“v— —’v vv- “ “v I'vw wv ‘-A»A._« ! THE EVALUATEON 0F BAGERML GROWTH RAYE CQNSETANTS? F09; MUNICIPAL WASTES Thanh for the Dawn 35 M. 5. IfiiCHEEAR STEE'E UI‘SWERSETY Jacob Nichotas Dick 1954 THESIS .‘ mminmmmr‘mnmjm .J LIBRARY 31293 00377 068 4 Michigan State Universuy This is to certify that the thesis entitled ‘ THE EVALUATION OF BACTERIAL GROWTH * RATE CONSTANTS FOR 3 MUNICIPAL WASTES presented by Jacob Nicholas Dick has been accepted towards fulfillment of the requirements for LA. S. degree in Sanitary Engineering {>9 1/ MIA? I Major professor f : Date MQLZZ. 1964 IAst ’66 .-—-== - ABSTRACT THE EVALUATION OF BACTERIAL GROWTH RATE CONSTANTS FOR MUNICIPAL WASTES by Jacob Nicholas Dick This thesis reports the results of a study made on several municipal wastes to determine the growth rate con- stants of bacteria and their relationship to B.O.D. The growth rate constants were determined by measuring the respiration rates per unit sample volume in the Warburg apparatus. A semi-logarithmic plot was then made of the respiration rates and the slope of that portion of the curve which displayed a straight line was taken to be the growth rate constant. It was found that an approximately linear relationship existed between the growth rate constant and the B O.D. at low B.O.D. concentrations and that the growth rate constant became independent of the B.O.D. at high concentrations. The maximum hourly respiration rates per unit sample volume were related to the B O.D. It was found that a linear relationship existed between the maximum hourly respiration rate per unit sample volume and the initial B.O.D. concentration. THE EVALUATION OF BACTERIAL GROWTH RATE CONSTANTS FOR MUNICIPAL WASTES By Jacob Nicholas Dick A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Civil Engineering 1964 m ACKNOWLEDGMENT The author wishes to express his indebtedness and appreciation to Dr. K. L. Schulze, Department of Civil Engineering, Michigan State University, whose generous help and encouragement made this thesis possible. ii TABLE OF CONTENTS ACKNOWLEDGMENT . LIST OF FIGURES. LIST OF TABLES LIST OF SYMBOLS. l. 2. CDNChU'I O O 0000 LITERATURE REVIEW PROCEDURE . . . . . 2.1 Sampling . . . . . . . . . 2.2 SuSpended Solids Determination. 2.3 Biochemical Oxygen Demand Determination. 2.31 Standard B.O.D5 . . . . . 2.32 Dissolved B.O.D . 2.4 Warburg Apparatus Procedure. THEORY . . RESULTS. 4.1 Respiration Rates 4.2 Growth Rate Values and B.O.D. DISCUSSION. CONCLUSION. APPENDICES. . . . . . . BIBLIOGRAPHY . . . iii Page ii iv vi \OKOKOKOKO 10 ll 15 17 17 21 2a 28 29 72+ Figure 1-8. 10. ll. 12. 13. 1A. 15-17. 18-22. LIST OF FIGURES Relationship of Respiration Rate and Time Typical Respiration Rate Plot of Unfiltered Sample with Glucose Added. Typical Respiration Rate Plot of Filtered Sample with Glucose Added Typical ReSpiration Rate Plot of Unfiltered Sample with Glucose Added. Typical Respiration Rate Plot of Filtered Sample with Glucose Added Typical ReSpiration Rate Plot of Unfiltered Sample with Glucose Added. Typical Respiration Rate Plot of Filtered Sample with Glucose Added Plot of Maximum Respiration Rate Versus B.O.D5 . . Plot of Growth Rate Constant klO and B.O.DS. . . . . . . . . . iv Page 29-36 37 38 39 40 Al 42 43-45 46—50 21—22. LIST OF TABLES Page Respiration Rates . . . . . . . . . 51-69 Type of Sample, S.S., B.O.D., and pH. . . 70 Growth Rates, Maximum Respiration Rate, and B.O.DS . . . . . . . . . . 71-73 10 mg/l mg/l/hr B.O.D.; B.O.D5 m1. ul COOOD. LIST OF SYMBOLS a constant defining the rate of deoxygenation growth rate constant to the base 10 growth rate constant to the base e milligrams per litre milligrams per litre per hour Biochemical Oxygen Demand 5-day Biochemical Oxygen Demand millilitres milligrams microlitres millimetres oxygen parts per million per hour suSpended solids hours dissolved oxygen inverse logarithm of the hydrogen ion concentration Chemical Oxygen Demand vi 1.0 LITERATURE REVIEW Phelps, as found in Clark (12, p. 9), was the first to establish a mathematical relationship describing the observed rate of deoxygenation of sewage. The law may be expressed as: d (L ‘ Lt) = Kt dt where, K = a constant defining the rate of deoxygenation. Lt = the organic matter at time (t), or the oxygen requirement of the sample at time (t). L = the organic load present at the beginning. Pleissner, as found in Clark (12, p. 12), also sought to derive an expression of the rate of deoxygenation of polluted waters on the assumption that the velocity of the reaction depended not only upon the concentration of organic matter but also upon the concentration of dissolved oxygen. Realizing the importance of referring to a standard point on the deoxygenation curve when comparing sewages, he proposed a forty-eight hour incubation period. Using this incubation period he derived a mathematical expression as follows: mt = 17.0 log2 t where, mt = a number that when divided into the oxygen demand value for the time t gives the standard hourly oxygen loss. t = incubation time in days. Mueller as found in Clark (12, p. 14), compared the relationship between bacterial count and hourly loss of oxygen in water using Bacillus fluorescens liquefaciens and Bacterium 0011. He found that a direct proportionality did not exist between the two parameters but that the maximum loss of oxygen per hour did correspond to the maximum bac- terial count. "The Oxygen Demand of Polluted Waters" written by Theriault (32) and published by United States Public Health Service as Public Health Bulletin Number 123_in 1927 ranks as one of the great classics of this field. The report con- sists of two parts: first a critical review of the pertinent literature to that date, and second an experimental verifi- cation of the rate of sewage deoxygenation. The first, or carbonaceous, stage of deoxygenation was found to conform to the monomolecular formula proposed by Phelps previously. Penfold (25) in 1912 stated that little attention had been paid to the influence of the concentration of the culture medium employed when considering the generation time of bacteria. Working with a culture of Bacillus typhosus, Penfold showed that the rate of growth was greatly influenced by a peptone concentration when it is below 0.4 per cent. The generation-time was inversely proportional to the con- centration of peptone at values below 0.2 per cent. Callow (lO) washed bacteria free from culture medium and investigated the oxygen uptake rate. Various cultures of bacteria were used and the oxygen uptake rates varied from 5 to 25 c.c. of oxygen per gram of dry weight under these conditions. Butterfield (6) explored the relationship between food concentration and bacterial growth in 1929. He found that in pure cultures of Bacterium aerogenes the relationship between the limiting bacterial numbers and the concentra- tion of food supply was logarithmic. In the case of grossly mixed cultures including plankton, the increase in the limiting bacterial numbers produced by an increase in food concentration was always less than in the case of pure cul- tures. Martin (21) measured the oxygen consumption of Escherichia coli during the lag and logarithmic phases of growth. He observed that the rate of oxygen consumption per cell increased rapidly from the time of inoculation to ’ a point of maximum respiration near the end of the lag phase of the growth curve. The maximum surface area per cell coin— cided with the point of maximum oxygen consumption per cell. Lea and Nichols (19) showed the importance of certain essential