DEPLETION OF OXYGEN BY MICROORGANISMS
IN ALASKAN RIVERS AT LOW TEMPERATURES
FEDERAL WATER QUALITY ADMINISTRATION
NORTHWEST REGION
ALASKA WATER LABORATORY
College, Alaska
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DEPLETION OF OXYGEN BY MICROORGANISMS
IN ALASKAN RIVERS AT LOW TEMPERATURES
by
Ronald C. Gordon, Ph.D.
Presented at the
International Symposium on
Water Pollution Control in Cold Climates
University of Alaska, July 1970
for the
FEDERAL WATER QUALITY ADMINISTRATION
DEPARTMENT OF THE INTERIOR
ALASKA WATER LABORATORY
COLLEGE, ALASKA
Working Paper No. 4
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A Working Paper presents results of investigations
which are to some extent limited or incomplete.
Therefore, conclusions or recommendations—expressed
or implied—are tentative.
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TABLE OF CONTENTS
SUMMARY . '•
INTRODUCTION
MATERIALS AND METHODS
River Description and Sampling Locations
Sample Collection and Handling ;
Handling of-Samples in the Laboratory for DO Analysis
Substrates Used for DO Depletion Studies
Enumeration and Isolation of Heterotrophic Bacteria
Chemical Analyses
Statistical Treatment of DO Depletion Data
RESULTS
Pure culture study of psychrophilic bacteria isolated from
a sub-Arctic river.
Effect of complex organic substrate concentration and incu-
bation temperature on" the dissolved oxygen (DO)
depletion in sub-Arctic river water.
The relative effect of complex organic substrates on DO
depletion in sub-Arctic river water.
The effect on DO depletion when nitrogen and phosphorus
were added to sub-Arctic river water in the presence
of substrates devoid of these nutrients.
Effect of sewage treatment plant effluents on DO depletion
in unpolluted sub-Arctic river water.
Effect of incubation temperature on DO depletion in Arctic
water in the presence of a complex organic substrate.
DISCUSSION AND CONCLUSIONS
ACKNOWLEDGEMENTS
REFERENCES
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LIST OF FIGURES
Figure 1. Apparatus for preparation and bottling of river water
samples for dissolved oxygen depletion studies.
Figure 2. Effect of the concentration of a complex organic sub-
strate on dissolved oxygen depletion in sub-Arctic river
•• water polluted with raw domestic sewage and effluents
from primary treatment plants.
Figure.3. Effect of incubation temperatures on dissolved oxygen
depletion when 120 mg/1 vitamin-free casamino acids was
added to sub-Arctic river water polluted with raw do-
mestic sewage and effluents from primary sewage treat-
ment plants.
Figure 4. Effect of incubation temperature on dissolved oxygen
depletion when 120 mg/1 vitamin-free casamino acids was
added to unpolluted sub-Arctic river water.
Figure 5. Relative effect of three complex organic substrates on
dissolved oxygen depletion in sub-Arctic river water
polluted with raw domestic sewage and effluents from
primary treatment plants.
Fibure 6, Relative effect of high levels (substrate not rate
limiting) of three complex organic substrates on dis-
solved oxygen depletion in unpolluted sub-Arctic river
water when incubated at 10°C.
Figure 7, Relative effect of low levels (substrate being rate
limiting) of three complex organic substrates on dis-
solved oxygen depletion in unpolluted sub-Arctic river
water when incubated at 10°C.
Figure 8. Effect of glucose on dissolved oxygen depletion in sub-
Arctic river water polluted with raw domestic sewage and
effluents from primary treatment plants.
Figure 9. Effect of glucose on dissolved oxygen depletion in unpol-
luted sub-Arctic river water when incubated at 10°C in
the presence and absence of added inorganic nitrogen and
phosphorus.
Figure 10. Effect of ethyl alcohol on dissolved oxygen depletion
. . in unpolluted sub-Arctic river water when incubated at
10°C in the presence and absence of added inorganic
nitrogen and phosphorus.
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LIST OF FIGURES Continued
Figure 11. Effect of sodium acetate on dissolved oxygen depletion
in unpolluted sub-Arctic river water when incubated at
10°C in the presence and absence of added inorganic
nitrogen and phosphorus.
Figure 12. Effect of incubation temperature on dissolved oxygen
depletion when effluent from the Fairbanks, Alaska city
primary sewage treatment plant was added to unpolluted
sub-Arctic river water.
Figure 13. Effect of incubation temperature on dissolved oxygen
depletion when effluent from a 0°-0.5°C bench scale
activated sludge sewage treatment system was added to
unpolluted sub-Arctic river water.
Figure 14. Effect of incubation temperature on dissolved oxygen
depletion when 120 mg/1 vitamin-free casamino acids was
added to unpolluted Arctic river water.
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LIST OF TABLES
Table 1. Effect of increased incubation 'temperature on the growth
of bacterial isolates from samples obtained from a sub-
Arctic river
Table 2. Relative distribution and the effects of increased incu-
bation temperature on the growth of two morphological
types of bacteria isolated from a sub-Arctic river
Table 3. Chemical analysis of water samples from two locations on
a sub-Arctic river
Table 4. Comparison of the rate of dissolved oxygen depletion when
a substrate was added to Arctic and sub-Arctic river water
samples
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SUMMARY
Plate counts indicated that a sub-Arctic river contained
heterotrophic bacteria capable of growth at 0°C on a complex
growth medium. Pure culture studies showed that two morpho-
logical types produced colonies, on this medium and that domestic
pollution apparently altered the composition of the population.
The ability of the natural, mixed, bacterial flora to utilize
dissolved oxygen (DO) was studied in polluted and unpolluted
sub-Arctic river water samples at incubation temperatures
ranging from 0° to 20°C. The results indicated that the in-
digenous bacteria were capable of extensive metabolic activity
when complex organic substrates were added. Incubation temperature
affected the lag phase, but not the extent of DO depletion. It
was also noted that one of the major activities of these bacteria
appeared to be proteolysis and that growth factors in two complex
substrates enhanced activity. Glucose, sodium acetate and ethyl
alcohol were poorly utilized as substrates, and the addition of
nitrogen and phosphorus enhanced activity. When primary sewage
treatment plant effluent was added to river water samples, there
was rapid and extensive DO depletion at all incubation temperatures.
Secondary effluent added to the system resulted in some activity
at 10° and 20°C, but essentially none at 0°C.
Extensive DO depletion was observed at all incubation tempera-
tures between 0° and 20°C when samples from an unpolluted
Arctic river were incubated with a complex organic substrate.
