TLEA1
WATER POLLUTION CONTROL RESEARCH SERIES • 16050 EQS 12/71
Enteric Bacterial Degradation
of
Stream Detritus
ENVIRONMENTAL PROTECTION AGENCY
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WATER POLLUTION CONTROL RESEARCH SERIES
The Water Pollution Control Research Series describes the
results and progress in the control and abatement of pollution
in our Nation's waters. They provide a central source of
information on the research, development and demonstration
activities in the Environmental Protection Agency, through
inhouse research and grants and contracts with Federal, State,
and local agencies, research institutions, and industrial
organizations.
Inquiries pertaining to Water Pollution Control Research
Reports should be directed to the Chief, Publications Branch
(Water), Research Information Division, R&M, Environmental
Protection Agency, Washington, B.C. 20460.
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ENTERIC BACTERIAL DEGRADATION
OF STREAM DETRITUS
by
Charles W. Hendricks, Ph. D.
Department of Microbiology
University of Georgia
Athens, Georgia 30601
for the
OFFICE OF RESEARCH AND MONITORING
ENVIRONMENTAL PROTECTION AGENCY
Project # 16050 EQS
December, 1971
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EPA Review Notice
This report has been reviewed by the Environmental Protection
Agency and approved for publication. Approval does not signify
that the contents necessarily reflect the views and policies of
the Environmental Protection Agency, nor does mention of trade
names or commercial products constitute endorsement or recom-
mendation for use.
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ABSTRACT
A laboratory and field investigation was conducted between August,
1968 and July, 1971 to relate basal nutrients in the water and on the
bottom of a warm, fresh water stream to their ability to support the
growth and multiplication of pathogenic and nonpathogenic enteric
bacteria. Three independent studies, including (1) a water quality
analysis of the Oconee River (Clark County, Georgia), (2) respiration
experiments, and (3) continuous culture experiments were designed
to provide useful information in this research.
The results of this investigation indicate that natural populations and
selected laboratory strains of enteric bacteria have the capacity to
metabolize substrates that were present in the Oconee River environ-
ment including autoclaved river water. These organisms, however,
lacked the ability to increase in numbers in continuous culture with
river water and suspended detritus recovered above a secondary
sewage treatment facility, but they did demonstrate positive growth
rates with substrates recovered below the plant.
Data from this study also demonstrated that the sands and clays forming
the stream bottom have the capacity to sorb substrates from the over-
layering water, and that sediment eluates will stimulate the respiration
rate of the study bacterial strains. These results suggest that the
stream bottom can provide a suitable environment for the growth of
bacterial species and perhaps control basal nutrient concentration in
the water itself.
This report was submitted in fulfillment of Project Number 16050 EQS,
under the sponsorship of the Environmental Protection Agency.
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CONTENTS
Section Page
I Conclusions 1
II Recommendations 3
III Introduction 5
IV Methods and Materials 7
V Results 31
VI Discussion 85
VII Acknowledgements 95
VIII References 97
IX Publications and Patents 103
X Glossary 105
v
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FIGURES
No. Page
1 Map of Athens, Georgia showing the three
study sites. 8
2 Study site 1. 9
3 Study site 2. 10
4 Study site 3. 11
5 Study locations below the Bailey St. sewage
treatment plant for Salmonella recovery. 15
6 Biological oxygen monitor (Yellow Springs
Instruments, Inc. ). 19
7 Schematic design of the continuous culture
system employed in this investigation. 22
8 100 ml. continuous culture device. 23
9 1.7 liter continuous culture device in duplicate. 24
10 Four possible cases of constant washout rates
seen in continuous culture studies. 27
11 Formic hydrogenlyase synthesis by Escherichia
coli ATCC 11775 and by Enterobacter aerogenes
ATCC 12658 at 30. 0 and 44. 5 C. 54
12 Formic hydrogenlyase synthesis at 44. 5 C by
various enteric bacteria. 55
13 Effect of pH and buffer concentration for elution
of hexoses and protein from river sediment. 59
14 Respiration of selected enteric bacteria in
minimal inorganic salts medium containing
various concentrations of glucose at 30 C,
20 C, and 5 C. 62
VI
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No. Page
15 Respiration rates of selected pathogenic and
nonpathogenic enteric bacteria in 0. 3 _M_
phosphate buffer extracted sediments and
river water. 66
16 Continuous culture of a natural heterotrophic
bacterial population in Oconee River water
from site 3 and 14 C. 69
17 Continuous culture of a natural heterotrophic
bacterial population in Oconee River water
from site 3 at 26 C. 70
18 Continuous culture of a natural heterotrophic
bacterial population in Oconee River water
from site 3 at 30 C. 72
19 Continuous culture of a natural heterotrophic
bacterial population at 30 C in minimal salts
medium diluted 1:1000. 73
20 Washout rate vs. dilution rate plot for Escherichia
coli in Oconee River water from sites 1, 2, and
3 at 30 C. 75
21 Growth curve of Escherichia coli and Bdellovibrio
bacteriovorus in N. B. Medium at 30 C. 79
22 Growth curve of Escherichia coli and Bdellovibrio
bacteriovorus in filtered and autoclaved site 3
Oconee River water at 30 C. 80
23 Growth curve of Escherichia coli and Bdellovibrio
bacteriovorus in site 3 river water that was
supplemented with nutrients after 4 days. 81
24 Continuous culture of Escherichia coli and Bdello-
vibrio bacteriovorus in Oconee River water from
site 3 at 30 C. 83
VII
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TABLES
No. Fa§e
1 Mean analysis of nutritional factors in the North
Oconee River at Site- 1 during 1968-1969 32
2 Mean analysis of nutritional factors in the North
Oconee River at Site 2 during 1968-19^9 33
3 Mean analysis of nutritional factors in the North
Oconee River at Site 3 during 1968-19^9 34
%
4 Mean bacteriological counts from the North Oconee
River at Site 1 during 1968- 19&9 35
5 Mean bacteriological counts from the North Oconee
River at Site 2 during 1968- 1969 36
6 Mean bacteriological counts from the North Oconee
River at Site 3 during 1968-1969 37
7 Mean analysis of nutritional factors in the North
Oconee River at site 1 during 1969-1970 38
8 Mean analysis of nutritional factors in the North
Oconee River at Site 2 during 1969-1970 39
9 Mean analysis of nutritional factors in the North
Oconee River at Site 3 during 1969-1970 40
10 Mean bacteriological counts from the North Oconee
River at Site 1 during 1969-1970 41
11 Mean bacteriological counts from the North Oconee
River at Site 2 during 19&9-1970 42
12 Mean bacteriological counts from the North Oconee
River at Site 3 during 1969-1970 43
13 Mean analysis of nutritional factors in the North
Oconee River at Site 1 during 1970-1971 44
14 Mean analysis of nutritional factors in the North
Oconee River at Site 2 during 1970-1971 45
viii
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No. Page
15 Mean analysis of nutritional factors in the North
Oconee River at Site 3 during 1970-1971 46
16 Mean bacteriological counts from the North Oconee
River at Site 1 during 1970-1971 47
17 Mean bacteriological counts from the North Oconee
River at Site 2 during 1970-1971 48
18 Mean bacteriological counts from the North Oconee
River at Site 3 during 1970-1971 49
19 Mean CHN analysis of suspended solids present in
river water from sites 1, 2 and 3 50
20 Gas production by enteric bacteria recovered from
river water on M-Endo MF medium at 44. 5 C 51
21 Gas production from glucose, lactose, and formate at
30. 0 C and 44. 5 C by aquatic forms of Enterobacter
aerogenes conforming to the "coliforml'designation 53
22 Formic hydrogenlyase activity of selected coliform
bacteria 56
23 Salmonella recovered from water and bottom sediments
of the North Oconee River 58
24 Basic nutrient analysis of Oconee River water and
extracts of river bottom sediments from the three
study sites 60
25 Respiration of various enteric bacteria in Oconee
River water and in extracts of river bottom
sediments from site 1 63
26 Respiration of various enteric bacteria in Oconee
River water and in extracts of river bottom sediments
from site 2 64
27 Respiration of various enteric bacteria in Oconee
River water and in extracts of river bottom
sediments from site 3 65
ix
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No. Page
28 Respiration rates of Enterobacter aerogen.es
(ATCC 12658) and Escherichia coll (ATCC
11775) using slime and stream detritus as
substrates 67
29 Basal nutrient concentration of autoclaved site 3
river water and dilute minimal salts-glucose
medium 68
30 Average growth rates of native bacterial populations
in site 3 Oconee River water and dilute minimal
medium 74
31 Growth rates of selected enteric bacteria in Oconee
River water from site 3 76
32 Growth rate of Escherichia coli at 30 C in concentrated
stream detritus from the Oconee River 77
33 Respiration rate of Escherichia coli at 30 C in detritus
samples and eluates of sediments present in
detritus samples from site 3 78
x
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SECTION I
CONCLUSIONS
1. Based upon water quality determinations for basal nutrients and
types and numbers of bacteria present, the North Oconee River above
Athens, Ga. does not appear grossly contaminated. However, the
Bailey St. sewage plant does contribute significantly to the basal
nutrient concentration and an unknown number of Salmonella to the
stream that is subsequently used for recreational purposes.
2. There appears to be more Salmonella associated with the bottom
sediments than are present in the overlaying water in the area below
the sewage plant effluent (Site 3).
3. The presence of an inducible formic hydrogenlyase system at 44. 5
C in fecal coliforms (E. coli) was found to be the basis for the Eijkman
fecal coliform concept.
4. The laboratory strains of enteric bacteria utilized in this study
could metabolize substrates present in river water, collected detritus
and extracts of bottom sediments.
5. The test bacterial strains were unable to grow in chemostats when
river water taken above the sewage plant was employed. River water
taken below the sewage plant effluent did support the growth of these
organisms.
6. Although the observed multiplication rates for the enteric bacteria
used in the study were small in comparison to that which can be
achieved in laboratory media, any process which would have an end
result of contributing oxidizable substrates to the system could in
effect substantially raise these growth rates.
7. Bdellovibrio bacteriovorus, an obligate bacterial parasite for Gram
negative bacteria, including Escherichia coli, can infect the host in
river water, but it is doubtful that the parasite is a major factor in
stream self-purification mechanisms.
8. Whether or not enteric bacteria grow in substrates from the aquatic
environment seems to be a function of the integrated environmental
parameters such as basal nutrient concentration, temperature, stream
flow, etc. That is, the concept of a growth limiting nutrient for enteric
bacteria may not be valid in a relatively non-polluted stream.
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SECTION II
RECOMMENDATIONS
This research project was limited to laboratory experiments using
natural substrates recovered from the Oconee River in attempts to
determine if they could be metabolized by enteric bacteria including
pathogenic species. The river water, sediment eluates, and pro-
cessed detritus substrates used in this study did receive mild labora-
tory treatment prior to use, and it is recommended that in situ
studies of a similar nature be made before data in this report is
extrapolated directly to the natural aquatic environment.
Based upon the data in this report and taking into consideration the
above statement on extrapolation of data to the environment, certain
general recommendations, however, can be made.
1. Bottom sediments should be included in routine sampling procedures,
especially during water quality surveys.
2. Depending upon the immediate use of the water and the area, in
question, provisions should be made to monitor water quality prior
to any disruption of the integrity of the sediment layer of a lake,
stream or estuary. Parameters that would be of importance and
perhaps predict problems of a public health nature are: a) presence
and numbers of specific organisms and b) a measure of microbial
activity such as respiration determinations.
3. Work is needed to determine the significance of the growth of
potentially dangerous organisms in fresh water. Questions that would
be of interest include: a) are other organisms besides enteric capable
of growth in fresh water, b) are such organisms virulent, c) are toxic
factors synthesized in quantity to be hazardous and d) under what
conditions could such growth be controlled.
4. Further work is also needed to elucidate the observed sediment-
nutrient interaction and to determine how this interaction affects the
microbiological quality of water.
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SECTION III
INTRODUCTION
The principles governing the growth and multiplication of bacteria in
nutritionally adequate media at near optimal temperatures are well
known. In general, a few bacterial cells can produce vast populations
in these media since the initial concentration of nutrients does not be-
come a critical factor for growth. However, in natural environments,
or in media where nutrient concentrations are marginal and tempera-
tures far from optimal, multiplication of mesophilic bacteria often
becomes erratic, and precise growth rates are ^ot easily determined.
A few investigators, including Kusnezow (1959) and Ruttner (19&4),
are convinced that many heterotrophic bacterial species are capable
of growth in fresh water lakes and streams. These bacteria seem to
be involved in organic dissimilation processes in which complex
organic compounds are returned to the biotope as inorganic and rela-
tively simple organic substance for recycling by photo- and chemo-
synthetic organisms.
Certainly, nutritional conditions present in the aquatic environment
are of great importance as determinants of whether or not a particular
bacterial species will survive and later multiply. Probably only a
very small fraction of the total bacterial growth occurs in the free
flowing water where an adequate supply of nutrients is not easily
obtained. However, on, or in, the bottom sediments, extensive
growth of bacteria can occur in the micro-environment where nutrients
can be in high concentration surrounded by a relatively vast area devoid
of nutrients. Temperatures in this environment would not fluctuate
as sharply as the diurnal variations of the stream proper, thus creat-
ing more stable conditions.
This study includes attempts to relate the types of organic material
in and on the bottom of a warm, fresh water stream to their ability
to support the growth and multiplication of pathogenic and nonpatho-
genic enteric bacteria. Once specific knowledge has been obtained
concerning the utilization of this material, we would be in a better
position to understand the roles of both the aquatic environment and
the bacteria in a polluted stream. Such data could also aid in the
classification of rivers and streams and form a foundation for the
construction of a model system which might be able to predict the
theoretical pollution limits of a given body of water.
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Previous investigations concerning the nutritional requirements for
enteric bacteria, especially for E. coii, have contributed greatly to
our understanding of the organism and its metabolism, but most of
this knowledge has been derived with the use of artificial culture
media containing organic materials in concentrations and in type far
exceeding those found in natural surface waters and bottom sediments.
It is conceivable that present knowledge concerning well-nourished
bacteria may not be applicable to studies dealing with microbial
growth and reproduction in aquatic environments, especially with
bacteria associated with pollution, and those which are not indigenous
to the environment.
Many intestinal disease-producing bacteria in man and higher animals,
including those within the family Enterobacteriaceae, are not fastidious
in their nutritional requirements. In fact, many of the prototrophic
species belonging to the genera Proteus, Salmonella, Paracolobactrum,
and Shigella are able to grow and reproduce in a basic inorganic salts
medium similar to that used to grow algae, providing a suitable carbon
source is present. McGrew and Mallette (1962) have shown that '
intestinal bacteria can reproduce in such a medium that contained less
than 5 (j.g/ml of-glucose, and this concentration approaches that of
hexoses commonly found in unpolluted river water.
If potentially pathogenic bacteria can obtain adequate nutrients in the
natural aquatic environment and are also capable of reproduction,
needed information could be obtained on mesophilic growth in natural,
low nutrient substrates at temperatures far removed from their
optimum 35-47 C range. Such research could also aid in the bacterio-
logical classification of waters capable of becoming polluted if dilution
purification factors were overcome and may clarify present ideas con-
cerning stream self-purification and the role of aquatic environments
in the dissemination of disease.
This investigation consists of three independent studies each of which
was designed to provide useful biochemical and bacteriological infor-
mation concerning the ability of enteric bacteria to survive and
multiply in the presence of low levels of natural aquatic substrates.
These studies are as follows;
1. Water quality analysis of the North Oconee River at three selected
sites at Athens, Georgia.
2. Evaluation of the ability of enteric bacteria to use natural aquatic
substrates by oxygen uptake experiments.
3. Evaluation of the ability of enteric bacteria to use natural aquatic
substrates by continuous culture experiments.
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SECTION IV
METHODS AND MATERIALS
Part 1
Water Quality Analysis of the North Oconee River at three selected
sites at Athens, Georgia
Chemical and bacterial assays
Site selection: Three sites were selected on the north branch of the
Oconee River that flows through Athens, Georgia (Fig. 1). Site one
is situated about 4 mi. northeast of the city and the river in this area
has been flowing through agricultural and undeveloped land (Fig. 2).