elements in a bacterial medium. They stated that maximum bacterial growth can only be obtained when sufficient cell-building elements, such as nitrogen, phosphorus, and potash are present in the substrate. Using pure cultures of bacteria isolated from activated sludge, Butterfield, Ruchhoft, and McNamee (7) found that the addition of fresh nutrient substrate to these pure culture activated sludges under aeration very greatly increased the quantities of oxygen utilized. Butterfield and Wattie (8) explained this fact by stating that the rate of oxidation of bacterial food during the first hours of incubation was dependent upon the number of living bacteria. The greater the number of bacteria, the more extensive was the initial oxidation. The rate of oxidation was also found to be influenced by the degree of dispersion of bacteria, or bacterial floc. Adequate dis- persion was necessary to produce extensive oxidation. In 1939 Sawyer and Nichols (28) studied the effect of various factors on the oxygen utilization rate of acti- vated sludge. They found that the oxygen uptake rate was directly proportional to the sludge concentration. At higher temperatures, the rate of oxygen utilization greatly exceeded that at the lower temperatures. The change in activity with temperature, however, was not linear. Grieg and Hoogerheide (17) determined in 1941 that the oxygen uptake of growing cultures of bacteria was directly proportional to the bacterial content and that rate of oxygen uptake constituted a convenient method for the measurement of the rate of growth. They measured the oxygen consumption in Warburg manometers and found it to be directly propor— tional to the bacterial content when the latter was deter— mined nephelometrically. Monod (23) reported extensive studies on the growth of bacteria under aerobic conditions in simple media con- taining a single carbohydrate. The increase in cell mass of Escherichia coli and Bacillus subtilis grown on many different carbohydrates was found to be directly propor- tional to the initial concentration of the nutrient, indicating that exhaustion of the food supply was the factor limiting growth. The rate of growth was studied in relation to the concentration of nutrient and was found to follow the relationship, k = km 0 c' + c where, km = maximum rate of growth c = concentration of nutrient remaining 0' = a constant In 1946 a comprehensive study was released by the Subcommittee on Sewage Treatment at Military Installations (24) on all phases of sewage treatment and on oxygen demand characteristics. The average value of K was found to be 0.18, but extreme values ranging from 0.10 to 0.30 were reported. Hinshelwood (18) in 1947 stated that the logarithmic law was found to be a very good approximation of bacterial growth over quite a wide range of conditions, but lacked absolute character. He also stated that over quite wide ranges of media concentrations the growth rate was almost independent of concentration and not until very dilute con- centrations was there a decrease in the growth rate. He pointed out that a quantitative effect of the media on growth would be difficult to establish since the growth rate also depended upon the degree of adaptation. Ruchhoft, Placak, Kackmar, and Calbert (26) found the value of K and L in a series of B.O.D. determinations to be dependent upon the sewage concentration to a large extent. They observed that in their specific case at a dilution of one per cent or greater consistent results were obtained, whereas below this dilution the data became inconsistent and were somewhat effected by seeding. Caldwell and Langelier (9), working at the University of California, used the direct oxygen utilization method for sewage analyses. They reported a proportionately higher rate of oxygen demand in undiluted sewage samples. They reported the concentration of the sewage to be a factor in the rate of oxidation but that a limit existed above which the reaction velocity was not proportionally increased. Garret and Sawyer (l6) 1J1 studies pertaining to the removal of soluble B.O.D. and oxygen utilization of mixed cultures developed from a sewage seed showed that the growth of the organisms in mixed cultures was in agreement with the laws of growth found by investigators to be applicable to pure cultures of bacteria. At high concentrations of B.O.D. the rate of growth was constant, and at low concentrations the rate of growth was directly proportional to the remain- ing soluble B.O.D. Smith (31) studied the respiratory activity of acti- vated sludge. Within a range of about 0.2 to 6 ppm of dis- solved oxygen the unit rate of oxygen utilization was not significantly affected. The oxygen uptake rate decreased with increasing sludge age and increased with increasing B.O.D. loading. Buswell, Mueller, and Van Meter (5) discussed the rate of oxygen consumption in the B.O.D. test. They found a direct relationship between maximum bacterial population and total pollutional load. They suggested that the oxygen consumption curve consists of two rates (1) a higher rate associated with cell multiplication and (2) a lower rate associated with resting or dying cultures. Longmuir (20) used a polarograph to determine the re- lation between respiration rate of bacteria and the concen- tration of dissolved oxygen. The relationship could be expressed in the form of the Michaelis-Menten equation. Balmat (2) discussed the rate at which settleable and supra-colloidal sewage solids underwent decomposition and found that the larger particles were characterized by a relatively low rate of biochemical oxidation due to the slow action of the hydrolyzing exoenzymes of the bacteria. Fisichelli and Palombo (14) in 1960 analyzed a total of 170 24-hour composite samples of raw and primary efflu- ent to determine the velocity reaction constant K. For raw sewage samples they found K to range from 0.05 to 0.29 with a mean of 0.16 and for primary sewage samples they measured K values from 0.055 to 0.29 with a mean of 0.15. Sawyer (27) stated that for a great many years the B.O.D. reaction was considered to have a rate constant K equal to 0.10 per day at 2000. As the application of the B.O.D. test spread to the analyses of industrial wastes and the use of synthetic dilution water became established, it was noted that the K values of different waste materials varied considerably from the accepted value of 0.10 per day. It was also noted that the K value varied from day to day as shown by Schroepfer, Robins, and Susag (29) who reported values of experimentally determined rate constants from the Minneapolis-St. Paul Sanitary District and Mississippi River. Schuller (30) using the Warburg technique in 1961 con- cluded that it was possible, by multiplication with a constant, to calculate the total oxygen demand after the highest hourly respiratory value had been determined. 2.0 PROCEDURE 2.1 Sampling Two sampling procedures were used in this study. The first procedure consisted of collecting a series of grab samples at one sewage treatment plant throughout the day. When this method was used the samples were collected in screw cap bottles and stored in a refrigerator until used. The second procedure consisted of obtaining grab samples from different sewage treatment plants for compar- ison. The samples in this case were obtained in gallon Jugs and stored in a pail of ice until used. All samples were collected from the primary effluent of the respective sewage treatment plants. 