In general, the results were similar to those found with
unpolluted sub-Arctic river samples. However, the lag phase
before the start of DO depletion was extended at all in-
cubation temperatures.
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INTRODUCTION
Several Arctic (20,37) and sub-Arctic (12,14,23,37) rivers
in Alaska (Alaskan rivers) have low concentrations of dissolved
oxygen (DO) during periods of total ice cover; conditions
which occur naturally without domestic or industrial pollution.
A similar oxygen deficit was noted in some unpolluted rivers in
the northern and central belt of the U.S.S.R.. (7). Data from
various sub-Arctic rivers in Alaska indicated that DO depletion
was a continuous process throughout most of the period of ice
cover, with an increase in DO concentration shortly before
Spring breakup (12,14,37). The extent of depletion increased
progressively toward the lower reaches of each river (13,14).
Investigations in the U.S.S.R. have shown that the low DO
concentration resulted from the ice cover which prevented
reaeration (7). Since there is essentially no open water during
the period of ice cover over many Alaskan rivers, there is little
chance for significant reaeration. Under natural conditions,
the extent of oxygen depletion is often sufficient to reduce
the DO concentration to a level far below the 7 mg/1 minimum
set by the Alaska water quality standards (38).. A DO con-
centration of 1 J mg/1 was measured in an unpolluted Arctic
river (20,37) and, 1,1 mg/1 (14) and KO mg/1 (35) in unpolluted
sub-rArctic rivers.
The aquatic biota of Alaskan rivers seem to survive the
extreme fluctuations in the amount of DO which they encounter
throughout the year under natural conditions. Problems arise
w_hen oxidizable domestic or industrial wastes enter these
rivers, When the biochemical oxygen demand (BOD) of these
wastes is added to the natural requirement for DO, the result
may be detrimental to the ecosystem,
Ingra,ham and Stokes Q7) discussed the numerous definitions
of psychrophilic bacteria and set forth what is probably the
most useful definition, "Psychrophiles are bacteria that grow
well at 0°C within 2 weeks".. These organisms appear to be
ubiquitous in nature since they have been found in soil, rivers,
lakes, mud and food (9,39,40), Psychrophiles have been studied
in both the Arctic and Antarctic and have been found in soil
and water (5?6,11 ,21 ,41)., These organisms, and their activity
at low, temperatures have been the subject of several reviews
(8 ,9, 16, 17, 22) and will not be discussed in detail here. Stokes
and Redmond (40) considered psychrophiles to be present in
large enough numbers in natural habitats to be important in the
cycling of matter. Wuhrmann, et^ a]_. (42) stated, "Self-
purification processes start at the microbial level..." and,
"Most of the work, is accomplished by heterotrophic micro-
organisms (bacteria, fungi, flagellates}"*.
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Active metabolism of organic material in a river during the
winter has been demonstrated in the U.S.S.R. (7). Plate counts
of heterotrophic bacteria indicated that Alaskan rivers have
'bacterial populations in the range of 10^-106 organisms/ml
which are capable of growth on a synthetic medium at low
temperatures (14). There is evidence that the number of
organisms capable of growth at low temperatures increases
progressively toward the lower.reaches of a sub-Arctic river
in Alaska (14) and in a river in the U.S.S.R. (7) during the
period of total ice cover.
It has been shown that psychrophilic bacteria are capable of
rapid metabolic activity at low temperatures. Since Alaskan
rivers have populations of these organisms, it appears that
they may be responsible for a significant portion of the DO
depletion observed under both natural and polluted conditions.
The subject of this report is DO depletion by the indigenous
bacteria in a sub-Arctic river. The effect of added organic
and inorganic nutrients, and incubation temperature on the rate
and extent of DO depletion was investigated. The data obtained
from a sub-Arctic river were compared to similar data from an
Arctic river.
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MATERIALS AND METHODS
D
River Description and Sampling Locations ;
Most of the experimental results were obtained from a sub-
Arctic river in interior Alaska. Because of the high level
of domestic pollution in the lower reach and the convenient
location, the Chena River was chosen for detailed study. It
is a non-glacial stream with many ground water sources, and
is approximately 150 miles in length (12)t Raw domestic
sewage and effluents from several primary sewage treatment
plants in the greater Fairbanks area enter the river in the
last 28 miles before it joins the Tanana River. Two sampling
locations were selected, one below all major sources of domestic
pollution and the other above any source.
Comparative data were obtained from an Arctic river in the
"-Arctic Slope" area of Alaska. The Sagavanirktok (Sag) River
was selected because it is the major river flowing through an
area of extensive oil development and is accessible for sampling.
It is a non-glacial stream originating in the Brooks Range, flows
north approximately 170 miles to the Beaufort Sea and receives
littla, if any, domestic pollution (14)% One sampling location
was selected approximately 85 river miles above the mouth of the
river near the settlement of Sagwon.
• . \ • > -
-•Sample •CollectlorKand-Handling
*\ """ * ' *,^1 1 ' \ ' ~J\^i f. \ V % \ * < ~V'~!~~~^ -•"~v~\~» . ' J ~ -
All sample locations had total ice cover and a water temperature
of essentially 0°C throughout the study period. Samples were
obtained through holes-drilled in the ice, Samples from the
Chena River for the dissolved oxygen (DO) depletion study
were collected in sterile five gallon polypropylene carboys
by dipping water from the hole in the ice. Because of the large
yolume required, no problem with increase of water temperature
was encountered during the 2 to 3 hour period between sample
collection and handling in the laboratory.
Samples from the Sag River for the DO depletion study required
s,omewhat different handling. These samples were collected in
new, clean, but not sterile, five gallon polyethylene carboys
which were sealed with tight screwcaps. It was not possible to
dip the water, so it was pumped into the carboys. The samples
were shipped by air freight to the laboratory. The water
temperature rose from approximately 0° to 3.5°C during the 9
.hour period between sample collection and handling in the
laboratory., This temperature rise did not appear to be ex-
cessive, and was not considered significant.
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Samples for chemical analysis were collected in small mouth .
25o'ml screwcap polyethylene bottles. These bottles were filled
by submerging them in the hole drilled in the ice and returned
to the laboratory without further field treatment. They were
frozen as soon as possible after arrival at the laboratory and
stored at -20°C until they were analyzed.
Samples for the determination of DO in the river were collected
in 300 ml biochemical oxygen demand (BOD) bottles. The bottles
were lowered on a rod sampler below the bottom of the ice,
allowed to fill completely, and the oxygen was fixed immediately
after being brought to the surface.