Site two is located near the center of Athens and well within the city
limits. The river at this point has received water from various
storm drains and creeks that drain the residential areas. No domes-
tic sewage is known to be added to the river in this area (Fig. 3).
Site three is situated about 750 meters below one of the two Athens
municipal sewage plants (Fig. 4). The sewage at this facility receive
both primary and secondary treatment, and the effluent is chlorinated
and allowed to settle prior to addition in the stream.
Sample collection and analysis: Water, sediments, and stream
detritus in various forms were collected by the techniques of
Morrison and Fair (1966) and returned to our laboratories for sub-
strate and bacteriological analysis. Specific parameters of interest
are types and numbers of bacteria, water temperature, protein,
orthophosphate, ammonia nitrogen, and carbohydrate content, pH,
and Carbon-Nitrogen-Hydrogen ratios. These determinations were
made on at least a biweekly basis and also when water was collected
for a particular growth study.
Assay procedures: Chemical and physical parameters of the Oconee
River were monitored by techniques approved by the American Public
Health Association and published in Standard Methods for the Examina-
tion of Water and Wastewater (APHA, 1971). Glucose and total hexose
were measured by the Glucostat (Worthington Biochem. Corp. ) and
anthrone (Morris, 1948) procedures, respectively, and protein content
was determined by the Folin-Ciocalteau and Biuret procedures (Colo-
wick and Kaplan, 1955). Enteric bacteria enumerations were made
according to the techniques of Morrison and Fair (1966), and species
identification made with the use of Bergey's Manual (Breed-et al. ,
1957), the taxonomic aid, The Identification of Enterobacteriaceae
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Fig. 1. Map of Athens, Georgia showing the
three study sites.
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V
Fig. 2. Study site 1,
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Fig. 3. Study site 2.
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•» l^t™
*"•- •" '^y^ ••
'•]iifj&-
Fig. 4. Study site 3.
>zj$®j$lj^m.
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(Edwards and Ewing, 1962) and then confirmation by NCDC, Atlanta, Ga.
Sampling procedure evaluations
Two separate field studies were initiated to evaluate the reliability
of the Eijkman fecal coliform concept and the recovery of salmonellae
in stream bottom sediments vs. surface waters.
Formic hydrogenlyase induction as a basis for the Eijkman fecal coliform
concept: There have been numerous attempts to differentiate between a
"fecal" and a "non-fecal" coliform, and the most successful of these are
those of the elevated temperature of incubation variety (Geldreich, 1966. )
These procedures are primarily modifications based upon the Eijkman
(1904) observation that members of the Escherichia group, as described
by Parr (1938a,b), could produce gas in a glucose medium at 46 C,
whereas Enterobacter (Aerobacter) could not. Perry and Hajna (1944)
improved upon the Eijkman technique by substituting lactose for glucose,
adding bile salts and a buffering system to the medium and lowering
the incubation temperature to 44. 5 C. Utilizing modification of this
basic fecal coliform procedure, Geldreich jJt^aL (1962), and more
recently Mishra_et ja.1. (1968), have observed a positive correlation
of gas production at the elevated incubation temperature with Parr's
Escherichia group of coliforms. More significantly, however, Mishra
_et _al. found that more than 52% of the Enterobacter types recovered
also gave positive elevated temperature reactions.
The basic mechanism by which gas is produced by Escherichia and not
by Enterobacter at elevated temperature is not clearly understood,
nor is it known with certainty which Enterobacter strains from aquatic
or terrestrial environments are capable of producing gas at these
temperatures. The present investigation was undertaken to determine
which particular gas-producing enzyme system in Enterobacter is
affected by the incubation at 44. 5 C and to test the basic concept of the
Eijkman procedure with known coliform strains and organisms recover-
ed from the aquatic environment.
Basic Warburg Procedure: The activity of induced formic hydrogen-
lyase was determined in a Warburg respirometer (Aminco) by the techni-
que of Quist and Stokes (1969). Duplicate vessels contained 2. 0 ml of a
washed cell suspension in the main compartment, and 0. 2 ml of a 20%
KOH solution in the center well for the absorption of COS. Substrates for
induction included in the side arms were 60 umoles of sodium formate
(0. 3ml), 20 ^.moles of glucose (0. 2ml), and 0. 1 ml of a 10% concentration
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of yeast extract (Difco). To ensure that the enzyme system was
being induced in the Warburg vessels and was not preformed in
the original culture flasks, 0. 03 mg of chloramphenicol (0. 3 ml)
was added to one of the Warburg vessels in each series to inhibit
Ae novo protein synthesis. The remaining flask received 0. 3 ml
of 0. 1 M phosphate buffer (pH 6. 9). The gas phase in each experi-
ment was N2, and the bath temperature was at either 30. 0 or 44. 5
C, depending on the particular experiment. Each vessel was
incubated with shaking until a constant rate of Ha evolution was
reached, and results were expressed in Q(Hs) values, as microliters
of H2 evolved per hour per milligram of dry cells. There was
virtually no endogenous H3 production and no Hg formed in control
vessels containing only cells and sodium formate.
Selection of organisms; Enteric bacteria employed in this study were
obtained from both the American Type Culture Collection (Rockville,
Md. ) and by recovery from the North Oconee River in Clark County,
Ga. River water samples were collected by techniques suggested
by the American Public Health Association (1966) and cultured on
M-Endo-MF Broth (BBL) by the use of the membrane filter procedure.
After incubation at 44. 5 ± 0. 1 C for 16 to 18 hr, all colonies were
counted that conformed to the "enteric" designation (lactose fermenta-
tion with the production of green, metallic sheen). Organisms
conforming to either the "enteric" or "coliform" designation were
further differentiated by their ability to produce gas at 44. 5 C in
MR-VP medium (BBL) and by indole: methyl red: Voges-Proskauer:
citrate (IMViC) classification (Geldreich, _e_t aJ. , 1966). The taxo-
nomic classification of the isolates was confirmed by techniques
suggested by Edwards and Ewing (1962) and the nomenclature used
in the remainder of this study follows that proposed by Ewing (1963)
which substitutes the generic name Enterobacter for Aerobacter.
After these preliminary tests were completed, all organisms,
including the stock cultures of Escherichia coli (ATCC 11775) and
Enterobacter aerogenes (ATCC 12658) were maintained on Nutrient
Agar (Difco) slants in the refrigerator for later use.
Gas production by Enterobacter: Each strain recovered was inoculated
into a duplicate series of Durham fermentation tubes containing 1%
concentrations of glucose (Difco), lactose (Difco), or sodium formate
(Fisher Scientific Co. , Pittsburgh, Pa. ) to qualitate gas production.
The basal medium was Nutrient Broth (Difco), and each tube was
preincubated to equilibrate the medium to the desired experimental
temperature. One set of fermentation tubes was incubated at 30. 0 C
and the other at 44. 5 C, and all tubes were observed for collected
gas in the inverted vials at 24 and 48 hr. After incubation, 1 ml of
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20% KOH solution was added to each culture tube containing gas to
absorb any evolved CO2, and after 30 min of incubation at room
temperature, the inverted vial was removed from the culture tube
and the open end was placed in a flame to determine if H2 was
present.
Formic hydrogenlyase assay: Pure culture cell suspensions of the
ATCC Escherichia and Enterobacter strains of aquatic enteric bacteria
were prepared by inoculating each into flasks containing 200 ml of
sterile, prewarmed Trypticase Soy Broth (BBL). These cultures
were grown aerobically with vigorous shaking to avoid inducing the
lyase system. Growth temperature was either 30. 0 C or 44. 5 C,
depending on desired experiment, and cells were harvested after 18
hr of incubation. The cells were collected by centrifugation and
washed three times in 0. 1 M phosphate buffer, pH 6. 8, and suspended
in buffer to give an optical density (OD) of 0. 2 at 540 nm with a
Spectronic 20 (Bausch St Lomb,' Inc. , Rochester, N. Y. ) colorimeter-
spectrophotometer. Dry weights of cells were obtained by comparing
the OD with a standard curve, which was prepared with organisms
washed with deionized water and then dried overnight at 105 C.
Increased recovery rate of salmonellae from stream bottom sediments
vs. surface waters: Much of our concern today for the presence of
enteric pathogens in our surface waters is largely due to the increas-
ing demands which are being placed upon this resource. Techniques
have been developed which increase recovery rates of the occasional
Salmonella or Shigella which can be found in high-quality surface
water (Spino, 1966; Fair and Morrison, 1967) but the lack of their
recovery does not preclude their presence at a particular sampling
point.
Sampling procedures: In light of the increasing number of reports
which suggest growth and multiplication of the coliform group of
bacteria in natural waters (Kusnezow, 1959; Kitrell and Furfari,
1963; Hendricks, 1970) and that river and lake bottom sediments
will stimulate their rate of growth (Boyd and Boyd, 1962; Hendricks
and Morrison, 1967), it was speculated that recovery yields of
pathogenic enteric bacteria might be higher than usually reported
for surface waters if river bottom sediments were concomitantly
sampled. For this study, several locations were selected on the
North Oconee River (Clark County, Ga. ) below an unchlorinated
treated sewage outfall for periodic water and bottom sediment col-
lection (Fig. 5). Samples were collected from each location by
techniques suggested by the American Public Health Association
(1966) and enriched for the Salmonella-Shigella group of organisms
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A *
Fig. 5. Study locations below the Bailey St. sewage
treatment plant for Salmonella recovery.
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in Selenite Broth (Difco). Ten grams (wet weight) of sediment were
inoculated into tubes containing 10 ml of the enrichment broth, where-
as one liter of river water was filtered through a 0. 45p. (HA)
filter disc (Millipore Corp. ). The filter disc was then placed into
a similar Selenite Broth tube. After 18 hr of incubation at 37 C,
samples of the enriched cultures were streaked to MacConkey Agar
(Difco) for primary isolation. The taxonomic classification of the
isolates was made by techniques suggested by Edwards and Ewing
(1962) and was confirmed serologically by the Enteric Bacteriology
Unit, Center for Disease Control, Atlanta, Ga.
Part 2
Evaluation of the Ability of Enteric Bacteria to Use Natural Aquatic
Substrates by Oxygen Uptake Experiments
Enteric bacterial metabolism of substrates in river water and in
stream sediment eluates
The concern today by environmentalists with the concentration of
nutrients in freshwater lakes and streams is largely the result of our
increasing demands which are being placed upon this resource. For
some time it has been known that terrestrial bacterial species can
grow and reproduce in extremely dilute nutrient concentrations
(Butterfield, 1929; McGrew and Mallette, l%2; Hendricks and
Morrison, 1967) of laboratory media, but most of these organisms
are not involved in pathogenesis of man or higher animals. Entero-
bacteriaceae, however, not only contains bacteria which are indicators
of fecal pollution, but others, such as Salmonella, Shigella, and
Arizona, which can produce serious intestinal disease.
It is suspected that bottom sediments of lakes and streams play a
major role in the recycling process of nutrients which allows for much
of the observed heterotrophic growth (Harter, 1968), but the nature of
the role is still quite vague. Studies by Malaney et al. (1962) and Boyd
and Boyd (1962) indicate that sediments will stimulate the growth of
bacterial species indigenous to freshwater lakes and streams. Work
by Hendricks and Morrison (1967), though, has shown that stream
sediments have the capacity to bind basal nutrients loosely and that
aqueous extracts of sediments will increase the rate of growth of
various enteric species in high-quality water at 15 C and less. It
was postulated by these investigators that this loosely bound material
was probably available for microbial use within the natural environ-
ment. The study is primarily concerned with nutrient binding by
river bottom sediments and conditions for its removal and use by
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enteric bacteria.
Study sites; Water and bottom sediments were collected for investi-
gation from sites on the North Oconee River, a typical stream of the
North Georgia piedmont in Clarke County, near Athens, Georgia.
These sites are identical to those described earlier.
Organisms: One strain each of Escherichia coli, ATCC 11775;
jCnterobacter aerogenes (Aerobacter aerogenes), ATCC 12658;
Proteus rettgeri, Arizona arizonae (Paracolobactrum arizonae),
Shigella flexneri Al, NCDC (Atlanta, Georgia); and Salmonella
senftenberg, CPHS (Ottawa, Canada), was grown in Trypticase Soy
Broth (BBL) at 30 C for 16 hours. Cells were then harvested by
centrifugation; washed 3 times in sterile, carbon-free, deionized
water; incubated at 30 C for 4 hr to expend endogenous metabolism;
and rested at 4 C for 18 hr before each experiment.
Experimental substrates; River water and stream bottom sediments
were collected from each study site. The river water was immediately
sterilized in an autoclave at 121 C for 15 min and then frozen after
samples were removed for chemical analysis. After collection,
sediments from each site were divided into 30 g lots, and each
aliquot was washed three consecutive times with 50 ml volumes of
carbon-free deionized water. These slurries were agitated for 30
min with a magnetic mixer and then clarified by centrifugation. Each
washing was collected, sterilized in the autoclave, and frozen for
later use.
After determining optimal pH and buffer ionic strength for elution, 30 g
aliquots of washed sediment from each site were eluted with 50 ml of
0. 3 M sodium phosphate buffer (Colowick and Kaplan, 1955), pH 7. 0,
in separate experiments, and with river water from the site where the
sediment was collected. These eluates were also autoclaved at 121 C
for 15 min and then frozen for later respiration studies and chemical
analysis.
Analyses for nutritional constituents were made on the sediment wash-
ings and eluates as well as river water from each site by chemical
procedures. Ammonia nitrogen and orthophosphate content was deter-
mined by procedures in Standard Methods (1966), hexose was measured
by anthrone (Morris, 1948), and protein content was determined by
the Folin-Ciocalteau procedure (Colowick and Kaplan, 1955).
Contamination of equipment by basal nutrients: In experiments of the
nature that are reported here, it is extremely important that the dilute
17
-------
nutrient substrates, including the river water, not be contaminated
by extraneous basal nutrients. Major sources of possible contamina-
tion include the utensils, water, air, filters, and many other materials
that may come in contact with the culture system.
In this investigation the major source of contamination was tap distilled
water so it was necessary to employ a deionizing system that rendered
the water carbon free as determined by CHN analyses on a one 1.
lyophilized water sample. All utensils used in this study were washed
with detergent, well rinsed with tap water, and incubated overnight in
a sulfuric acid-dichromate bath. The utensils were then rinsed with
copious amounts of carbon free water, capped with paper and auto-
claved. All membrane filters used to filter sterilized media were
washed with hot carbon free water to remove soluble basal nutrients,
and no culture device was employed with out suitable air filtration.
Bacterial respiration studies with eluted sediments: Cell suspensions
of each rested bacterial culture were prepared in deionized water
and standardized to 0. 9 optical density at 540 nm with a Spectronic
20 (Bausch and Lomb, Rochester, N. Y. ) colorimeter- spectro-
photometer. Dry cell weights were obtained by comparing the
optical density with a standard curve •which was prepared with orga-
nisms washed with deionized -water and then dried overnight at 105 C.
Respiration studies were carried out with the use of a Biological
Oxygen Monitoring System (Cole-Farmer, Chicago, 111. ) in which
4 ml of a particular substrate were placed into the monitor with 2 ml
of the standardized bacterial suspension, and oxygen uptake •was
measured for 15 min (Fig. 6). Individual experiments for each
organism and substrate were run in duplicate at temperatures of
30 C, 20 C, and 5 C. Control substrates consisting of deionized
water, river water from each site, varying dilutions of minimal
salts-glucose medium (Davis, 1950), and minimal medium containing
0. 1, 0. 2, and 0. 3 M phosphate were run in each temperature series.
Temperature of incubation was controlled within ± 0. 2 C with a Lauda-
Brinkman K-2R Circulator (Westbury, N. Y. ) and respiration was
calculated as mg atoms O/hr. /mg dried cell weight (Pomeroy and
Johannes, 1968) after correcting for endogenous activity.
Utilization of substrates in detritus samples and from slime capsules
of Enterobacter aerogenes by the coliform bacteria Escher'ichia coli
and Enterobacter aerogenes
Detritus: Detritus samples for respiration studies were recovered
from the three study sites on the Oconee River by two different
18
-------
Fig. 6. Biological oxygen monitor (Yellow Springs
Instruments, Inc.).