2.2 Suspended Solids Determination The total suspended matter was determined in this study as described in Standard Methods (1). 2.3 Biochemical Oxygen Demand Determination 2.31 Standard B.O.D:§.--The standard 5—day Biochem- ical Oxygen Demand was determined as described in Standard Methods (1). An equivalent amount of potassium nitrate, however, was utilized as a nitrogen source instead of the ammonium chloride as stated in Standard Methods to prevent errors due to nitrification. Gaffney and Heukelekian (15) 10 have recommended a nitrate source of nitrogen rather than an ammonium source to reduce nitrification. The dilution water control was performed by filling four B O.D. bottles with the dilution water and determining the dissolved oxygen content of two of these bottles im- mediately. The other two bottles were set into the B.O.D. incubator for 5 days and on the fifth day the dissolved oxygen content was determined for these bottles. In all cases the Alsterberg azide modification of the Winkler method as found in Standard Methods (1) was employed. The two initial D.0. determinations were repeated until two consecu- tive determinations within 0.1 mg/l of D.0. were observed. If either of the incubated dilution water control bottles showed an oxygen uptake of 0.2 mg/l, or above, the B.O.D. determinations were considered unsatisfactory. A11 B.O.D. bottles were washed with chromic acid solution and then rinsed several times with distilled water before being used. The incubated B.O.D bottles were examined from time to time to insure a good water seal. 2.32 Dissolved B.O.D.—-The dissolved B.O.D. was deter- mined by filtering the sample through a Gooch crucible. The filter was made up as described in Section 2.2. The B.O.D. was determined on the filtrate as described in Section 2.31. 11 2.4 Warburg Apparatus Procedure Before the Warburg flasks were used the grease from the glass joints was removed with a piece of cheese cloth dipped in unleaded gasoline. The flasks were next placed in a Hemosol soap solution for 12 hours, rinsed with water, washed in chromic acid solution, and rinsed 5 times with distilled water. A 20 per cent potassium hydroxide solution was placed in the centre well of the flask to absorb the carbon dioxide that was given off. To increase the surface area of the potassium hydroxide a small folded piece of filter paper was placed into the centre well. As previously mentioned two sampling procedures were used. When the first procedure was used duplicate flasks were set up each containing 5 ml of the sample. The dupli- cation was made to determine the reproduction of results. When the second procedure was used a part of the sample was filtered through a Gooch crucible as described in Section 2.2. This was done to eliminate any particulate matter from contributing to the B.O.D. so that only soluble B.O.D. would contribute to the growth rate. Then ten 100 m1 graduates were set in a row, five were filled with the filtered sample and the remaining 5 filled with the unfil— tered sample. To four graduates, both of the filtered and unfiltered sample, the following amounts of glucose were added: 6.7 mg, 13.4 mg, 26.8 mg, and 53.6 mg. These 12 quantities of glucose were added to increase the original B.O.D5 by 50, 100, 200, and 400 mg per litre respectively. The five filtered samples were next seeded with 1 ml of the original primary effluent per 100 m1 of sample. Five ml of sample was carefully transferred from each of the 10 gradu- ates to 10 Warburg flasks. The pipette was cleaned with distilled water after each 5 ml sample had been transferred. Stop-cock grease was next applied to the side-arm stopper of the flask and the manometer arm to insure that no air leaks would develop. The flasks were attached to the manometer, with the valves on the manometer open, placed in the water bath, and allowed to shake for approximately 15 minutes. This was done to allow the temperature and pressure to equalize. The shaker was stopped, all valves closed, the manometer fluid adjusted to read 150 mm on each leg and the apparatus again started. The shaking rate employed in this study was 85 oscillations per minute. The manometers were read at approximately 1-hour intervals and the results recorded. When the 150 microlitre supply was almost consumed, the manometer cocks were opened, the manometer again filled with air, and the fluid carefully adjusted to read 150 on each leg. Then the manometer cocks were closed and the vibrator again started. During the night hours, 12:00 P.M. to 7:00 A.M., it happened on occasion that the oxygen consumed was greater 13 than the 150 microlitre capacity of the manometer. Cheng (11) developed a formulation whereby two settings of the manometer under these conditions would still give results with errors of only one per cent. To achieve this the mano- meter was adjusted so that the liquid level in the open leg was just on the scale and the readings on both of the legs recorded. The reading on the closed leg above the 150 mark was observed. The manometer was next adjusted so that the closed leg reading would be twice the value of the previous reading of the closed leg. The readings on both legs were again recorded and by the following formulation the oxygen consumed evaluated. y = We f - e where, y = the distance below the scale of the liquid level in the open leg. e = the distance above zero that the fluid has moved in the closed leg when the open leg is just on zero. f = the distance above zero that the fluid has moved in the closed leg when the fluid in the open arm is some distance on the open leg. W = the difference of the two readings on the open leg. If f were chosen to equal 2e, then y = W. 14 Each test was conducted at a temperature of 200C. The temperature was maintained at this level by circulating water from the University water system through the water bath. An overflow mechanism was installed into the water tank to maintain a constant water level. Since the Univers- ity water supply had a nearly constant temperature of 1700., the heater in the water bath maintained the temperature at a constant 2000. i 0.05%. A thermobarometer was employed to correct for atmos- pheric pressure changes. The logs of the observed oxygen uptake rates were plotted versus time for each sample. The growth rate con- stant klO was then evaluated from the slope of the straight line portion of this plot. 3.0 THEORY The commonly accepted equation for the growth of organisms can be expressed as: y = yoekt y = the organism concentration at time t y0 = the original organism concentration k = the growth rate constant t = the interval of time .1. = ekt lo 8;10 (FOL): 2.31 kt = klot Burk and Milner (4) working at the Fixed Nitrogen Research Laboratory in Washington, D. C., studied the nitrogen fixation of micro—organisms with the Warburg tech— nique. They stated that nitrogen fixation was indicated by an increase in the rate of oxygen consumption, which was caused by the growth of organisms, and this in turn was de- pendent on nitrogen fixation. These quantities are, ordinary conditions, proportional to growth increases as measured by dry weight, cell number, or turbidity. 