPHY
1. Albrecht, C. R., "Bacterial Air Pollution Associated With the Sewage Treatment Process," unpublished master's thesis, University of Florida, 1958.
2. Brown, R. M., Jr., "The Collection and Cultivation of Airborne Algae for a Biology Lab Exercise," American Journal of Botany, LII (1965), 653.
3. , "Notes on Hawaiian Airborne and Soil Algae," American Journal of Botany, LII (1965), 644.
4 . , D. A. Larson, and H. C. Bold, "Air-borne Algae: Their Abundance and Heterogeniety," Science, CXLIII (February 7, 1964), 583-585.
5. Darwin, Charles, "An Account of the Fine Dust Which Often Falls on Vessels in the Atlantic Ocean," Quarterly Journal of the Geological Society of London* XI (1844), 26-30.
6 . , "Yellow Rain," Gardiner's Chronicle., July 18, 1863, p.675.
7. Dias, F. F., and J. V. Bhat, "Microbial Ecology of Activated Sludge, I. Dominant Bacteria," Applied Microbiology,, XII (1964), 412-417.
8. Dixon, F. R., and L. J. McCabe, "Health Aspects of Waste-water Treatment," Journal of the Water Pollution Control Federation. XXXVI (1964), 984-9897" ~
9. Ehrenberg, C. G., "Bericht uber die zur Bekanntraachung Geeigneten Verhandlunger der IConigl," Preussische Akademie der Wissenschaften Berlin, IX (1844), 194-197.
•̂0* . > "Passatstaub und Blutregen ein Grosses Organisches Unsichtbares Wirken und Leben in der Atmosphare," Akademie der Wissenschaften Berlin Physikalische Abhandlungen. XXIII (1849), 269.
10
11
11. Eliassen, R., and C. A. Vath, "Air Pollution Control in Sewage Treatment Plants," Journal of the Water Pollution Control Federation, XXXII (1960), 424-427.
12. Fair, G. M., and W. F. Wells, "Measurement of Atmospheric Pollution and Contamination by Sewage Treatment Works," Proceedings, 19th Annual Meeting of the New Jersey-Sewage Works Association, 1934.
13. Glaser, J. R., and J. 0. Ledbetter, "Size and Numbers of Aerosols Generated by Activated Sludge Aeration," Water and Sewage Works, CXIV (1967), 219-221.
14. Gregory, P. H., The Microbiology of the Atmosphere, New York, Interscience Publishers, Inc., 1961.
15 . , E. D. Hamilton, and T. Serramula, "Occurence of the Alga Gleocapsa in the Air," Nature, CLXXVI (1955), 1270.
16. Horrocks, W. H., "Experiments to Determine the Conditions Under Which Specific Bacteria Derived From Sewage May Be Present in the Air of Ventilating Pipes, Drains, Inspection Chambers, and Sewers," Proceedings of the Royal Society of London, Series B. LXXIX (1907), 236.
17. Kelly, C. D., and S. M. Paddy, "Microbiological Studies of Air Over Some Nonarctic Regions of Canada," Canadian Journal of Botany, XXXI (1953), 90-106.
18. Lackey, J. B., "Biology of Sewage Treatment," Sewage Works Journal, XXI (1949), 659-665. "
19. , "The Microscopic Flora and Fauna of Tree Holes," Ohio Journal of Science, XL (1939), 186-192.
20. Ledbetter, J. 0., "Air Pollution from Aerobic Waste Treatment," Water and Sewage Works, CXI (1964), 62-63.
— . » and C. W. Randall, Bacterial Emissions from Activated Sludge Units," Industrial Medicine and Surgery, XXXIV (1965), 130-133~
12
22. Mackenthum, K. M., and W. M. Ingram, Biological Associated Problems in Freshwater Environments, Cincinnati, Federal Water Pollution Control Administration, 1967.
23. Mahoney, J. L., Ill, "Airborne Algae in the Vicinity of a Trickling Filter," unpublished paper, Department of Biology, North Texas State University, Denton, Texas 1967.
24. McElhenney, T„ R., and others, "Algae: A Cause of Inhalant Allergy in Children," Annals of Allergy, XX (1962), 739-743.
25. McKinney, R. E., and A. Gram, "Protozoa and Activated Sludge," Sewage and Industrial Wastes, XXVIII (1956), 1219-1231.
26. Meier, F. C., and C. A. Lindberg, "Collecting Microorganisms from the Arctic Atmosphere," Scientific Monthly, XL (1935), 5-20.
27. Molisch, H., Populare Biologische Vortrage, Jena, Gustav Fischer, 1920.
28. Napolitano, P. J., and D. R. Rowe, "Microbial Content of Air Near Sewage Treatment Plants," Water and Sewage Works, CXIII (1966), 480-483.
29. Overeem, M. A. van, "On Green Organisms Occuring in the Lower Trophosphere," Recueil des Travaux Botaniques Neerlandais. XXXIV (1937), 389-439.
30 . , "A Sampling Apparatus for Aero-plankton," Proceedings of the Royal Academy of Amsterdam, XXXIV-(1936)7 981-990.
31. Paddy, S. M., and C. D. Kelly, "Studies on Microorganisms in Arctic Air During 1949 and 1950," Canadian Journal of Botany, XXXI (1953), 107-122.
32. Proctor, B. E., "The Microbiology of the Upper Air, I," Proceedings of the American Academy of Arts and Sciences, LXIX (1934), 315-340.
33 . , "The Microbiology of the Upper Air, II," Journal of Bacteriology, XXX (1935), 363-375.
13
34. Proctor, B. E., and B. W. Parker, "The Microbiology of the Upper Air, III," Journal of Bacteriology, XXXVI (1938), 175-184.
35 . , "Microorganisms in the Upper Air," Aerobiology, edited by F. R. Moulton, Washington, American Association for the Advancement of Science, 1942, 48-53.
36. Randall, C. W., and J. 0. Ledbetter, "Bacterial Air Pollution from Activated Sludge Units," American Industrial Hygiene Association Journal^ XXVII (1966), 506-519.
37. Salisbury, J. H., "On the Cause of Intermittent and Remittent Fevers, with Investigations which Tend to Prove that These Affections Are Caused by Certain Species of Palmellae," American Journal of Medical Science, LI (1866), 51-75.
38. Schlichting, H. E., Jr., "Meterological Conditions Affecting the Dispersal of Airborne Algae and Protozoa," Lloydia, XXVII (1964, 64-78.
39. , "The Role of Waterfowl in the Dispersal of Algae" doctoral dissertation, Michigan State University, University Microfilms, Ann Arbor, 1958.
40. , "Viable Species of Algae and Protozoa in the Atmosphere," Lloydia, XXIV (1961), 81-88.
41. Smith, D. T., N. F. Conant, and J. R. Overman, Zinsser Microbiology. 13th ed., New York, Appleton-Century-Crofts, 1964.
42. Spendlove, J. C., "Production of Bacterial Aerosols in a Rendering Plant Process," Public Health Reports, LXXII (1957), 176-180,
43. Stevenson, R. E., and A. Collier, "Preliminary Observations On the Occurrence of Air Borne Marine Phytoplankton," Lloydia, XXV (1962), 89-93.
14
44. Woodcock, A. H., "Bursting Bubbles and Air Pollution," Sewage and Industrial Wastes." XXVII (1955), 1189-1192.
45. , "Note Concerning Human Respiratory Irritation Associated With High Concentrations of Plankton and Mass Mortality of Marine Organisms," Journal of Marine Research, VII (1948), 56-62.