19
-------
processes:
Aliquots of river water were dried at 105 C for 24 hrs and others
lyophilized directly. The remaining solid material in each case was
termed Suspended detritus and separately stored.
Particulate material present in river water was concentrated with the
use of a device similar to the marine plankton concentrator of
Pomeroy and Johannas (1968). This detritus was lyophilized and the
remaining solid material was termed Concentrated detritus.
Slime: Polysaccharide capsular material (Slime) was produced from
Enterobacter aerogenes by culturing the organism in a minimal salts-
glucose medium (Davis, 1950). The slime that was produced in 18-Z4
hr was recovered by ethanol precipitation-cold centrifugation procedures
(Colowick and Kaplan, 1955) and then lyophilized for later use.
Respiration studies; The detritus and slime samples were diluted
with deionized water and used in respiration studies by techniques
outlined above.
Part 3
Evaluation of the Ability of Enteric Bacteria to Use Natural Aquatic
Substrates by Continuous Culture Experiments
It is generally assumed that basal nutrient concentrations present
in most fresh water and marine systems are not sufficient for sus-
tained bacterial growth. Although nitrogen and phosphorus are often
in very short supply, growth of these microorganisms in water seems
to be primarily limited by low concentrations of a suitable carbon and
energy source. Studies of McGrew and Mallette (1962) have shown
that approximately 5. 0 [j.g/ml glucose is required just for maintenance
of a bacterial culture, but this concentration is becoming more common
in nature. These and other data by Herbert (1961) and by Tempest
and Hunter (1965) suggest that a significant portion of the aquatic
bacterial population may not be metabolically active, but it is also
not inconceivable that a portion of the prototrophic bacterial popula-
tion could be growing in situ at very low rates which cannot be easily
detected.
To test this hypothesis in a fresh water system, the chemostat, a
continuous culture apparatus has been employed which, when run at
rate limiting substrate concentrations, allows for a growth rate
equal to the rate at which the nutrient is diluted in the culture vessel.
20
-------
Figure 7 is a schematic representation of the continuous culture
system that was employed in this series of experiments while
Figs. 8 and 9 consist of photographs of two different culture de-
vices that were designed for these studies.
Preliminary experiments
The first experiments in this series were designed to answer four
basic questions concerning the chemostat system and these included;
1. Of what significance is bacterial attachment to the glass walls
of the chemostat? 2. Can very dilute minimal medium support the
growth of a natural population of enteric bacteria from the Oconee
River? 3. Can river water support the growth of natural populations
of enteric bacteria? 4. At what dilution rates does bacterial growth
take place?
Organisms; Bacteria present in an unsterilized aliquot of river
water taken from below the sewage plant effluent (Site 3) served as
an inoculum for these preliminary studies. 1. 7 liters of the water
was centrifuged to sediment the bacteria present, the supernatant
was then poured off and the cells were then resuspended in an equal
volume of the test substrate. These cells were then introduced into
the chemostat to initiate the experiments.
Experimental substrates: The two experimental substrates that were
used in these studies included dilute minimal medium (Davis, 1950)
and Oconee River water from site 3.
1. The minimal salts-glucose medium was prepared and sterilized
and then diluted 1:1, 000 with sterile .carbon free, deionized water.
This medium was then introduced into the sterile chemostat for
growth studies.
2. River water was collected by techniques mentioned previously
and returned to the laboratory immediately for autoclaving (121 C
for 20 minutes). After basal nutrient concentrations were determined,
the water was ready for use as an experimental substrate.
Calculations: Herbertjet _al. (1956) has reported that during the
transient state in a chemostat, a population (x) changes:
dx = u:x - Dx
dt
or
21
-------
A
D
B
H
C
T2J
A. Air Pump
B. Medium Pump
C. Waste Pump
D. Medium Reservoir
E. Waste Reservoir
F. Continuous Culture Apparatus
6. Magnetic Mixer
H. Circulator
Power Source and Coil
Fig. 7. Schematic design of the continuous culture
system employed in this investigation.
22
-------
Fig. 8. 100 ml. continuous culture device.
Z3
-------
.
Fig, 9. 1. 7 liter continuous culture device in duplicate.
24
-------
x = x0e
Where XQ = initial population (t=o), p. = growth rate, and
D = dilution rate.
In solving the previous equation for the growth rate
where
= D +J_ In (X/XQ)
t
_L In (x/x0) = -A
and A equals the washout rate.
Therefore:
= D - A
Jannasch (1969) has postulated four different cases of constant washout
rates are conceivable in continuous culture studies:
1. The population doubles in one retention time and a steady state
results.
2. The population grows at a rate greater than the washout rate.
3. The population does not grow and is washed out of the culture
device.
25
-------
4. The population disappears at a rate faster than the washout rate
(death of the population).
These four cases are shown graphically in Fig. 10.
Bacterial attachment studies; In this series of experiments, the
chemostat was run at 14 C and 26 C to more closely resemble in
situ conditions. The dilution rate used at both experimental tempera-
tures was 0. 058 hr. "\ In addition to determining suspended bacterial
number and basal nutrient concentration, a series of glass microscope
cover slips were placed into the chemostat for the enumeration of
attached bacteria. Aliquots of water and a cover slip were removed
at intervals over a 24 hour period. Counting procedures for the
suspended bacteria were identical to those used previously, but the
cover slips were first ground in a Waring blender with 100 ml sterile
deionized water before aliquots were plated.
Dilute minimal medium and river water experiments: The chemostat
experiments utilizing (Davis, 1950) Minimal Medium diluted 1:1, 000
and river water from site 3 were run at an incubation temperature of
30 C and at a dilution rate of 0. 012 hr. ~1. After the organisms had.
been introduced into the culture vessel, total enteric and coliform
bacterial counts were determined daily on M-Standard Plate Count
Broth (Difco) and M-Endo Broth (Difco) by the Millipore procedure,
and duplicate plates were incubated at both 30 C and 44. 5 C to estimate
both nonfecal and fecal coliforms. Aliquots of water were also removed
from the chemostats for analysis of basal nutrients when the counts
were made.
Continuous culture experiments using river water and the collected^
detritus from the three sites as substrates
Data obtained in the preliminary experiments served as a basis to
quantitate growth rates of six selected enteric bacterial species in
river water and suspended and concentrated detritus samples from the
three study locations on the North Oconee River.
Organisms; The enteric bacteria used in this portion of the investiga-
tion were prepared by the following procedure. One strain each of
Escherichia coli, ATCC 11775; Enterobacter aerogenes (Aerobacter
aerogenes) ATCC 12658; Proteus rettgeri, Arizona arizonae (Para-
colobactrum arizonae), Shigella flexneri Al, NCDC (Atlanta, Georgia);
and Salmonella senftenberg, CPHS (Ottawa, Canada), was grown in
Trypticase Soy Broth (BBL) at 30 C for 16 hours. Cells were then
harvested by centrifugation; washed 3 times in sterile, carbon-free,
26
-------
T
H
1
(I) A«0
X
o
o
TIME
Fig. 10. Four possible cases of constant washout rates seen
in continuous culture studies. A = washout rate,
D = dilution rate, ^ = growth rate, R = amount of
time required to eliminate 1/2 (H) of the population.
Jannasch, 1969.
27
-------
deionized water; incubated at 30 C for 4 hr to expend endogenous
metabolism; and rested at 4 C for 18 hr before an experiment.
Experimental substrates: River water was collected from the
three study sites and immediately autoclaved (121 C for 20 min)
upon returning to the laboratory. River water that was to be used
as substrate immediately received no further treatment, but aliquots
of the water from each site were processed for the collection of sus-
pended and concentrated detritus by procedures previously mentioned.
Experimental procedures; The same basic procedures for operation
of the chemostat were used in the studies with both the river water
and detritus samples.
1. River water: In separate experiments, each of the washed and
rested test organisms (usually 1x10 cells) -were introduced into
chemostats containing sterile water from each of the 3 sites, and
each experiment was continued until a growth rate or a washout rate
could be obtained with confidence at temperatures of 30 C, 20 C, and
5 C. The elapsed time was generally 200-450 hours for any particular
experiment. Each chemostat was sampled daily for bacteriological
counts and basal nutrients by procedures previously mentioned.
2. Suspended and concentrated detritus; Samples of suspended and
concentrated detritus that had been collected over a two year period
were pooled according to site, suspended in 5 1. of deionized water
and sterilized in the autoclave. This material was then used as
substrate for culture of E. coli at growth temperature of 30, 20, and
5 C. Each experiment was allowed to continue until either a growth
or a washout rate could be calculated.
Parasitic activity of Bdellovibrio bacteriovorus against E. coli in
river water
Even though enteric bacteria are capable of growth in fresh •water
systems, their numbers certainly do not increase continually without
limit. In fact, their numbers decrease dramatically down stream from
sewage outfalls. Interest in the bacterial parasite Bdellovibrio
bacteriovorus initially began through reports of this organism attacking
Gram negative bacteria and primarily cells of E. coli (Stolp and Starr,
1965; Varon and Shilo, 1969; Gillis and Nakamura, 1970). It was con-
ceivable to us that such an organism could be involved in various
mechanisms of biological stream self-purification if the organism could
be maintained in the fresh water environment and a study was begun to
evaluate its response to E. coli in the chemostat.
28
-------
Organisms: The strain of _B. bacteriovorus used in this study was
recovered from bottom sediments at site 2, and the organism is an
obligate parasite for E. coli , ATCC 11775.
The technique for isolating the B. bacteriovorus strain was essentially
that of Stolp and Starr (1963) and consisted of suspending 200 g of
sediment (wet wt) in 200 ml of deionized water and shaking vigorously
for 1 hour. The suspension was centrifuged for 5 minutes at 200 rpm,
and then the supernatant (containing all types of microbes extracted
from the soil) was submitted to a series of differential filtrations.
The stepwise filtration through Millipore filters of different porosities
was started with filters of 3 p. average pore size diameter, and continued
with filters of 1. 2, 0. 8, 0. 65 and 0. 45 [j. pore size. Portions of the
last 2 or 3 fractions, which have a diminishing content or ordinary
bacteria, were then mixed with a suspension of the host strain. These
mixtures were then plated in the same way that is done in phage
isolation experiments by using the double-layer technique. If the
added fraction of the soil filtrate contains the parasitic organisms,
B. bacteriovorus lytic spots appear after 2-4 days which, in their
initial stages, are externally identical to phage plaques. The isolation
of Bdellovibrio from sewage or other material can be undertaken in a
fashion similar to that described here.
Culture media: The technique for culturing the parasite is very much
like that used to culture bacteriophages. The basic yeast peptone (YP)
medium consists of a solid base layer and a semi-solid top layer. The
media used are as follows:
1. Base layer medium: 1000 ml deionized water; 3 g Difco yeast
extract; 0. 6 g Difco peptone; 15 g agar; pH 7. 2.
2. Top layer medium: 1000 ml deionized water; 3 g Difco yeast
extract; 0. 6 g Difco peptone; 6 g agar; pH 7. 2.
3. YP Solution: 1000 ml deionized water; 3 g Difco yeast extract;
0. 6 g Difco Peptone; pH 7. 2.
After the bottom layer solidified a top layer was overlaid with 0. 5
ml portions of a washed E. coli cell suspension and of the filtrate which
is to be checked for Bdellovibrio mixed with about 4 ml of the molten
semi-solid agar YP medium. After overnight growth, the plates are
checked for phage plaques. Although the phage content of river water
or sewage samples is usually extremely low, phage plaques occur
occasionally and must be marked at this point in order to avoid con-
fusion with the parasite plaques which develop somewhat more slowly.
Bdellovibrio normally requires at least 2 days in order to develop
visible plaques in isolation experiments since the single parasitic cells
29
-------
embedded in the lawn of growing bacteria have to overcome their
own lag phase before they can start multiplying. Plaques in isolation
plates, which are suspected to be caused by Bdellovibrio, were cut
out from the top layer, suspended in YP solution, and plated in a
lawn of the host bacteria by using a dilution series to get single plaque
formation. If lytic spots developed after 2 days of incubation, some of
the plaques were checked microscopically for the presence of parasites.
Purification: Parasites from a single plaque were suspened in sterile
YP solution, which was then passed through a 0. 45 p. Millipore filter,
diluted, and plated for plaque formation with an excess of the homolo-
gous host. After 3 successive single plaque isolations, the strain was
regarded as pure in the sense of representing the descendants of a
single cell (i. e. , a clone). The organisms recovered were maintained
in a YP medium culture of JE. coli at 5 C until use.
B. bacteriovorus activity in river water: After preliminary experi-
ments to compare the growth of B. bacteriovorus in a nutritionally
rich YP medium with that achieved in river water on E. coli, the
parasite was introduced into chemostat containing an actively growing
culture of E. coli at 30 C. Counts of both organisms were determined
daily by techniques outlined above, and basal nutrient concentrations
were also monitored.
30
-------
SECTION V
RESULTS
The following results were obtained during the investigation of Enteric
Bacterial Degradation of Stream Detritus which was active from
August, 1968 through July, 1971. These results will be presented as
follows:
1. Water quality analysis of the North Oconee River at three selected
sites at Athens, Georgia.
2. Evaluation of the ability of enteric bacteria to use natural aquatic
substrates by oxyge.n uptake experiments.
3. Evaluation of the ability of enteric bacteria to use natural aquatic
substrates by continuous culture experiments.
Part 1
Water Quality Analysis of the North Oconee River at three selected
sites at Athens, Georgia
Routine analyses
Chemical and bacterial assays: Tables 1 through 18 contain data
obtained from chemical and bacteriological analyses of the North
Oconee River water at three study sites near Athens, Georgia. These
data are presented in the form of monthly means of at least two samples.
Concentrations of basal nutrients are expressed in terms of mg/1 while
bacteriological counts are shown as the count/ml of river water. In
each case, the means have been rounded off to the first experimental
significant digit. Table 19 contains results from the Carbon-Hydrogen-
Nitrogen analyses of the dried dissolved solids from each of the study
sites over the complete study period.
Procedure evaluations
Formic hydrogenlyase induction as a basis for the Eijkman fecal
coliform concept; A total of 629 isolates were recovered from the
North Oconee River (Site 3) over a 1-year period (1968-1969) and
were broadly categorized into either the "coliform" or "enteric"
groups on the basis of their cultural reactions on Endo medium.
These organisms were further differentiated into Escherichia, Entero-
bacter, and intermediate groups by the IMViC procedure and for gas
production in glucose at 44. 5 C (Table 20). Geldreich (1964) reported
that enteric bacterial stains producing gas at the elevated temperature
31
-------
Table 1. Mean analysis of nutritional factors in the North Oconee
River at Site 1 during 1968-1969
Test
Temp(°C)
pH
DO(%Sat. )
BOD
Nitrogen
Hexose
Phosphate
Protein
Solid sd
Month
1968
Aug
23. 5
7. 0
64. 0
2. la
c
2. 5
2. 0
10. 0
77. 0
Sept
19.5
6.9
56.5
2. 3
-
1. 8
2.0
11. 5
105. 0
Oct
17. 0
6. 9
65. 0
2. 2
-
1.4
0. 8
14. 0
60. 0
Nov
9.0
6. 8
93. 0
2. 2
2. 0
3. 0
1. 7
18. 0
126. 0
Dec
4. 5
6.9
87. 0
2. 2
0. 5
1.6
0.9
12. 5
56. 0
1969
Jan
8. 0
7. 0
59. 0
1. 0
3.9
0. 8
3.6
21. 7
56. 0
Feb
7. 0
6.9
48. 0
2. 0
0. 5
1. 8
0. 2
5. 0
447. 0
Mar
10. 5
7. 0
47. 0
2. 1
0. 5
1. 1
0.4
3. 0
127. 0
Apr
16. 5
7. 2
80. 0
1. 8
0. 5
1. 0
0.6
2.0
54. 0
May
17. 0
6. 1
63. 0
2. 1
0. 5
1. 0
1. 2
20. 0
591. 0
June
23. 5
7. 2
85. 0
2.4
4. 3
2. 1
0.6
6.0
205. 0
July
24. 5
7. 0
75, 5
3. 2
1.6
0.8
0. 2
1. 0
45. 0
Mean
68-69
15. 0
6.9
68. 5
2. 1
3. 2
1.6
1.2
10.4
158. 0
tv
Concentration expressed in Mg/liter.
