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,

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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.

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to
20     30     40
 TIME (HOURS)
50

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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.

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II
                            TIWE (HOURS)

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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.

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20
40     60     80
 TIME (HOURS)
100
120

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 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

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                               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.

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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.

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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.

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     100     120
140
160    160    2OO
TIME (HOURS)

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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.

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  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

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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

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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.

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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.

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~   J2 '
o
p
o
CO
CO

o
            40
120
160    200    240


    TIME  (HOURS)
230    320     360    40O'430

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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.

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0  40
80   120     160   200
240    280      500    540



    TIME  (HOURS)
580    620    630    700

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 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.

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                              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.

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                      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

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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.

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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.

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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.

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                           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.

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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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                               REFERENCES
 1.   Adams, J.C.  and Stokes,  J.L.,  "Vitamin  Requirements  of
     Psychrophilic Species  of Bacillus,"  Journal  of Bacteriology,
     95_, pp 239-240 (1968).

 2.   American Public Health Association,  Standard Methods for
     the Examination of Water and Wastewater,  12th Edition,
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