46. ZoBell, C. E., "Microorganisms in Marine Air," Aerobiology, edited by F, R. Moulton, Washington, American Asso-ciation for the Advancement of Science, 1942, 55-68.
CHAPTER II
MATERIALS AND METHODS
Air Sampling Location
The Denton, Texas sewage treatment plant is located
at Mayhill Road and Woodrow Lane, three and one-half miles
southeast of the Denton County courthouse. The plant
receives sewage from the north, south, and west parts of
Denton. This plant, built in 1962, uses the sludge acti-
vation method in the treatment of sewage. Aeration of the
sewage-sludge mixture is effected by bubbling air through
this mixture by sumberged jets in the center of a large
basin (151 feet long, 61 feet wide) near the center of the
plant.
The treatment plant is situated on a grassy, clay sand
hill, bordered on the north, east, and west sides by trees
and pasture. Open pasture with a few trees extends for some
distance to the south of the plant. Pecan Creek, which
receives the effluent of the treatment plant, is located
about 200 yards north and northeast of the aeration basin.
Engineer's drawings and construction details (5) indicate
15
16
that the elevation at the aeration basin is 570 feet and that
the creek and land surrounding the 18 acre plant is generally
less than 555 feet. The grounds of the plant are covered
with grasses and ornamental shrubs. Only a few large trees
are found on the plant grounds and many small ones have
recently been planted.
With the exception of two small equipment buildings and
utility poles, there are no structures taller than ten feet
within 400 feet of the aeration basin (fig. 1). The return
sludge lift station and operator's office is located 40 feet
north of the eastern end of the basin. Meterological instru-
ments were placed on the roof and inside this building
during this study,, The blower building, where equipment
was stored between sampling periods, is 40 feet north of the
western end of the basin. These buildings are 11 feet high
and 102 feet apart. The space between the two structures
consists of a small, paved parking area and grass lawn.
Other large structures at the plant are the raw sewage pump
station, about 400 feet north of the basin, and the sludge
digester and drying beds, about 500 feet southwest of the
aeration basin. Immediately east and west of this basin
are the open settling units or clarifiers.
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The 55-foot diameter primary clarifier for raw sewage
is centered 80 feet west of the aeration basin (figs. 1,2).
Sewage that has been screened and chopped to eliminate
large, undissolved solids enters the clarifier at its center.
As suspended solids settle to the bottom where they are
removed for digestion and drying, the liquid portion passes
over a peripheral weir and is piped to the aeration basin.
This mixture of liquid sewage and unsettlable particles is
agitated by air jets in the large basin. This aeration and
agitation with the aid of microorganisms in the sewage
increases the size of the particles and converts them to
insoluble, non-putrescible solids known as activated
sludge (19). The activated sewage then passes a weir and is
piped to the center of the final clarifier, which is 65 feet
wide and centered 60 feet east of the aeration basin. The
activated sludge-sewage mixture is settled; sewage plant
effluent passes a peripheral weir, is chlorinated, and dis-
charged into Pecan Creek. The settled activated sludge or
return sludge is sent back to the aeration basin or to the
digester and drying beds. On the average, 2,800,000 gallons
of sewage are processed daily at the Denton sewage treatment
plant.
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Air passing over this aeration basin was sampled using
a Gelman Model 24001 Sequential Sampler by the liquid
impingment method. Samplers were placed on portable
structural aluminum stands and sampled air at a height of
eighty-seven inches above the ground. The sampling stations
(fig. 2) were on opposide sides of the basin on a north-south
line at the center of the basin, seventy-five and a half feet
from either end. The stations were fifteen feet from the
north or south side of the aeration unit. Power for the
samplers was provided by electrical cables terminating in
the blower building.
Air Sampling Procedure
Low velocity liquid impingers were used throughout
this study. These "bubblers" were constructed with eight
dram vials fitted with two-hole, rubber stoppers and glass
tubes (fig. 3). The longest tube served as the air intake
during operation. The shorter tube was connected to the
sampler vacuum pump and sequential valve with rubber tubing.
The orifice of the intake tube was submerged in the collecting
medium and adjusted to a distance of fifteen millimeters from
the vial bottom. Fifteen bubblers were prepared prior to
each sampling run. The outer tube openings were plugged with
21
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cotton and the empty bubblers sterilized in the autoclave
at 121 degrees centigrade and 15 pounds pressure for 15
minutes. After sterilization and cooling, fifteen milli-
liters of the collection medium were added aseptically to
each bubbler sampler. Twelve of these were used in each
sampling run; one was designated as. a sampling medium
control; the remaining two were used to replace any bubblers
damaged in transit to the sewage plant, if necessary.
The collection medium used was Bristol's Solution (3).
Six stock solutions, 1000 milliliters each in volume, were
prepared. Each contained one of the following salts in the
amount listed:
Sodium nitrate 25.0 grams
Calcium chloride 2*5
Magnesium sulfate 7.5
Dipotassium phosphate 7.5
Potassium phosphate 17.5
Sodium chloride 2.5
Ten milliliters of each stock solution were added to 940
milliliters of glass distilled water. One drop of a 1.0
per cent ferric chloride solution was then added to the
mixture. The solution was then sterilized by filtration
(Millipore, pore size 0.22 microns) into a sterile, double
23
side-arm, 1500 milliliter flask. The lower are was con-
nected to a sterile syringe set-up that was used to deliver
fifteen milliliters of the solution to each bubbler.
Each week a sampling run was made, beginning March 22,
1967. The two sequential samplers were loaded with six
bubblers each in the laboratory and taken to the sewage plant,
There, the samplers were placed on their stands at the
sampling stations (fig. 2). These were connected to their
power supply and simultaneously started after the cotton
plugs were removed from the exposed intake tubes. Each run
consisted of six, two-hour air sampling periods, separated
by two-hour wait periods. The total time for each run was
twenty-two hours. The air sampling rate was 0.25 cubic feet
of air per minute. For each two-hour period, 30 cubic feet
of the air was sampled at each station. After completion of
the run, the openings of the intake tubes were sealed with
autoclave tape. The samplers and bubblers were then returned
to the laboratory for analysis.
Microbiological Analysis
The contents of each bubbler were aseptically added to
individual 125 milliliter flasks of Soil-water Medium. The
soil-water culture medium, modified after Pringsheim (13),
24
•was prepared as follows: One-quarter-inch of seived loam
soil (pH 6.5 to 7.0) was placed in forty-eight clean,
125 milliliter flasks. Flasks were then filled to about
two inches from the top with glass-distilled water and
plugged with cotton. All flasks were then sterilized by
steaming at atmospheric pressure for one hour on three
consecutive days. For each group of soil-water culture
flasks prepared, three were chosen at random as culture
medium controls.
For bacteriological analysis, triplicate one-tenth
milliliter samples were taken from the soil-water medium
cultures and plated out in triplicate on plates of Bacto-
Tryptic Soy agar, Eosin Methylene Blue (EMB) agar, and
Brain Heart Infusion agar, using sterile pipets and the
spread-plate method. For each set of twenty plates that
was poured, three were set aside as controls. The collection
medium control bubbler and soil-water medium control flask
were also plated out at this time. Tryptic Soy agar plates
were incubated at room temperatures, 28 to 30 degrees
centigrade, for 24 to 48 hours. EMB and Brain Heart Infusion
agar plates were incubated at 35 to 37 degrees centigrade
for 24 hours.