Ammonia Nitrogen.
"Data not collected.
Dissolved solids.
-------
Table 2. Mean analysis of nutritional factors in the North Oconee
River at Site 2 during 1968-1969
Test
Temp(°C)
pH
DO(%Sat. )
BOD
Nitrogen
Hexose
Phosphate
Protein
Solidsd
Month
1968
Aug
24. 0
6.9
69. 0
2. la
c
2. 5
2. 8
10. 0
120. 0
Sept
20. 0
7. 0
59. 0
2. 3
2. 5
1. 2
15. 5
84. 0
Oct
17. 0
7. 0
46. 0
2. 2
3. 2
0.6
16. 2
62. 0
Nov
9. 5
6. 6
53. 0
2. 2
2. 5
3. 5
1. 9
17. 5
88. 0
Dec
4. 5
7. 0
57. 0
1.9
0. 5
2. 5
1.0
• 9. 7
51. 0
1969
Jan
7. 5
7. 0
35. 0
1. 5
2.9
1. 2
3.6
21. 7
69. 0
Feb
7. 0
6.9
46. 0
2. 3
0. 5
1. 8
4. 0
3. 0
423. 0
Mar
10. 0
7. 0
50. 0
1. 7
0. 5
1. 0
1. 8
7. 5
180. 0
Apr
17. 0
7. 4
52. 0
1.6
1. 2
1. 5
1.6
S. 0
72. 0
May
17. 0
7. 0
53. 0
1. 2
7. 5
5. 5
2. 1
25. 0
202. 0
June
24. 0
7. 0
98. 0
2.4
3. 1
2. 5
0. 7
7. 0
185. 0
July
24. 5
6.9
80. 0
2.4
2. 1
2. 0
0. 4
2. 2
94. 0
Mean
68-69
15. 2
7. 0
58. 2
2. 0
2. 3
2. 5
1. 8
11. 7
134. 0
J.
Concentration expressed in Mg/liter.
-»
D
Ammonia nitrogen.
Data not collected.
•d
Dissolved solids.
-------
Table 3. Mean analysis of nutritional factors in the North Oconee
River at Site 3 during 1968-1969
Test
Temp(°C)
PH
DO(%Sat. )
BOD
Nitrogen"
Hexose
Phosphate
Protein
Solidsd
Month
1968
Aug
24. 5
6.9
60. 0
1. 8a
c
3. 0
1. 9
10. 0
107. 0
Sept
20. 5
7. 0
48. 0
3. 0
1. 0
1.6
15. 0
144. 0
Oct
17. 5
7. 0
35. 0
3. 6
-
1. 8
2. 8
18. 5
91.5
Nov
9.0
6.4
54. 0
3. 5
7. 0
2. 2
2. 7
27. 0
126. 5
Dec
5. 0
6. 3
47. 0
3.9
3. 2
2. 1
3. 3
17. 0
74. 0
1969
Jan
8.0
7. 1
30. 0
3. 8
7.2
1. 3
9.4
22. 3
94. 0
Feb
8.0
6.9
33. 0
2. 8
1.9
1. 1
0. 8
5. 5
500. 0
Mar
11. 0
7. 3
36. 0
3. 1
2. 2
1. 0
3. 2
11.8
223. 0
Apr
17. 0
7. 0
39. 0
2. 5
4.4
2. 0
2. 0
7. 0
66.0
May
17. 0
7.4
43. 0
2. 9
6.4
3. 0
15. 2
25. 0
152. 0
June
23. 5
7. 2
74. 5
5. 7
5. 1
1. 0
0. 5
11. 0
83. 0
July
24. 5
7. 0
47. 5
3. 7
4. 8
0.6
0. 7
3. 5
107. 0
Mean
68-69
15. 5
7. 0
45. 6
3.4
4. 7
1. 7
3. 7
14. 5
147. 0
Concentration expressed in Mg/liter.
Ammonia Nitrogen.
"Data not collected.
Dissolved solids.
-------
Table 4. Mean bacteriological countsa from the North Oconee River
at Site 1 during 1968-1969
Heterotrophic Bacteria
30°C
Aug 68 10> 000
Sept 9, 700
Oct 1,800
Nov 2, 000
Dec 370
Jan 7,400
Feb 730
Mar 430
Apr .150
May 1,300
June 320
July 2, 500
Mean
68-69c 3,100
44. 5°C
_b
116
149
87
66
200
60
68
30
10
15
545
122
Enteric Bacteria
Total
30 °C
1, 300
290
1,700
3,200
19
5, 200
1
40
30
1,200
28
400
1, 200
Coliforms
30°C
200
100
315
400
0
2, 820
0
25
20
0
1
90
330
Total
44. 5°C
_
283
61
40
16
3
4
0
1
0
2
100
46
Coliforms
44. 5°C
_
126
34
15
6
3
3
0
1
0
2
0
17
Counts/ml of river water.
Data not collected.
'Means have been rounded off to the first significant digit.
35
-------
Table 5. Mean bacteriological counts3" from the North Oconee River
at Site 2 during 1968-1969
Heterotrophic Bacteria
3QOC
Aug 68 40, 000
Sept 3,400
Oct 2, 900
Nov 40, 000
Dec 1,800
Jan 69 5,400
Feb 140
Mar 300
Apr 430
May 1,200
June 380
July 2, 000
Mean
68-69c 8,200
44. 5°C
_b
590
730
1,200
447
197
33
14
32
100
52
120
320
Enteric Bacteria
Total
30°C
1,500
450
1,700
18, 000
625
1,900
40
60
350
1,400
19
750
2, 200
Coliform
30 °C
300
230
625
10, 000
470
800
10
40
100
0
3
80
1, 100
Total
44. 5°C
-
100
650
950
98
1
2
0
7
4
55
30
170
Coliform
44. 5°C
-
44
460
210
43
1
1
0
7
0
18
0
70
Count/ml river water.
Data not collected.
Means have been rounded off to the first significant digit.
36
-------
Table 6. Mean bacteriological countsa from the North Oconee River
at Site 3 during 1968-1969
Heterotrophic Bacteria
30 °C
Aug 68 200, 000
Sept 35, 000
Get 280,000
Nov 150,000
Dec 12,000
Jan 69 13, 000
Feb 3,400
Mar 5, 600
Apr 7, 800
May 150,000
June 30, 000
July 80, 000
Mean
68-69° 74,000
44. 5°C
_b
27, 000
2,000
5,400
590
360
610
170
300
350
390
1, 300
3, 500
Enteric Bacteria
Total
30 °C
10, 000
7, 800
3, 300
15, 000
12, 000
11, 000
2, 800
4,400
1,400
2, 800
2, 500
1, 800
6, 200
Coliform
30 °C
1, 000
1, 900
610
4,400
1, 200
1, 100
975
3, 800
1, 100
63
280
300
1,900
Total
44. 5°C
-
1, 500
1,400
630
227
50
331
1
80
10
38
1,200
450
Coliform
44. 5°C
-
290
800
321
92
36
260
0
72
0
31
430
210
Count/ml river water.
Data not collected.
'Means have been rounded off to the first significant digit.
37
-------
Table 7. Mean analysis of nutritional factors in the North Oconee
River at site 1 during 1969-1970
Test
Temp(°C)
PH
DO(%Sat. )
BOD
Nitrogen"
Hexose
Phosphate
Protein
Solids0
Month
1969
Aug
22. 5
7. 0
98. 0
6. 8a
0. 5
1. 0
0. 7
2. 0
69. o
Sept
22. 0
6.9
107. 0
3. 1
0. 5
1. 2
0.6
1. 5
60. 0
Oct
14. 0
6. 8
93. 0
3. 0
0. 1
0. 8
0. 4
1. 4
164. 0
Nov
9. 0
7. 0
95. 5
1. 5
0. 2
0. 5
0. 2
1. 2
183. 0
Dec
5. 0
6. 7
83. 0
1. 5
0. 5
0. 0
0. 2
1-9
354. 0
1970
Jan
0. 5
7. 1
92. 5
3. 4
0. 4
0. 5
0.4
1. 5
60. 0
Feb
6. 5
7. 0
102. 0
2. 7
0. 6
0. 8
0. 4
7. 1
95. 0
Mar
12. 5
6.9
90. 0
2.4
0. 6
0. 8
0. 4
2. 0
63. 0
Apr
17. 5
7. 2
78. 0
1. 5
0.6
0. 6
0.6
2. 8
75. 0
May
17. 0
7. 0
90. 0
2. 3
0. 8
0. 2
0. 5
1. 9
50. 0
June
23. 5
7. 3
75. 0
3. 5
0. 9
5. 5
0. 8
2. 2
38. 0
July
22. 5
6. 7
78. 5
0. 8
0.4
0. 1
0. 5
4. 9
91.5
Mean
69-70
14. 4
7. 0
90. 2
2. 7
0. 5
1. 0
0. 4
2. 5
108. 5
OO
00
Concentration expressed in Mg/liter.
Ammonia nitrogen.
'Dissolved solids,
-------
Table 8. Mean analysis of nutritional factors in the North Oconee
River at Site 2 during 1969-1970
Test
Temp(°C)
PH
DO(%Sat. )
BOD
Nitrogen"
Hexose
Phosphate
Protein
Solidsc
Month
1969
Aug
23. 0
6.7
67. 0
4. 5a
0. 8
1. 8
0. 8
4. 0
93. 0
Sept
22. 5
6. 8
92. 0
1. 7
0. 6
1. 7
0. 6
1. 5
84. 0
Oct
15. 0
6. 5
100. 0
2. 0
0. 2
0. 8
0. 7
1. 4
165. 0
Nov
10. 5
6.7
102. 0
7. 0
0. 3
1. 1
0. 1
1. 2
172. 0
Dec
6. 0
6.6
82. 5
4. 9
0.9
0. 7
0. 7
3. 3
364. 0
1970
Jan
1. 5
6.9
94. 0
3.6
0. 4
1. 0
0. 3
2. 2
61. 0
Feb
8. 0
7. 0
102. 5
2. 0
0. 7
0. 4
0. 4
6. 6
58. 0
Mar
12. 5
6. 8
88. 0
1. 8
0. 6
1. 0
0. 6
2. 8
69. 0
Apr
18. 0
7. 2
90. 5
2. 0
0.6
0. 7
0. 5
2. 7
78. 0
May
17. 0
7. 0
96. 0
1. 4
0. 8
0. 2
0. 6
3.4
60. 0
June
24. 0
7. 2
82. 0
1. 0
0. 8
0. 5
0. 6
1. 2
30. 0
July
23. 2
6. 8
80. 5
2.4
0. 8
0. 0
0. 5
1. 4
78. 0
Mean
69-70
15. 1
6. 8
89. 8
2. 8
0. 6
0. 8
0. 5
2. 6
109. 3
Concentration expressed in Mg/liter.
Ammonia nitrogen.
'Dissolved solids.
-------
Table 9. Mean analysis of nutritional factors in the North Oconee
River at Site 3 during 1969-1970
Test
Temp(OC)
PH
DO(%Sat. )
BOD
Nitrogen"
Hexose
Phosphate
Protein
Solidsc
Month
1969
Aug
23. 0
6. 8
81.5
6. 8a
1. 8
1. 7
1. 0
4. 0
82. 0
Sept
22. 5
6.9
67.5
4. 2
2. 0
2. 1
1.6
6. 5
91. 0
Oct
15. 5
606
87. 5
7.6
1. 1
1. 2
1. 2
3. 2
187. 0
Nov
10. 5
6. 7
92. 5
4. 5
1. 0
0.9
0. 4
2.4
160. 0
Dec
6. 0
6. 6
83. 0
3.4
1.9
0. 8
1.9
9-9
385. 0
1970
Jan
1. 5
6. 8
80. 5
5. 0
1. 2
0.8
0. 9
5. 1
95. 0
Feb
7. 5
6.8
90. 0
4.6
9.2
1. 4
1. 0
6.9
70. 0
Mar
13. 0
6. 8
90. 0
2.9
1. 1
0. 8
0. 8
3. 2
71. 0
Apr
18. 0
6.2
69. 0
1.9
1.6
1. 1
1. 1
4. 7
78. 0
May
17. 0
7. 0
86. 5
2.2
1. 2
1.7
0. 9
4. 3
63. 0
June
23. 5
7.0
57. 0
4. 7
2. 1
1. 1
1. 3
4. 5
31. 0
July
23.2
6. 8
50: 0
4. 1
2. 8
0.6
2. 0
5.8
86.5
Mean
69-70
15. 1
6.7
71.2
4. 3
2. 3
1. 1
1.2
5. 0
116.6
lConcentration expressed in Mg/liter.
Ammonia nitrogen.
'Dissolved solids.
-------
Table 10. Mean bacteriological counts3- from the North Oconee River
at Site 1 during 1969-1970
Heterotrophic Bacteria
30°C
Aug 69 3, 100
Sept 1,300
Oct 775
Nov 515
Dec. 1,100
Jan 70 2,800
Feb 2,000
Mar 575
Apr 375
May 880
June 1,200
July 10,000
Mean
69-70b 2,100
44. 5°C
100
125
190
80
160
10
780
110
10
240
260
3, 000
420
Enteric Bacteria
Total
30°C
350
700
265
130
154
330
115
200
210
650
200
8, 800
1,000
Coliform
3o°g
0
200
0
15
60
30
25
30
30
60
0
2, 800
270
Total
44. 5oc
35
205
55
5
0
5
0
0
0
100
40
.2, 000
200
Coliform
44. 5°C
0
55
14
5
0
5
0
0
0
20
10
200
25
Counts /ml river water.
Means have been rounded off to the first significant digit.
41
-------
Table 11. Mean Bacteriological counts a from the North Oconee River
at Site 2 during 1969-1970
Heterotrophic Bacteria
30°C
Aug 69 3,900
Sept 12, 000
Oct 2,700
Nov 11,000
Dec 5,000
Jan 70 2,400
Feb 1,800
Mar 995
Apr 535
May 2, 100
June 1,700
July 6,000
Mean
69-70b 4, 100
44. 5°C
250
160
280
1, 800
380
20
145
105
55
160
500
3, 600
620
Enteric Bacteria
Total
30 °C
1,700
1, 600
800
570
940
320
135
245
270
880
400
10, 000
1,500
Coliform
30°C
100
100
150
170
700
100
30
55
25
60
400
3, 300
430
Total
44. 5°C
375
325
310
750
70
10
0
0
0
150
40
3, 000
420
Coliform
44. 5°C
110
105
140
61
45
5
0
0
0
0
10
200
56
Count/ml river water.
Means have been rounded off to the first significant digit.
42
-------
Table 12. Mean bacteriological counts a from the North Oconee River
at Site 3 during 1969-1970
Heterotrophic Bacteria
30°C
Aug 69 10, 000
Sept 250,000
Oct 7,500
Nov 3,400
Dec 5,100
Jan 70 5, 100
Feb 10,000
Mar 2, 100
Apr 91,000
May 3,000
June 4, 700
July 800, 000
Mean
69-70b 100,000
44. 5°C
300
18, 000
4, 500
4, 000
3,400
200
725
330
245
200
1, 900
34, 000
5, 700
Enteric Bacteria
Total
30 °C
8, 000
30,000
955
1, 500
5, 600
3, 500
5,700
780
4, 900
980
4, 900
240, 000
26, 000
Coliform
30°C
100
4,600
500
700
2, 000
1, 800
2,200
370
3,200
190
1, 100
37, 000
4, 500
Total
44. 5°C
430
1,600
900
1,200
700
105
80
25
50
900
5, 000
13,000
2, 000
Coliform
44. 5°C
170
1, 100
500
730
400
100
60
15
30
300
1, 100
7,600
1, 000
Count/ml of river water.
Means have been rounded off to the first significant digit.