Handling of Samples in the Laboratory for DO Depletion Studies
After the samples were returned to the laboratory, they were
taken directly into the 10°C cold room. The DO depletion study
was set up immediately, using pre-cooled glassware to mini-
mize any adverse effect on the natural distribution of the
microorganisms in the samples. A predetermined volume of river
water, 24-36 liters, and the substrate being studied were
placed in a 2 1/2 or 3 1/2 gallon, sterile, glass carboy. The carboy
was placed on the aoparatus shown in Figure 1-A, The water was
stirred rapidly with a magnetic stirrer while the temperature
Was raised 1°T.1,5°C above the intended incubation temperature
with a, thermostatically controlled, 1000 watt, Vicor glass,
ijwnersion heater. The increase in temperature above that
selected for incubation prevented supersaturation of the water
wjth. DO., When the desired temperature was reached, stirring
was continued and the water was aerated vigorously for 10 minutes
using a gas dispersion tube to bring the DO level to or near
saturation. After temperature adjustment and aeration, the
river water was pumped into BOD bottles as shown in Figure 1-B.
Th.e bottles were filled from the bottom to prevent entrainment
of additional DO.. The initial DO level was determined by
immediately fixing the oxygen in three of the BOD bottles.
The rest of the BOD bottles were placed in incubators at 0°,
5°, 10°, 15° or 20°C., Time intervals were selected to permit
the depletion of DO to Be followed. The DO was determined in
three bottles at each time interval.
.Substrates Used for DQ^Depletion Studies
Several laboratory substrates of varying complexity were used.
Vitamin-Free Casamino Acids, Control 534363 (Difco) was used to
compare rates of DO depletion at several temperatures, with
water from various sources, and as a control for other studies.
Yeast Extract, Control 523143 (Difco) and Beef Extract, Control
495576 (Difco) were used as complex substrates containing growth
factors. Growth factors are defined as organic compounds,
generally in minute amounts, required for growth by an or-
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Figure 1. Apparatus for preparation and bottling of river
water samples for dissolved oxygen depletion studies.
(A) Sample was stirred vigorously while the temperature
was equilibrated at 1°-1,5°C above the desired incubation
temperature with a thermostatically controlled, 1000 watt,
Vicor glass, immersion heater; followed by dissolved
oxygen equilibration at or near saturation by aeration
with a gas dispersion tube. (B) Equilibrated sample was
pumped into biochemical oxygen demand bottles for in-
cubation.
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ganism in addition to the principal sources of carbon and
energy. Glucose (Dextrose, Control 527712, Difco) was used to
represent the carbohydrates. Ethyl alcohol (dehydrated, N.F.,
Federal Government stock no. 6505-105-0000) was the only alcohol
used. Sodium acetate (^02^02.3^0, Mallinckrodt analytical
reagent) was used to represent the organic acids.
Primary and secondary sewage treatment plant effluents were
also studied. Primary effluent was obtained from the Fair-
banks city plant before the effluent entered the chlorine
contact chamber. Secondary effluent was obtained from a bench
scale activated sludge system being operated in the Alaska
Water Laboratory at 0°-1.0°C.
Enumeration and Isolation of Heterotrophic Bacteria
The membrane filter method and a broth culture medium prepared
from components [2.5 g/1 Yeast Extract (Difco), 5 g/1 Tryptone
(Difco), and 1 g/1 Dextrose (Difco) made up in glass dis-
tilled water and adjusted to pH 7.0 at 25°C before autoclaving]
were used to enumerate bacteria at 0°C. This medium was found
to give higher numbers on membrane filters at 0°C than any other
medium tried (14). However, this does not mean that these were
the only bacteria present in the water. All membrane filter
preparation was done in the 10°C cold room, using pre-cooled
equipment and materials. Incubation of filters was continued
until there was no further increase in numbers on consecutive
counts.
Isolation of pure cultures was accomplished by picking in-
dividual colonies from the membrane filter after the number
of colonies had stopped increasing. The colonies were placed
in tubes of the same broth medium used for initial enumeration,
and incubated at 5°C because growth was more rapid than 0°C.
After growth appeared, material from the broth cultures was
streaked on Plate Count Agar (Difco) and incubated at 5°C.
Individual colonies were picked.and grown in broth. This
procedure was repeated as a final check of culture purity.
The pure cultures were maintained for further study by monthly
transfer to fresh broth and incubation at 5°C.
Chemical Analyses
The Technicon Auto Analyzer was used for the following analyses:
Orthophosphate phosphorus by the Technicon ammonium molybdate
industrial method; ammonia nitrogen by the sodium phenol ate
method (10); nitrite nitrogen by diazotization (10); nitrate
nitrogen by hydrazine reduction (10).
Total nitrogen and total carbon were determined with the
Perkin-Elmer model 240 Elemental Analyzer.
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Total phosphorus was determined by the persulfate digestion
method (10), except for glucose. Glucose samples were ashed
[AOAC Methods, section 29.013 (3) ], followed by the ortho-
phosphate phosphorus determination previously described.
Chemical oxygen demand was determined as described in the 12th
edition of Standard Methods for the Examination pjf Water and
Wastewater (2).
DO was determined by the azide modification of the iodometric
method (2).
Statistical Treatment of DO Depletion Data
Each set of 3 DO measurements was evaluated by the 0 Test to
reject questionable results. The remaining measurements were
averaged to obtain the reported result. To compare rates of
DO depletion, an attempt was made to establish a rate con-
stant with one substrate at each incubation temperature. Data
obtained during the period of most rapid DO depletion were
treated with first and second order kinetics, and did not fit
either form. The arithmetic form, DO vs time, provided the
most useful treatment of the data. A straight edge was laid
along the slope of the DO depletion curve, and the data points
on the portion of the curve which appeared to have the most
rapid rate of change were used to establish an approximate
rate (mg/l/hr) for the purpose of comparing data within this
study.
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RESULTS
Pyre culture study of psychrophilic bacteria isolated from
a sub-Arctic river.
Mater samples were collected from a polluted and an unpolluted
location in a sub-Arctic (Chena) river on December 17, 1968
and contained, respectively, 9000 and 550 heterotrophic bacteria
per ml which were capable of growth at 0°C on the complex
organic medium as described in the Materials and Methods
section. All colonies on a representative membrane filter
from each location were isolated in pure culture. Broth
tubes inoculated with the pure cultures were incubated as
shown in Table 1. All cultures from both locations grew at
0°, 5°, and 10°C, but not at higher temperatures. The per-
centage of the total number which did grow at 20°C and 25°C
was the same from both locations. At 30°C and above, the
percentage of cultures from the polluted location which grew
decreased much more slowly than from the unpolluted location.
