"L I B RAFLY OF THE UN IVLR.5ITY Of ILLINOIS 628 no37-4<3 ENGINEERING 001* noon ^ iiB^j *«w The person charging this material is re- sponsible for its return on or before the Latest Date stamped below. Theft, mutilation, and underlining of books are reasons for disciplinary action and may result in dismissal from the University. University of Illinois Library •_,rrj1-|W- % CIVIL ENGINEERING STUDIES SANITARY ENGINEERING SERIES NO. 38 CONTROLLING FACTORS IN METHANE FERMENTATION By R. E. SPEECE and R. S. ENGELBRECHT FINAL REPORT SEPTEMBER 1, 1962 THROUGH JANUARY 31, 1966 Supported By DIVISION OF WATER SUPPLY AND POLLUTION CONTROL U. S. PUBLIC HEALTH SERVICE RESEARCH PROJECT WP-00394 DEPARTMENT OF CIVIL ENGINEERING UNIVERSITY OF ILLINOIS URBANA, ILLINOIS JUNE, 1966 CONTROLLING FACTORS IN METHANE FERMENTATION By R. E. Speece - Principal Investigator Presently with New Mexico State University, University Park, New Mexico R. S. Engelbrecht - Co- Principal Investigator Iwao Iijima V. Kothandaraman Jan A. Kern Final Report September 1, 1962 Through January 31, 1966 Supported By Division of Water Supply and Pollution Control U. S. Public Health Service Research Project WP- 00394 Department of Civil Engineering University of Illinois Urbana, Illinois June 1966 Digitized by the Internet Archive in 2013 http://archive.org/details/controllingfacto38spee TABLE OF CONTENTS Page List of Figures .......................... iii Organization of Report. ....................... v Personnel .......... ......... vi Assay of Trace Metals, Trace Organics, and Physical and Chemical Factors. ........................ 1 Fractionation and Assay of Digested Sludge Supernatant and Cattle Rumen Liquor .................... 4 Dilution Studies. ........................ 6 The Effect of Frequent Temperature Variation on Methane Production 8 Introduction. 9 Procedure ........ ..... 10 Results 12 Discussion of Results ......... ..... 14 Engineering Significance. 18 ■, Bibliography 31 si The Effect of Temperature and Detention Time on the Activity , (j of Methane Formers and Acid Formers ........ 32 Procedure. 33 ;•* Results 33 jH Discussion 34 ;> The Effects of Constant Detention Time and Varied Loading Rates on Anaerobic Digestion ...... 37 Introduction 38 Literature Review . 39 Procedure „ 40 Results 43 Discussion 44 Summary 47 Bibliography 50 Appendix 51 Destruction of the Protein and Lipid Components of Raw Sludge During Anaerobic Digestion ......... 54 Introduction 55 Procedure 55 Discussion of Results ........... 56 Summary 63 References ...... ............. 83 Appendix 84 ii LIST OF FIGURES Figure No. Page 1 Proposed Anaerobic Contact Stabilization Process 20 2 Schematic Digestion Apparatus 21 3 Relative Gas Production vs Temperature 22 4 Dependence of Gas Production on Temperature 23 5 Gas Production vs Temperature 24 6 " " " " 25 -i ll !l ll II nc o II II II ll 27 q ti ii it it 28 10 Comparative Digester Performance at 35 C and 45 C 29 11 Temperature Dependence of Acid Forming and Methane Forming Microorganisms in Anaerobic Digestion of Sewage Sludge 30 Activity and Gas Production 36 1 Activity and Gas Production - Constant Detention Time and Varied Loading 48 2 Activity and Gas Production - Constant Detention Time and Varied Loading 49 1 Gas Production with Varied Loading and Varied Detention at 35°C 65 2 Gas Production with Varied Loading and Varied Detention at 25°C 66 3 Gas Production with Constant Loading and Varied Detention at 35°C 67 4 Gas Production with Constant Loading and Varied Detention Time at 25°C 68 5 Effect of Varied Loading Rate and Varied- Detention Time on Digestion of Raw Sludge 69 6 Effect of Detention Time on Digestion of Raw Sludge with Constant Loading Rate 70 7 Effect of Solids Detention Time on Effluent Characteristics for Varied Loading and Varied Detention 71 8 Effect of Solids Detention Time on Effluent Characteristics with Constant Loading and Varied Detention 72 9 Activity in COD with Constant Loading of Raw Sludge and with HAc Feed at 35°C 73 10 Activity in COD with Varied Loading and Varied Detention of Raw Sludge with HAc Feed at 35°C 73 11 Activity in COD with Constant Loading of Raw Sludge with HAc Feed at 25°C 74 12 Activity in COD with Varied Loading and Varied o Detention of Raw Sludge with HAc Feed at 25 C 74 iii List of Figures, Continued 13 Activity and Gas Production at 35 C, Raw Sludge and HAc Feed 75 14 Activity and Gas Production at 25 C, Raw Sludge and HAc Feed 76 15 Effect of Varying the Concentration of Substrate in the Inflowing Medium (S ) on the Steady-State Relationship 77 16 The Variation of the Bacterial and Substrate Concentration with Detention Time 78 17 Protein Concentration in the Effluent Sludge vs Dilution Rate (Varied Loading) 79 18 Protein Concentration vs Detention Time Constant o Loading and Varied Detention at 35 C 80 19 Output vs. Dilution Rate 81 20 Output vs. Detention Time 81 21 Effect of Acetate Supplementation on Gas Production 82 IV Organization of Report In presenting this final