25
Bergey1s Manual of Determinative Bacteriology (4) was
used to identify bacteria collected. Colony morphology of
bacteria appearing after the incubation period was noted.
Colonies representing the majority of the bacterial population
were isolated by streaking on additional plates. Gram
stains (1) and agar slant cultures were prepared of these
organisms. Bacteria were isolated and identified using
staining techniques, differential and selective media, and
microscopic examination. Coliform bacteria were readily
isolated by their characteristic colony morphology on EMB
agar plates (6). Other differential tests used were
motility, carbohydrate fermentation reaction, indole production,
the methyl red test, Voges-Proskauer reaction, growth on
Simmon's Citrate agar, urea decomposition, litmus milk
reaction, gelatin liquifaction, nitrate reduction, production
of hydrogen sulfide in Kligler's Iron Agar, and characteristic
growth on Bismuth Sulfite or Salmonella-Shigella agars (1,2,
4,6,8,16,21).
After removing samples for bacteriological determinations,
soil-water media cultures were kept in an algal culturing
unit (12) at 21 to 30 degrees centigrade for nine months.
Throughout this period, the laboratory temperature rose on
four occasions due to air conditioning failures, but the
26
temperature remained at 21 degrees most of the time. The
cultures were examined at the end of the first, second, and
third week by preparing wet-mount slides from each flask.
Then, cultures were routinely examined every five weeks.
When possible, unialgal or uniprotozoan cultures were made
in additional flasks of soil-water medium. Cells were
sketched and measurements made using the compound micro-
scope and an ocular micrometer. Iodine-potassium iodide
stain (21) was used to test for starch production. India
ink was used as a negative stain for matrix and other
organelles (15). Living protozoa were studied with the aid
of several reagents: nickel sulfate, methyl cellulose,
butacaine sulfate solution, neutral red, Lugol's solution,
and iodine-potassium iodide (9,10,15,21). Six-three-one
solution was used occasionally on slides to kill organisms
for study (15). Permanent slide preparations following
Kudo's techniques were unsuccessful (10). Algae were
identified using Smith, Prescott, Fritsch, and Tiffany and
Britton (17,12,7,18) Protozoa were identified using Kudo,
Jahn, Ward and Whipple, and Ludin and West (10,9,20,11).
27
Monitoring Meterological Conditions
As an essential part of this research, weather instruments
were installed at the sewage treatment plant to measure
several parameters. These were mounted on the roof and
inside the return sludge lift station. Wind direction was
obtained using a Gelman-Gill bivane and recorder. Wind
velocity was obtained using an anemometer and recorder.
Both instruments were mounted on the roof of the building
at a height of fourteen feet above the ground, attached to
a vent pipe about three feet above the building. Recorders
for these instruments and a recording barograph were
installed in the chlorinator room of the station. A Taylor
hygrothermograph was installed and was- supposed to record
the temperature and relative humidity during the air sampling
periods, but was not operational or erratic throughout the
study. As a result, this information was obtained from the
Texas A and M University Agricultural Experiment Station,
Substation 6, located about five and one-half miles west-
northwest of the sewage treatment plant. The amount of
precipitation at the plant was determined using a rain gauge.
Reports on the general Denton weather conditions on radio
station KDNT were used to supplement personal observations
and recorded data.
CHAPTER BIBLIOGRAPHY
1. American Public Health Association, American Water Works Association, and Water Pollution Control Federation, Standard Methods for the Examination of Water and Wastewater, 12th ed., New York, American Public Health Association, Inc., 1965.
2. Bailey, W. R., and E. G. Scott, Diagnostic Microbiology, 2nd ed., St. Louis, C. V. Mosby Company, 1966.
3. Bold, H. C., "The Morphology of Chlamydomonas chlamy-dogama," Bulletin of the Torrey Botanical Club,LXXVI (1949), 101-108.
4. Breed, R. S., E. G. D. Murray, and N. R. Smith, Bergey's Manual of Determinative Bacteriology, 7th ed., Baltimore, Williams and Wilkins Company, 1957
5. Denton, Texas Sewage Treatment Plant, Freese, Nichols, and Endress Consulting Engineers, Fort Worth, Texas, August, 1961.
6. Difco Laboratories, Difco Manual, 9th ed., Detroit, Difco Laboratories, Inc., 1953.
7. Fritsch, F. E., The Structure and Reproduction of the Algae, Vol. I, New York, The MacMillian Company, 1936.
8. Geldreich, F. E., Sanitary Significance of Fecal Coliforms in the Environment. Water Pollution Control Research Series Publication WP-20-3, Cincinnati, Federal Water Pollution Control Administration, 1966.
9. Jahn, T. L., and F. F. Jahn, How to Know the Protozoa, Dubuque, William C. Brown Company, 1949.
10. Kudo, R. R., Protozoology, 5th ed., Springfield, Charles C. Thomas, 1966.
28
29
11. Ludin, F. C., and L. S. West, Key to the Free-living Protozoa of the Upper Peninsula, Marquette, Northern Michigan College Press, 1963.
12. Prescott, G. W., Algae of the Western Great Lakes Area, Dubuque, William C. Brown Company, 1962.
13. Pringsheim, E. G., Pure Cultures of Algae, Cambridge, Cambridge University Press, 1946.
14. Schlichting, H. E., Jr., "Construction of an Inexpensive Plant Growth Chamber," Turtox News, XLI (1963), 214-215.
15. , unpublished lecture notes, Department of Biology, North Texas State University, Denton, Texas, 1967.
16. Smith, D. T., N. F. Conant, and J. R. Overman, Zinsser Microbiology, 13th ed., New York, Appleton-Century-Crofts , 1964.
17. Smith, G. M., The Fresh-water Algae of the United States, 2nd ed., New York, McGraw-Hill Book Company, Inc., 1950.
18. Tiffany, L. H., and M. E. Britton, The Algae of Illinois, Chicago, University of Chicago Press, 1952.
19. Torpy, W. N.? and A. H. Chasick, "Activated Sludge Principles," Sewage and Industrial Wastes, XXVII (1955), 1217-1233.
20. Ward, H. B., and G. C. Whipple, Freshwater Biology, 2nd ed., edited by W. T. Edmondson, New York, John Wiley "and Sons, Inc., 1963.
21. Williams, 0. B., Laboratory Manual for Bacteriology, Austin, University of Texas Press, 1955.
CHAPTER III
RESULTS
In Table I, thirteen genera of algae and protozoa and
twelve groups of bacteria identified from air sample cultures
are listed.
Organisms were enumerated only on the basis of the
frequency of samples in which they were found. No attempt
has been made to determine the number of cells present in
the air during the sampling period because the collecting
and culturing methods employed did not permit quantitative
estimates. The collection medium was not examined immediately
after sampling but was added to soil-water medium to culture
algae and protozoa. Descriptions of the viable algae, protozoa,
and bacteria are based on this second medium and represent
the majority populations present. Because of the large
number of samples and the difficult taxonomic problems of
certain groups, only generic classification? have been
attempted in the case of most of these organisms. Genus-like
names were given to those organisms closely resembling a
certain organism but differing in some aspects. Organisms
30
31
TABLE I
MICROORGANISMS CULTURED FROM AIR SAMPLES
Organism Number of Samples*
Algae and Protozoa Chlamydomonas sp Chlorella ellipsoidea . . . . Chlorella sp . Chlorococcum-like sp Chromulina sp.. . . . . . . . Cryptoglena sp Cryptomonas sp Euglena sp Microcystis sp . Peranema sp Pleuromonas sp. Uronema-like sp Westella botryoides unidentified hypotrich. . . . unidentified protozoan cyst .
