DEPLETION OF OXYGEN BY MICROORGANISMS IN ALASKAN RIVERS AT LOW TEMPERATURES FEDERAL WATER QUALITY ADMINISTRATION NORTHWEST REGION ALASKA WATER LABORATORY College, Alaska ------- 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 ------- A Working Paper presents results of investigations which are to some extent limited or incomplete. Therefore, conclusions or recommendations—expressed or implied—are tentative. ------- 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 ------- 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. ------- 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. ------- 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 ------- 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. ------- 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}"*. ------- 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. ------- 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. ------- 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- ------- 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. ------- ------- 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. ------- 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. ------- 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 ------- 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. ------- 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. ------- 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. ------- 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. ------- TIME (HOURS) ------- 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. ------- 0 yo 40 60 80 100 120 TIME (HOURS) 140 160 180 200 ------- 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. ------- 40 80 120 160 200 TIME (HOURS) 240 280 ------- 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. ------- 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, American Public Health Association, Inc., New York (1965). 3. Association of Official Agricultural Chemists, Official Methods of_ Analysis p_f the Association of_ Agricultural Chemists. 10th Edition, Association of Official Agricultural Chemists, Washington, D.C.(1965). 4. 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