report, all the various factors which were studied with regard to their effect on methane stimulation are discussed. In the first part of the report, a series of studies are summarized. Some of these factors indicated a low magnitude of stimulation of the methane fermentation. Other factors showed no effect. In the remaining body of the report, a series of separate studies is presented of the factors studied which gave strong, positive results. Each of these studies is written up as a complete entity with separate figure numbers, table numbers, and bibliography. Personnel The Principal Investigator and Co-Principal Investigator for the entire period of the project has been R. E. Speece and R, S. Engelbrecht, respectively. Personnel employed on this project and their period of employment, were as follows: Professional Personnel C. V. RamaRao C. V. RamaRao C . V . RamaRao Kazune Ihda Edward Persha V. Kothandaraman V. Kothandaraman Jan Kern R. E. Speece R. E. Speece V. Kothandaraman V. Kothandaraman V. Kothandaraman I. Iijima I . I i j ima I. Iijima R. E. Speece R. E. Speece Title Research Assistant Research Assistant Research Assistant Research Assistant Research Assistant Research Assistant Research Assistant Research Assistant Ass't Prof, of San. Eng, Ass't Prof, of San. Eng, Research Ass Research Ass Research Ass Research Ass Research Ass Research Ass Assoc. Profe Assoc. Profe istant istant istant istant istant istant ssor ssor eriod oi : Appointment 7, of Time 9-16-62 to 6-15-63 50% 9-16-63 to 8-31-63 100% 9-16-63 to 9-31-63 1007o 9-16-62 to 6-15-63 507o 6-16-63 to 8-31-63 1007. 9-16-63 to 6-15-64 507c 6-16-64 to 8-31-64 1007c 9-16-63 to 6-15-64 507c 6-16-63 to 8-15-63 1007 6-16-64 to 8-15-64 1007c 9-1-64 to 6-15-65 507c 6-16-65 to 9-1-65 1007 o 9-16-65 to 2-1-66 507c 9-1-64 to 6-15-65 507c 6-16-65 to 8-15-65 1007c 9-16-65 to 2-1 -66 507c 6-16-65 to 8-16-65 1007c 9-16-65 to 2-1 -66 257o The contributions made by these persons in carrying out the objectives of this study are sincerely acknowledged. VI Assay of Trace Metals, Trace Organics And Physical And Chemical Factors Assay of Trace Metals and Trace Organics The initial phases of the project were designed to evaluate the effects of a number of compounds which had produced stimulation in a previous study. These compounds were: iron, cobalt, thiamine, proline, glycine and benzimidazole. The addition of these separate compounds and combinations thereof allowed acetate utilization rates of 1000 mg/l/day. Whereas, the control, which received none of these compounds, operated at from 200 to 500 mg/l/day . However, even the maximum rate achieved was much lower than is commonly experienced in the anaerobic digestion of sewage sludge. Obviously, something was still lacking in the environment which inhibited the rates of methane production. During this study period, ammonium bicarbonate was substituted for the sodium bicarbonate buffer system. This resulted in increasing the ammonia nitrogen content from 60 to 800 mg/1. However, no noticeable stimulation of methane production resulted. Assay of Physical and Chemical Factors Asbestos has been reported to stimulate methane production. In a study to evaluate this effect, it was found that a digester containing 30 grams per liter of asbestos resulted" in doubling of the methane production over that of a control which had no asbestos. In an attempt to elucidate the effect of asbestos on methane production, asbestos was extracted under anaerobic conditions and the extract was assayed. The addition of the anaerobic asbestos extract resulted in no significant increase in methane production over the control digester. References were found in the literature of bacteria which multiply more rapidly in the presence of material which increases the surface area, such as glass beads or an inert, finely divided precipitate. A slight, positive stimulation was found when powdered CaCOo was added to digesters, but the effect was only minor, 2- The effect of volatile acids concentration was evaluated and found to control the rate of methane production. The Michaelis-Menten Model has since been found by other investigators to reasonably predict acetate utilization rates. The effect of detention time on acetate utilization rates was evaluated. At longer detention times, acetate utilization rates were proportionately greater due to the greater standing crop of organisms maintained. However, at increasing detention times, the unit activity of mg/1 acetate utilized per gram of organisms per day decreases. There appears to be a relationship whereby the activity of the organisms decreases with mean cell age. The following results were obtained: Acetate Utilization Rates 3.15 gm acetate/day at 6 day detention time. gm cells 1.00 gm acetate/day at 100 day detention time. gm cells Net Synthesis Rate 5.3% synthesis of acetate at 6 day detention time. 1.0% synthesis of acetate at 100 day detention time. The effect of mixing was strongly evident. Continuous mixing resulted