This suggested that domestic pollution caused a change in pop-
ulation composition.
Parrel! and Rose (9) pointed out in their review that Gram
negative rods are the most common psychrophilic bacteria isolated,
both qualitatively and quantitatively. Gram negative rods have
been isolated from littoral and marine sediments in the Canadian
Arctic (21) and were the most common bacteria isolated from
water in sub-Arctic Alaska (11). Parrel! and Rose (9) referred
to phychrophilic members of the genus Vibrio (spiral bacteria)
as not being as common as the Gram negative rods, but were still
isolated regularly.
Further study of the pure cultures revealed that only Gram
negative rod and spiral morphological types of bacteria had
produced colonies on the original membrane filters. The effect
of incubation temperature on the two types of bacteria from
each location is shown in Table 2. The data, from the un-
polluted location, indicated that increasing the incubation
temperature above 25°C caused a more rapid decrease in the
percentage of the spiral than of the rod shaped bacteria which
grew. The results from the polluted location were similar
except that the more rapid decrease of spiral bacteria took
place above 30°C rather than 25°C. An additional point of
interest was that all the spiral bacteria grew at 20°C, but
some of the rods from both locations were inhibited at this
temperature.
Examination of Table 2 showed that the ratio of rod to spiral
bacteria changed from 1.5:1 at the unpolluted location to
2.9:1 at the polluted location. This twofold increase of rods
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TABLE 1
Effect of increased incubation temperature on the growth of bacterial
isolates from samples obtained from a sub-Arctic river a>"
Incubation
Temperature
0° - 10°C
20
25
30
35
45
Total
Pol
Number of
Isolates
66
63
52
49
22
7
Sample Location
lutedc
% of Total
Isolates
100
95.5
78.8
74.2
33.3
10.6
66
Unpol
Number of
Isolates
38
35
30
11
6
1
lutedd
% of Total
Isolates
100
92.1
78.9
28.9
15.8
2.6
38
a. Samples were taken on December 17, 1968, when the river had total ice
cover and the water temperature was 0°C.
b. The isolates were obtained by picking all colonies from a membrane
filter which had been incubated at 0°C until there was no further increase
in numbers on consecutive counts.
c. The polluted location was below a reach of the river receiving raw
domestic sewage and effluents from primary sewage treatment plants.
d. The unpolluted location was upstream from any source of domestic
or industrial pollution.
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TABLE 2
Relative distribution and the effects of increased incubation temperature on the
growth of two morphological types of bacteria isolated from a sub-Arctic river a>b
Incubation
Temperature
•
0-10°C
20 ,.
25 '
30
35
45
Total
Poll
No. of
Isolates
49
46
46
35
19
7
Rod
Sample Location
utedc
% of
Isolates
100
93.9
93.9
71.4
38.8
14.3
49
Morphological Type
Unpollutedd
No. of
Isolates
22
20
18
10
5
1
% of
Isolates
100
90.9
81.8
45.5
22.7
4.5
22
Poll
No. of
Isolates
17
17
16
14
3
0
Spiral
Sample Location
uted
% of
Isolates
100
100
94.1
82.4
17.6
0.0
17
Unpol
No. of
Isolates
15
15
12
1
1
0
luted
% of
Isolates
100
100
80.0
6.7
6.7
0.0
15
a. Samples were taken on December 17, 1968, when the river had total ice cover and the water tempera-
ture was 0°C.
b. The isolates were obtained by picking all colonies from a membrane filter which had been incubated
at 0°C until there was no further increase in numbers on consecutive counts.
c. The polluted location was below a reach of the river receiving raw domestic sewage and effluents
from primary sewage treatment plants.
d.;l The unpolluted location was upstream from any source of domestic or industrial pollution.
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relative to spiral bacteria was further indication that domestic
pollution altered the composition of the bacterial population.
Effect of complex organic substrate concentration and incubation
temperature on the dissolved oxygen (DO) depletion in sub-Arctic
river water.
Vitamin-free casamino acids had been used previously in pure
culture studies of Pseudomonas fluorescens at low incubation
temperatures (19,27) and was found to give excellent growth,
which was not enhanced by the addition of yeast extract. In
view of these earlier reports, some preliminary results from
this laboratory and the relatively simple composition of the
substrate, vitamin-free casamino acids was selected as the base- •
line and comparative substrate.
The effect of substrate concentration on DO depletion in polluted
river water is shown in Figure 2. These data indicated that a
vitamin-free casamino acids concentration of 120 mg/1 was
sufficient to eliminate the substrate as a rate limiting factor
in DO depletion. There was no lag phase at 20°C and the DO
concentration in the water was reduced to nearly 0 mg/1 in
15-16 hours. A similar effect of substrate concentration was
observed at 10°C, but the time required to deplete the DO
from near saturation to 0 mg/1 was approximately 50 hours.
Since the water temperature in the Chena River rarely, if ever,
rises above 20°C (12,14), temperatures between 0° and 20°C were
selected for incubating samples. The effect of incubation
temperature on DO depletion in polluted Chena River water is
shown in Figure 3. The volume of water obtained from the river
was large enough to supply samples for all incubation temperatures.
This provided directly comparable temperature effect data when
the samples were incubated in the presence of 120 mg/1 vitamin-
free casamino acids. The results indicated that the length
of the acceleration phase increased and the rate of DO depletion
was reduced as the incubation temperature was decreased, and
there was a short lag phase at the 0°C incubation temperature.
However, the extent of DO depletion did not appear to be temperature
dependent.
Comparative results on the effect of incubation temperature
were obtained with unpolluted river water (Fig. 4). The results
showed.that there was a lag phase at the lower incubation
temperatures (0°, 5°, and 10°C) before the acceleration phase
began. This is in contrast to the lack of a lag phase with
samples from the polluted location. The extent of the DO
depletion, as found with the sample from the polluted location,
did not appear to be temperature dependent. However, the total
elapsed time was increased 50-100 percent.
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Figure 2. Effect of the concentration of a complex organic
substrate on dissolved oxygen depletion in sub-Arctic river
water polluted with raw domestic sewage and effluents from
primary treatment plants. Samples were incubated at 20°C.
Symbols: © , 90 mg/1; A, 120 mg/1; and'A, 150 mg/1
vitamin-free casamino acids; o , river water blank.
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TIME (HOURS)
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Figure 3. Effect of incubation temperature on dissolved
oxygen depletion when 120 mg/1 vitamin-free casamino
acids was added to sub-Arctic river water polluted with
raw domestic sewage and effluents from primary sewage
treatment plants. A river water blank (& ) was incubated
at 20°C.