-------
Table 13. Mean analysis of nutritional factors in the North Oconee
River at Site 1 during 1970-1971
Test
Temp(°C)
pH
DO(%Sat. )
BOD
Nitrogen"
Hexose
Phosphate
Protein
Solidsd
Month
1970
Aug
24. 5
6. 8
92. 0
i.oa
0. 6
1. 0
0. 7
1. 5
73. 5
Sept
24. 0
6.7
83. 0
1. 5
0. 9
1. 0
0. 3
2.9
112. 5
Oct
15. 0
6. 8
119. 0
2. 0
0. 4
0. 3
1.2
1. 1
83. 0
Nov
9- 5
6.9
100. 0
3. 8
0. 1
2. 7
0. 5
1. 2
58. 0
Dec
7. 0
7. 0
81. 5
1. 2
0. 7
_ c
0.4
2. 3
95. 0
1971
Jan
2. 5
6. 8
95. 0
2. 2
0. 3
0. 5
0. 2
2. 0
37. 5
Feb
6.0
6. 7
88. 0
1. 1
0. 3
0. 4
0. 3
1. 2
78. 0
Mar
5. 0
7. 8
93. 0
1. 5
0.6
2. 4
0.6
3. 2
104. 0
Apr
15. 0
7. 3
79. 0
1.0
1. 1
7. 0
1. 2
4.9
35. 0
May
17. 5
7. 6
79. 5
0. 8
1. 1
4. 3
2. 0
4. 5
94. 5
June
23. 0
7. 2
82. 5
0. 7
0. 8
3.4
2. 2
3. 8
90. 0
July
22. 5
7.4
74. 5
0.9
0. 9
4. 7
2. 8
6. 2
143. 5
Mean
70-71
14. 5
7. 1
88. 9
1. 5
0. 6
2. 5
1. 0
2.9
83. 7
b
Concentration expressed in Mg/liter.
Ammonia Nitrogen.
'Data not collected.
Dissolved solids.
-------
Table 14. Mean analysis of nutritional factors in the North Oconee
River at Site 2 during 1970-1971
Test
Temp(OC)
pH
DO(%Sat. )
BOD
Nitrogen"
Hexose
Phosphate
Protein
Solidsd
Month
1970
Aug
25. 0
6. 6
83. 0
0. 6a
0. 8
1.4
0. 9
1. 5
73. 5
Sept
24. 0
6. 7
76. 0
1. 2
1. 0
1.4
0. 5
3. 2
125. 5
Oct
15. 5
6. 8
113. 5
2. 0
0.6
0. 4
1.9
1. 6
88. 0
Nov
9.5
6. 8
101. 5
3. 3
0.6
2. 2
2. 8
1. 9
74. 0
Dec
7. 0
7. 0
87. 5
1. 9
0. 9
c
0. 4
4. 2
92. 0 :
1971
Jan
3. 0
6.9
96. 5
1. 9
0. 4
0. 2
0. 2
1. 8
49. 0
Feb
6. 5
6.4
90. 0
2.6
0.4
1. 4
0. 3
1. 3
53. 5
Mar
6. 0
7. 3
97. 0
2. 8
0. 7
1. 9
0. 8
3. 7
105. 0
Apr
15. 5
6.9
86. 5
0. 8
1. 1
6. 0
1. 3
5. 2
39. 0
May
18. 5
7. 1
85. 5
1. 4
1. 0
5. 0
1. 6
3. 5
79. 5
June
24. 0
7. 4
86. 0
0.6
0. 7
2.4
1. 8
3. 0
91. 0
July
23. 5
6.9
82. 0
2.4
1. 0
4.2
4. 4
9.8
179. 5
Mean
70-71
15. 0
6.9
90. 4
1. 8
0. 8
2.4
1.4
3. 4
84. 2
Concentration expressed in Mg/liter.
D
Ammonia Nitrogen.
c
Data not collected.
d
Dissolved solids.
-------
Table 15. Mean analysis of nutritional factors in the North Oconee
River at Site 3 during 1970-1971
Test
Temp(OG)
PH
DO(%Sat. )
BOD
Nitrogen
Hexose
Phosphate
Protein
Solids0
Month
1970
Aug
25. 0
6.9
66.0
4. 0*
2. 2
1. 5
1. 9
4.6
74. 0
Sept
25. 0
6. 8
75. 0
3. 0
1. 4
1. 7
1. 0
5. 5
129. 5
Oct
16. 0
7. 0
1E7. 0
3. 6
1.2
1. 2
4. 5
1.4
121.0
Nov
9.5
7. 1
96. 0
4. 9
1. 0
2. 0
2. 1
1. 9
77. 0
Dec
7. 0
6.9
83. 0
3.6
1. 0
2. 0
0. 8
2.9
90. 0
1971
Jan
3. 0
6.9
90. 0
1. 3
0. 9
1. 3
0. 4
3. 8
51. 0
Feb
6. 5
6.4
88. 5
2. 8
0. 8
0. 7
0. 5
4. 8
70. 0
Mar
6.0
7. 0
90. 5
2.9
1. 1
1. 7
1. 0
5. 1
115. 0
Apr
15. 0
7. 2
83. 5
2. 3
1.4
8. 2
1. 3
6.5
54. 5
May
18. 5
7. 2
77. 5
3. 5
1. 4
4. 3
2. 0
5. 6
89. 5
June
24. 0
7. 0
71. 5
2. 7
1.4
3. 0
3. 4
6. 3
92. o
July
23. 5
6.7
74. 0
3. 1
1. 4
5. 7
5.6
11. 3
212. 0
Mean
70-71
15. 0
6.9
85. 2
3. 1
1. 3
2. 8
2. 0
5. 0
98. 0
Concentration expressed in Mg/liter.
Ammonium nitrogen.
'Dissolved solids.
-------
Table 16. Mean bacteriological counts3- from the North Oconee River
at Site 1 during 1970-1971
Heterotrophic Bacteria
30 °C
Aug 70 2, 600
Sept 14,000
Oct 9, 000
Nov 18,000
Dec 5,000
Jan 71 572
Feb 1,200
Mar 2,200
Apr 1,500
May 30, 000
June 1 , 400
July 15,000
Mean
70-71 8,400
44. 5°C
95
55 700
500
30
100
144
35
152
525
170
75
1, 600
757
Enteric Bacteria
Total
30 °C
470
3, 000
1, 000
15, 000
2, 800
288
112
279
966
1, 100
595
5, 500
2, 600
Coliform
30 °C
220
1, 100
200
20
320
29
12
74
32
85
40
930,
255
Total
44. 5°C
10
550
10
2
20
11
2
7
10
13
7
470
102
Coliform
44. 5°C
5
530
8
0
0
1
1
4
1
3
1
330
73
aCount/ml river water.
^Means have been rounded off to the first significant digit.
47
-------
Table 17. Mean bacteriological countsa from the North Oconee River
at Site 2 during 1970-1971
Heterotrophic Bacteria
30 °C
Aug 70 2, 800
Sept 55,000
Oct 24, 000
Nov 15,000
Dec 16,000
Jan 71 6, 600
Feb 1,800
Mar 2,400
Apr 6, 600
May 2, 100
June 3, 000
July 18, 000
Mean
70-71b 13,000
44. 5°C
695
2, 100
1,000
1,400
410
971
86
63
150
148
234
930
682
Enteric Bacteria
Total
30 °C
1,300
18,000
3, 500
2, 100
5,900
292
209
480
2,100
1,300
525
6,200
3, 500
Coliform
30 °C
200
5,000
900
300
700
51
44
107
270
85
55
1,200
746
Total
44. 5°C
155
750
80
70
600
22
5
8
24
16
22
225
164
Coliform
44. 5°C
100
250
10
10
100
4
3
5
8
3
3
131
52
aCount/ml river water.
"Means have been rounded off to the first significant digit-
48
-------
Table 18. Mean bacteriological counts3- from the North Oconee River
at Site 3 during 1970-1971
Heterotrophic Bacteria
30 °C
Aug 70 450, 000
Sept 100,000
Oct 120,000
Nov 85,000
Dec 26,000
Jan 71 11, 000
Feb 21,000
Mar 14,000
Apr 31,000
May 43,000
June 4, 500
July 9, 300
Mean
70-71b 76,000
44. 5 °C
26,000
9, 500
60,000
3,700
1, 100
686
495
415
565
960
306
500
8, 700
Enteric Bacteria
Total
30 °C
190,000
76,000
24,000
20, 000
9, 500
1,900
4,400
2,300
5,700
8, 500
63
2, 700
29, 000
Coliform
30 °C
60, 000
3, 000
13, 000
6,400
3,800
104
1, 700
1,400
1,400
1, 500
16
375
7, 700
Total
44. 5°C
7, 700
5, 000
1, 500
1, 900
400
203
178
178
252
47
2
82
1, 500
Coliform
44. 5°C
3,600
1, 500
300
900
200
125
70
99
49
14
1
13
558
aCount/ml river water.
Means have been rounded off to the first significant digit.
49
-------
Table 19. Mean CHN analysis of suspended solids present in river water
from sites 1, 2 and 3
Date
1968-69
1969-70
1970-71
Site
1
2
3
1
2
3
1
2
3
Carbon
%
24. 15
23. 22
32. 14
23. 33
24. 77
33. 38
33. 23
33.93
34.69
mg/mg
solid
0. 1571
0. 1584
0. 2178
0. 1566
0. 1831
0.2338
0. 2000
0. 2036
0. 2219
Hydrogen
%
2.35
2.48
2.57
2.93
2, 85
2. 93
2.69
2.70
2.67
mg/mg
solid
0. 0157
0. 0162
0. 0174
0. 0209
0. 0196
0. 0181
0. 0176
0. 0187
0. 0169
Nitrogen
%
0.58
0.51
0. 58
0.58
0.58
0.74
0. 74
0.79
0. 83
mg/mg
solid
0. 0035
0. 0031
0. 0039
0. 0039
0. 0041
0. 0057
0. 0048
0. 0053
0. 0070
Inert
%
72. 92
73.79
64. 71
73. 24
71. 88
62.95
63. 34
62.58
61. 81
mg/mg
solid
0.4726
. 0.4539
0.4175
0.4824
0. 4833
0. 4205
0.4526
0.4680
0.4315
-------
Table 20. Gas production by enteric bacteria recovered from river water on M-Endo MF medium at 44. 5 C
Parr's IMViC*
group
Escherichia group
(+ + --. + ,
Enterobacter group
Intermediate group
Coliform isolates
Organisms
recovered
226
130
86
Gasd
221
23
44
Per cent
(group total)
97. 8
17. 7
51. 2
Enteric isolates0
Organisms
recovered
86
28
73
Gas
77
15
66
Per cent
(group total)
89.5
53.6
90.4
Total isolates
Organisms
recovered
312
158
159
Gas
298
38
110
Per cent
(group total)
95. 5
24. 0
69- 2
a. Indole, methyl red, Voges-Proskauer, citrate reactions observed.
b. Organisms exhibiting lactase utilization and a metallic, green sheen.
c. Organisms exhibiting lactose utilization.
d. Production of gas in MR-VP medium at 44. 5 C.
-------
and having + + --,_+--, or + - - - IMViC reactions may be
considered to be Escherichia species and of fecal origin, whereas
--++, -_+-, or - - - + strains, which lack the ability to produce
gas at 44. 5 C, are Enterobacter and are probably of soil or vegetative
origin.
The results of these experiments (Table 20) show that 70. 9% of all
organisms recovered produce gas, which proved to be a mixture of
Hs and CO2, but less than 50% of the isolates were Escherichia
species. Of all Enterobacter, 24% also had the capacity for gas
production. Much the same correlation pattern of gas production at
44. 5 C with IMViC types was observed when only the "coliform11
isolates were considered. Of significant importance is the observation
that 17. 7% of the Enterobacter species in the coliform group produced
gas from glucose.
Formate and lactose were observed to serve satisfactorily as sub-
strates for gas production by those aquatic Enterobacter species which
could produce gas at 44. 5 C from glucose. The aquatic strains and
E. coli could produce gas from the three substrates at both 30. 0 and
44. 5 C, whereas the ATCC E. aerogenes culture could not at the
higher temperature. These data indicate that the evolved CO3 and H2
result from an active formic hydrogenlyase and that this particular
enzyme system can be functional in Enterobacter at the temperature
specified for the Eijkman procedure (Table 21).
Data from the Warburg experiments (Fig. 11) suggest that formic
hydrogenlyase is synthesized by both the _E_. coli and_E_. aerogenes
stock cultures at 30. 0 C and by_E. coli at 44. 5 C. The induction time
for formic hydrogenlyase synthesis by the stock cultures was about
30 min (Fig. 11) in each case, but when coliform organisms recovered
from the aquatic environment were used in .'Similar experiments, times
of induction were variable and ranged from 30 min to approximately
2 hr. (Fig. 12). None of the aquatic organisms used in these experi-
ments could be biochemically grouped with the Escherichia species,
but all organisms used, with the exception of one (+-++), synthesized
formic hydrogenlyase. These included an organism selected from the
23 gas-producing _E. aerogenes strains. Evidence for formic hydro-
genlyase induction was further demonstrated by the lack of enzymatic
activity in control experiments, in which chloramphenicol was added
to the substrates for induction. These data indicate that the formic
hydrogenlyase system was induced and not simply an activation of the
system which might have been present.
Table 22 shows specific activity of the synthesis of formic hydrogenlyase
52
-------
Table 21. Gas production from glucose, lactose, and formate at 30. 0 C and 44. 5 C by aquatic
forms of Enterobacter aerogenes conforming to the "coliform" designation3-
Organism
Enterobacter aerogenes
- -++
-- + +
-- + -
+
Escherichia coli
No. of
Strains
1
12
3
8
1
Source
ATCC 12658
Oconee R.
Oconee R.
Oconee R.
ATCC11775
Gas production from
Glucose
30. 0 C
+
+
+
+
+
44. 5 C
+
+
+
+
Lactose
30. 0 C
+
+
+
+
+
44. 5 C
+
+
+
+
Formate
30. 0 C
+
+
+
+
+
44. 5 C
+
+
+
+
LThese organisms produced gas in MR-VP medium at 44. 5 C.
-------
30CH
Q
LU
IE
or
o
U-
M
200-
100-
HOURS
Fig. 11. Formic hydrogenlyase synthesis by Escherichia.
coli ATCC 11775 at 30. 0 C (A), 44. 5 C (A), and
Enterobacter aerogenes ATCC 12658 at 30 C (O),
44.5 C (•).
54
-------
300-
Q
UJ
^
-------
Table 22. Formic hydrogenlyase activity of selected coliform
bacteria
Organism
Escherichia coli
++--b
Enter obacter
aerogenes
--++
--++
-- + -
Intermediate coliform
group
+++-
-++-
- + - +
+ -++
Source
ATCC 11775
ATCC 12658
Oconee R.
Oconee R.
Oconee R.
Oconee R.
Oconee R.
Oconee R.
Assay
Temp C
30. 0
44. 5
30. 0
44. 5
44. 5
44. 5
44. 5
44. 5
44. 5
44. 5
Activity3-
2, 360
360
2, 680
0
2, 860
2, 500
3, 300
2, 250
2, 260
0
"Expressed in microliters of H2 per hour per "milligram
(dry weight) of cells.
Parr's IMViC grouping.
56
-------
by using data frorn the Warburg experiments. Known stock cultures
of E_. coli produced approximately 10% of the activity at the elevated
temperature, whereas E. aerogenes failed to synthesize the enzyme.
However, at 44. 5 C, the aquatic strains of E. aerogenes were capable
of synthesizing formic hydrogenlyase at a rate comparable to the
stock culture at the lower temperature. In general, the intermediate
forms were quite active at 44. 5 C and formed formic hydrogenlyase
at a rate equivalent to that of the stock cultures at 30. 0 C.
Increased recovery of salmonellae from stream bottom sediments vs.
surface waters: The data presented in Table 23 demonstrate that
higher recovery yields of Salmonella can be achieved from bottom
sediments than from surface waters at site C (see Fig. 5). Of the
195 samples of sediments and river water taken over a 1-year period
(1968-1969), approximately 90% of the Salmonella species recovered
were found in,the bottom sediments. Explanations for this observation
are difficult since a variety of both physical and biological phenomenon
could be responsible for the recovery. It is entirely possible that
sedimentation and sorption of the organisms to the sand and clays could
concentrate bacteria on the stream bottom. This phenomenon could
in itself increase the recovery yields of any desired bacterial species
and, if the organism could find suitable nutrients present, growth might
occur to further increase recovery yields. It is interesting to note that
8 of the 10 Salmonella were recovered when mid-day water temperatures
were above 24. 0 C, since we have demonstrated in the laboratory that
prototrophic strains of Salmonella senftenberg and Shigella flexneri can
metabolize substrates present in aqueous extracts of bottom sediments
at this temperature and below ( see parts 2 and 3 of this report).