Bacteria Gram negative bacilli
Achromobacter-Alcaligenes spp. Aerobacter sp Escherichia sp Flavobacterium sp Klebsiella sp Proteus sp Pseudomonas sp. . . . . . . . Salmonella sp.. . Serratia sp uncertain
Gram positive bacilli Gram positive cocci
3 4 5 4 2 1 3 3 1 2 1 3 2 2 4
102 105 147 157 83 18 5 1
153 10
121 146
*Total number of samples = 192.
32
were studied in mixed and unialgal or pure culture. Several
keys and reference works were consulted for the identification
of the algae (5,8,10,12,13,14), protozoa (7,8,9,10,12,14),
and bacteria (1,2,3,4,6,11).
Chlorococcum-like cells (Table I) were the most frequently
encountered alga in this survey. These cells were in large
masses of hundreds of cells that easily covered the soil
surface in the soil-water medium flasks. Strict
identification could not be made because colony cell numbers
were usually higher than that described. Cell wall and
chloroplast characteristics and the formation of biflagellated
zoospores identified this as a species of Chlorococcum or
similar organism (5,10). The flagellates reported were
easily identified using several keys, but the ciliates
encountered presented some difficulty. The Uronema-like
organism appeared to be rather uniformly ciliated, lacked
a caudal filament or cilium, and had a rather indistinct
oral ciliature (8,14). The hypotrich found was characterized
by an unusually small size (25 microns long), typical
flattened body form, and sparce, irregular, ventral cirri.
In general, this ciliate closely resembled both Balladyna
and Psilotricha species (8,14). A number of unidentifiable
protozoan cysts were found in four collections, usually
33
occurring in the same collection with the Uronema-like
ciliates. These cysts were spherical or ovoid and were
about eight microns in diameter.
Great numbers of bacterial colonies developed from
media that was plated out. Studying plates of EMB agar
allowed for quick identification and isolation of coliform
bacteria. Species of Escherichia obtained included both
citrate positive (Citrobacter, 2) and citrate negative forms.
Klebsiella was identified and distinguished from Aerobacter
by capsule formation and lack of motility. Urease and methyl
red positive organisms were classified as Proteus species.
One collection on May 18, 1967 contained several paratyphoid
Salmonella organisms that were determined by growth on bismuth
sulfite agar plates and by slide agglutination with Group B
antiserum. Serratia, Pseudomonas, F1avobacterium, Achromobacter,
and Alcaligenes organisms were identified by colonial char-
acteristics and reactions in various media. Organisms
grouped as uncertain included a number of Paracolobactrum-
like organisms with slow and variable biochemical reactions.
Gram positive organisms were of the aerobic, spore-forming
Bacillus type and a great variety of cocci, including Sarcina,
Staphylococcus, Micrococcus, and others. Colonies representing
less than five per cent of those observed on similar plates
34
for each sample were not identified. Molds and yeasts, as
well as an occasional actinomycete, were also cultured from
the air samples; these were disregarded in this survey.
The frequency with which the algae, protozoa, and
bacteria were found in the air with respect to wind direction
and time of day is presented in Table II.
A total of 192 samples were taken during the four month
survey. Of the samples positive for algae and protozoa,
eight were taken on the upwind side of the aeration basin.
Seven samples taken at night contained these organisms,
whereas five were positive during the day collections.
Only twelve of the 192 samples contained algae or protozoa.
More genera of algae and protozoa were identified from the
upwind-day collections (Table II). Only one sample was
positive on the downwind side of the basin during the day.
This collection was made on July 13 and contained only the
Chlorococcum-like alga. On only one occassion, June 22, algae
and protozoa were found in both the upwind and downwind
air samples.
Bacteria were found in all of the 192 samples taken in
this survey. More genera were found in the downwind samples
than in upwind samples (Table II). Genera found most often
during the day were Flavobacterium and Escherichia. At night,
35
TABLE II
COLLECTION FREQUENCY DISTRIBUTION OF
VIABLE ORGANISMS*
Organism Upwind Samples Downwind Samples
Organism Day Night Day Night
Chlamydomonas sp. 1 0 0 2 Chlorella ellipsoidea 1 2 0 1 Chlorella sp. 2 3 0 0 Chlorococcum-like sp. 0 2 1 1 Chromulina sp. 0 1 0 1 Cryptoglena sp. 1 0 0 0 Cryptomonas sp. 0 2 0 1 Euglena sp. 2 0 0 1 Microcystis sp. 1 0 0 0 Peranema sp. 0 1 0 1 Pleuromonas sp. 0 0 0 1 Uronema-like sp. 0 3 0 0 Westella botryoides 1 0 0 1 unidentified hypotrich 0 2 0 0 unidentified protozoan cyst 0 3 0 1
Achromobacter-Alcaligenes spp 5 14 20 63 Aerobacter sp. 16 31 22 36 Escherichia sp. 24 29 41 53 Flavobacterium sp. 37 33 36 51 Klebsiella sp. 12 15 28 28 Proteus sp. 1 7 1 9 Pseudomonas sp. 1 1 1 2 Salmonella sp. 0 0 0 1 Serratia sp. 21 58 30 44 uncertain Gram negative 2 3 1 4 Gram positive bacilli 18 28 35 40 Gram positive cocci 19 28 38 61
*Total number of samples = 192,
36
Serratia, Flavobacterium, Escherichia, and the Achromobacter-
Alcaligenes group were found most often. Serratia was the
only organism found more often in upwind samples than in
downwind ones. The groups found most often in downwind
collections were the Escherichia, Flavobacterium, and Gram
positive cocci.
A summary of prevailing meterological conditions during
the air sampling runs is given in Table III.
Three samples contained algae and protozoa on March 30
during the morning hours. These organisms were found in
five collections during the June 22 and 23 sampling period.
On July 13 and 14, the first three sequential samples contained
algae and protozoa. Only one collection on July, 21 contained
these microorganisms. No samples were taken on April 28 and
August 5 as a result of equipment failure. Controls for the
cultures taken May 10 and 11 were found to be badly contaminated
with Microcystis and bacteria; the results of samples taken
on this date were discarded. For the majority of samples
the wind was out of the south at a velocity between 10 and
20 miles per hour. Thus, most of the downwind samples were
those taken on the north side of the aeration unit. The
skys were partly cloudy throughout the survey and traces of
rain were recorded at the sewage treatment plant on only
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four occassions during the weekly sampling runs. In
between sampling runs, traces of rain that could not be
recorded occurred in the last part of May, early June,
and the last part of July. The weather was quite
variable during July. The prevailing winds were from
the north and unusually low temperatures were recorded
throughout the North Central Texas area in the second week
of that month.
CHAPTER BIBLIOGRAPHY
1. American Public Health Association, American Water Works Association, and Water Pollution Control Federation, Standard Methods for the Examination of Water and Wastewater, 12th ed., New York, American Public Health Association, Inc., 1965.