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0
yo
40
60
80 100 120
TIME (HOURS)
140
160
180
200
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Figure 4. Effect of incubation temperature on dissolved
oxygen depletion when 120 mg/1 vitamin-free casamino
acids was added to unpolluted sub-Arctic river water.
A river water blank (A) was incubated at 20°C.
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40
80
120 160 200
TIME (HOURS)
240
280
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The relative effect of complex organic substrates on DO
depletion in sub-Arctic river water.
Most of the psychrophilic bacteria are found in a few genera
(9) and some have been isolated from water in sub-Arctic Alaska
(11). Nutritional studies have shown that the growth requirements
vary over a wide range from the simple need for a carbon and
energy source to the need for vitamins and other preformed growth
factors (1,19,24,27,30,32,). Vitamin-free casamino acids contains
18 amino acids and essentially no other growth factors, while
yeast and beef extracts contain many amino acids, vitamins and
other water soluble growth factors. The use of these three sub-
strates for DO depletion studies permitted an examination of the
effect of added growth factors.
All the bacteria isolated from both locations were capable of
growth at 10°C on a complex medium containing a variety of pre-
formed growth factors (Table 1), and DO depletion with river
water samples took place in a reasonable time at 10°C with
vitamin-free casamino acids as the substrate (Figs. 3 and 4).
Since 10°C appeared to be adequate for growth and metabolic
activity, it was selected as the incubation temperature for
additional studies.
Water samples from both sample locations were incubated at 10°C
with the three complex organic substrates in quantities contain-
ing the same amounts of carbon. The results are presented in
Figures 5 and 6. The acceleration phase of the DO depletion
curve was shorter with samples from both locations when the
yeast or beef extract was used as the substrate. This suggested
that preformed growth factors either enhanced overall metabolic
activity or were required by a portion of the bacterial popu-
lation. The results indicated that there was a difference in
the relative effect of yeast and beef extracts on DO depletion
at each location. The yeast extract caused a very pronounced
decrease in the acceleration phase as related to either of the
other substrates when incubated with water from the unpolluted
location (Fig. 6), while the effect in water from the polluted
location did not become apparent until later (Fig. 5). This
could mean (a) that one or more growth factors were added with
the sewage or (b) that the bacteria enhanced by domestic
pollution (Table 1) did not require the growth factors in yeast
extract and that those which required growth factors needed a
much longer time to utilize significant DO.
Results similar to those obtained with a high level of substrates
in unpolluted water (Fig. 6) were obtained with a low substrate
level, shown in Figure 7. In all cases, this low level of sub-
strate limited the amount of DO utilized. Growth factors added
in the yeast and beef extracts shortened the acceleration phase,
-------
Figure 5. Relative effect of three complex organic
substrates on dissolved oxygen depletion in sub-Arctic
river water polluted with raw domestic sewage and
effluents from primary treatment plants. Samples
were incubated at 10°C. Symbols: o , river water blank;
A, 120 mg/1 vitamin-free casamino acids; • , 106 mg/1
beef extract; D, 80 mg/1 yeast extract. All three
substrates contained equal amounts of carbon.
-------
to
20 30 40
TIME (HOURS)
50
-------
Figure 6. Relative effect of high levels (substrate not
rate limiting) of three complex organic substrates on
dissolved oxygen depletion in unpolluted sub-Arctic
river water when incubated at 10°C. Symbols: o , river
water blank; •, 120 mg/1 vitamin-free casamino acids;
A, 106 mg/1 beef extract; A , 80 mg/1 yeast extract.
All three substrates contained equal amounts of carbon.
-------
II
TIWE (HOURS)
-------
Figure 7. Relative effect of low levels (substrate being
rate limiting) of three complex organic substrates on
dissolved oxygen depletion in unpolluted sub-Arctic
river water when incubated at 10°C. Symbols: o , river
water blank; © , 30 mg/1 vitamin-free casamino acids;
D, 26 mg/1 beef extract; a , 20 mg/1 yeast extract.
All three substrates contained equal amounts of carbon.
-------
20
40 60 80
TIME (HOURS)
100
120
-------
but the extent of DO utilization with these.substrates was less
than with the vitainin-f.ree casamino acids. This suggested that
one or more ami no acids were required by a large portion of the
bacterial population and that there was a limiting amount
present in the extracts. Similar results were obtained at 0°,
5°, 15° and 20°C with this low substrate level. Since these
data would be redundant, they have not been shown.
The effect on DO depletion when nitrogen and phosphorus were
added to sub-Arctic river water in the presence of substrates
devoid of these nutrients.
Ammonia, nitrite and nitrate nitrogen and orthophosphate phosphorus
concentrations were determined by chemical analysis each time
samples were taken from either location, and the ranges of values
obtained are shown in Table 3. Both ammonia nitrogen and ortho-
phosphate phosphorus were increased by domestic pollution.
The results presented in Figures 8 and 9 indicated that glucose,
which contained the same amount of carbon as the vitamin-free
casamino acids control, was poorly utilized as a substrate
for DO depletion in Chena River water. Nitrogen and phosphorus,
in amounts equal to the amounts in vitamin-free casamino acids,
were added to the river water, which contained glucose. When
nitrogen alone was added to the system, little effect on the
DO depletion was observed with either polluted (Fig. 8) or
unpolluted (Fig. 9) water. The same was true for phosphorus
in the polluted water. However, when phosphorus was added to
the unpolluted water, DO depletion appeared to be enhanced to
some extent. This suggested that the amount of phosphorus
.naturally present was a limiting factor. When phosphorus and
nitrogen were both added, a very marked effect on DO depletion
in the presence of glucose was observed with either polluted or
unpolluted river water. This effect was more pronounced with
the unpolluted (Fig. 9) than with the polluted water (Fig. 8).,
because it altered both the extent of DO depletion and the time
span, while only the time span was changed in the polluted
water. These results also suggested that a portion of the
bacterial population was not active in DO depletion with
glucose as the substrate, possibly because the necessary growth
factors were not provided.