in more than doubling of the acetate utilization rate as compared to a nonmixed control. The surface charge of the methane producing organisms was altered by adding AICI3 and a cationic polyelectrolyte. There was no significant stimulation or repression in either case. However, it did indicate that the addition of a coagulant for removal and recycle of the bacterial mass in an anaerobic waste treatment process is feasible. A chelating agent, EDTA, was added to a "washed-out" digester to assay its effects on methane production. There was no significant effect after several slug additions of 100 mg/1 of the sodium salt of EDTA. This indicates that the limited rates of methane production were not due to the inhibiting action of a toxic metal which was able to be chelated. In summary,, a number of factors were elucidated which stimulated the rate of methane fermentation. These were: Iron alone Iron and cobalt in combination Asbestos Calcium carbonate solids Increased volatile acids concentration Increased sludge retention time Continuous mixing Increased temperature to an upper limit of 45 C. Fractionation and Assay of Digested Sludge Supernatant and Cattle Rumen Liquor An extensive study of the nutritional requirements of a pure culture of methanogenic bacterium Methanobacterium ruminant ium has been underway by Professor Marvin P. Bryant, Department of Dairy Science, University of Illinois, Urbana, Illinois. This organism grows in a liquid media containing H2-CO2 as an energy source. An unidentified growth factor found in rumen fluid and digested sewage sludge was required for growth. This growth factor was not found in yeast extract, cell-free extract of E^ coli or many other crude materials commonly used to grow nutritionally exacting bacteria. Therefore, a study was undertaken in the Sanitary Engineering Laboratory to fractionate cattle rumen fluid and digested sludge supernatant and assay its stimulation capacity in an enriched culture of acetate utilizing methanogenic bacteria. Very little energy is available to the microorganisms from anaerobic acetate utilization as compared to the very high amounts of energy available from hydrogen utilization. Consequently, much higher growth rates are possible to -4- organisms utilizing hydrogen as substrate. However, even though considerably lower growth rates would be anticipated with an acetate substrate, qualitative stimulation was considered to be sufficient to indicate the presence of growth factors. The University of Illinois maintains several fistulated cattle from which the samples of rumen fluid were withdrawn. The digested sludge supernatant was taken from the Urb ana -Champaign Sewage Treatment Plant. The procedures used to fractionate the rumen fluid were as follows and were used singularly or in combinations. Centrifugation - 12,000 x g for 10 minutes. Activated Carbon Adsorption - using chloroform extraction in Soxhlet apparatus with vacuum distillation for removal of chloroform. Ion Exchange - Hydrogen cycle, hydroxyl cycle and mixed bed deionization. Dialysis - Membrane used permitted dialysis of inorganic ions and organic coloring matters, but not proteins, lipids, carbohydrates or other macromolecules . Solvent Extraction - Butanol and petroleum ether soluble fractions. Ashing of Residue The conclusions of this study were: 1. There was a limiting concentration for the addition of evaporated residue from digested sludge supernatant, beyond which the addition was inhibitory. This inhibition probably was related to salt toxicity. 2. The stimulatory factors in digested sludge supernatant were not eliminated by centrifuging at 12,000 x g for 10 minutes. 3. Solvent extraction of digested sludge supernatant with butanol indicated that the greater fraction responsible for stimulation was insoluble in butanol. 4. Salts responsible for reducing the activity of the methane organisms were dialysable and exist as organic chelates. 5. The ionic species of the dialysable inorganics in the digested sludge supernatant inhibited methane fermentation. ■5- 6. Anionic exchange of the centrifuged digested sludge supernatant at pH 7.0 did not alter the stimulation capacity. 7. Some component in the digested sludge supernatant with a carboxyl or phosphate fuctional group caused a slightly increased rate of methane fermentation. 