2. Bailey, W. R., and E. G. Scott, Diagnostic Microbiology, 2nd ed., St. Louis, C. V. Mosby Company, 1966.
3. Breed, R. S., E. G. D. Murray, and N. R. Smith, Bergey's Manual of Determinative Bacteriology, 7th ed., Baltimore, Williams and Wilkins Company, 1957.
4. Difco Laboratories, Difco Manual, 9th ed., Detroit, Difco Laboratories Inc., 1953.
5. Fritsch, F. E., The Structure and Reproduction of the Algae, Vol. I., New York, Macmillan Company, 1935.
6. Geldreich, E. E., Sanitary Significance of Fecal Collforms in the Environment, Publication WP-20-3, Cincinnati, Water Pollution Control Administration, 1966.
7. Jahn, T. L., and F. F. Jahn, How to Know the Protozoa, Dubuque, William C. Brown Company, 1949.
8. Kudo, R. R., Protozoology, 5th ed., Springfield, Charles C. Thomas, 1966.
9. Ludin, F. C. and L. S. West, Key to the Free-living Protozoa of the Upper Peninsula, Marquette, Northern Michigan College Press, 1963.
10. Prescott, G. W., Algae of the Western Great Lakes Area, Dubuque, William C. Brown Company, 1962.
11. Smith, D. T., N. F. Conant, and J. R. Overman, Zinsser Microbiology, 13th ed., New York, Appleton Centoury-Crofts, 1964.
39
40
12. Smith, G. H., The Fresh-water Algae of the United States, 2nd ed., New York, McGraw-Hill Book Company, Inc., 1950.
13. Tiffany, L. H., and M. E. Britton, The Algae of Illinois, Chicago, University of Chicago Press, 1952.
14. Ward, H. B., and G. C. Whipple, Freshwater Biology, 2nd ed. edited by W. T. Edmondson, New York, John Wiley and Sons, Inc., 1963.
CHAPTER IV
DISCUSSION
It is well documented in the literature that algae,
protozoa, and bacteria are found in the air. Gregory (7)
has discussed the physical environmental phenomena of air
movements that cause dust and microscopic organisms to become
airborne. The atmosphere is layered, that is, at different
elevations there exist differences in the air temperature,
density, viscosity, and pressure. These differences cause
the motion of air in our environment. One common mechanism
for the addition of dust particles to the air is the action
of winds and small eddy currents in the air flow as it passes
over and around objects. These currents lift dust and the
possible microorganisms contained therein into the atmosphere.
Other mechanisms are simple splashing of water or the bursting
of bubbles at a water surface, as reported by Mason (15) and
Woodcock (29). Tiny droplets form at the water surface because
of the cohesive properties of the water molecules. These
droplets and any objects that might be contained in them are
carried into the air. Gregory (7) suggests that even drying
41
42
and crumbling of the thallus of an alga or the soredia of a
lichen may contribute to the algal flora of the air. Airborne
microbes have been found in practically all parts of the
atmosphere. Bacteria have been collected from the air only
inches over the surface of sewage aeration basins (11,22).
Algae and protozoa have been found in the air over a football
stadium (24) and along highways (3). Airborne microbes have
also been cultured from airplane collections at thousands of
feet in the atmosphere (18,20). However, most of the many
workers in aerobiology have neglected the algae ..and protozoa
in their search for airborne microorganisms. Although these
organisms are also found in sewage (1,4,12), none of the
recent studies of airborne organisms at sewage plants (11,19,
22) have reported these organisms in the air. A crude
preliminary survey has been made (14) in which two genera of
algae were found in the air near a sewage treatment trickling
filter.
Methods of survival of these airborne microbes have been
extensively studied. The adverse effects of drying, radiation,
and temperature are quite different among the various organisms
because of survival mechanisms like cysts, spores, and gelatinous
sheaths around cells. Loss of water from proteins and increased
survival time with lower or higher humidity is reported as one
43
factor to be considered by Wells (26). Gislen (6) maintains
that bacteria survive less often when high temperature and high
humidity are combined. Ultra-violet radiations are most
detrimental to airborne organisms on clear days when no
natural atmospheric shield exists (6). According to Gislen (6),
many bacteria and protozoa are capable of withstanding
ultra-violet longer on cloudy days. In the survey presented
in this research, most of the algae and protozoa were found
on cloudy days or at night when the humidity was high and the
temperatures were moderate.
The .case for disease transmission through the atmosphere
has been well documented (26). Algae have become important as
possible causitive agents in various respiratory ailments,
as shown by several persons (8,9,16,23,29) and reported by
Mackenthum (13). It has not been conclusively shown that
some of the reported symptoms are not produced by the
synergistic action of the algae and bacteria. Many blue-green
algae that have been found in the air (14,24) have a matrix
which may contribute to the dispersal of bacteria embedded in
this matrix. Perhaps the algae and protozoa in sewage can
become airborne like the bacteria (11,19,22) and constitute
a health hazard themselves. Wells reports that organisms of
less than five microns in size are responsible for most of the
44
respiratory infections (26). Many of the non-bacteria
organisms isolated in the air near the sewage aeration basin
are in this size range.
Several weaknesses are evident in this survey of the
airborne organisms at the sewage treatment plant. First,
quantitative data as to the number of cells in the air has
not been determined. One reason for this is that the liquid
impingment method of air sampling breaks up clusters of cells
and dust (28). Other sampling techniques might have been
used (24, 28) to give a quantitative estimate of the airborne
microorganisms. No single sampling device has been shown to
be of equal quality in collecting and culturing different
types of cells. In qualitative surveys, the low velocity
bubbler impinger has been reported to give satisfactory
results (24,28). Also, the original samples were not plated
out for cell counts but were immediately subcultured for the
determination of viable algae and protozoa.
Next, the location of the samplers and sampling stations
should be considered, since this would effect the numbers
and kinds of organisms collected. Survival distances of
airborne organisms vary greatly within short distances from
the source of emission (11,19,22) generally depending on the
wind velocity. Eddy currents in the air created by the
45
aeration basin would affect the dispersal direction and
distance traveled by these living aerosols. More quantitative
background and experimental counts might be obtained by-
using a number of samplers and different locations for
sampling the air. Another factor is the length of the
sampling period and length of time that the samples remain at
the sampling station before analysis. In this study, the
first sample taken remained at the sewage plant until the
twenty-two hour run was completed. During this time, changes
in sample content could certainly occur. Temperature
fluctuations on the inside of the sequential sampler case
might have been detrimental to some organisms collected.
The sampler intake tubes remained open to the atmosphere
throughout the sampling run, perhaps permitting additional
organisms to enter the samplers. Not only could these
parameters affect the number of viable cells but they could
also change the kinds of organisms found on analysis.
The choice of the collection and culture media determine
which organisms are reported as viable (28). Bristol's
solution and the soil-water medium were used in this study
because they were convenient and have been successfully used
by previous investigators (2,24) for the cultivation of the
algae and protozoa. Perhaps some organisms actually removed
46
from the air were not viable in these media. In this study,
control media were also cultured as a check for sterility.
This precaution proved that on one occasion algal and
bacterial contamination had occurred.
Some other sources of error may have occurred at the
sewage plant,, For example, no record of frothing or foaming
due to detergents in the sewage--or control of this—was
kept at the plant. To control this action, water is sprayed
on the surface of the aeration tank (21,25). This fine
spray could cause additional turbulence that could affect
in turn the air sampled and its content. Sewage treatment
plant personnel working in the vicinity of the. sampling stations
could have some effect on the result of the aerial sampling.