Although procedures used in this study were adequate for the recovery
of ahigellae, none could be detected on the primary isolation media from
any of the study sites.
Part 2
Evaluation of the Ability of Enteric Bacteria to Use Natural Aquatic
Substrates by Oxygen Uptake Experiments
Enteric bacterial metabolism of substrates in river water and in stream
sediment eluates; Preliminary experiments indicated that 0. 3 M phos-
phate buffer (pH 7. 0) eluted the basal nutrients from the stream sediments
in maximum concentration (Fig. 13). Table 24 presents data from experi-
ments to determine the basic nutrient concentration of both the river
water and extracts of sediments from each study site. Attempts to elute
57
-------
Table 23. Salmonella recovered from water and bottom sediments of the North Oconee
Rivera
Source
River water
Site C
Bottom sediments
Site C
Site E
Samples
taken
195
39
195
39
39
Salmonella recovered
No.
1
1
9
8
1
Per cent
0. 6
2. 6
4. 6
20. 1
2. 6
Salmonella
1 S. enttsritidis
3 S. enteritidis
4 S. enteritidis
1 S. enteritidis
1 S- enteritidis
sp. isolated
ser. anatum
ser. anatum
ser. indiana
ser. meleagridis
ser. indiana
t_n
00
Thirty-nine samples of water and of sediments were recovered from each of
the five sites (195 samples each total).
-------
tiro-
's.
0»
E 15
UJ
(O
O
X 5-
UJ n
l
5
6
I
7
I
8
-80 CT ~ 40-
\ V
o» c
eo E £
UJ
K
-20 O
(T.
OL
9
PH
UJ
o
X
UJ
20-
*£5
- too ^
o>
k 75 P
- 50
UJ
ac
o.
0.0 1.0 2.0 3.O 4JO 5.O
BUFFER CONCENTRATION
(-log molar cone.)
Fig. 13. Effect of pH and buffer concentration for elution of hexose (O)
and protein (•) from river sediment. The pH was standardized
to 7. 0 in studies to determine optimal buffer concentration for
maximal elution.
-------
Table 24. Basic nutrient analysis of Oconee River water and
extracts of river bottom sediment from the three study sites
Nutrient3
assayed
Ammonium nitrogen
Folin protein
Hexose
Orthophosphate
PH
Site
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
River
•water
1.6
2. 3
4. 7
13.6
13. 8
1.8. 3
1.6
2. 8
1. 0
1. 5
2.0
4. 1
6.9
7. 0
7. 0
Bottom sediment
Washed
0. 6
0. 2
1. 0
1. 5
0. 2
0. 2
1. 0
0. 6
2. 4
0. 0
0. 0
0. 4
7. 0
7. 0
7. 0
Buffer
elutedc
9. 0
8. 5
25. 0
45. 0
63. 0
120. 0
12. 0
22. 5
36. 0
_d
-
-
7. 0
7. 0
7. 0
Concentrations expressed in mg/1 of sample.
Concentration of nutrients present in the 3rd successive washing
of deionized water-
c Sediment eluted with 0. 3 M phosphate buffer (pH 7. 0).
"Phosphate concentrations were 0. 3 M.
60
-------
measurable quantities of the basal nutrients from the sediments with
river water above those already present in the river water were with-
out success.
Control experiments demonstrating basal respiration levels by the
nonpathogenic enteric bacteria (E. coli, E. aerogenes, and P. rettgeri)
with diluted minimal inorganic salts - glucose media are presented in
Fig. 14. No uptake of oxygen was observed when the test organisms
were run in deionized water before each respiration rate determination,
and no apparent phosphate effect upon respiration •was observed at the
final experimental concentration level of 0. 2 M.
Use of the substrates present in the river water and in washed and
buffer eluted bottom sediments is shown in Tables 25 through 27 and
in Fig, 15. Tables 25, 26 and 27 express the respiration rates of
the nonpathogenic enterics at 30, 20 and 5 C with the test substrates
from sites 1, 2 and 3 respectively. In Fig. 15 are compared the
respiration rates of the nonpathogenic enterics with pathogenic species
of S. flexneri, S. senftenberg, and_A_. arizonae in buffer eluted sedi-
ments and river water from below the sewage plant (site 3).
Utilization of substrates in detritus samples; Table 28 demonstrates
the results obtained when suspended and concentrated stream detritus
samples were used as substrates for metabolism by coliform bacteria.
Slime (polysaccharide capsular material) produced by E. aerogenes
was used as a control substrate.
The device of Pomeroy and Johannas (1968) was an effective means
for concentrating the detritus used in this portion of the study, A 5. 7
fold increase in concentration over the suspended (lyophilized) detritus
being observed, while the overall concentration factor for hexose in the
concentration detritus as compared to river water was 61 fold.
Part 3
Evaluation of the Ability of Enteric Bacteria to Use Natural Aquatic
Substrates by Continuous Culture Experiments
Preliminary experiments: The basal nutrient concentration of the
river water from site 3 and that of the control substrate dilute minimal
medium is presented in Table 29. Sufficient river water was collected
and processed before the studies were initiated so that the basal nutrient
concentration for each experiment was identical. Figures 16 and 17
show results of short term continuous culture experiments (24 hrs. )
61
-------
Enterobocter oerogentt
O.I 0.01 0.001 0.0001
GLUCOSE
(O/L)
Fig. 14. Respiration of selected enteric bacteria in minimal
inorganic salts medium containing various concentra-
tions of glucose at 30 C, (O); 20 C, (+); and 5 C, (•).
Respiration levels have been corrected for endogenous
activity.
62
-------
Table 25. Respiration of various enteric bacteria in Oconee River
water and in extracts of river bottom sediments from site 1
Organism
Escherichia coli
Enterobacter
aerogenes
Proteus rettgeri
Temp.
C
30
20
5
30
20
5
30
20
5
River
watera
0. 19d
0. 00
0. 00
0. 22
0. 00.
0. 00
0. 26
0. 00
0. 00
Bottom sediment
Washedb
0. 34
0. 00
0. 00
0. 46
0. 09
0. 16
0. 12
0. 00
0. 00
Buffer
eluted
1. 98
0. 44
0. 44
1. 60
1. 13
0. 00
1. 64
1. 38
0. 00
aRespiration rates expressed as mg atoms oxygen (O)/h mg
dry cell weight.
After third successive wash.
C0. 3 M phosphate buffer (pH 7. 0).
All respiration rates have been corrected for endogenous
activity.
63
-------
Table 26. Respiration of various enteric bacteria in Oconee River
•water and in extracts of river bottom sediments from site 2
Organism
Escherichia coli
Enterobacter
aerogenes
Proteus rettgeri
Temp.
C
30
20
5
30
20
5
30
20
5
River
water a
0. 43d
0. 21
0. 00
0. 53
0. 00
0. 00
0. 25
0. 00
0. 00
Bottom sediment
Washedb
0. 23
0. 52
0. 00
0. 37
0. 00
0. 24
0. 00
0. 21
0. 00
Buffer
elutedc
1. 81
1. 68
0. 18
1. 26
1. 27
0. 10
1. 68
0. 80
0. 00
aRespiration rates expressed as mg atoms oxygen (O)/h mg'
dry cell weight.
"After third successive wash.
C0. 3 M phosphate buffer (pH 7. 0).
'-'All respiration rates have been corrected for endogenous activity.
64
-------
Table 27. Respiration of various enteric bacteria in Oconee River
water and in extracts of river bottom sediments from site 3
Organism
Escherichia coli
Enterobacter
aerogenes
Proteus rettgeri
Temp.
C
30
20
5
30
20
5
30
20
5
River
water3"
0. 58d
0. 33
0. 00
0..45
0. 00
0. 00
0. 36
0. 17
0. 00
Bottom sediment
Washed b
0. 22
0. 10
0. 00
1. 34
0. 90
0. 00
0. 36
0. 12
0. 00
Buffer
elutedc
2. 58
1. 73
0. 34
4. 25
1. 13
0. 10
3. 23
1. 07
0. 00
aRespiration rates expressed as mg atoms oxygen (O)/h dry
cell weight.
After third successive wash
C0. 3 M_ phosphate buffer (pH 7. 0).
All respiration rates have been corrected for endogenous
activity.
65
-------
Fig. 15. Respiration rates of selected pathogenic and nonpathogenic
enteric bacteria in 0.3 M_ phosphate buffer-extracted sediments
(S) and river water (W).
66
-------
Table 28. Respiration rates of Enterobacter aerogenes (ATCC 12658)
and Escherichia coli (ATCC 11775) using slime and stream
detritus as substrates
Substrate
Slimeb
Concentrated
detritus
Suspended
detritus
(lyophilized)
Suspended
detritus
(oven driedc)
Hexose
concentration
(Hg/ml)
31. 8
116. 0
28. 4
38. 2
Respiration
Rate
(mgO/hr. /mgdcwa)
E. aerogenes
0. 63
3. 77
0. 95
1. 27
E. coli
0. 10
2. 80
0. 10
0.40
Respiration
Rate/(j.g hexose
E. aerogenes
0. 020
0. 033
0. 033
0. 033
E. coli
0. 003
0. 024
0. 006
0. 010
amgO/hr. /mgdcw = milligrams molecular oxygen per hour per milligrams of dried
(105 C for 24 hrs) cell ^weight.
" Polysaccharide capsular material of E. aerogenes.
c Dried at 105 C for 24 hours.
-------
TABLE 29. Basal nutrient concentration of autoclaved site 3
river water and dilute minimal salts-glucose mediuma
Concentration
Assay
Ammonia nitrogen
Protein
Hexose
Phosphate
PH
Source
Site 1
Site 2
Site 3
Minimal Medium
Site 1
Site 2
Site 3
Minimal Medium
Site 1
Site 2
Site 3
Minimal Medium
Site 1
Site 2
Site 3
Minimal Medium
Site 1
Site 2
Site 3
Minimal Medium
Mg/1
1. 1
1. 5
3. 0
0. 5
2. 9
3.2
5. 1
0. 0
2. 1
2.4
2.8
10. 0
1. 3
1.6
3. 2
5. 0
7. 0
6.9
6.9
7. 0
aMinimal medium diluted 1:1, 000.
68
-------
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7.5 -
ui
2* 6.5
2 E
o
v
9
4.5-
3.5
6.5
CC
U
t-
O
ffl £
V
O 9
Ul O
O -I
5.5-
(0
3.5-
2.5
Total heterotrophic bacteria
Enteric bacteria
Coliform bacteria
12 16
HOURS
20
24
Fig. 16. Continuous culture of a natural heterotrophic
bacterial population in Oconee River water from
site 3 at 14 C. Dilution rate = 0. 058 hr. "\
69
-------
<
cc
9.5^
8.5-
2 "
< E
CD o
\
o o 7.5
ui -J
x
o
6.5-
5.5
cc
UI
t-
o
00 •=
Q
UI
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z
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a_
co
13
E
v.
o>
o
6.5-
5.5-
4.5
3.5
Total heterotrophic bacteria
Enteric bacteria
Coliform bacteria
I
4
I
8
I
12
HOURS
16
I
20
24
Fig. 17. 'Continuous culture of a natural heterotrophic bacterial
population in Oconee River water from site 3 at 26 C.
Dilution rate = 0. 058 hr. "1.
70
-------
conducted at 14 and 26 C respectively in site 3 river water and at
dilution rate of 0. 58 hr. " . Over the 24 hour period, oxygen con-
centration was reduced 12% in the 14 C experiment and 50% in the
device run at 26 C. Basal nutrient concentrations in both series of
experiments did not appear to fluctuate greatly and showed only a
slight decrease in hexose and ammonia nitrogen content, no change
in phosphate and an elevated protein concentration as compared to the
nutrient concentration in the resevoir. Figure 19 contains results from
420 hr. continuous culture experiment using dilute minimal salts and
glucose as nutrient source, while Fig. 1$ shows the results of a
similar experiment -with river water as a nutrient source. Both of
these experiments •were run at dilution rates of 0. 012 hr. ~1 and
incubation temperatures of 30 C. Table 30 shows the growth rates
for these experiments.
Prior to the initiation of the continuous culture experiments using the
six enteric test, strains, a standard dilution rate of 0. 012 hr. -1 was
found to yield an.adequate growth rate for_E_. coli in site 3 river water
culture experiments at 30 C (Fig. 20). This flow rate was used in all
subsequent continuous culture experiments at each of the three standard
incubation temperatures (30, 20 and 5 C).
Continuous culture experiments using river water and the collected
detritus from the three sites as substrates: River water from sites
1 and 2, in each case, failed to provide any of the six test organisms
with sufficient nutrients to allow for growth at the three selected
temperatures. Table 31, however, contains data on growth rates of
the six bacterial test strains in water from site 3.
In the continuous culture experiments using both concentrated and
suspended detritus, no significant growth for E. coli was observed
at 20 or at 5 C, but low growth rates were observed at 30 C for this
organism (Table 32). Attempts to determine why higher growth rates
were not observed in these experiments demonstrated that basal
nutrients had adsorbed to the inorganic particulate material which has
formed a part of the concentrated detritus sample (Table 33).
Bdellovibrio bacteriovorus - Escherichia coli interactipns
Activity in river water: A typical growth curve at 30 C for B.
bacteriovorus in NB Medium is presented in Fig. 21, while Fig, 22
shows the same experiment using river water from s'ite 3 as a nutrient
source. Figure 23 is the result of an experiment designed to demon-
strate that if additional nutrients are added to E. cpli-B. bacteriovorus
system, numbers of both organisms would be stimulated. Results of
71
-------
6.0 -
O 5.0
o
U)
_l
03
< 4.0.
3.0 -
2.0
30.0 C
IOO 200 300
HOURS
4OO
Total heterotrophic bacteria
Enteric bacteria
ColUorm bacteria
44.
0.0
Fig. 18.
100 200 300
HOURS
400
Continuous culture of a natural hetero-
trophic bacterial population in Oconee
River water from site 3 at 30 C. Dilution
rate = 0. 012 hr. -1.
72
-------
o
o
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CD
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H-
o
6.0 -
3.0
5.0 -
4.0 -
3.0 -
2.0
30.0 C
200 300
HOURS
400
Total heterotrophic bacteria
Enteric bacteria
Coliform bacteria
44. 5 C
I
100
200 300
HOURS
4OO
Fig. 19. Continuous culture of a natural heterotrophic bacterial
population at 30 C in minimal salts medium diluted
1:1000. Dilution rate = 0. 012 hr. ~1.
73
-------
Table 30. Average growth rates of native bacterial populations in site 3
Oconee River water and dilute minimal medium
Nutrient
Source
Minimal Medium
1:1, 000 dilution
(Davis, 1950)
River Water
(Site 3)
Bacterial
Group
Heterotrophs
Enterics
Coliforms
Heterotrophs
Enterics
Coliforms
Growth Rate
hr.-1
30 C Population
0. 017
0. 018
0. 019
0. 007
0. 007
0. 006
44. 5 C Population
0. 019
0. 015
0. 012
0. 013
da
d
"Death of the culture
-------
0.10 -j
0.08 H
J— 0.06 H
LU
J—
<
cc
o
f-
13
_J
0
0.04 H
0.02
0.00
+ Site I
o Site 2
• Site 3
0.02 ' 0.0 0.02 0.04 0.06 0.08 0.10
WASHOUT RATE (hr"1)
Fig. 20. Washout rate vs. dilution rate plot for Escherichia
coli in Oconee River water from sites 1, 2, and 3
at 30 C.