8. Rumen liquor and the various fractions and combinations thereof, except the centrifuged rumen liquor solids, inhibited the rate of acetate utilization. Dilution Studies In the previous sections, the acetate utilization rates never approached the high rates reported by McCarty and Vath. The rates would rise to a certain value and reach a plateau. Some limitation prevented higher rates. This was verified in a study in which a 3-liter digester was fed acetate and buffered nutrient solution containing inorganic nutrients. Excess acetate substrate was always maintained in the system to keep from limiting methane production and the hydraulic and solids detention time was 15 days. At equilibrium, the completely mixed contents were equally divided between two digesters. One digester was mainr tained at a volume of 1.5 liters to serve as a control while the other digester was diluted back to the original volume of 3 liters with buffered nutrient solution, Operation of both digesters was continued on a 15 day detention time with excess acetate maintained at all times. At the subsequent equilibrium, both digesters reached the same acetate utilization rate per unit volume of approximately 1000 mg/l/day. However, the total acetate utilization was consequently double in the digester, which had been diluted 100 percent with buffered nutrient solution. Therefore, this strongly indicates either the accumulation of toxic end products or a limiting nutrient concentration. Studies at Purdue in a dialysed system have also confirmed this observation. They noted gas production reached a maximum and decreased. However, after placing fresh media outgide the dialysed system, gas production would reach a new maximum which was double the initial maximum. Thus, either a toxic end -6- product was lost from the system or a new supply of limiting nutrient was added when the old media was replaced by fresh media. In an attempt to determine whether solubility of a required ion was limiting the rate of methane production, eight 10-liter digesters containing one liter of digested sludge were fed for 10 days with raw sludge- All digesters were then diluted to 10 liters volume with the following dilution waters: 1. Tap water 2. Buffered nutrient solution 3. Demineralized water 4. Stream water 5. Stream water - zeolite softened 6. Tap water - zeolite softened 7. Buffered nutrient solution - zeolite softened, 8. Centrifuged digester supernatant. Zeolite softening treatment was used to remove any multivalent cations which may precipitate anions in the digester environment. There was no significant difference in the acetate utilization rates of any of these digesters after dilution. This indicates that the presence or absence of multivalent cations in the dilution water had no effect on the system. However, this study is to be repeated in a "washed out" system which is more clearly defined as to the chemical constituents as opposed to this present preliminary study where raw sludge was initially fed. _7_ THE EFFECT OF FREQUENT TEMPERATURE VARIATION ON METHANE PRODUCTION -8- THE EFFECT OF FREQUENT TEMPERATURE VARIATION ON METHANE PRODUCTION By R. E. Speece, Associate Professor of Sanitary Engineering and Jan A. Kern, Research Assistant, University of Illinois Presented at the Annual Meeting of Central States Water Pollution Control Federation June 1964 Urbana, Illinois Introduction The methane- forming microorganisms are generally considered to be more sensitive to physical and chemical changes than the acid-forming microorganisms involved in the anaerobic digestion process. Also, because the methane -forming microorganisms utilize the volatile acid end-products produced by the acid forming microorganisms, failure of the methane- formers to utilize the volatile acids at approximately the same rate as they are produced, can result in a "stuck" digester. As a result, the fastidious nature of the methane- forming organisms combined with the critical position they occupy in the anaerobic digestion scheme, makes it very important that satisfactory environmental conditions be maintained for them in order to promote good digestion. However, in normal anaerobic digestion, the volatile acid concentration is low, indicating that the methane- forming organisms are capable of utilizing the volatile acids at least as fast as they are being formed. In other words, the acid formation is the rate-limiting step in normal digestion. A number of studies have been made of the effect of temperature on the anaerobic digestion process (1) , (2) , (3) . These studies have been made using a complex feed such ag primary sludges. When using complex substrates, both the acid-formation and methane- format ion rates are involved and the net overall effect being observed is actually controlled by whatever the rate-limiting step in the process happens to be. If the volatile acid concentration is low, then the acid farming step determines the overall rate which is observed. In this study, acetate was fed as the sole substrate. Therefore, only the rate of activity of the me thane -forming organisms was observed, since only these organisms can utilize acetate under anaerobic conditions. The objectives of this study were to determine the effect on the methane- forming organisms in a mesophilic sludge under the following conditions" 1. A temperature change sustained for a number of hours as would occur if the temperature of the digester contents rose or fell over a 24 hour interval. 2. A temperature drop for a 15 minute interval as would occur when digested sludge is pre-mixed with the raw sludge before it is pumped to the digester. 