15 16 Burk (3) in 1934 stated that the growth of Azotobacter may be measured by either cell nitrogen, cell number, dry matter, turbidity, respiration rate, or amount of azotase. Under any set of reasonably favorable physiological condi- tions each of these quantities increases logarithmically with time. Owing to this simple relationship the initial velocity of growth or nitrogen fixation at unit cell concen- tration can easily be obtained as a function of any particu- lar variable, such as temperature, pH, concentration of sugar, nitrogen pressure or source of fixed nitrogen. It is measured by the first order velocity constant g: g = 2.30 d log (a +y) dt where, a = the initial Azotobacter concentration y = the increase in t hours Burk used g as a notation for the growth rate constant and stated that it was conveniently evaluated experimentally from the lepe of a plot of the logarithm of the respiration rate against time. Grieg and Hoogerheide (17) studied the growth of micro- organisms in the Warburg apparatus and found the oxygen uptake of growing cultures of bacteria to be proportional to the bacterial content. They further stated that since the oxygen uptake is directly proportional to bacterial content, the measurement of the rate of oxygen uptake constitutes a convenient method for the measurement of the rate of growth. 4.0 RESULTS 4.1 Respiration Rates In experiments No. 1 and 2 a series of grab samples collected from the primary effluent of the East Lansing sewage treatment plant on September 25 and on October 3, 1962 were analyzed. The results are shown in Tables 1 to 13 and in Figures 1 to 8. The curves show that the loga- rithmic and declining growth phases were of relatively short duration and approximately of equal length. This can be observed from the symmetry displayed in most of the respir- ation rate curves. The curves in Figures 1 to 8 inclusive indicate a logarithmic phase of approximately 4 hours dura- tion. As shown in Figures 1 to 8 inclusive all East Lansing samples demonstrated a great similarity. They all approached a maximum hourly rate of 10 mg Og/l/hr. The minimum respir- ation rate at the beginning of each sample appeared to be 2 or 3 mg 02/l/hr. These curves did not indicate the presence of a lag phase. In experiments No. 3, 4, and 5 grab samples from the primary effluent of the East Lansing, Lansing, and William- ston sewage treatment plants were prepared according to procedure 2 described in Section 2. The data from the East Lansing sample are listed in Tables 14 and 15 and shown graphically in Figures 9 and lO. 1? 18 The curve in Figure 9 represents a typical sample of East Lansing unfiltered primary effluent dated November 5, 1962, where 6.7 mg of glucose was added. The curve is quite similar to those in Figures 1 to 8 inclusive. The curve in Figure 10 resulted from a typical filtered sample with 6.7 mg glucose added; however, it shows some distinct differ- ences. A considerable lag phase is observed as compared to the curve in Figure 10 and the respiration rates are quite small at the beginning. The log growth phase appears to be somewhat longer than in the previously mentioned curves. The data for the samples from the Lansing plant are listed in Tables 16 and 17 and shown graphically in Figures 11 and 12. Figure 11 represents a typical unfiltered Lansing sample with 6.7 mg of glucose added. The initial respiration rate was approximately 1 mg 02/l/hr which increased to a maximum of approximately 3.5 mg 02/l/hr. Figure 12 shows a curve typical for a filtered Lansing sample with 6.7 mg of glucose added. The very low initial oxygen uptake rate con- tinued for approximately 60 hours before it increased to approximately 5 mg 02/1/hr. The curves of Figures 11 and 12 showed a very distinct lag phase. The unfiltered sample showed a lag phase of ap- proximately 20 hours duration whereas in the filtered sample logarithmic growth started only after approximately 60 hours. The curves further showed a different form possi- bly due to an inhibitory effect. In Figure 11 the 19 logarithmic growth phase appeared to be 20 hours long and the declining growth phase had a duration of approximately 10 hours. Figure 12 showed a good symmetrical curve with shorter logarithmic and declining growth phases. This dif- ference may be due to the inhibitory agents adhering to the particulate matter which were being removed by filtration. The data for the samples from the Williamston plant are listed in Tables 18 and 19 and shown graphically in Figures 13 and 14. Figure 13 represents a typical unfiltered sample with no glucose addition. The respiration rate began at approximately 1 mg 02/1/hr and increased to a maximum of 8 mg 02/1/hr. The curve was very similar to those obtained for the unfiltered samples from the East Lansing plant. In Figure 14 the curve for a typical filtered sample is shown also with no glucose addition. The respiration rate began at a rather low value and increased to approximately 6.5 mg 02/l/hr. The curves of Figures 13 and 14 are very similar t except for the lower initial respiration rate of the filtered sample. The respiration rate curves obtained from those samples where 13.4, 26.8, and 53.6 mg glucose were added are not shown; however, the curves were very similar to the ones depicted except that higher maximum respiration rates were obtained. The higher respiration rates were caused by the fact that a larger microbial population developed from the higher concentration of substrate. 20 The respiration rates per unit volume of sample varied considerably. As shown in Table 22 the maximum respiration rate of 45 mg 02/1/hr was observed when the initial B.O.D. of the sample was 477 ppm. This value was obtained from a filtered sample of the Williamston plant with 536 mg glucose added. The lowest respiration rate of 23A) mg 02/l/hr. was obtained from an unfiltered sample of the Lansing plant. Some lag phase was observed with all the filtered samples. In this case the lag phase was possibly caused by a new environment or due to the very low bacterial cell con- centration at the start. Table 20 is a listing of the data on pH, suspended solids and B.O.D5 which were obtained on the original samples from the East Lansing, Lansing, and Williamston plants. In addition this Table contains the B.O.D. values measured for the filtered samples where no glucose was added. The B.O.D5 values for those samples where glucose was added were assumed to be higher by 50, 100, 200, or 400 mg/l according to the amount of glucose added to the filtered or unfiltered samples. In Figures 15, 16, and 17 the maximum respiration rates observed for the East Lansing, Lansing, and Williamston samples with glucose addition were plotted against the respective B.O.D5 data for these samples. The data used for these graphs are listed in Table 22. The curves indicate a definite relationship between the initial B.O.D. concentration 21 and the maximum hourly respiration rate per unit volume of sample. It should also be noted that the slope of the line differed with each sample. This was probably caused by the characteristics of the particular type of waste. Schuller (30) working in Germany in 1961 compared the maximum hourly respiration rate with the 24 hour B.O.D. as obtained from Warburg measurements and the chemical oxygen demand (C.0.D.) whereas in this study the 5-day B.O.D. was used. Schuller (30) obtained maximum hourly respiration rates from primary effluentsranging from 7 mg 02/1/hr to 30 mg Og/l/hr. These values are quite similar to those reported in this study. 4.2 Growth Rate Values and B.O.D. Figure 18 is a plot of the klO values obtained for the unfiltered samples from the East Lansing plant as listed in Table 21 versus the B.O.D5 concentration of these same samples. The curve shows the specific growth rate to be linear with respect to the B.O.D. for most of the data. It is possible however that at B.O.D. values above 200 ppm the specific growth rate constant k approached a maximum value. 