75
-------
Table 31. Growth rates of selected enteric bacteria in Oconee River
water from site 3
Organism
Escherichia coli
Enterobacter aerogenes
Proteus rettgeri
Arizonae arizona
Salmonella senftenberg
Shigella flexneri
Wash out
Temperature C Rate
(hr.-1)
30
20
5
30
20
5'
30
20
5
30
20
5
30
20
5
30
20
5
0. 017
-0. 009
-0. Oil
0. 018
-0. 006
-0. 018
-0. 002
-0. 001
-0. 017
-0. 001
-0. Oil
-0. Oil
0. 001
-0. 020
-0. 032
0. 001
-0. 007
-0. 010
Growth
Rate
(hr.-1)
0. 029
0. 003
0. 001
0. 030
0. 006
-
0. 012
0. 010
-
0. Oil
0. 001
0. 001
0. 013
-
-
0. 013
0. 005
0. 002
Generation
Time (hrs. )
34. 5
333. 3
1, 000. 0
33. 3
166.6
-
83. 3
100. 0
-
90. 0
1, 000. 0
1, 000. 0
76. 9
_
-
76. 9
200. 0
500. 0
Dilution rate was 0. 012 hr. in each case.
-------
Table 32. Growth rate of Escherichia coli at 30 C in concentrated stream
detritus
Growth Parameter
Retention time (hrs)
Dilution Rate (hr"1)
Wash out Rate (hr-1)
Growth Rate (hr"1)
Generation time (hrs)
from the Oconee
Site 1
83. 3
0. 012
-0. Oil
0. 001
1, 000
River
Site 2
83. 3
0. 012
-0. 016
-0. 004
-
Site 3
83. 3
0. 012
-0. 003
0. 009
111. 1
-------
Table 33. Respiration rate of Escherichia coli at 30 C in
detritus samples and eluates of sediments present in
detritus samples from site 3
Substrate
Respiration Rate
Mg atoms O/hr. /mg dry cell wt.
Detritus Sample
Eluates
Wash ai
Wash 2
Wash 3
0. 3 M PO,
buffer
1.25
0. 70
0.85
0. 85
2. 50
Deionized water wash.
-------
CO
LU
O
*4—
O
O
8.
coil
boctcrlovorus
0 I 33456789 JO
DAYS
Fig. 21. Growth curve of Escherichia coll
and Bdellovibrio bacteriovorus in
N. B. medium at 30 C.
79
-------
C/)
bJ
O
(D
O
_J
5-
4 -
E. coll in
autoclaved river water
filter sterilized river water
B. bacteriovorus
in
autoclaved river water
filter sterilized river water
2 -
DAYS
Fig. 22. Growth curve of Escherichia coll and Bdellovibrio
bacteriovorus in filtered and autoclaved Site 3
Oconee River water at 30 C.
80
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u
O
**-
O
O
5 -i
4 -
E. colj in river water
E. coli after addition of peptone and yeast extract
B. bacteriovorus in river water
B. bocteriovorus after addition of peptone and
yeast extract
1 1
D 2
' i
4
I ]
6
1 1 !
8
| 1
10
DAYS
Fig. 23. Growth curve of Escherichia coli and Bdellovibrio
bacteriovorus in site 3 river water at 30 C. Pep-
tone and yeast extracts were added to the culture
after 4 days.
81
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the continuous culture of the E. coli and B. bacteriovorus parasitic
system in river water from site 3 (dilution rate 0. 012 hr. ~1 30 C)
are shown in Fig. 24.
82
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o
o
UJ
_J
CO
8.
7.
6.
5.
4.
3.
2.
• Control (E.coli)
o £ coli host
+ B. bocteriovorus
100
200
300
HOURS
400
500
Fig. 24. Continuous culture of Escherichia coli and Bdello-
vibrio bacteriovorus in Oconee River water from
site 3 at 30 C.
83
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SECTION VI
DISCUSSION
From August, 1968 through July, 1971, three integrated studies were
undertaken to study enteric bacterial metabolism of various substrates
found in a fresh -water stream environment. These studies are as
follows:
1. Water quality analysis of the North Oconee River at three selected
sites at Athens, Georgia.
2. Evaluation of the ability of enteric bacteria to use natural aquatic
substrates by oxygen uptake experiments.
3. Evaluation of the ability of enteric bacteria to use natural aquatic
substrates by continuous culture.
Part 1
Water Quality Analysis of the North Oconee River at Three Selected
Sites at Athens, Georgia
Chemical and bacteriological assays: River water from the three
study sites was sampled at least twice monthly and analyzed for basal
nutrient concentration and for numbers and types of organisms present.
Little deterioration of the Oconee River in terms of water quality was
observed between sites one and two, but the most dramatic change in
water quality did occur in the site three area downstream from the
sewage plant effluent. The water at this location, however, does not
appear to be grossly polluted at this time and perhaps the only potential
hazard of an immediate nature might be from the pathogenic .organisms
•which are present in the stream. (Table 23).
There appears to be little or no correlation between basal nutrient
concentration and bacterial numbers, but this is not surprising since
both determinations are "standing crop" estimations and yield little
information, concerning bacterial activity or turnover of the nutrients
within the system.
Procedure evaluations
Formic hydrogenlyase induction as a basis for the Eijkman fecal
coliform concept: Gray and Gest (1965) have postulated that the
hydrogenlyase system present in Enterobacteriaceae contains two
distinct enzymes, a hydrogenase and a formate dehydrogenase.
85
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This system is inducible and anaerobic, with carbon dioxide and
molecular hydrogen as reaction end products. The jL coli and E.
aerogenes cultures used in this investigation had the capacity to
induce formic hydrogenlyase synthesis at 30. 0 C, but E. aerogenes
was unable to synthesize the enzyme system at 44. 5 C. These
results are consistent with those of Quist and Stokes (1969). The
recovery of biochemically sound strains of E. aerogenes from the
aquatic environment, which have an inducible formic hydrogenlyase
system at 44. 5 C, is not surprising (Table 21, 22), since biochemical,
morphological and serological variations in enteric bacteria as
laboratory phenomena are well known (Edwards and Ewing , 1962;
Hayes, 1968). Reports of such phenomena occurring under natural
conditions are rare, although Velaudapillai (1961) and Hendricks
and Morrison (1967a) have suggested that exchange of genetic
material between bacteria can occur in various natural environments
to give rise to biochemically aberrant organisms.
Explanations for the presence of a high temperature-insensitive
hydrogenlyase system in some of the aquatic strains of E. aerogenes
are difficult. It is entirely possible that the wild type, as represented
by this ATCC culture, cannot synthesize the enzyme system or
synthesizes a portion that is inactivated at elevated temperature and
that the selective pressures of the aquatic environment allow a particu-
lar mutant population to survive. This hypothesis is consistent with
observations of Peterson and Gunderson (I960) and Morita and Burton
(1963). It is also reasonable to suggest that a formic hydrogenlyase
system active at an elevated temperature may be irreversibly lost upon
prolonged storage as are virulent factors of certain pathogenic organisms.
Considerable evidence has recently been discovered which indicates
that the positive correlation between gas production at an elevated temper-
ature of incubation and the presence of fecal coliforms (E. coli) may be
restricted to environments that are grossly contaminated by feces of man
and certain warm-blooded animals (Geldreich et al. 1962; Mishra et al. ,
1968). In this particular environment, it is obvious that the gas-
producing coliforms are indicative of fecal pollution, since E. coli
assumes dominant proportions among those organisms capable of synthe-
sizing an active formic hydrogenlyase. However, in environments where
fecal contamination and numbers of E. coli are minimal, other gas-
producing enteric bacteria, which are not fecal coliforms, can reach
significant proportions to alter the statistical fecal conform relationship.
The results of this study substantiates the latter hypothesis, in which
significant numbers of Enterobacter species (24%) from relatively clean
river water had the capacity to produce H2 and CO2 at 44. 5 C (Table 20).
86
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Although Enterobacter species may be recovered from the feces of
man and animals (Lofton £tal. , 1962), these organisms are not
considered to be indicators of fecal pollution, but rather they are
associated with soil and vegetation. The present data becomes
significant when one considers the increasing reports (Boyd and Boyd, 1962;
Hendricks and Morrison, 1967b), of growth and multiplication of the
E. coli-E. aerogenes group of coliforms in natural waters. It is
entirely possible that water of questionable quality may be needlessly
rejected if differentiation between fecal and nonfecal coliforms is made
solely on the basis of an elevated-temperature fecal coliform test.
Increased recovery of salmonellae from stream bottom sediments vs.
surface waters: Although Salmonella can be recovered below sewage
treatment facilities by a variety of techniques, it is especially inter-
esting that more of these enteric pathogens can be recovered from
bottom sediments than surface water by a relatively unsophisticated
procedure. These data suggest that there is at least a migration of the
organisms from the surface water to the bottom sediments where perhaps
a more favorable condition exists for survival and later multiplication.
It is also of interest that nine Salmonella were recovered from the site
C location. The lack of adequate mixing and dispersal of the organisms
present in the sewage effluent with the river water was probably
responsible for the lack of Salmonella recovery at the upper two sites.
No explanation is readily available for lower recovery rates at sites
below the C location, unless substantial precipitation and sorption of
the organisms to the bottom sediments occurred in the site C area to
preclude recovery downstream.
Part 2
Evaluation of the Ability of Enteric Bacteria to Use Natural Substrates
by Oxygen Uptake Experiments
Enteric bacterial metabolism of substrates in river water and in
stream sediment water: Previous studies (Butterfield, 1929; McGrew
and Mallette, 1962; Hendricks and Morrison, 1967b) have indicated
that basal nutrients in concentrations approximately equal to Davis' (1950)
minimal salts-glucose medium diluted 1:1000 were sufficient for the
maintenance and limited growth of prototrophic enteric bacteria. Data
obtained in this study (Table 24) demonstrated that the ammonia nitrogen,
carbon, and orthophosphate present in the three test substrates were
comparable in concentration to those media. With the exception of
hexose present in the river water, concentrations of the basal nutrients
87
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contained in both the river water and buffer-extracted bottom
sediments were maximal at the site located below the sewage out-
fall (site 3).
Table 24 also demonstrates that bottom sediments from the Oconee
River can be washed relatively free from loosely associated material
and that a very high concentration of ammonia nitrogen, protein, and
hexose can be sorbed onto the river bottom sediments. This observa-
tion becomes even more significant because three successive washings
with deionized -water and trial elution with river water could not remove
the tightly bound material. Elution, though, was accomplished with
buffer of ionic strengths which might be common to only the most
severely polluted aquatic environments. These data suggest that the
basal nutrients were very tightly sorbed on the sands and clays forming
the stream bottom sediments, and that they may not be readily avail-
able for metabolism by aquatic microorganisms. We have tested this
concept with BOD studies in our laboratory and found that washed
sediments had no stimulating effect on the oxygen demand. This is in
agreement with Weber and Coble (1968), who have found that cationic
pesticides which were subject to microbial degradation could be
adsorbed on various clays and were then no longer subject to decompo-
sition or even readily available for plant uptake.
Respiration of organisms in the aquatic environment has been used as
a means of estimating in situ activity (Olson and Rueger, 1968; Rueger
£t al. , 1968; Schroeder, 1968.) Control experiments (Fig. 13) suggest
that a respiration rate of about 0. 5-3. 5 mg atoms 0/h/mg dried cell
weight could be achieved if the carbon (hexose and protein) analyses
of the river water and extract bottom sediments represent readily
oxidizable substrates. Similar rates would be expected with the pre-
pared natural substrates at both the 30 and 20 C incubation temperatures,
but respiration at 5 C should be minimal or nonexistent. Results of the
respiration rate studies using river water and extracted sediments
confirmed this hypothesis (Table 25, 26, and 27) and reflected the basal
nutrient concentrations. Respiration rates above endogenous levels
for all organisms tested in river water were lowest at site 1, but as
nutrient concentration increased in the water from sites 2 and 3,
respiration rates at both 20 and 30 C approached the predicted values
(Tables 26 and 27). Sediment eluted with phosphate buffer in all cases
yielded respiration rates far exceeding those observed with river water.
With the exception of Enterobacter aerogenes at site 1, both Escherichia
coli and Enterobacter aerogenes could use the substrates present in
the eluates from all sites at 5 C. Buffer eluted sediments from below
the sewage plant (site 3) demonstrated the highest respiration rates
-------
achieved at 30 C with rates at 20 and 5 C approximating those at the
other two sites. When these rates were compared with those for the
pathogenic species (Shigella flexneri, Salmonella senftenberg, and
Arizona arizonae), equivalent respiration was observed (Fig. 15).
These data indicate that both pathogenic as well as nonpathogenic
bacteria could use substrates that were present in the river water
and those sorbed on the bottom sediments after elution by relatively mild
laboratory treatment.
Utilization of substrates in detritus samples-. The data in Table 28
indicates that coliform bacteria can metabolize substrates present
in mildly concentrated detritus from the Oconee River. It is inter-
esting to note that E. aerogenes, an organism associated with soil,
can metabolize the detritus at a faster rate than_E. coli (0. 02-0. 03
to 0. 00-0. 02 mg 0/mg dried cell weight/g hexose/hr. ) These data and
those obtained with river water and stream sediment eluates suggest
that while both E. coli and E. aerogenes can metabolize substrates
present in the aquatic environment, _E_. aerogenes would be more
successful in competition.
Part 3
Evaluation of the Ability of Enteric Bacteria to Use Natural Aquatic
Substrates by Continuous Culture
Preliminary experiments with native populations
Bacterial attachment studies: From the data obtained in the water
quality studies (Tables 1-19, 29) and in the respiration experiments
(Tables 24-28, Fig. 14), it was concluded that initial experiments to
demonstrate bacterial growth in river water could best be accomplished
with a natural bacterial population and river water from site-3. These
experiments were designed around the incubation temperatures of 14
and 26 C; 14 C being the mean of the water temperature in the fall and
spring, and 26 C occurring during the summer. For these experiments
the dilution rate was arbitrarily set at 0. 058 hr ~\
Figure 16 shows the results of a 14 C experiment. An initial drop in
the suspended organisms was observed, and this was attributed mainly
to organisms leaving the suspended population and attaching to the glass
surfaces. The organisms then decreased sharply and, at nine hours
again reached a concentration comparable to that of the culture vessel.
These data indicate that the organisms were attaching to the glass,
multiplying and releasing cells into the suspension for subsequent wash-
89
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out. Although the enterics and coliforms actively grew and metabo-
lized, the proportion of these populations in the culture diminished
with respect to time.
Figure 17 shows the results of an experiment run at 26 C. There
appeared little difference between the percentage of attached enterics
and coliforms and those in suspension, but the entire culture seemed
to fluctuate more than was observed at 14 C. As predicted .growth and
attachment of organisms in the culture vessel occurred much more
rapidly at the higher temperature.
Analysis of the basal nutrient concentration data from these experiments
is difficult since the precision of the tests doesnot allow for the detection
of very small differences in concentration. In general, though, there
was a decrease in hexose and ammonia nitrogen in these experiments
at both incubation temperatures, little change in phosphate and an
increase in protein concentration, and these data are indicative of a
metabolizing culture and agrees with data by Herbert, et al. (1956).
These results and those of Sanders (1966, 1967) demonstrate that
bacterial attachment to the glass surfaces in a continuous culture
device is an important consideration where the results of the sus-
pended population are analyzed. However, such data, as evidenced
by the coliforms in our system, suggest that attachment may be a
mechanism by which low growth-rage organisms manage to contribute
significantly to the culture. This group of slow-growing organisms, in
the attached state, could account for otherwise puzzling increased in
the suspended population that might be labeled as "bursts", of growth.
Bacterial growth studies: The growth of native bacterial populations
in dilute nutrient systems can be clearly shown if longer termed
experiments are employed than in the attachment studies. Data in
Figs. 18 and 19 demonstrate this observation. Figure 19 shows the
results of an experiment run with dilute minimal salts-glucose medium
(Dilution rate = 0. 012 hr -1at 30 C). With the exception of the coliform
bacteria counted at 30 C (upper panel), all groups of organisms increased
in number during the first 24 hrs of growth. After that time, the
various populations of organisms grew at a rate equal to or greater
than the flow rate (Table 30) but began to oscillate with periodicity of
about 100 hrs. The fecal coliforms (estimated by the 44. 5 C coliform
counts in the lower panel) in this system were not as competitive as
the other populations and these data suggest that they would eventually
be washed out of the system.
90
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When river water from site 3 was substituted for dilute minimal
medium as a nutrient source, it was observed that the population of
organisms counted at 30 C also increased in number during the first
24 hrs of growth (Fig. 18). After that time, the population decreased
in magnitude but yet demonstrated a positive growth rate (Table 30).