3. A temperature drop for a 2 hour interval as would occur if a proposed "anaerobic" contact stabilization process were feasible.. Presently, cold, diluted wastes are uneconomical to treat by anaerobic digestion due to excessive heat requirements. However, if the wastes could be adsorbed on a bacterial or inert surface and thus concentrated, it may prove economical to heat this more concentrated form of the sludge and stabilize it by anaerobic digestion. Such a system is shown schematically in Figure 1. Procedure A 2 1 Erlenmeyer flask was placed in a temperature controlled bath as shown in Figure 2. The digester was purged with nitrogen gas and 1.8 1 of sludge from a well operating 35 C digester was transferred to the flask. The digester contents were mixed by a magnetic stirrer placed underneath the bath. The temperature of the bath was controlled by a thermoregulator and it was kept in circulation by an air diffuser. At the beginning of a run, the volatile acid concentration was determined. Sufficient acetate was then added to bring the volatile acid concentration to 700 to 1500 mg/1 , . The acetate was fed as the neutral salt in the form of calcium acetate. Acetate was continuously fed to the digester with an -10- electrolytic pump at approximately the same rate at which it was being utilized. Thus, feed concentration never limited the rate of gas production. At the start of a temperature drop study, the temperature was maintained for about an hour at the level to which it was to be raised after the drop. Gas production was recorded at 10-minute intervals during this first hour, during the drop, and for a sufficient period after the drop to reach equilibrium. These readings were continued during the temperature drop. Test runs were made to observe the effects of both temperature increases and decreases on the rate of gas production. Observations were made on gas production for the following temperature schedules: o Initial Temperature- ( C) Drop Temperature ( C) Duration of Temperature Drop (Min) __- 35 10 15 35 10 120 35 20 15 35 20 120 50 10 120 It was desirable in the course of this study to confirm the effect of temperature on both the acid-formation rate and gas production rate. To accomplish this, an actively digesting sample of sludge was taken from the primary digester at the Champaign-Urbana Sewage Treatment Plant. This insured that both groups of microorganisms were functioning well. An increase in volatile acids concentration was simulated by adding sufficient calcium acetate to raise the volatile acids concentration in the sludge to 1800 mg/1. Then 10% by volume of primary sludge was added to insure adequate substrate for the acid-forming organisms. The sludge was then divided into 2 portions and placed in flasks which had been purged of oxygen by flushing with nitrogen and carbon dioxide gas. ■11- The contents of the one flask were maintained at 35 C and the contents of the second flask were maintained at 45 C. Both flasks were continually mixed and once a day 1/20 of the flask contents was withdrawn and replaced by an equal amount of raw sludge. The raw sludge feed simulated normal digester operation and provided a food source for the acid forming microorganisms s and the volatile acids provided a food source for the methane- forming microorganisms „ Thus s by comparing the volatile acids concentration and gas production in the 45 C digester, with that in the 35 C digester, the effect of a temperature increase on the relative rates of activity of the two groups of microorganisms (the acid - formers and the methane- formers) could be determined. If the activity of both groups of microorganisms was dependent to the same o o degree on temperature, the volatile acids concentration in both the 35 C and 45 C digesters should be equal, because the increased volatile acid utilization would be matched by a corresponding increased volatile acid formation. However, if the o o volatile acids concentration was higher in the 45 C digester than in the 35 C digester, this would signify that the rate of production of volatile acids by the acid-formers increased to a greater degree with temperature than the rate of volatile acid utilization by the methane-formers. Finally, if the volatile acids concentration was lower in the 45 C digester than in the 35 C digester, this would indicate that the activity of the methane- formers increased at a greater rate with temperature than the acid-formers. Results The experimental results fall very much in line with those which would be predicted from theoretical considerations of the response of microorganisms to various temperature levels and temperature, changes. Table 1 and Figure 3 give the relative gas production rates for a 35 C (mesophilic) acclimated sludge when the temperature is changed as indicated. -12- TABLE 1 GAS PRODUCTION RATES FOR EXTENDED INTERVALS OF TEMPERATURE Temperature Time Interval Gas Production Relative Rate ( C) (Hrs) (ml/hr/1) 20 20.5 Nil 25 20.8 24 36 30 25.7 46 68 35 68* 100 40 5.9 90 