10 In Figures 19, 20, and 21 the klO values for the East Lansing, Lansing, and Williamston samples with glucose addi- tion were plotted against the respective B.O.D5 values as listed in Table 22. Figure 19 demonstrates an approximately linear rela- tionship between the specific growth rate constant k10 and 22 the B.O.D. up to a growth rate value of 0.13 per hour. The concentration of soluble B.O.D. at which the growth rate became B.O.D. independent appeared to be approximately 200 ppm. In the unfiltered sample klO appeared to reach its maximum value at B.O.DS of 400 mg/l. It should also be noted that the unfiltered curve is identical to that of the filtered curve except that the unfiltered curve is shifted to the right on the graph. For the Lansing sample as shown in Figure 20 the curves representing the filtered and unfiltered portion were quite different. The curve for the filtered portion began very much like the curves shown in Figures 18 and 19; however, the specific growth rate did not exceed a value of 0.11 and decreased with increasing glucose concentrations. The unfiltered curve had the general appearance of the already mentioned curves but the specific growth rate did not exceed a value of 0.06 per hour. In Figure 21, representing the Williamston sample, - the unfiltered sample portion produced extremely scattered data, probably due to a malfunction of the Warburg apparatus. The filtered portion of the sample was similar to those shown in Figures 18 and 19. The maximum growth rate value appeared to be 0.13 per hour at approximately 200 mg/l B.O.D. Figure 22 is a graph prepared by plotting the klO values obtained for all filtered samples versus their 23 respective soluble B.O.D5 concentrations. This plot demon- strates clearly that the specific growth rate approaches a maximum value of 0.13 at a B.O.D. concentration of 200 mg/l. Under the most ideal conditions as shown in Figure 19, there was no appreciable difference between the filtered and unfiltered sample portions except that the curve for the filtered portion was moved more to the right on the graph. Figure 20, however, showed a clear difference in the two curves. The curve for the unfiltered portion reached a maximum growth rate klo of 0.06 per hour whereas the filtered curve obtained a maximum growth rate klO of 0.105 per hour. It appeared that certain inhibiting effects were removed by the filtering process. Another interesting phenomenon was the inhibiting effect of higher glucose con- centrations demonstrated for the filtered portion of the sample. 5.0 DISCUSSION In a study such as this it is extremely important to be able to duplicate the results obtained. In Figures 2, 3, 5, 6, and 7 data from duplicate samples are plotted together. It should be noted that the same slope has been obtained for either of the two samples. It was originally the intent of this study to measure the mass of sludge growth by weight and to obtain a growth rate value by plotting the logarithm of the sludge increase against time. Due to the low concentrations of suspended solids in the samples and the difficulty experienced in determining the suspended solids precisely, this method proved unsatis- factory. It was then decided to determine the sludge growth by utilizing respiration rate as a measure of the sludge growth rate. All biological systems need certain chemical substances which are required for their growth and normal function. In this study a reagent grade glucose was added to a number of sewage samples as an additional carbon source. The nitrogen source and other nutritional elements necessary for growth had to be supplied by the sample. Garret and Sawyer (16) who also evaluated growth rates from mixed bacterial cultures utilized a synthetic nutrient solution where all essential elements were present to obtain a maximum growth rate. In 24 25 this study the maximum growth rate was produced by increasing only the concentration of the carbon source. Garret and Sawyer (l6) determined the growth rate con- stant by plotting the accumulated oxygen consumption versus time on a semi—logarithmic scale and by measuring the slope of the straight line portion of this curve. The writer does not believe this to be a true determination of the growth rate constant. It is generally assumed that the oxygen con- sumed in a biological oxidation reaction is used partly for cell synthesis and partly for direct oxidation of the organic matter. Therefore a measure of the total oxygen consumed is not a measure of the cell increase but a value which is larger than the specific growth rate constant. It should be noted that the respiration rate unit employed was a unit of volume rather than a unit of weight. Respiration rate units commonly employed are mg 02 per gram of sludge per unit of time. The unit employed in this study was mg 02 per unit volume of original sample per unit of time. Figures 15, 16, and 17 were graphs of maximum respir- ation rates as related to the standard 5—day B.O.D. It should be observed that each plot has a different slope. The slope of the line was characteristic of the particular waste and would be different for each municipality. The greater the slope of the line the more readily the waste was broken down and conversely the smaller the slope the more difficult it was to break down the organic matter. 26 As shown in Table 22, certain samples did not obtain a logarithmic growth phase. This occurred in samples of relatively low B.O.D where the margin of error inherent in the equipment was exceeded. All the filtered samples, as demonstrated in Figures 10, 12, and 14, showed small initial respiration rates. This may be due at least in part to insufficient seeding of the sample; however, caution had to be observed to avoid too much seeding and thereby eliminating the log growth phase. Soluble organic matter can be assimilated directly by biologically active bacterial cell material whereas particu- late matter must first be hydrolyzed to be available to the cells. Consequently the soluble B.O.D. fraction will be depleted directly and the particulate matter removed by coagulation, entrainment, adsorption, and only later on by oxidation of components made soluble by enzymes. The loga- rithmic growth rate constant klo as determined in this study was related to the soluble B.O.D. as demonstrated in Figures 19, 20, 21, and 22. The data indicate that a maximum growth rate would be reached if the soluble B O.D. fraction should exceed 200 ppm B.O.D. At lower soluble B.O.D. concentrations the growth rate of the bacteria would be dependent on the substrate concentration. Normally activated sludge plants are designed to oper- ate at an aeration period of 4 to 8 hours, an air supply of 27 0.5 to 2.0 cubic feet per gallon of sewage, and a return sludge capacity of 25 per cent of the sewage flow. Hasel- tine (22) in 1955 criticized this design procedure because no mention of the amount of activated sludge to be carried in the aeration tanks was stipulated. He proposed to design the plant around the Sludge Volume Index, B.O.D. to solids loading, and the return sludge capacity. In this procedure the total volume of the aeration tanks is set by the above mentioned criteria. This study would indicate a further criterion to be used, namely the growth rate k. It should be mentioned here, however, that at high growth rates the activated sludge becomes dispersed and the coagulant growth disappears. This would be an important factor in the utilization of large growth rates. 