Disturbances such as mowing, as suggested by Brown (3), would
greatly alter the natural air flora and fauna. At the Denton
sewage plant, little activity occurred near the air samplers
during the sampling periods. This type of variable was not
recorded, but certainly not overlooked, in this survey.
Many of the algae, protozoa, and bacteria cultured from
the air samples in this survey have also been found in the
air by previous investigators (3,22,24). At least one isolation
Salmonella was made in this studyj recent surveys by others
have not reported this organism. The non-enteric bacteria
47
found in this survey appear to be rather common in studies
of this type. In his 1962 studies, Schlichting (24) reported
twenty-three genera of algae and protozoa in his air samples
obtained during a similar period (March 22 to August 6).
He was working at a different air sampling height and location
and did not find many of the organisms cultured from the
sewage plant air samples, such as Westella. Chlamydomonas.
Peranema. Cryptoglena, Chromulina, Cryptomonas. Pleuromonas„
Uronema-like species, and hypotrich-like organisms. Perhaps
this difference was a result of sampling air near the
sewage aeration unit, as well as the other differences in
the sampling techniques and procedures.
At the Denton sewage plant, samples of the activated
sludge-sewage mixture from the surface at various places in
the aeration basin contain large numbers of the organisms
collected in the air near the basin, as well as many not
found in the samples. The sewage in the basin contains
about twice as many flagellates as it does ciliates or amoeba.
No coccoid algae have been observed in these sewage samples.
Five-minute air exposure agar plate collections have been
made on the service walk in the center of the basin. On a
cool, humid night after a few days of rain, high concentrations
of coliform and other bacteria were collected in this manner.
48
A wide variety of different media was used, including EMB
agar, Brain Heart Infusion agar, Tryptic Soy agar, bismuth
sulfite agar, modified synthetic sewage agar, and sterilized
sewage agar. These cultures arid those made by direct plating
of sewage produced bacteria that were also isolated from the
air collections performed in this survey. This means that
the sewage aeration basin could have been the source of
microorganisms found in the air samples.
In this survey, no associations among airborne organisms
could be demonstrated. Bacteria were found more often than
the algae or protozoa. As more genera were collected at night,
it would seem that daylight, humidity, and temperature had
affected the viability and dispersion of these organisms.
Fairly consistent weather conditions throughout this survey
cannot be reflected in the air samples. Under similar conditions
at similar times, dissimilar populations were found. Rain-
fall is usually thought to greatly change the number of
bacteria in the air (7) for a period after the rain. At the
Denton plant some samples were positive for algae and protozoa
and bacteria after short showers. Perhaps the sampling
stations were close enough to the aeration basin that collection
of microorganisms emitted by the aeration could be affected
after the rain had stopped. Downwind samples containing
49
algae and protozoa were found when the wind was from the
north. Perhaps the changing wind direction and eddy currents
caused by the sewage treatment plant buildings caused an
increase in the amount of dispersion of these organisms.
Although not quantitative, this survey has shown that
airborne algae and protozoa can be found in the air at this
type of sewage plant under these sampling conditions.
It does appear that the aeration unit can emit these organisms
to the air as they can be found in the liquid at the surface
of the aeration basin. More extensive quantitative studies
of this phenomenon should be made to obtain a better idea
of the role of sewage treatment in the dispersion of these
organisms.
CHAPTER BIBLIOGRAPHY
1. Baines, S., and others, "Protozoa As Indicators in Activated Sludge Treatment," Sewage and Industrial Wastes, XXV (1953), 1023-1033.
2. Bold, H. C., "The Morphology of Chlamydomonas chlamy-dogma," Bulletin of the Torrey Botanical Club, LXXVI (1949), 101-108.
3. Brown, R„ M., Jr., D. A. Larson, and H. C. Bold, "Airborne Algae: Their Abundance and Heterogeniety," Science, CXLIII (February 7, 1964), 583-585.
4. Denoylles, Frank, Jr., "Factors Affecting Phytoplankton Distribution in a Double-Cell Sewage Lagoon," Journal of Phycology, III (1967), 174-181.
5. Fair, G. M., and W. F. Wells, "Measurement of Atmospheric Pollution and Contamination by Sewage Treatment Works," Proceedings, 19th Annual Meeting of the New Jersey Sewage Works Association, 1934.
6. Gislen, Torsten, "Aerial Plankton and Its Conditions of Life," Biological Reviews of the Cambridge Philosophical Society. XXIII (1948), 109-126.
7. Gregory, P. H., The Microbiology of the Atmosphere, New York, Interscience Publishers, Inc., 1961.
8. Heise, H. A., "Symptoms of Hay Fever Caused by Algae," Journal of Allergy, XX (1949), 383-384.
9 . ., "Symptoms of Hay Fever Caused by Algae, II," Annals of Allergy. IX (1951), 100.
10. Jenkins, S. H., "The Role of Protozoa in the Activated Sludge Process," Nature, CL (1942), 607-610.
11. Ledbetter, J. 0., and C. W. Randall, "Bacterial Emissions From Activated Sludge Units," Industrial Medicine and Surgery, XXXIV (1965), 130-133.
50
51
12. Lackey, J. B., "Biology of Sewage Treatment," Sewage Works Journal, XXI (1949), 659-665.
13. Mackenthum, K. M., and W. M. Ingram, Biological Associated Problems in Freshwater Environments, Cincinnati, Federal Water Pollution Control Administration, 1967.
14. Mahoney, J. L., III, "Airborne Algae in the Vicinity of a Trickling Filter," unpublished paper, Department of Biology, North Texas State University, Denton, Texas, 1967.
15. Mason, B. J., "Bursting of Bubbles at the Surface of Sea Water," Nature, CLXXIV (1954), 470-471.
16. McElhenney, T. R., and others, "Algae: A Cause of Inhalent Allergy in Children," Annals of Allergy, ~ XX (1962), 739-743.
17. McKinney, R. E., and R. G. Weichlein, "Isolation of Floe Producing Bacteria From Activated Sludge," Applied Microbiology, I (1953), 259.
18. Meier, F. C., and C. A. Lindberg, "Collecting Micro-organisms from the Arctic Atmosphere," Scientific Monthly, XL (1935), 5-20,,
19. Napolitano, P. J., and D. R. Rowe, "Microbial Content of Air Near Sewage Treatment Plants," Water and Sewage Works, CXIII (1966), 480-483.
20. Overeem, M. A. van, "A Sampling Apparatus for Aero-plankton," Proceedings of the Royal Academy of Amsterdam, XXXIV (1936), 981-990.
21. Polkowski, L. B., and others, "Evaluation of Frothing in Sewage Treatment Plants," Sewage and Industrial Wastes, XXXI (1959), 1004-1015.
22. Randall, C, W., and J. 0. Ledbetter, "Bacterial Air Pol-lution from Activated Sludge Units," American Industrial Hygeine Association Journal, XXVII (1966), 506-519.
52
23. Salisbury, J. H., "On the Cause of Intermittent and Remittent Fevers, with Investigations which Tend to Prove that These Affections Are Caused by Certain Species of Palmellae," American Journal of Medical , Science. LI (1866), 51-75.
24. Schlichting, H. E., Jr., "Meterological Conditions Affecting the Dispersal of Airborne Algae and Protozoa," Lloydia, XXVII (1964), 64-78.