132 45 54.0 117 171 ^Calculated Value Figure 4 indicated the relative gas production rates of a digester at 34 C which is then dropped to 27 C, raised back to 33 C and finally raised on up to 40°C. Figures 5 through 8 shows the response of a mesophilic sludge held at approximately 35 C and then dropped to 10 C for both 15 minutes and 2 hours and o also dropped to 20 C for the same two time intervals. Figure 9 shows the corresponding effect of a temperature drop to 1.0 C for a 2 hour interval on a mesophilic sludge held at approximately 50 C before and after the temperature drops. Figure 10 indicates the effect of an increase in temperature from ° 35 C to 45 C on the relative rates of activity of the acid- formers and the methane formers. Figure 11 is the curve of rate of activity vs. temperature for acid- forming and methane- forming organisms as hypothesized from the results of this study. -13- Discussion of Results Figures 3 through. 9 reveal the effect of frequent temperature variations on just the methane production stage of anaerobic digestion, because acetate was fed as the sole substrate . Under anaerobic conditions , acetate can be used by only the methane-f ormers . The amount of volatile acids in the digester was continually maintained at sufficiently high concentrations which would not limit the rate of gas production. This is not the case in normal sewage sludge digestion which characteristically maintains a low and thus rate-limiting concentration of volatile acids. The neutral calcium salt of acetic acid was fed so that the only gas which would evolve from a digester held at constant temperature would be that due to the activity of the methane - formers . No carbon dioxide evolution would be encountered when this neutral salt was fed to the digester. Also,, the calcium would continually precipitate from solution as the acetate was consumed and there would be no cation toxicity problem. Finally, it was necessary to insure an unlimited food source for the methane- formers at all times and with the neutral calcium acetate being fed, there was no danger of an accidental drop in pH„ Feeding calcium acetate as the sole substrate results in a gas composition of 75% CH. and 25% CO . The CO introduces an error into the gas production values with each temperature change, however, due to its high solubility in water and the great dependence of CO solubility on temperature. Thus, following each temperature increase, there is an initial marked increase in gas production, after which gas production comes to an equilibrium at a somewhat lower rate. This spike in the gas production is accounted for by the decreased solubility of CO., at higher temperatures and therefore the release of C0 9 from solution. This correction was not made because, the exact percentage of CO in the digester atmosphere would have to be known both during the temperature drop when C0„ would have been sucked -14- back into solution and after the raise in temperature when the CCL would be somewhat above the theoretical 25%. The CCL equilibrium of the digester contents and atmos- phere was established soon after a temperature increase occurred because the volume at the top of the Erlenmeyer flask was small and was rapidly flushed out by the gas production. Figure 3 shows the clear response of gas production from mesophilic sludge which had been incubated at 35 C and was then incubated for a number of hours at the temperatures shown. Figure 3 indicates gas production bears a linear response to temperature as opposed to the traditional logarithmic response. Below a tem- o perature threshold of 20 C, gas production was nil. This observation is also o borne out by later figures. Gas production could not be sustained at 50 C for more than a few hours before it commenced dropping off as noted in Figure 9 ° o in which a mesophilic digester was raised to 50 C, then lowered to 10 C for 2 hours and finally raised back to 50 C. From Figure 3, it is seen that a decrease in digester temperature from 37 C to 30 C reduces the gas production to approximately 50%. Golueke (1) found that the destruction of volatile matter decreased from 50% to 40% when the digestion temperature was decreased from the range of 35 C - 55 C down to 30 C. Thus, while volatile matter destruction at 30 C decreased to only 80% of that at 37 C, gas production would suffer much more severely by decreasing to 50% of the original. Golueke ' s results (1) were based on long term digester operation. As shown in Figure 4, the response of the methane- formers to a temperature change is immediate. As the temperature was dropped over an hour's period of time from 34 C to 27 C, the gas production decreased to about 12% of that at 34 C. As the temperature was increased from 27 C back to 33 C, the gas production resumed at approximately 100% of that before the temperature drop occurred. A subsequent increase in temperature to 40 C was accompanied by gas production rates of about 140% of those at 34 C . -15- Figures 