The effect of added nitrogen and phosphorus on DO depletion,
with ethyl alcohol (Fig. 10) and sodium acetate (Fig. 11) as
the substrates, was studied in unpolluted water. The carbon
content of both substrates and the amount of nitrogen and
phosphorus added were the same as in the vitamin-free casamino
acids control. Both of these substrates were even more poorly
utilized for DO depletion than was the glucose (Fig. 9) without
the addition of nitrogen and phosphorus. Again, as with glucose,
the addition of nitrogen and phosphorus enhanced the utilization
-------
TABLE 3
Chemical analysis of water samples from
two locations on a Sub-Arctic river
Determination
Dissolved Oxygen
Ammonia Nitrogen
Nitrate Nitrogen
Nitrite Nitrogen
Orthophosphate
Phosphorus
Range of Values (mg/l/hr)
Polluted Sample
Location3
2.5 - 5.9
0.40 - 0.83
0.02 - 0.09
0.003 - 0.007
0.02 - 0.08
Unpolluted Sample
Location'3
3.5 - 8.0
0.06 - 0.18
0.03 - 0.12
0.001 - 0.004
<0.01 - 0.02
a. The polluted location was below a reach of the river receiving .
raw domestic sewage and effluents from primary sewage treatment
plants. The range of values.is from 10 samples taken between
December 16., 1969 and April 7, 1970.
b. The unpolluted location was upstream from any source of domestic
or industrial pollution. The range of values is from 7 samples taken
between December 10, 1969 and April 14,1970.
-------
Figure 8. Effect of glucose on dissolved oxygen depletion
in sub-Arctic river water polluted with raw domestic sewage
and effluents from primary treatment plants. Incubation at
10°C in the presence and absence of added inorganic nitrogen
and phosphorus. Symbols: O , river water blank; A , 120 mg/1
vitamin-free casamino acids as a control; B , 80 mg/1 glucose;
D, 80 mg/1 glucose, KH?PO, (0.33 mg/1 phosphorus) and
K2HP04 (0.33 mg/1 phospRorfts); • , 80 mg/1 glucose, (NH4)?SO,
(3.33 mg/1 nitrogen) and KN03 (10 mg/1 nitrogen); A , 80 mg/1
glucose, I<2HP04, KH2P04, (NH4)2S04 and KN03 (nitrogen and
phosphorus in same amounts as above). The glucose, KoHP04,
KH2P04, (NH4)2S04 and KNOa were added to give the same level
of carbon, phosphorus, and nitrogen as found in the casamino
acids control.
-------
II
10
20
30
40 50 60
TIME (HOURS)
7 0
8 0
90
100
-------
Figure 9. Effect of glucose on dissolved oxygen depletion in
unpolluted sub-Arctic river water when incubated at 10°C in the
presence and absence of added inorganic nitrogen and phosphorus.
Symbols: o , river water blank; A , 120 mg/1 vitamin-free casa-
mino acids as a control; • , 80 mg/1 glucose; A , 80 mg/1
glucose, KHgPO^ (0.33 mg/1 phosphorus) and K2HP04 (0.33 mg/1
phosphorus); n , 80 mg/1 glucose, (NH4)2S04 (3.33 mg/1 nitrogen)
and KN03 (10 mg/1 nitrogen);.. • , 80 mg/1 glucose, K2HP04,
KH2P04 and KNO^ (nitrogen and phosphorus in same amounts as
above). The glucose, K2HP04, KH2P04, (NH4)2S04 and KN03 were
added to give the same level of carbon, phosphorus, and nitrogen
as found in the casamino acids control.
-------
100 120
140
160 160 2OO
TIME (HOURS)
-------
Figure 10. Effect of ethyl alcohol on dissolved oxygen depletion
in unpolluted sub-Arctic river water when incubated at 10°C in
the presence and absence of added inorganic nitrogen and phos-
phorus. Symbols: © , river water blank; A , 120 mg/1 vitamin-
free casamino acids as a control; ©, 60 mg/1 ethyl alcohol;
a , 60 mg/1 ethyl alcohol, K2HP04 (0.33 mg/1 phosphorus),
KH2P04 (0.33 mg/1 phosphorus), (NH4J2S04 (3.33 mg/1 nitrogen)
and KNOa (10 mg/1 nitrogen). The ethyl alcohol, K2HP04, KH2P04,
(^4)2804, and KNOg were added to give the same level of carbon,
phosphorus and nitrogen as found in the casamino acids control.
-------
II
10
•s,
o
t-
UJ
_l
o.
Ul
o
z
UJ
o
>"
X
o
o
UJ
O
CO
CO
20 40 60 80
TIME (HOURS)
100
120
-------
Figure 11. Effect of sodium acetate on dissolved oxygen depletion
in unpolluted sub-Arctic river water when incubated at 10°C in the
presence and absence of added inorganic nitrogen and phosphorus.
Symbols: O , river water blank; A , 120 mg/1 vitamin-free casamino
acids as a control; •, 180 mg/1 sodium acetate; A , 180 mg/1
sodium acetate, K2HP04 (0.33 mg/1 phosphorus), KH2P04 (0.33 mg/1
phosphorus), (NHJ2S04 (3.33 mg/1 nitrogen) and KN03 (10 mg/1 nitrogen).
The sodium acetate, K2HP04, KH2P04, (NH4)2S04 and KN03 were added
to give the same level of carbon,'phosphorus, and nitrogen as found
in the casamino acids control.
-------
40 60
TIME (HOURS)
100
120
-------
of ethyl alcohol and sodium acetate. The DO depletion with the
-vitamin-free casamino acids control was still greater even
though the utilization in the presence of these substrates was
enhanced. This is added support for the role of growth factors
in the metabolic activity of the bacterial population.
Both yeast and beef extracts contained slightly less ammonia
nitrogen than did the vitamin-free casamino acids. The addition
of ammonia nitrogen had no effect on the utilization of DO with
either extract in polluted or unpolluted water, since the results
were identical to those shown in Figures 5 and 6.
Effect of sewage treatment plant effluents on DO depletion in
unpolluted sub-Arctic river water.
The primary sewage treatment plant effluent contained 24 mg/1
ammonia nitroqen, 0.01 mg/1 nitrite nitrogen, 0.15 mg/1 nitrate
nitrogen, 3.4 mg/1 orthophosphate phosphorus and 235 mg/1
chemical oxygen demand (COD). This effluent was added to un-
polluted river water in an amount which gave a final COD of 59
rng/1. These results are shown in Figure 12. Oxidizable substrate,
growth factors and inorganic nutrients in the effluent permitted
rapid DO depletion at all incubation temperatures. This DO
depletion was more rapid than with a high level of vitamin-free
casamino acids (Fig. 4). Since the indigenous population in the
river water had no discernible effect on DO depletion at any in-
cubation temperature, it appeared that the effluent had a bacterial
population capable of rapid and extensive activity.
The effect of effluent from an activated sludge sewage treat-
ment system on DO depletion in unpolluted water is shown in
Figure 13. Effluent from the activated sludge system operating
at 0°-1.0°C was added to unpolluted river water, giving a final
COD of 16 mg/1. The results showed that DO depletion activity
increased with increasing incubation temperature. This suggested
that either a change in growth factor requirements or different
enzyme systems made more substrate available for utilization
at the higher incubation temperatures. The bacterial population
in the river water appeared to have some effect on the extent
of DO depletion at 10° and 20°C, since the rate and extent
of depletion was increased when the effluent was incubated in
river water.