6.0 CONCLUSIONS Some municipal wastes have an extended lag phase before the logarithmic growth phase begins. There is a linear relationship between the maximum hourly respiration rate per unit liquid volume and the initial B.O.D concentration. The duration of the logarithmic growth phase under normal conditions is approximately 5 hours; however, it is depen- dent on the initial substrate concentration. The curve relating B.O.D and growth rate constant is characteristic of each waste. This relationship could change; however, throughout the day depending on the various components that make up the waste at different times during the day. The growth rate of bacteria in sewage is B.O.D. dependent below approximately 200 ppm dissolved B.O.D. Above 200 ppm dissolved B.O.D the growth rate is independent of the B.O.D. concentration and at its maximum rate. The maximum growth rate value k at 200C. was found to be approximately 0.30 per hour in this study. The mean generation time corresponding to this k value is 2.31 hours. 28 7 . 0 APPENDICES OXYGEN UPTAKE RATE mq/I/hr 30 20 LEGEND East Lansinq S.T.P. primary effluent 1000 am Sept 25,1962 SS. - 74 mq/l 10 Sday BOD -145mq/| l \\ k6 0.036 hr-1 1 8 7 I \ . f g 5 w A \h 4 8 12 16 20 24 28 TIME HOURS Fiqure1.- Relationship of Respiration Rate and Time 3O LEGEND East Lansinq S.T. F.’ primary effluent 12:00 am. Sept. 25,1962 5.5. - eomq/I E3 RIG: O. 081 h I:1 5day BOD -160mq/I Duplicate samples at 03900 N OXYGEN UPTAKE RATE mq/l/hr‘ ‘3: \ 4 ‘\ \ . \ 3 \ \6\ \ 0 \ “I..." ~ :tfl 2 -,- 4 8 12 16 20 TIME HOURS 24 28 Fiqure 2.- Relationship of Respiration Rate and Time 60 50 40 to O N O OXYGEN UPTAKE RATE mq/I/hr 8 01 me .5 ._.-._—_( f LEGEND \ East Lansnnq S.T. P. primary effluent 2‘00p.m. Sept. 25,1962 SS- 102 mq/I ; l l l T g _J_ l ! 5day BOD ~188mq/l k,o= 0.119 hr’1 Duplicate sampbs ' 5 j . l + \I /i b o; x I \ + \ % \ o C \\ 4. l ‘ * 't 4 ' 8 12 16 20 24 28 TIME HOURS Fiqure 3.-Relationship of Respirction Rate and Time 60 50 40 U 0 to O l OXYGEN UPTAKE RATE mq/I/hr 01 0) \l a) 8 K0. A LEGEND East Lansinq ST P primary effluent 4:00pm. Sept.25,1962 SS - 106 mq /l 5 day BOD - 176 mq/I kw: 0.098 hr’ V—‘dh—I ____- 1_ 1? \ l> N \~ ‘ 0 8 12 16 TIME HOURS 20 24 28 Fiqure4 Relationship of Respiration Rate and Time OXYGEN UPTAKE RATE mq/I/hr‘ 60 i 1 50 l l . 4o 1 la 1 LEG END ‘ 3 0 East Lansinq S.T. F? primary effluent 6=00 pm. Oc t.3,1962 5.3- 202 mq/l 20 / 5day BOD - 164 mq/I k,o= 0:09 7 hr" Duplicate samples J 10 1 fl 1 0101\lm E *z / b A + <9 \ 1 \J\ + l N 4 8,1216 20 24 28 TIME HOURS Fiqure 5 Relationship of Respiration Rate and Time OXYGEN UPTAKE RATE mq/l/hr 60 50 40 LEGEND 30 East Lansinq SIP . primary effluent 8: 00 pm. Oct.3,1962 5.3- 192 mq/l 20 5 day BOD - 191 mq/I . / k.o= 0.087 hr‘1 ‘ Duplicate samples 10 f / \ / 1: 0|me \°*\ 43+ 4 .1: / 4 8 12 16 20 24 28 TIME HOURS Fiqure 6,-Relationship of ReSpiration Rate and Time OXYGEN UPTAKE RATE mq/I/hr 60 l 50 I l t l 40 , LEGEND I 30 East Lansinq S.T.F? primary effluent 10: 00 p. m. Oct 3,1962 S.S.- 88 mall 20 5 day BOD - 191 mq/I / = 0.092hr'1 uplicate samples 0 + (i + 10 9° l 8 x“ 1 7 l! \ 6 7; + ' 9’ 5 ° I 0 . 4 \ \ 3 + v + _. 2 4 8 12 16 20 24 28 TIME HOURS Fiqure 7.-Relationship of Respiration Rate and Time OXYGEN UPTAKE RATE mq/I/hr‘ 1 , 60 __.~11 . _.__-._fi.______ J» -IwamL- __ 40 " __,_____ — ,____ 7 - LEGEND : 30 East Lansinq STE 1 primary effluent 1 12:00 p.m.Oct 3,1962 1 S.S.- 184 mall I 20 J 6 day BOD - 151 mq/I ——:——. k,o= 0.066 hr’1 T 0 “ Ti 8 mm *———t-— n. 7 74 l i 6 ° a — l 1 l 4 o \ o 3 s 2 4 8 12 16 20 24 28 TIME HOURS- Fiqure 8.- Relationship of Respiration Rate and Time 30 20 —h OXYGEN UPTAKE RATE mq/I/hr‘ LEGEND East Lansinq S.T.P. 7:30am. Nov. 5 1962 5 day BO D - 239 mq/l klo= 0. 036hr‘1 O / 9 8 // 7 I 6 n /\\ o O 5 o a o o 4 / o 3 o 0 ) o I? o \ 2 \ o \1 o I o o 5 10 15 20 25 30 35 40 TIME HOURS Fiqure 9-Typicol Respiration Rate plot of unfiltered sample with qlucose added OXYGEN UPTAKE RATE mq/l/hr 01 mflmtDO 0.9 0.8 0.7 0.6 TIME HOURS ._.._. ‘ I / LEGEND / — East Lansinq S.T.P. I ,__ 7:300.m.Nov.5,1962 5 day BOD - 93 mq/I ‘ klo=0.0625hr-1 o I o P o 0 fl \ /’ \ 1’0 A / v o 5 10 15 20 25 30 35 40 Fiqure10-Typical Respiration Rate plot of filtered sample with qlucose added Donna 80020 53> oEEom accoEE: Lo 63 ovum cofiogfimom 629:. 150.591.. 09 WKDOI w: :. om em on on on ov om ON 0. / 000 0 j // H. r 0 DI //0 o o LEI / \ 7 £906.90. \\. a .8an - a 0m goo n 8918625808 mam ocean... ozmomn m0 v.0 m0 Q0 m0 r «ILI/l/bllJ Elva BM‘Vidn NBOAXO 4.1 00—. tonne mucosa 53> 03an 8.10:: 6 63 8.0m cozacfimom 60631905211 Om Om Oh mmDOI Om wit. on 0? 0m 0m 0.. / / C 7.308 n 0:. 3055 1o om sou m 89.8.6onon me aEmcaq oszw... . NO NO v.0 m .0 0.0 m0 Jll/l/bw aiva EMT/1cm NBOAXO OXYGEN UPTAKE RATE mq/I/hr‘ 20 LEGEND [ Williamston S.T.P. I 8=OOa.m.Nov.26,1962 h Sday BOD -118mq/l kg = 0.184 hr“ U'l OTVQCOO Q 2/ .‘N o o \ O \ O 0.9 0.8 Q 4 8 12 16 20 24 28 TIME HOURS Fiqure 13 ~Typica| Respiration Rate plot of unfiltered sarnple with qlucose added IO 9 I 8 - l/ l 7 - I I . 6 ~ ' 5 iv i I ' ’ 7, LEGEND l E 4 Williamston S.T.F? ' > I I 8=OOa.m.Nov.26,1962 E 3 Sday BO D-77mq/l _. 1 CI k,o=0.108hr"1 LU '2 0: 2 DJ K < . .— a. 3 I z , 111 O 1 50.9 0.8 I 1 0.6 I I 0.5 I 0.4 4 8 12 16 20 24 28 TIME HOURS Fiqure14-Typical Respiration Rate plot of filtered sample with qlucose added 00$ 00? .38 mo 0 m 00m OON moOm mamLo> 30¢ co_BL_amom E:E_xo2 3 «CE. 9232... 00—. \ \ \ \ \ \ \ \l ( $92962 .Ed 02 Donna 3820 5:5 uEEum 8.23:2: Uc_mco._ pmow ozmwwq V N F O N no N mm V? JH/I/DUJ BlVH NOIlVHIdSBH WOWIXVW L; 1; OOO moOm mamgo> 30m coghummm E:E_xo_2 20 3E- 92:9... 000 00V .38 mo 0 m 00m cow co, \ \fiuli \o\- \ \o\ ~8an .82 .Ed oonm novuo omens? 5_>> oEEcm vogogtca UEmcS Dzwmuw... v N P O N m N mm v v JU/l/buJ 31w NOIlVHIdSBa WDWIXVW 000 moOm mamgo> 30m co_8.__amom E:E_x02 .0 SE... 5932... 000 00? <9: mo 0 m 00m 00m 09 i\ V Nwmnmw .>OZ .Ed OOHm vmvvo vaUBU 53> oEEom 8.3%: coemEai; ozmom: V N ‘— CO 0 N N JH/l/bUJ 31w NOllValdSBH WHWIXVW (D C") v v .14 _s “—3 Q N '0 on b A GROWTH RATE CONSTANT klo hr‘1 ~o O ' N 03 l LEGEND East Lansinq unfiltered / samples of Sept.25 and ,/ , Oct.3,1962 E o O 1 o F 50 100 150 200 250 B O 05 mq/l Fiqure18-Plot of Growth Rate Constant kloand BOD5 COO moOm .28 0; 2228 3am 526.5 .0 8.9923: c o m 00m 00V :9: m 00m OON OO— oEEom D803: +|..|+ 29:3 83ng: oilo wanna meuac 53> NmmBSOZ *0 29:3 ccficg 78w ozwmumu. \ \ *- - 0, Q 0. 0. 0. Flu °'>i .LNVLSNOD 31w Hlmoaa £2 000 moOm. oco 0.x 63300 30w. 530.0 .0 #0.“... -ON m£22.... :9: mo 0 m oom oov oom OON OOP \o K \ 29:8 8.0:: TI; oEEom 8.85:5 o o Dunno 303.0 5.2, «monwgoz .o 0683. 6290.. DZmeMJ _ _ _ PO. m0. mo. 50. mo. F ‘-. m P: :9. L_.lu °'>. iNVlSNOO 31w HiMOHO moom .85 o; 2388 82. 5380 .0 so_¢;mm.su_... :9: me o m ooo oom oov oom ‘ 00m 00. oEEom no.6: C +l+ oEEom no.3...c: 9|... nonnn om0oa.o 5.3 mouQNSO—L .0 0.9.5.5 cmeEO:=>> OZMOMJ (O 9-9. «‘9. 3. Elf 53. Q 8 Lyu 0'). iNViSNOI) aiva HiMOH‘E) O N. COO moom .80 or. E3200 «Em 5380 no 63. -mmosu... m .\UE n. O m 00m 00v. 00m OON OO.‘ nonno mo; 303.0 3055 moEEom no.3... :< OZUOUJ m ‘— 0) f\ If) m \- "- "' 0. O. 0. 0. 0. Flu °'>i .LNVlSNOD 31w HiMOth-D l0 ‘_. TABLE 1 RESPIRATION RATES Thermobaro- Respiro— Corrected Oxygen ReSpira- Time meter meter Reading Consumed tion Rate Hrs. Reading Reading pl mg/l mg/l/hr 1.0 0 +13 13 4.3 4.3 2.0 — 4 +15 11 3.6 3.6 3.0 0 +15 15 4.9 4.9 4.0 — 2 +21 19 6.3 6.3 5.1 - 2 +31 29 9.5 9.5 6.0 — 4 +22 18 6.0 6.0 8.0 -16 +27 11 3.6 1.8 9.25 0 +17 17 5.6 4.5 10.75 - 5 +20 15 4.9 3.3 20.75 - u +60 56 18.5 1.8 25.5 +12 + 8 20 6.6 1.4 27.25 +15 — 3 12 4.0 2.3 29.25 + 5 + 7 12 4.0 2.0 Type of sample — primary effluent — 10:00 a.m. Sept. 25, 1962 — East Lansing Sewage Treatment Plant Date Place . 