25. Sparr, A. E., "Froth Control at Bay Park," Sewage and Industrial Wastes, XXX (1958), 305-312.
26. Wells, W. F., and M. W. Wells, "Air Borne Infection," Journal of the American Medical Association, CVII (1936)," 1698-1703.
27. Wolfe, F. T., "The Microbiology of the Upper Air," Bulletin of the Torrey Botanical Club, LXX (1943), 1-14.
28. Wolfe, H„ W., and others, Sampling Microbial Aerosols, Public Health Service Monograph No. 60, Fort Detrick, U. S. Public Health Service, 1959.
29. Woodcock, A. H., "Note Concerning Human Respiratory Irritation Associated with High Concentrations of Plankton and Mass Mortality of Marine Organisms," Journal, of Marine Research, VII (1948), 56-62.
CHAPTER V
SUMMARY AND CONCLUSIONS
1. This survey has shown that airborne algae and
protozoa are found in the air at the Denton sewage treatment
plant and that these may be emitted by the activated sludge
aeration basin.
2. Thirteen genera of algae and protozoa and twelve
groups of bacteria were identified from cultures of the air
samples taken at the sewage treatment plant.
3. Algae and protozoa were found in twelve of the
192 air samples taken over the four month survey; bacteria
were found in all samples.
4. Algae and protozoa were collected more often in
air samples taken at night and on the upwind side of the
aeration basin.
5. A greater variety of bacteria was collected in
air samples taken at night and on the downwind side of the
aeration basin.
6. There was no apparent relationship found between
the airborne bacteria and the algae and protozoa at this
location.
53
54
7. The algae, protozoa, and bacteria cultured from
the air samples were also found in surface samples taken
from the aeration basin and on plates of sterile media
exposed for five minutes on the service walk in the center
of the basin, twelve inches above the bubbling mixture of
sewage and activated sludge.
8. Many studies have shown that bacteria are emitted
to the air at sewage treatment plants; this survey has
shown that algae and protozoa may also be found in this
habitat.
9. In addition to the other microorganisms, the algae
and protozoa have been shown to be of medical importance as
a cause of some respiratory irritations and other ailments;
it is suggested by this survey that more extensive and
quantitative studies need to be made in areas of suspected
high airborne concentrations of these organisms.
10. More thorough studies should be made of the role
of sewage treatment in the dispersal of algae and protozoa.
BIBLIOGRAPHY
Books
American Public Health Association, American Water Works Association, and Water Pollution Control Federation, Standard Methods for the Examination of Water and Wastewater. 12th ed., New York, American Public Health Association, Inc., 1965.
Bailey, W. R., and E. G. Scott, Diagnostic Microbiology. 2nd ed., St. Louis, C. V. Mosby Company, 1966.
Breed, R. S., E. G. D. Murray, and N. R. Smith, Bergey's Manual of Determinative Bacteriology. 7th ed., Baltimore, The Williams and Wilkins Company, 1957.
Difco Laboratories, Difco Manual. 9th ed., Detroit, Difco Laboratories, Inc., 1953.
Fritsch, F. E., The Structure and Reproduction of the Algae, Vol. I, (2 volumes), New York, The M a c M i l i n n ^ m i ^ S ^ ' 1935.
Geldreich, E.̂ E., Sanitary Significance of Fecal Coliforms ZH jfflyirorimexit, Water Pollution Control Research Series Publication WP-20-3, Cincinnati, Federal Water Pollution Control Administration, 1966.
Gregory, P. H., The Microbiology of the Atmosphere. New York Interscience Publishers, Inc., 1961. '
Jahn,^T. L., and F. F. Jahn, How to Know The Protozoa. Dubuque, William C. Brown Company, 1949. '
K U d ° V R " ^ " ' P r°| :? Zf o l o g Y. 5 t h ed-' Springfield, Charles C. Thomas, 1966.
Ludin, F. C. and L. S. West, Keg to the Free-living Protozoa Uffier Peninsula, Marquette, Northern Michig^
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56
Mackenthum, K. M., and W. M. Ingram, Biological Associated Problems in Freshwater Environments, Cincinnati, Federal Water Pollution Control Administration, 1967.
Molisch, H., Populare Biologische Vortrage, Jena, Gustav Fischer, 1920,
Prescott, G. W., Algae of the Western Great Lakes Area, Dubuque, William C. Brown Company, 1962.
Pringsheim, E. G., Pure Cultures of Algae, Cambridge, Cambridge University Press, 1946.
Proctor, B. E., and B. W. Parker, "Microorganisms in the Upper Air," Aerobiology, edited by F. R. Moulton, Washington, American Association for the Advancement of Science, 1942.
Smith, D. T., N. F. Conant, and J. R. Overman, Zinsser Microbiology, 13th ed., New York, Appleton-Century-Crofts, 1964.
Smith, G. M., The Fresh-water Algae of the United States, 2nd ed., New York, McGraw-Hill Book Company, Inc., 1950.
Tiffany, L. H., and M. E. Britton, The Algae of Illinois, Chicago, University of Chicago Press, 1952.
Ward, H. B., and G. C. Whipple, Freshwater Biology, 2nd ed., edited by W. T. Edmondson, New York, John Wiley and Sons, Inc., 1963.
Williams, 0. B., Laboratory Manual for Bacteriology, Austin, University of Texas Press, 1955.
Wolfe, H. W., and others, Sampling Microbial Aerosols, Public Health Service Monograph No, 60, Fort Detrick, U. S. Public Health Service, 1959.
ZoBell, C. E., "Microorganisms in Marine Air," Aerobiology, edited by F. R. Moulton, Washington, American Association for the Advancement of Science, 1942.
57
Articles
Baines, S., and others, "Protozoa As Indicators in Activated Sludge," Sewage and Industrial Wastes, XXV (1953), 1023-1033.
Bold, H. C., "The Morphology of Chlamydomonas chlamydogama," Bulletin of the Torrey Botanical Club, LXXVI (1949), 101-108.
Brown, R. M., Jr., "The Collection and Cultivation of Air-borne Algae for a Biology Lab Exercise," American Journal of Botany, LII (1965), 653.
_, "Notes on Hawaiian Airborne and Soil Algae," American Journal of Botany, LII (1965), 644.
Brown, R. M., Jr., D. A. Larson, and H. C. Bold, "Airborne Algae: Their Abundance and Heterogeniety," Science CXLII (February 7, 1964), 583-585.
Darwin, Charles, "An Account of the Fine Dust which often Falls on Vessels in the Atlantic Ocean," Quarterly Journal of the Geological Society of London. XI (1844), 26-307
"Yellow Rain," Gardiner1s Chronicle, July 18, 1863, p. 675.
Denoyelles, F., Jr., "Factors Affecting Phytoplankton Distribution in a Double-Cell Sewage Lagoon," Journal of Phycology, III (1967), 174-181.
Dias, F. F., and J. V. Bhat, "Microbial Ecology of Activated Sludge, I. Dominant Bacteria," Applied Microbiology, XII (1964), 412-417.
Dixon, F. R.,and L. J. McCabe, "Health Aspects of Wastewater Treatment," Journal of the Water Pollution Control Federation. XXXVI (1964), 984-989.
Ehrenberg, C. G., "Bericht iiber die zur Bekanntmachung Geeigneten Verhandlunger der Konigl, Preussische Akademie der Wissenschaften Berlin. IX (1844), 194-197.