Effect'of incubation temperature on DO depletion in Arctic
water in the presence of a complex organic substrate.
Vitamin-free casamino acids at a concentration of 120 mg/1 was used
as the substrate for DO depletion studies in Arctic river water
(Sag River). The results, given in Figure 14, showed a lag
phase at all incubation temperatures before DO depletion began.
-------
Figure 12. Effect of incubation temperature on dissolved oxygen
depletion when effluent from the Fairbanks, Alaska city primary
sewage treatment plant was added to unpolluted sub-Arctic river
water. Symbols: O , 25% effluent and 75% river water; • ,
25% effluent and 75% sterile glass distilled water; • , 25%
sterile glass distilled water and 75% river water.
-------
20
40
60 80
TIME (HOURS)
100
120
140
160
-------
V
Figure 13. Effect of incubation temperature on dissolved oxygen
depletion when effluent from a 0°-0.5°C bench scale activated
sludge sewage treatment system was added to unpolluted sub-
Arctic river water. Symbols: A, , 25% effluent and 75% river
water; O , 25% effluent and 75% sterile glass distilled water;
O , 25% sterile glass distilled water and 75% river water.
-------
~ J2 '
o
p
o
CO
CO
o
40
120
160 200 240
TIME (HOURS)
230 320 360 40O'430
-------
Figure 14. Effect of incubation temperature on dissolved oxygen
depletion when 120 mg/1 vitamin-free casamino acids was added to
unpolluted Arctic river water. A river water blank (o) was in-
cubated at 0°, 10° and 20°C.
-------
0 40
80 120 160 200
240 280 500 540
TIME (HOURS)
580 620 630 700
-------
The lag phase was extremely long at the lower temperatures,
particularly at 0°C. However, the extent of DO depletion did
not appear to be temperature dependent. It was shown previously
(14) that the Sag River had a large population of heterotrophic
bacteria capable of growth at low temperatures. Since only one
large volume sample was available from the Sag River, the reason
for the extended lag phase remains to be determined.
The same substrate concentration and incubation temperatures
made it possible to relate the results from both the Sag
(Fig. 14) and Chena (Figs. 3 and 4) rivers. One outstanding
point was the relative time before the start of DO depletion.
There was a lag phase only at the 0°C incubation temperature
with samples from the polluted location on the Chena River
and the lag phase was apparent only at 0°, 5° and 10°C with
samples from the unpolluted location. An extended lag phase
at all temperatures was observed with samples from the Sag
River. A point of similarity with all samples was that the
extent of the DO depletion did not appear to be temperature
dependent.
The results shown in Figures 3, 4 and 14 did not fit either
the first or second order kinetic forms so a rate constant
.was not obtained. Approximate rates (mg/l/hr) of DO depletion
were obtained directly from the depletion curves, and the re-
sults are presented in Table 4. It must be stressed that these
results are approximations and have value only in the context
of this study. It would seem reasonable to have found the
highest rates of DO depletion with polluted Chena River water.
However, unpolluted Chena River water apparently gave higher
rates than the polluted equivalent at 15° and 20°C. The rates
from both Chena River samples were nearly the same at 0°, 5°
and 10°C. The sample from the Sag River gave lower rates at
10°, 15° and 20°C than either Chena River sample. This
suggested that the bacteria from the Sag River were more
adversely affected by the higher incubation temperatures than
those from the Chena River. Additional support for this
suggestion was the -nearly equal rates found at 15° and 20°C
with Sag River water. The results showed that the source of
the sample had little or no effect on the rate of DO depletion
at 0° and 5°C. This suggested that all or part of the
bacterial population from each source had the same ability
to utilize an organic substrate at low temperatures.
-------
TABLE 4
Comparison of the rate of dissolved oxygen depletion when a
substrate was added to Arctic and sub-Arctic river water samples
Incubation
Temperature
20°
15°
10°
5°
0° :
Rate of Dissolved Oxygen Depletion9 (mg/l/hr)
Sub-Arctic River
Polluted Sample
Location'3
1.36
0.92
0.63
• 0.22
0.22
Unpolluted Sample
Locationc
1.73
1.13
0.53
0.26
0.25
Arctic River
Unpolluted Sample
Location
0.62
0.65
0.35
0.20
0.20
a. 120 mg/1 Vitamin-Free Casamino Acids (Difco) was added to each
river water sample.
b. The polluted location was below a reach of the river receiving
raw domestic sewage.and effluents from primary sewage treatment
plants.
c. The unpolluted location was upstream from any source of
domestic or industrial pollution.
-------
DISCUSSION AMD CONCLUSIONS
A sub-Arctic (Chena) river had a population of heterotrophic
bacteria capable of growth at 0°C on a complex medium. With
dissolved oxygen (DO) depletion as the measurement, there
appeared to be little metabolic activity in a closed, station-
ary river water system. When vitamin-free casamino acids was
added to the stationary system, there was rapid and extensive
DO depletion at all incubation temperatures (Fig. 4). The
rate of DO depletion appeared to still be increasing at the
lower incubation temperatures when the oxygen was exhausted,
which suggested the maximum rate had not been reached. Thus,
oxygen may have been limiting.
Jezeski and 01 sen (19) found that shake cultures increased
growth rate and maximum growth level of Pseudomonas f1uorescens
at 4° and 10°C as compared to stationary cultures. In the
shake cultures, oxygen was no longer a limiting factor, and the
bacterial cells were kept in a constantly changing micro-
environment which removed metabolic end products and brought
the cells in contact with new substrate. Such a dynamic
system more nearly simulates environmental conditions in a
river than does a stationary system. The indigenous bacteria
had the potential for rapid metabolic activity in a stationary
system. Next, a dynamic system must be studied to more
accurately assess the role of these bacteria in the natural
environment and the effects of added substrates, such as sewage
effluents.
Metabolic activity observed with the protein derivative,
vitamin-free casamino acids, as the substrate was not as rapid
as with yeast or beef extract (Fig. 6). These extracts con-
tained growth factors, and carbohydrates in addition to
proteinaceous material. It was apparent that one or more growth
factors were responsible for the increased metabolic activity.
However, it is not known whether the growth factors enhanced
the activity of all or part of the bacterial population, or
whether a portion of the population had an absolute growth
factor requirement. An understanding of the role of growth
factors is necessary as an aid in developing design criteria
for sewage treatment plants that will provide sewage effluents
which minimize the demand for DO.