52 TABLE 2 RESPIRATION RATES Thermobaro— Respiro— Corrected Oxygen ReSpira- Time meter meter Reading Consumed tion Rate Hrs. Reading Reading pl mg/l mg/l/hr 1.0 0 +16 16 5.1 5.1 2.0 - 4 +25 19 6.1 6.1 3.0 0 +28 28 8.9 8.9 4.0 - 2 +30 28 8.9 8.9 5.1 - 2 +21 19 6.1 6.1 6.0 — 4 +19 15 4.8 4.8 8.0 -16 +29 13 4.2 2.1 9.25 - 1 8 +22 21 6.7 5.3 10.75 _ 5 +20 15 4.8 3.2 20.75 - 4 +75 71 22.7 2.3 25.5 +12 +16 28 9.0 1.9 27.25 +15 - 3 12 3.8 2.2 29.25 + 5 + 9 14 4.5 2.2 Type of Sample - primary effluent Date - 12:00 a.m. Sept. 25, 1962 Place - East Lansing Sewage Treatment Plant 53 TABLE 3 RESPIRATION RATES Thermobaro- ReSpiro- Corrected Oxygen ReSpira— Time meter meter Reading Consumed tion Rate Hrs. Reading Reading pl mg/l mg/l/hr 1.0 0 +16 16 5.7 5.7 2.0 - 4 +23 19 6.7 6.8 3.0 0 +24 24 8.5 8.5 4,0 - 2 +24 22 7.8 7.8 5.1 - 2 +18 20 7.1 7.1 6.0 - 4 +17 13 4.6 4.6 8.0 -16 +25 9 3.2 1.6 9.25 - 1 +19 18 6.4 5.1 10.75 - 5 +19 14 5.0 3.3 20.75 - 4 +64 60 21.0 2.1 25.5 +12 +10 22 7.8 1.6 27.25 +15 - 4 11 3.9 2.2 29.25 + 5 + 7 12 4.3 2.1 Type of Sample - primary effluent Date - 12:00 a.m. Sept. 25, 1962 Place - East Lansing Sewage Treatment Plant 54 TMflE4 RESPIRATION RATES Thermobaro- ReSpiro- Corrected Oxygen ReSpira- Time meter meter Reading Consumed tion Rate Hrs. Reading Reading pl mg/l mg/l/hr 1.0 0 +16 16 5.5 5.5 2.0 - 4 +21 17 5.8 5.9 3.0 0 +22 22 7.8 7.8 4.0 — 2 +31 29 10.0 10.0 5.1 - 2 +42 40 13.7 13.7 6.0 - 4 +35 31 10.6 10.6 8.0 -16 +28 12 4.1 2.1 9.25 - 1 +32 31 10.6 8.5 10.75 - 5 +32 27 9.3 6.2 20.75 - 4 +91 87 30.0 3.0 25.5 +12 +23 35 12.0 2.5 27.25 +15 - 1 14 4.8 2.7 29.25 + 5 +10 15 5.2 2.6 Type of sample - primary effluent Date - 2:00 p.m. Sept. 25, 1962 Place - East Lansing Sewage Treatment Plant 55 TABLE 5 RESPIRATION RATES Thermobaro- Respiro- Corrected Oxygen ReSpira- Time meter meter Reading Consumed tion Rate Hrs. Reading Reading pl mg/l mg/l/hr 1.0 0 +13 13 4.6 4.6 2.0 - 4 +19 15 5.5 5.5 3.0 0 +19 19 7.0 7.0 4.0 — 2 +26 24 8.9 8.9 5.1 - 2 +37 35 12.8 12.8 6.0 - 4 +30 26 9.6 9.6 8.0 -16 +27 11 4.0 2.0 9.25 - 1 +27 26 12.8 10.2 10.75 - 5 +27 22 8.1 5.4 20.75 - 4 +81 77 28.5 2.8 25.5 +12 +19 31 11.4 2.4 27.25 +15 '- 3 12 4.4 2.5 29.25 + 5 + 9 14 5.2 2.6 Type of Sample + primary effluent Date — 2:00 p.m. Sept. .5, 1962 Place - East Lansing Sewage Treatment Plant 56 TABLE 6 RESPIRATION RATES Thermobaro- Respiro- Corrected Oxygen Respira- Time meter meter Reading Consumed tion Rate Hrs. Reading Reading pl mg/l mg/l/hr 1.0 0 +14 14 4.8 4.8 2.0 — 4 +21 17 5.8 5.8 3.0 0 +22 22 7.5 7.5 4,0 _ 2 +30 28' 9.6 9.6 5.1 - 2 +37 35 12.0 12.0 6.0 — 4 +25 21 7.2 7.2 8.0 -16 +28 12 4.1 2.1 9.25 - 1 +28 27 9.3 7.4 10.75 - 5 +27 22 7.6 5.1 20.75 - 4 +76 72 24.7 2.5 25.5 +12 +19 31 10.6 2.2 27.25 +15 - 1 14 4.7 2.7 29.25 -+ 5 + 8 13 4.4 2.2 Type of sample - primary effluent Date - :OO p.m. Sept. 25, 1962 Place - East Lansing Sewage Treatment Plant 57 TABLE 7 RESPIRATION RATES Thermobaro— Respiro— Corrected Oxygen ReSpira— Time meter meter Reading Consumed tion Rate Hrs. Reading Reading p1 mg/l mg/l/hr 1.0 + 3 + 2 5 1.6 1.6 2.2 + 7 + 7 14 4.6 4.0 4.0 + 7 +25 32 10.6 5.8 5.25 + 2 +31 . 33 10.9 8.7 6.0 + 4 +15 19 6.3 8.4 7.0 + 2 +17 19 6.3 6.3 8.0 - 2 +12 10 3.3 3.3 10.0 - 3 +34 31 10.2 5.1 11.0 - 3 +15 12 4.0 4.0 12.0 0 +14 14 4.6 4.6 23.0 + 8 +76 84 28.0 2.5 25.0 + 1 + 5 6 2.0 1.0 Type of Sample - primary effluent Date — 6:00 p.m. Oct. 3, 1962 Place - East Lansing Sewage Plant 58 TABLE 8 RESPIRATION RATES Thermobaro- Respiro- Corrected Oxygen ReSpira- £321.“? £233. 322313. R833” 0512??“ 327.55.? 1.0 + 3 + 3 6 2.1 2.1 2.2 + 7 + 9 A 16 5.6 4.8 4.0 + 7 +27 34 12.0 6.6 5.25 + 2 +31 33 11.7 9.3 6.0 + 4 +15 19 6.7 8.9 7.0 + 2 +13 15 5.3 5.3 8.0 - 2 +14 12 4.3 4.3 10.0 - 3 +31 28 9.8 4.9 11.0 - 3 +14 11 3.9 3.9 12.0 0 +12 12 4.3 4.3 23.0 + 8 +71 79 27.7 2.5 25.0 + 1 + 3 4 1.4 .7 Type of Sample - primary effluent Date — 6:00 p.m. Oct. 3, 1962 Place - East Lansing Sewage Treatment Plant 59 TABLE 9 RESPIRATION RATES Thermobaro— Respiro- Corrected Oxygen Reapira- Time meter meter Reading Consumed tion Rate Hrs. Reading Reading pl mg/l mg/l/hr 1.0 + 3 + 7 10 3.2 3.2 2.2 + 7 +13 20 6.4 5.5 4.0 + 7 +36 43 13.8 7.5 5.25 + 2 +39 41 13.0 10.4 6.0 + 4 +21 25 8.0 10.7 7.0 + 2 +46 48 15.4 15.4 8.0 — 2 +20 18 5.7 5.7 10.0 - 3 +45 42 13.3 6.7 11.0 - 3 +18 15 4.8 4.8 12.0 0 +18 18 5.7 5.7 23.0 + 8 +98 106 34.0 3.1 25.0 + 1 + 5 6 1.9 .9 Type of Sample - primary effluent Date — 8:00 p.m. Oct. 3, 1962 Place - East Lansing Sewage Treatment Plant 60 TABLE 10 RESPIRATION RATES Thermobaro- ReSpiro- Corrected Oxygen ReSpira- Time meter meter Reading Consumed tion Rate Hrs. Reading Reading pl mg/l mg/l/hr 1.0 + 3 +10 13 4.6 4.6 2.2 + 7 +12 19 6.7 5.8 4.0 + 7 +31 38 13.5 7.4 5.25 + 2 +34 36 12.7 10.2 6.0 + 4 +18 22 7.9 10.5 7.0 + 2 +34 36 12.7 12.7 8.0 - 2 +19 17 6.0 6.0 10.0 - 3 +41 38 13.5 6.7 11.0 - 3 +15 12 4.3 4.3 12.0 0 +14 14 5.0 5.0 23.0 + 8 +87 95 34.0 3.1 25.0 + 1 + 4 5 1.8 .9 Type of Sample — primary effluent Date - 8:00 p.m. Oct. 3, 1962 Place — East Lansing Sewage Treatment Plant 61 TABLE 11 RESPIRATION RATES Thermobaro- ReSpiro- Corrected Oxygen ReSpira- Time meter meter Reading Consumed tion Rate Hrs. Reading Reading 'pl mg/l mg/l/hr 1.0 + 3 +12 15 5.2 5.2 2.2 + 7 +12 19 6.5 5.6 4.0 + 7 +30 37 12.8 7.0 5.25 + 2 +32 34 11.7 9.4 6.0 + 4 +17 21 7.2 9.6 7.0 + 2 +39 41 14.0 14.0 8.0 - 2 +38 36 12.4 12.4 10.0 — 3 +39 36 12.4 6.2 11.0 - 3 +18 15 .2 5.2 12.0 0 +14 14 4.8 4.8 23.0 + 8 +93 101 34.6 3.1 25.0 + 1 + 6 7 2.4 1.2 Type of Sample — primary effluent Date - 10:00 p.m. Oct.3,l962 Place — East Lansing Sewage Treatment Plant 62 TABLE 12 RESPIRATION RATES Thermobaro- Respiro- Corrected Oxygen ReSpira- Time meter meter Reading Consumed tion Rate Hrs. Reading Reading ‘pl mg/l mg/l/hr 1.0 + 3 + 5 8 2.9 2.9 2.26 + 7 +10 17 6.4 5.5 4.0 + 7 +26 33 11.8 6.5 5.25 + 2 +27 29 10.6 8.5 6.0 + 4 +14 18 6.6 8.8 7.0 + 2 +33 35 12.5 12.5 8.0 — 2 +35 33 11.8 11.8 10.0 - 3 +37 34 12.0 6.0 11.0 - 3 +15 12 4.3 4.3 12.0 0 +14 14 5.0 5.0 23.0 + 8 +80 88 31.5 2.8 25.0 + l + 5 6 2.2 1.1 Type of Sample - primary effluent Date — 10:00 p.m. Oct. 3, 1962 Place - East Lansing Sewage Treatment Plant 63 TABLE 13 RESPIRATION RATES Thermobaro- Respiro- Corrected Oxygen Respira- Time meter meter Reading Consumed tion Rate Hrs. Reading Reading ’pl mg/l mg/l/hr 1.0 + 3 + 8 11 3.8 3.8 2.1 + 7 +14 21 7.3 6.3 4.0 + 7 +30 37 12.8 7.0 5.25 + 2 +33 35 12.0 9.6 6.0 + 4 +15 19 6.5 8.7 7.0 + 2 +16 18 6.2 6.2 8.0 - 2 +15 13 4.5 4.5 10.0 - 3 +33 30 10.4 5.2 11.0 - 3 +13 10 3.5 3.5 12.0 0 +13 13 4.5 4.5 23.0 + 8 +71 79 27.0 2.4 25.0 + 1 + 5 6 2.1 1.1 Type of Sample - primary effluent Date — 12:00 p.m. Oct. 3, 1962 Place - East Lansing Sewage Treatment Plant 64 TABLE 14 RESPIRATION RATES Thermobaro- Respiro- Corrected Oxygen ReSpira— Time meter meter Reading Consumed tion Rate Hrs. Reading Reading ‘pl mg/l mg/l/hr 1.0 0 +13 13 4.8 4.8 2.0 0 +10 10 3.7 3.7 3.25 + 2 +15 17 6.3 5.0 4.25 + 1 +14 15 5.5 5.5 5.25 — 4 +16 12 4.4 4.4 6.25 - 4 +21 17 6.3 6.3 7.25 — 4 +18 14 5.2 5.2 8.41 - 4 +23 19 7.0 6.0 9.25 - 4 +10 6 2.2 2.6 10.25 - 6 +14 8 2.9 2.9 11.5 -10 +15 5 1.8 1.4 12.25 + 1 + 8 9 3.3 4.4 21.5 - 3 +46 43 16.0 1.7 22.75 - 3 +13 10 3.7 2.9 24.5 + 9 + 1 10 3.7 2.1 27.0 +20 ~11 9 3.3 1.3 28.25 + 4 — 2 2 .7 .6 29.25 + 5 + 3 8 2.9 2.9 30.25 + 3 + 1 4 1.5 1.5 32.75 +10 + 1 11 4.0 1.6 34.25 + 4 + 3 7 2.6 1.7 36.25 + 9 + 6 15 5.5 2.8 45.75 +42 +13 54 20 0 2 1 47.25 —13 +12 0 - - 49.25 + 9 +14 23 8.5 4.2 21" VI...