5 through 8 indicate that as long as the digester temperature is below 20 C, gas production is nil. However, as soon as the temperature is raised back to the normal range of anaerobic digestion, gas production resumes at a rate proportional to the temperature within the range 20 C to 45 C . There appears to be no carryover of the adverse effect on the methane-formers from exposure to temperatures of 10 C and 20 C for 15 minute periods. Gas production resumed at essentially the same rate as soon as the temperature was restored to the initial level. Figure 6 showed a slight lag in recovery of gas production after maintaining a sludge at 10 C for two hours. It is noted that temperature recovery was also slow in this case due to the type of water bath used during this run. This equipment was modified in later experiments to allow more rapid temperature recovery. Figure 9 shows no gas production lag for a sludge held at 10 C for 2 hours and then raised to 50°C. Thus, the methane -forming microorganisms appear to be very adaptable to frequent temperature changes with no adverse effects resulting from temperature drops. However, Golueke (1) reported in his studies on the anaerobic digestion of sewage sludge at various temperature levels that once a digester was well established, it became very sensitive to any abrupt temperature drop. He cited the case of a 35 C digester in which the temperature dropped to 25 C for about 16 to 18 hours, and resulted in a reduction in breakdown from 52.8% down to 44%. It is hypothesized that such a situation would develop when the methane- formers ' rate of gas production is seriously retarded at the lower temperature. Thus, the volatile acids which are formed and accumulated are not converted to a gas and since the volatile acids are not volatile under the neutral pH conditions under which the solids are evaporated, they are measured as part and parcel of the volatile solids not destroyed. Table II and Figure 10 revel a very significant comparison. It vividly shows the beneficial effect of increased temperature on a digester in which the methane- -16- formers are not inhibited by anything but temperature. That is to say, there are no adverse environmental or physiological conditions restricting the rate of activity of the. methane-formers except temperature. A digester with high volatile acids concentrations and continued raw sludge addition was inoculated by taking 3.6 liters of well digesting sludge from the primary digester at the Champaign-Urbana Sewage Treatment Plant and adding 0.4 of raw sludge. The volatile acids concentration in the primary digester at the time was approximately 300 mg/1 as acetic acid. To this mixture of raw and digested sludge, sufficient calcium acetate was added to increase the volatile acids con- centration to approximately 1800 mg/1. This 4 1 volume was thoroughly mixed and divided into 2-2 1 volumes and incubated at 35 C and 45 C respectively. Each day 100 ml of raw sludge was added to each digester to simulate a 20 day detention time. TABLE II COMPARISON OF VOLATILE ACIDS REDUCTION AT 35°C AND 45°C 35< D c 45°C Time Vol. Acids Cone Comulative Gas Production Vol. Acids Ci one. Cumulative Gas Production (Hrs.) (mg/1) (ml) (mg/1) (ml) 1840 Cms Liquefied 1800 Gms Liquified 5.5 2040 240 (0.8) 1500 1000 (1.07) 7.5 1920 750 (1.41) 700 2840 (2.55) 8.5 1620 1250 (1.64) 340 4000 (3.75) 1.5 1440 1940 (2.43) 280 4610 (4.66) Figure 10 shows that in less than 30 hours, the volatile acid concentration in the 45 C digester had dropped about 1500 mg/1, to the original level. However, in the 35 C digester the volatile acids concentration had dropped only to about 200 mg/1 lower than it was initially. -17- It would be expected that the increase in temperature would not only increase the rate of activity of the methane- formers , but also the rate of activity of the acid-formers. This is borne out by calculations on the data from each 2 1 digester. o At 28.5 hours, 4000 ml of gas was produced by the 45 C digester and the volatile acids had decreased by 2.92 grams in the 2 1 digester. Assuming 600 ml of gas is produced per gram of volatile acids destroyed, then 4000 ml of gas represents the destruction of 6.67 gms of volatile acids. Since the amount of volatile acids in the digester was only reduced by 2.92 grams, then 6.67 - 2.92 = 3.75 gm of volatile acids must have been contributed by the acid-formers during this time. By similar calculations, it is found that only 1.64 gms of volatile acids were contributed by the acid-formers in the 35 C digester after 28.5 hours. It is concluded from Figure 10 that while an increase in temperature does increase the activity of both the methane- forming and acid-forming organisms, the methane- formers increase their activity at a considerably greater rate per C. . Thus, not only can the methane-formers consume the additional volatile acids contributed by the acid-formers at higher temperatures, but in addition they are able to reduce the pool of