In a review of psychrophilic bacteria, Ingraham and Stokes
(17) pointed out that they could carry out nearly all meta-
bolic activities at low temperatures, but at a slower rate
than at higher temperatures. Several psychrophilic and
-------
mesophilic Arthrobacter species were studied for effect of
temperature on growth by Roth and Wheaton (36). Rather than
a sharp cut-off point, there was a continuous gradation with
a decreasing lag phase at 0°C and an increasing one at 37°C.
The longest lag phase they measured at 0°C was about 300 hours
before the start of fairly rapid growth. They concluded that
the number of generations of a specific bacterium was not
temperature dependent, but the time to attain a certain number
was extended at lower temperatures.
The decreasing rate of metabolic activity with decreasing in-
cubation temperature which was reported previously (17) appeared
to be borne out by the results reported here (Table 4). This
was true with samples from above and below the polluted reach
of the Chena River. Several significant effects on metabolic
activity in the samples were noted after the Chena River had
flowed through the polluted reach. The lag phase before the
start of DO depletion was much shorter (Fig. 3) than with
samples from above (Fig. 4), which resulted in a much shorter
elapsed time from the start of incubation until all of the
DO had been utilized. The apparent effect of growth factors
on the rate of DO depletion was reduced (Figs. 5 and 6), and
glucose was more effectivley utilized as a substrate (Figs. 8
and 9). These effects on metabolic activity indicated that
raw sewage and primary treatment plant effluents added a high
level of organic substrates, growth factors, nitrogen and phos-
phorus to the river water. In addition to the nutrients, the
results presented in Figure 12 showed that bacteria capable
of rapid metabolic activity at low temperatures were present
in the primary treatment plant effluent. Because of these
factors, raw sewage and primary effluents would probably
significantly increase the DO demand under ice cover.
McDonald, et^ al_. (21) found proteolytic bacteria in Arctic
littoral and marine sediments. They found that proteolytic
enzymes were highly active at low temperatures and proposed
that these enzymes might be significant in protein degradation
in the Arctic. Rapid and extensive DO depletion was found in
Chena River water at low temperatures with protein derivatives
as the substrates. This suggested that proteolysis is one of
the major metabolic activities of the bacteria in the Chena
River.
A large variety of proteolytic bacteria have been found in
sewage treatment systems (15). Since proteolytic activity
has been found at low temperatures, there are probably similar
bacteria present in sewage treatment systems operating at low
temperatures. Support for this suggestion was obtained from
an activated sludge sewage treatment system operating at 0°C.
-------
This system reduced the DO requirements of domestic sewage to
a level that appeared to be of minimal influence in Chena River
water at 0°C (Fig. 13).
Earlier work with pure cultures of Pseudomonas fluorescens
showed that glucose was a poor substrate for DO utilization
at low temperatures (19) and that the generation time was much
longer than with vitamin-free casamino acids as the substrate
(27). This may have resulted from a change in the metabolic
pathway of glucose utilization at low temperatures (19,29), or
that more glucose was consumed for cell maintenance at low
temperatures (28). Inoue, et_ al_. (18) found that acetate-
oxidizing bacteria were vital in self-purification of rivers.
When substrates (glucose, sodium acetate and ethyl alcohol)
which did not contain nitrogen, phosphorus or growth factors
were added to the closed, stationary river water system, a
low level of metabolic activity resulted. Adding nitrogen
and phosphorus resulted in a marked increase in activity
(Figs. 9, 10 and 11). Even with these nutrients present, the
rate of DO depletion was greater with the vitamin-free casamino
acids. This suggested that a portion of the bacterial popu-
lation either required additional growth factors, or was not
capable of utilizing these substrates. Even though bacteria
capable of utilizing these substrates were present in the
Chena River, activity would probably be at a low level because
of the limited amount of nitrogen and phosphorus present under
natural conditions.
Bacteria were found to have an important role in the cycling
of phosphorus in the aquatic environment (31), and both phos-
phorus and nitrogen appeared to be effective in limiting
metabolic activity in the Chena River under natural conditions
(Fig. 9). Nitrogen and phosphorus present in raw sewage and
primary sewage treatment plant effluents reduced the limiting
effect of these nutrients (Fig. 8). Therefore, a method must be
found to control nitrogen and phosphorus in effluents entering
Arctic or sub-Arctic waters. Barth, et^ al_. (4) demonstrated that
it is feasible to remove both nitrogen and phosphorus on a pilot
plant scale using a combined chemical-biological removal system.
Since several methods are available (26), the "State of the Art"
of phosphorus removal is probably much more advanced than nitrogen
removal. Perhaps the initial efforts should be directed toward
adapting a phosphorus removal method.
Throughout this study, nitrogen was supplied in the form of
ammonia and nitrate at the levels present in the vitamin-
free casamino acids. It is necessary to determine if the form
nitrogen is in has any effect, and what concentration is actually
required. This should aid in determining what could be done to
control the effect of nitrogen on receiving waters.
-------
Results obtained with Arctic river water were far too limited
to be conclusive. However, there are some general similarities
between the Arctic (Fig. 14) and sub-Arctic rivers (Figs. 3
and 4). More detailed study is necessary before the effects
of pollutants on Arctic rivers can be defined.
It is becoming increasingly obvious that the 5 day, 20°C BOD
(biochemical oxygen demand) has very limited usefulness in the
Arctic or sub-Arctic because the receiving waters rarely reach
this temperature. Previous studies by Murphy and Miller (25),
Reid and Benson (34), and Reid (33) showed that a 20 day BOD,
incubated at a low temperature with receiving water or seed
culture acclimatized at a low temperature, gave more realistic
results with raw sewage. The results presented here (Figs. 12
and 13) showed that incubation temperature and diluent had an
effect on DO depletion with sewage treatment plant effluents.
Therefore, it is suggested that the receiving water should be
used as the diluent and the incubation temperature should be
at or near the temperature of the receiving water.
-------
ACKNOWLEDGMENTS
Mrs. Becky L. Quimby for her able assistance in the laboratory.
Mr. Ernst W. Mueller and his staff for providing the chemical
data presented here.
Mr. Michael A. Angelo for assistance in the mathematical
treatment of the data.
Mr. Sidney E. Clark for help in developing the equipment
used for aeration and temperature equilibration of the
samples.
-------
Use of product and company names is for identification only
and does not constitute endorsement by the U.S. Department
of the Interior or the Federal Water Quality Administration.
-------
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11. Fournelle, H.J., "Soil and Water Bacteria in the Alaskan
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-------
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