“ . when -_ .4 Type of Sample primary effluent (unfiltered) Date - 7:30 a.m. Nov. 5, 1962 Place - East Lansing Sewage Treatment Plant Glucose added — 67 mg/l 41.1 55 TABLE 15 RESPIRATION RATES Thermobaro- ReSpiro- Corrected Oxygen ReSpira- Time meter meter Reading Consumed tion Rate Hrs. Reading Reading ‘pl mg/l mg/l/hr moor: I o o '0 o. \i-D'CDI O\O\J‘:I + '_J H \OCDNONWJZ’UORDH l0 UW CDJ‘ICDI }_.I I 00. l 00. Ul-IZ‘CDONJECDMJE‘MUWL‘ONI CD-D'U‘IOIDOIDi—‘NOL‘Ml—JO i—‘UW 18 7. h) \owmxo JZ'OKJUUT JI'OKOUJUO l—‘O mttttti-J [\DOO '._.I 28.25 29.25 30.25 32.75 34.25 36.25 45.75 47025 49.25 man-Janina) F-J . .. .-++—+-++—+-++—+-++—+-++—+ Hi: I m UTU'IKOMHmmmwflmHflkmmtmmmtwOl—JH UT I o o o o o o O\l woomoxJz-ooxlowmoo I +l+++++++++ll+lllllll++ i—‘U'lfDl—‘l—J +-+ an primary effluent (filtered) 7:30 a.m. Nov. 5, 1962 East Lansing Sewage Treatment Plant 67 mg/l Type of Sample Date Place Glucose added 66 TABLE 16 RESPIRATION RATES Thermobaro- Respiro- Corrected Oxygen Respira- Time meter meter Reading Consumed tion Rate Hrs. Reading Reading ‘pl mg/l mg/l/hr 1.5 + 7 — 2 5 1.7 1.2 3.0 + 8 - 3 5 1.7 1.2 4.0 - l + 2 1 .3 .3 5.0 - 4 + 5 1 .3 .3 6.0 - 3 + 4 1 .3 .3 7.0 - 3 + 6 3 1.0 1.0 8.5 — 5 + 6 1 .3 .2 10.0 - 5 + 7 2 .7 .5 11.5 - 5 + 6 1 .3 .2 13.0 + 2 + 2 4 1.3 .9 20.0 - 4 +19 15 5.2 .7 22.33 0 +15 15 5.2 2.0 24.5 -11 +12 1 .3 .2 26.5 + 8 + 3 11 3.8 1.9 28.0 — 1 + 7 6 2.1 1.4 29.5 — 2 +11 9 3.1 2.1 32.0 -13 +30 17 5.8 2.3 34.0 - 5 +20 15 5.2 2.6 36.0 —12 +26 14 4.8 2.4 44.5 ~12 +63 51 17.5 2.1 47.0 - l + 6 5 1.7 .7 48.5 + 3 0 3 1.1 .7 50.0 +19 ~14 5 1.7 1.1 52.5 +27 -23 4 1.4 .6 54.0 + 1 + 2 3 1.1 .7 56.5 +19 —12 7 2.4 1.0 58.5 + 6 - 3 3 1.1 .5 60.0 + 7 - 4 3 1.1 .5 68.0 +50 —36 14 4.8 .7 70.5 - 5 + 6 1 .3 .1 72.5 + 6 — 4 2 .7 .4 74.5 +11 - 9 2 -7 -“ 77.0 + 3 0 3 1.1 .4 81.0 —11 +16 5 1.7 .4 82.5 + 1 0 1 .3 .2 84.5 - l + 2 1 .3 .2 85,5 0 + 2 2 .7 .7 93.5 +25 -17 8 2.8 .3 96.0 - 1 + 4 3 1.1 .4 Type of Sample rimary effluent (unfiltered) Date - :OO a.m. Nov. 12, 1962 Place Lansing Sewage Treatment Plant Glucose added 67 mg/l 67 TABLE 17 RESPIRATION RATES Thermobaro— ReSpiro- Corrected Oxygen Respira— Time meter meter Reading Consumed tion Rate Hrs. Reading Reading {pl mg/l mg/l/hr 1.5 + 7 - 3 4 1.5 1.0 3.0 + 8 - 8 0 —- -- 4.0 - l + 3 2 7 .7 5.0 — 4 + 4 0 -- -- 6.0 - 3 + 3 0 -- -- 7.0 — 3 + 3 O -- ~- 8.5 — 5 + 5 0 -— —— 10.0 - 5 + 5 O -— -- 11.5 - 5 + 4 O -— -— 13.0 + 2 - 1 1 .4 .3 20.0 - 4 + 5 1 .4 .1 22.5 0 +13 13 4 9 2 0 24.5 —11 + 5 O -- —- 26.5 + 8 - 6 2 .8 .4 28.0 — l + 4 3 1.1 .7 29.5 — 2 + 6 4 1.5 1.0 32.0 —13 +22 9 3.4 1.4 34.0 — 5 +12 7 2.6 1.3 36.0 -12 +15 3 1.1 .6 44.5 -12 +23 12 4.5 .5 47.0 - 1 + 2 1 .4 .2 48.5 + 3 — 2 1 .4 .3 50.0 +19 —17 2 8 5 52.5 +27 -27 0 -- -- 54.0 + 1 0 1 .4 .2 56.5 +19 —14 5 1.8 .8 58.5 + 6 - 4 2 .8 .4 60.0 + 7 — 4 3 1.1 .7 68.0 + 50 -18 32 12.0 1.5 70.5 - 5 +23 18 6.7 2.7 72.5 + 6 +16 22 8.4 4.2 74.5 +11 +10 22 8.4 4.2 77.0 + 3 + 1 4 1.5 .6 81.0 -11 +15 4 1.5 4 82.5 + 1 — 1 0 -- -- 84.5 - 1 + 2 1 .4 .2 85.5 0 + 1 1 .4 .4 93.5 +25 —22 3 1.1 .1 96.0 - 1 + 4 3 1.1 .4 primary effluent (filtered) 8:00 a.m. Nov. 12, 1962 Lansing Sewage Treatment Plant 67 mg/l Type of Sample Date Place Glucose added 68 TABLE 18 RESPIRATION RATES Thermobaro- Respiro- Corrected Oxygen ReSpira- Time meter meter Reading Consumed tion Rate Hrs. Reading Reading ‘pl mg/l mg/l/hr 1.5 + 7 - 1 6 2.1 1.4 2.5 + 9 0 9 3.2 3.2 3.5 + 8 + 4 12 4.3 4.3 5.0 +17 +15 32 11.3 7.5 6.5 + 1 +25 26 9.2 6.2 8.0 -10 +25 15 5.3 3.5 9.5 - 6 +19 13 4.6 3.1 11.25 - 1 + 9 8 2.8 1.6 12.5 + 2 + 4 6 2.1 1.7 14.0 + 1 + 5 6 2 1 1.4 15-0 + 5 0 5 1.7 1.7 16.0 0 + 1 1 .4 .4 23.25 -11 +35 24 8.5 1.2 25.25 - 2 + 7 5 1.7 .9 27.0 +15 - 8 7 2.5 1.4 29.0 + 3 + 2 5 1.8 .9 Type of Sample - primary effluent (unfiltered) Date - 8:00 a.m. Nov. 26, 1962 Place - Williamston Sewage Treatment Plant No glucose added 69 TABLE 19 RESPIRATION RATES Thermobaro- Respiro- Corrected Oxygen ReSpira- Time meter meter Reading Consumed tion Rate Hrs. Reading Reading pl. mg/l mg/l/hr 1.5 + 7 -15 0 __ __ 2.5 + 9 —11 0 __ -— 3.5 + 8 - 5 3 1.0 1.0 5.0 +17 -11 6 1.9 1.3 6.5 + l + 7 8 2.6 1.7 8.0 —10 +28 18 .9 3.9 9.5 - 6 +27 21 6.9 4.6 11.25 - 1 +33 32 10.5 6.0 12.5 + 2 + 8 10 3.3 2.6 14 0 + 1 + 9 10 3.3 2.2 15 O + 5 0 5 1.6 1.6 16 0 0 + 4 4 1.3 1.3 23.25 —11 +32 21 6.9 1.0 25.25 — 2 + 4 2 .6 .3 27.0 +15 —13 2 .6 .3 29.0 + 3 O 3 1.0 .5 Type of Sample - primary effluent (filtered) Date - 8:00 a.m. Nov. 26, 1962 Place - Williamston Sewage Treatment Plant N0 glucose added TYPE OF SAMPLE, S.S., B.O.D., AND pH 70 TABLE 20 Type of Date Time Sample S.S. .0.D. pH East Lansing Sept.25,1962 10:00 a.m. primary 74 145 7.6 effluent Sept.25,1962 12:00 a.m. ” 60 160 7.3 Sept.25,1962 2:00 p.m. " 102 188 7.1 Sept.25,1962 4:00 p.m. ” 106 176 7.0 Oct. 3,1962 6:00 p.m. " 202 164 7.8 Oct. 3,1962 8:00 p.m. ” 192 191 7.3 Oct. 3,1962 10:00 p.m. ” 88 191 7.3 Oct. 3,1962 12:00 p.m. " 184 156 7 8 Nov. 5,1962 7:30 a.m. unfiltered 252 189 —- primary effluent Nov. 5,1962 7:30 a.m. filtered —- 43 primary effluent Lansing NOV. 12,1962 8:00 a.m. unfiltered 153 86 7.7 primary effluent NOV. 12,1962 8:00 a.m. filtered -— 34 primary effluent Williamston NOV. 26,1962 8:00 a.m. unfiltered 74 118 —— primary effluent Nov. 26,1962 8:00 a.m. filtered -— 77 primary effluent GROWTH RATES, MAXIMUM RESPIRATION RATE, AND BODS 71 TABLE 21 Growth Rate Max. Resp. Rate BOD Sample klO’ hr‘l mg/l/hr mg/T East Lansing Sept. 25, 1962 10:00 a. 0.036 10.0 145 12:00 a. 0.081 10.0 160 2:00 p. 0.119 14.0 188 4:00 p. 0.098 14.0 176 East Lansing Oct. 3, 1962 6:00 p. 0.097 9.5 168 8:00 p. 0.087 12.5 191 10:00 p. 0.092 13.0 191 12:00 p. 0.066 10.0 156 GROWTH RATES, MAXIMUM RESPIRATION RATE, AND B0D5 72 TABLE 22 Growth Rate Max. ReSp. Rate BOD: Sample klO, hr-l mg/l/hr mg/1 East Lansing Nov. 5, 1962 unfiltered * * 189 no glucose added unfiltered 0.036 6.5 239 +67 mg/l glucose unfiltered 0.078 11.5 289 +134 mg/l glucose unfiltered 0.118 28.0 389 +268 mg/l glucose unfiltered 0.126 38.0 589 +536 mg/l gulcose filtered * * 43 no glucose added . - filtered 0.062 6.0 93 +67 mg/l glucose filtered 0.112 15.0 143 +134 mg/l glucose filtered 0.125 25.0 243 +268 mg/l glucose filtered 0.128 32.0 443 +536 mg/l glucose Lansing NOV. 12, 1962 unfiltered * * 86 no glucose added unfiltered 0.019 3.3 136 +67 mg/l glucose unfiltered 0.026 5.9 186 +134 mg/l glucose unfiltered 0.059 12.0 286 +268 mg/l glucose unfiltered 0.053 17.0 486 +536 mg/l glucose filtered * * 34 no glucose added filtered 0.100 4.9 84 +67 mg/l glucose 73 TABLE 22-—Continued Growth Rate Max. Resp. Rate BOD Sample R10: hr-l mg/l/hr mg/T filtered 0.106 11.5 134 +134 mg/l glucose filtered 0.055 18.0 234 +268 mg/l glucose filtered 0.072 35.0 434 +536 mg/l glucose Williamston Nov. 26, 1962 unfiltered 0.184 8.0 118 no glucose added unfiltered 0.132 15.0 168 +67 mg/l glucose unfiltered 0.196 18.5 218 +134 mg/l glucose unfiltered 0.104 25.0 318 +268 mg/l glucose unfiltered 0.093 42.0 518 +536 mg/l glucose filtered 0.108 6.5 77 no glucose added filtered 0.128 13.0 127 +67 mg/l glucose filtered 0.127 20.0 177 +134 mg/l glucose filtered 0.140 44.0 277 +268 mg/l glucose filtered 0.123 44.0 477 +536 mg/l glucose *Samples for which X10 could not be Obtained. 8.0 BIBLIOGRAPHY American Public Health Association, American Water Association, and Water Pollution Control Federation. "Standard Methods for the Examination of Water and Waste Water." 11th Ed., Amer. Pub. Health Assoc., New York, N. Y. (1960). Balmat, J. 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