The coliforms and enteric populations at 44. 5 C did not grow but rather,
died at a rate faster than the -washout rate; however, the heterotrophic
group of organisms at this temperature were by far the best competitors.
It is interesting to note that by the 420th hour of the experiment, the
population counted at 30 C was approximately equal in magnitude to
the heterotrophic population counted at 44. 5 C.
Oscillations with approximately a 100 hr periodicity were also
observed in the river water experiments, and they seemed much more
severe than those observed with the dilute minimal medium experi-
ments. Explanations for the periodicity in these experiments are
difficult, but it is possible that those organisms which have attached
to the glass surfaces could be sloughed off at particular times. Sanders
(1966) has shown that the bacterial slime in chemostats can be sloughed
off if the underlying portions go sufficiently anaerobic, and even though
these experiments -were constantly aerated, the system -was depleated
•with oxygen rapidly after initiation of an experiment.
Continuous culture experiments using the stock enteric cultures with
river water and collected detritus
River water experiments: River water from sites 1 and 2 would not
support the growth of any of the six test bacterial species used.
Representative data for E. coli at 30 Q is summarized in Fig. 20.
Water from site 3 (below the sewage plant effluent) did, however,
support the growth of all six strains, including pathogens, at 30 C.
Growth of the test organisms at 20 and 5 C was sparse or non-existent.
Although the growth rates achieved at 30 C are small and probably not
significant as compared to those achieved with full-strength laboratory
media, they are comparable to those reported by Jannasch (1969) for
Achromobacter in seawater. These data reported here suggest that
some enteric species including pathogens are capable of growth in
river water at temperatures representative of the environment.
Detritus studies: Attempts to demonstrate growth of the enteric
bacterial strains in the detritus samples were generally unsuccess-
ful. Table 32 does, however, contain data for E. coli in continuous
culture (Dilution rate = 0. 012 hr "1) with concentrated detritus as the
nutrient source. The observed growth rate of 0. 009 hr is low
91
-------
compared to that which can be achieved in water alone (0. 029 hr 1).
By working with the hypothesis that a significant portion of the nutrients
present in the concentrated detritus has sorbed to contaminating sands
and clays in the sample, it was demonstrated that nutrients could be
made available by ehition of 0. 3 M phosphate buffer as was shown in
the respiration studies (Table 33).
Bdellovibrio bacteriovorus-Escherichia coli interactions in continuous
culture
Activity in river water: After demonstrating that the_B_. bacteriovorus
recovered from the Oconee River will feed upon the stockedJE. coli
used in the continuous culture experiments (Fig. 21), it was observed
that the bacterial parasite was also active in static cultures of river
water that was either sterilized by membrane filtration or in the
autoclave from site 3 (Fig. 22). It was also demonstrated (Fig. 23)
thatJBd. bacteriovorus activity could be stimulated if nutrients were
added to the system, but numbers of infecting Bd. bacteriovorus would
not keep pace with the developing^, coli population. These results
indicate that, while the Bdellovibrio population remains viable in
river water and capable of responding to situations where the growth
of the host organism is favored, the bacterial parasite is unable to
successfully eliminate or drastically reduce the host population. This
observation is further substantiated by the continuous culture data in
Fig. 24 which indicated that an increasing portion of Bdellovibrio
population capable of attacking the EL coli host is lost by the 350th
hour of the experiment. .Reasons for this loss in population are
speculative, but either a development of resistance on the part of
either the host or the parasite could account for the loss. The latter
hypothesis favored because a third population of a yellow colony forming
organism was beginning to develop within the chemostat at this time
which other investigators have shown to be a heterotrophic form of the
Bdellovibrio bacteriovorus (Stolp and Starr, 1963; Shilo and Bruff,
1965, Simpson and Robinson, 1968).
The experiments in this investigation strongly suggest that more
research is needed to fully elucidate the interaction between micro-
organisms and the underlying sediment portion of the stream. Although
these data cannot be directly extrapolated to give information on the
fresh water ecosystem, a model can be proposed which suggests bottom
sediments can control the nutrient material which is suspended in the
water. Work by Wright and Hobbie (1965) has shown the rate at which
nutrients turn over to be significant for sustaining heterotrophic
92
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bacterial growth in lake water, and Hendricks and Morrison (1967a,b)
have speculated that only a minor component of the total bacterial
growth and reproduction can probably occur "within free flowing water
of a high-quality stream since nutrients are normally in low concentra-
tion. However, estimations of nutrient concentration and numbers of
organisms are not constant within fresh or marine environments and may
vary considerably from time to time. The observations (Morrison and
Fair, 1966; Low _et _al. 1968; Anthony, 1970) can be explained on the
basis that a portion of the sediment-nutrient complex can be removed
by aqueous extraction which could then be re suspended or dissolved in
the water. As the apparent nutrient concentration increased in the
river water, increased heterotrophic bacterial growth might also be
observed. However, once the adsorptive capacity of the sediments
has been reached, as perhaps exists around sewage plant effluents,
stream nutrients then could not be removed from the system and much
growth of aquatic organisms could result. An occurrence such as this
would seriously affect the aerobic component of the system and lead
to an altered self-purification potential for some distance below a
sewage outfall.
In summary, the presence of coliform bacteria in water for domestic
consumption is often considered as evidence for fecal contamination by
public health authorities. These organisms, while they primarily do
not produce intestinal disease, do serve as indicators for potentially
infectious microorganisms. Since these bacteria can gain access to
water supplies by a variety of means, any growth by either the coli-
form group of bacteria or the disease producing organisms in the
natural aquatic environment could significantly alter our present con-
cepts of the detection and surveillance of these organisms.
Rivers and streams are constantly involved in a process of partial
self purification in which complex materials are broken down by
microorganisms for later reuse by other organisms. While most
of the nutrient material is present in the water for subsequent
utilization, a significant portion is trapped by the sands and clays
forming the scream bottom in many areas.
Using tests -which are involved in detecting and analyzing polluted
waters and from levels of the self-purification potential, we have
found that water of the Oconee River, a typically non-polluted stream
of the North Georgia piedmont, is capable of supporting the growth
of bacteria including coliforms. We have also found that both organic
and inorganic compounds can be adsorbed by stream bottom sediments
and some of these compounds after removal can be then used as food
93
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for both coliforrn and intestinal disease producing bacteria.
The significance of our research is two fold and the immediate
problem is of a public health nature. If intestinal disease producing
bacteria such as Salmonella, Shigella, and Arizona can gain access
to our streams and multiply, then there are increasing chances of
human disease from the untreated or partially treated water. The
long range implications are potentially more serious. Pollution of
water resources by partially treated sewage, agricultural and certain
industrial wastes and wastes of wild or domestic animals feeding along
drainage areas, not only can contribute bacteria but also nutrient
material that both the coliforrn and disease producing enteric bacteria
can use for growth -which then would increase any immediate public
health problem that might exist.
Since the presence of coliforrn bacteria in water is only indicative
of fecal pollution, any increase in numbers of these organisms above
a minimal level might necessitate expensive additional treatment or
cause rejection of the water for a particular purpose. Substantial
changes in our laboratory differentiation procedures for the identifi-
cation of harmful bacteria in water supplies, and changes in our think-
ing are going to be required in the coming decade if we are going to
best use our aquatic resources.
94
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SECTION VII
ACKNOWLEDGEMENTS
Much of the laboratory work in this investigation was performed by
Martha Fleeman, Susan Cubillas, Gay Walter, Flossie Bonner, and
Cynthia Carr, while Doyle Anderson, David Lewis, David Mize and
Tom Fisher participated in the field studies. Their help is gratefully
acknowledged.
Valuable assistance has been rendered by Allen Cherer, Marcia Durso
and Stephanie Sanders who each •worked on portions of this investigation
and received a stipend from the NSF-SSTP program.
The support of the project by the Environmental Protection Agency and
the help provided by Pat C. Kerr of the Southeastern Water Laboratory,
and by Drs. Walter M. Sanders and Russell L. Todd, the Grant Project
Officers, is acknowledged with sincere thanks.
95
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SECTION VIII
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Olson, T. A. , and M. E. Rueger. 1968. Relationship of oxygen
requirements to index-organism classification of immature aquatic
insects. J. Water Pollut. Contr. Fed. 40:(Res Suppl. ) R 118-R202.
Parr, L. W. 1938a. The occurrences and succession of coliform
organisms in human feces. Amer. J. Hyg. 27:67- 87.
Parr, L. W. 1938b. Coliform intermediates in human feces. J.
Bacteriol. 3,6:1-15.
Perry, C. A. , and A. A. Hajna. 1944. Further evaluation of EC
medium for the isolation of coliform bacteria and Escherichia
coli. Amer. J. Public Health Nat. Health. 34:735-748.
99
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Peterson, A. C. , and M. F. Gunderson. I960. Some characteristics
of proteolytic enzymes from Pseudornonas flourescens. Appl.
Microbiol. _8^98-104.
Pomeroy, L. R. , and R. E. Johannes. 1968. Occurrence and respira-
tion of ultraplankton in the upper 500 meters of the ocean. Deep-Sea
Res. 1_5:381-391.
Quist, R. G. , and J. L. Stokes. 1969. Temperature range for formic
hydrogenlyase induction and activity in psychrophilic and mesophilic
bacteria. Antonie van Leeuwenhoek. J. Microbiol. Serol. 35:1-8.
Rueger, M. E. , T. A. Olson, .and J. L. Scofield. 1968. Oxygen
requirements of benthic insects as determined by manometric and
polarographic techniques. Water Res. _3:99-120.
Ruttner, F. 1964. Fundamentals of limnology. Translated from German
by D. G. Frey and F. E. J. Fry. University of Toronto, Press,
Toronto.
Sanders, W. M. 1966. Oxygen utilization by slime organisms in
continuous culture. Air Wat. Pollut. Int. J. 10:253-276.
Sanders, W. M. 1967. The growth and development of attached
stream bacteria, part 1. Theoretical growth kinetics of attached
stream bacteria. Water Resources Res. 3:81-87.
Schroeder, E. D. 1968. Importance of the BOD plateau. Water Res.
2,:803-809.
Shilo, M. , and B. Bruff. 1965. Lysis of gram negative bacteria by
host-independent ectoparasitic Bdellovibrio bacteriovorus isolates.
J. Gen. Microbiol. •40:317-328.
Simpson, F. J. , and J. Robinson. 1968. Some energy producing
systems in Bdellovibrio bacteriovorus, strain 6-5-S. Can. J.
Biochem. 46:865-873.
Spino, D.' F. 1966. Elevated-temperature technique for the isolation
of Salmonella from streams. Appl. Microbiol. 14:591-596.
Stolp, H. , and M. P. Starr. 1963. Bdellovibrio bacteriovorus gen. et.
sp. n. , a predatory, ectoparasitic and bacteriolytic microorganism.
Antonie van Leewenhoek J. Microbiol. Serol. 29:217-248.
Stolp, H. , and M. P. Starr. 1965. Bacteriolysis. Ann. Rev. Microbiol.
_19:79-104.
Tempest, D. W. , and J. R. Hunter. 1965. The influence of tempera-
ture and pH value on the macromolecular composition of magnesium-
limited and glycerol-limited Aerobacter aerogenes growing in a
chemostat. J. Gen. Microbiol. 41:267-273.
100
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Varon, M. , and M. Shilo. 1969- Interaction of Bdellovibrio
bacteriovorus and host bacteria. II. Intracellular growth and
development of Bdellovibrio bacteriovorus in liquid cultures.
J. Bacteriol. 99:136-141.
Velaudapillai, T. 1961. Of the naming of salmonellas, is there no
end? Intern. Bull. Bacteriol. Nomencl. Taxon. 11:1-4.
Weber, J. B. , and H. D. Coble. 1968. Microbial decomposition
of diquat adsorbed on montmorillonite and kaolinite clays. J.
Agr. Food Chem. 16:475-478.
Wright, R. T. , and J. E. Hobbie. 1965. The uptake of organic
solutes in lake water. Limnol. Oceanogr. 10:22-28.
101
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SECTION IX
PUBLICATIONS AND PATENTS
Publications:
1. Hendricks, Charles W. 1970. Formic hydrogenlyase induction as
a basis for the Eijkman fecal coliform concept. Appl. Microbiol. 19:
441-445.
2. Cubillas, Susan, and Charles W. Hendricks. 1970. Aquatic
bacterial respiration in a continuous culture system. Bacteriol. Proc.
p. 32.
3. Hendricks, Charles W. 1970. Enteric bacterial metabolism of
stream sediment eluates. Bacteriol. Proc. p. 32.
4. Hendricks, C. W. 1971. Continuous culture of natural bacterial
populations from a fresh water stream. Bacteriol. Proc. p. 52.
5. Hendricks, Charles W. 1971.. Enteric bacterial metabolism of
stream sediment eluates. Can. J. Microbiol. 17:551-556.
6. Hendricks, Charles W. 1971. Increased recovery rate of salmonel-
lae from stream bottom sediments versus surface waters. Appl.
Microbiol. 21:379-380.
Patents: None, but either one or both of the chemostats could be
patented.
103
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SECTION X
GLOSSARY
Basal Nutrients - A combination of simple inorganic and org-anic
compounds that -will support the growth of prototrophic bacteria.
Biotope - A place of life; the totality of the environmental conditions
under which a community of organisms exist.
Chemosynthetic - Organisms that synthesize organic matter from
mineral or organic substances with the aid of chemical energy.
Detritus - Finely divided settleable material suspended in the water.
Dilution Rate - The rate at which nutrients are added to a continuous
culture device, and -wastes are removed from the system.
Enzyme induction - The initiation of enzyme synthesis within an
organism.
Heterotrophic - The nutrition of plants ,and animals that are dependent
on organic matter for food.
Mesophilic - A mid-range temperature requiring organism (20-45 C).
_M - Moles or Molar
p. - Micro or Micron
Pathogenic - The ability of an organism to produce disease or
infections.
Photo synthetic - Organisms that produce organic matter from CO2 and
H2O with the aid of the energy of light.
Protrotrophic - The ability of organisms to grow on mixture of inorganic
salts, water, and a simple carbohydrate (i. e. glucose minimal medium).
Substrate - A chemical compound that is suitable for metabolism by an
organism.
--. U. S. GOVERNMENT PRINTING OKFICK : 1972— 4B4-<.84/179
105
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1
A cc&xsfon Number
5
2
Subjei-t Fii-ld & Group
05E
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Organ izati on
University of Georgia
Athens, Georgia
Title
ENTERIC BACTERIAL DEGRADATION OF STREAM DETRITUS
10
Authors)
Hendricks, Charles W.
16
21
Project Designation
16050 EQS
Note
22
Citation
23
Descriptors (Starred First)
* # *
Enteric bacteria, Degradation, Detritus, Biodegradation,
Streams, Growth rates, Respiration, Water analysis
'Pathogenic Bacteria,
25
Identifiers (Starred First)
^North Oconee River, Athens, Georgia, Sediment eluates
27
Abstract
An investigation was initiated to relate basal nutrients in the water and on the
bottom of a warm, fresh water stream to their ability to support the growth
and multiplication of pathogenic and nonpathogenic enteric bacteria. The results
of this study indicated that enteric bacteria have the capacity to metabolize
substrates that were present in the stream environment including autoclaved
river water. These organisms, however, lacked the ability to increase in
numbers in continuous culture with river water and suspended detritus recovered
above a secondary sewage treatment facility, but they did demonstrate positive
growth rates with substrates recovered below the plant. Data from this study
also demonstrated that the sands and clays forming the stream bottom have
the capacity to sorb substrates from the overlayering "water, and .that sediment
eluates will stimulate the respiration rate of the study bacterial strains. These
results suggest that the stream bottom can provide a suitable environment for
the growth of bacterial species and perhaps control basal nutrient concentration
in the water itself. {Hendricks - University of Georgia)
Abstractor
c.
Hendricks
/nsC
tution
Univer
sity
of
Georgia,
Athens,
Georgia
WR: 102
WRSI C
(REV. JUL. Y 19691
SEND TO: WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON, D. C. 2024O
Gf>o: 196B-35S-339
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Enteric Bacterial Degradation of Stream Detritus • 16050 feJ. ,-
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