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"Passatstaub und Blutregen ein Grosses Organisches Unsichtbares Wirken und Leben in der Atmosphare," Akadenie der Wissenschaften Berlin Physikalische Abhandlungen, XXIII (1849), 269.
Eliassen, R., and C. A. Vath, "Air Pollution Control in Sewage Treatment Plants," Journal of the Water Pollution Control Federation, XXXII (1960), 424-427.
Fair, G, M., and W. F. Wells, "Measurement of Atmospheric Pollution and Contamination by Sewage Treatment Works," Proceedings, 19th Annual Meeting of the New Jersey Sewage Works Association, 1934. .
Gislen, Tors ten, "Aerial Plankton and Its Conditions of Life," Biological Reviews of the Cambridge Philosophical Society, XXIII (1948), 109-126.
Glaser, J. R., and J. 0. Ledbetter, "Sizes and Numbers of Aerosols generated by Activated Sludge Aeration," Water and Sewage Works, CXIV (1967), 219-221.
Gregory, P. H., E. D. Hamilton, and T. Seeramula, "Occurrence of the Alga Gleocapsa in the Air," Nature, CLXXVI (1S55), 1270.
Heise, H. A., "Symptoms of Hay Fever Caused by Algae," Journal of Allergy, XX (1949), 383-384.
"Symptoms of Hay Fever Caused by Algae, II," Annals of Allergy, IX (1951), 100.
Horrocks, W. H., "Experiments to Determine the Condition Under Which Specific Bacteria Derived From Sewage May Be Present in the Air of Ventilating Pipes, Drains, Inspection Chambers, and Sewers," Proceedings of the Royal Society of London, Series B, LXXIX (1907), 236.
Jenkins, S. H., "The Role of Protozoa in the Activated Sludge Process," Nature. CL (1942), 607-610.
Kelly, C. D., and S. M. Paddy, '"Microbiological Studies of Air over Some Nonarctic Regions of Canada," Canadian Journal of Botany, XXXI (1953), 90-106.
59
Lackey, J. B., "Biology of Sewage Treatment," Sewage Works Journal, XXI (1949), 659-665.
, "The Microscopic Flora and Fauna of Tree Holes,'" Ohio Journal of Science, XL (1939), 186-192.
Ledbetter, J. 0., "Air Pollution from Aerobic Waste Treatment," Water and Sewage Works, CXI (1964), 62-63.
Ledbetter, J. 0., and C. W. Randall, "Bacterial Emissions from Activated Sludge Units," Industrial Medicine and Surgery, XXXIV (1965), 130-133.
Mason, B. J., "Bursting Bubbles at the Surface of Sea Water," Nature, CLXXIV (1954), 470-471.
McElhenney, T. R., and others, "Algae: A Cause of Inhalent Allergy in Children," Annals of Allergy, XX (1962), 739-743.
McKinney, R. E., and A. Gram, "Protozoa and Activated Sludge," Sewage and Industrial Wastes, XXVIII (1956), 1219-1231.
McKinney, R. E., and R. G. Weichlein, "Isolation of Floe Producing Bacteria from Activated Sludge," Applied Microbiology, I (1953), 259-260.
Meier, F. C., and C. A. Lindberg, "Collecting Microorganisms from the Arctic Atmosphere," Scientific Monthly, XL (1935), 5-20.
Napolitano, P. J., and D. R. Rowe, "Microbial Content of Air Near Sewage Treatment Plants," Water and Sewage Works, CXIII (1.966), 480-483.
Overeem, M. A. van, "On Green Organisms Occurring in the Lower Trophosphere," Recueil des Travaux Botaniques Neerlandais. XXXIV (1937), 389-439.
, "A Sampling Apparatus for Aeroplankton," Proceedings of the Royal Academy of Amsterdam. XXXIV £1936), 981-990.
60
Paddy, S. M., and C. D. Kelly, "Studies on Microorganisms in Arctic Air During 1949 and 1950," Canadian Journal of Botany, XXXI (1953), 107-122.
Polkowski, L. B., and others, "Evaluation of Frothing in Sewage Treatment Plants," Sewage and Industrial Wastes, XXXI (1959), 1004-1015.
Proctor, B. E., "The Microbiology of the Upper Air, I," Proceedings of the American Academy of Arts and Sciences, LXIX (1934), 315-340.
, "The Microbiology of the Upper Air, II," Journal of Bacteriology, XXX (1935), 363-375.
Proctor, B. E., and B. W. Parker, "The Microbiology of the Upper Air, III," Journal of Bacteriology, XXXVI (1938), 175-184.
Randall, C. ¥., and J. 0. Ledbetter, "Bacterial Air Pollution from Activated Sludge Units," American Industrial Hygiene Association Journal, XXVII (1966), 506-519.
Salisbury, J. H., "On the Cause of Intermittent and Remittent Fevers, with Investigations which Tend to Prove that These Affections Are Caused by Certain Species of Palmellae," American Journal of Medical Science, LI (1866), 51-75.
Schlichting, H. E., Jr., "Meterological Conditions Affecting the Dispersal of Airborne Algae and Protozoa," Lloydia, XXVII (1964), 64-78.
, "Construction of an Inexpensive Plant Growth Chamber," Turtox News, XLI (1963), 214-215.
, "Viable Species of Algae and Protozoa in the Atmosphere," Lloydia, XXIV (1961). 81-88.
Sparr, A. E., "Froth Control at Bay Park," Sewage and Industrial Wastes, XXX (1958), 305-312.
61
Spendlove, J. C., "Production of Bacterial Aerosols in a Rendering Plant Process"Public Health Reports, LXXII (1957), 176-180.
Stevenson, R. E., and A. Collier, "Preliminary Observations on the Occurrence of Air Borne Marine Phytoplankton," Lloydia, XXV (1962), 89-93.
Torpey, W. N., and A. H. Chasick, "Principles of Activated Sludge Operation," Sewage and-Industrial Wastes, XXVII (1955), 1217-1233."
Wells, W» F„, and M. W. Wells, "Air Borne Infection," Journal of the American Medical Association, CVII (1936)," 1698-1703.
Wolfe, F. T., "The Microbiology of the Upper Air," Bulletin of the Torrey Botanical Club, LXX (1943), 1-14.
Woodcock, A. H., "Bursting Bubbles and Air Pollution," Sewage and Industrial Wastes, XXVII (1955), 1189-1192.
"Note Concerning Human Respiratory Irritation Associated with High Concentrations of Plankton and Mass Mortality of Marine Organisms," Journal of Marine Research, VII (1948), 56-62.
Unpublished Materials
Albrecht, C. R., "Bacterial Air Pollution Associated with the Sewage Treatment Process," unpublished master's thesis, University of Florida, 1958.
Denton, Texas Sewage Treatment Plant, Freese, Nichols, and Endress Consulting Engineers, Fort Worth, Texas, 1961.
Mahoney, J. L., III, "Airborne Algae in the Vicinity of a Trickling Filterj" unpublished paper, Department of Biology, North Texas State University, Denton, Texas, 1967.
62
Schlichting, H. E., Jr., "The Role of Waterfowl in the Dispersal of Algae," doctoral dissertation, Michigan State University, Ann Arbor, University Microfilms, 1958.
, unpublished lecture notes, Department of Biology, North Texas State University, Denton, Texas, 1967.
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