volatile acids which is initially present. This explains the rapid decrease in volatile acids in the 45 C digester as compared to the less rapid decrease in the 35 C digester. Engineering Significance Figure 11 is hypothesized as a characteristic plot of the temperature dependence of the acid- forming and methane- forming microorganisms in a favorable anaerobic digestion environment. Above a certain temperature, X, the methane-formers are capable of consuming volatile acids at a rate greater than the rate at which they are supplied by the acid-formers. Therefore, the volatile acids concentration remains low. Below this temperature, X, the rate of activity of the methane- formers -18- is lower than the acid-formers. The net result is that volatile acids start to accumulate in the digester. Experience seems to indicate that the temperature, X, at which the rates are equal is approximately 30 to 35 C. There appears to be only a minor advantage to be gained by maintaining the temperature of a sewage sludge digester above this range, since the rate of liquefaction (acid-formation) is not appreciably increased according to Golueke (1). Above temperature, X, acid formation is the lower and thus the rate-limiting step in the two-step digestion process, and the increased potential capacity of the methane -formers cannot be exploited because they have only a limited food supply. However, in the anaerobic digestion of a soluble industrial waste, the rate of acid formation may be appreciably greater than with a complex solid like sewage sludge. In such a case, advantage could be taken of the markedly greater rate of methane formation at the 40 to 45 C level as compared to the 35 C commonly used. This would result in a much smaller digester. Pre-mixing of digested sludge with raw sludge before pumping the mixture into the digester appears to stop methane production if the temperature of the mixture would be lowered to less than 20 C. However, no retardation of gas production results as soon as the temperature is restored to normal inside the digester. The flow diagram proposed in Figure 1 appears to be feasible with respect to the methane- forming organisms being able to adapt to the changing temperatures. The adsorptive concentration of the pollutant would be critical and require considerable study. This study was supported by Grant WP-Q0394 from the Division of Water Supply and Pollution Control, United States Public Health Service. -19- *tp- 0) o 3 o o o LU =Q>= CO 3 \- < < Qu CL < o h- to UJ Q O < LU X o CO CsJ LU ID •21' o o m c o o •n o 10 o c « O Q. uu 150 / too < *r> to 20 30 Temperature 40 50 FIGURE 3. RELATIVE GAS PRODUCTION vs TEMPERATURE 22 (O E UJ a: 85 a. UJ »- c o o o tic a. < o z UJ Q UJ Ql UJ Q UJ o • uoipnpojcj SD9 |o;|iu| jo juso j©j $ # m- •J*t»JtdMitj a o X I E UJ tr 3 < UJ CL 2 UJ CO > z o H O z> a o oc CL to < UJ oc SUS£ »V uotpnpOJd 809 JO J^D j«^ -J14- 3 a — 8Jn|0J9dujaj[ M w O I I LU CC UJ Q. 2 UJ h- > o o 3 o o cc a CO < <0 UJ tr D CO u. OeQ£ IV U0U3ftp0J<4 so© |o * ua D *»d •25- Q a — 9Jn^0J9duia_L M V. 3 O I I e UJ a: 3 !5 or Ul UJ h- c/> > o 3 a o a: o. C/) < Hi AC DoQ£ IV uotpnpoi^ sdo lOvi^O J »d -26- o — ajnjoJOduia^ w i I LU QC Z> fee q: LU o. 2 LU H (/) > O Q O QC Ol 00 LU a: O u. -17 - o - «Jn*oJ8dujax M o I i I < a. S til co > o D Q O a: a. < 0) q: D CD OoOS W uouonpoj^ S09 ^0 * u »0 J3 d -28- I lu -uoinnpoJd S09 9AUO|nujn3 o o o m 3 O o o o o o (SI o o o spp\/ 9|uo|OA ox p»m»auoo e6pn|s JO sluojq paiDinojDQ O o m ^r o z < o o IO ro !i UJ o z < QC O Ul 0: OL 0: co UJ > < a. o o UJ QC D u. |/6iu - UOUDJJU93UO0 Sppy 9|U0|0A -29- Q Z < z o CD O q: uj < z < 2 CD Uu o z < >■ > 1- CO u UL co < UJ 2 or uj a: 2 o - K < Z CO a UJ tr t- UJ o UJ H C\J a CO UJ a o z g o m ro 2 S >- t- < Z Q UJ O UJ CNJ Q CO or UJ z co o UJ r- o >- z n < UJ U 1- o to UJ Q o lO CVJ >- < z UJ t- UJ Q O O O o o o ro O O O CvJ O o o (Aop/|/|UJ) NOIlonGOdd SV9 ( Q O QC Q_ < < >- > LU S H e> Z z O Q LU < O _J K Q UJ LU Q CC 1- Z § < Q 1- Z z < o CJ Nononaodd svo Sd3KIU0d 3NVH13IN su3iaiuoj aiov o o o o o o in *- ro I o o o (^Dp/r/y^-NCMlDnaOdd SV9 (Xop/jy6uj aO0)-AHAI10V CD Lu -«»- (fop/*yyuj-NoiiDnaoad svs (XDp/jy6uj aO0)-AHAI10V CM -»». 1. Agardy, F. J., Cole, R.D., and Pearson, E.A., "Kinetic and Activity Parameters of Anaerobic Fermentation System," SERL Report No. 63-2, Sanitary Engineering Research Laboratory, University of California, Berkeley, California; 1963, 2. Andrews, J.F., Cole, R.D., and Pearson, E.A., "Kinetics and Characteristics of Multistage Methane. Fermentations , " SERL Report No. 64-11, Sanitary Engineering Research Laboratory, University of California, Berkeley, California; 1964. 3. Banerji, S. K. , "Biological Removal of Colloidal Waste in the Activated Sludge Process," Ph.D. Thesis, University of Illinois, Champaign, Illinois; 1965. 4. Buswell, A. M. , and Hatfield, W. D . , "Anaerobic Fermentations," Illinois State Water Survey Bulletin No. 32, Urbana, Illinois; 1936. 5. Golueke, C.A., "Temperature Effect on Anaerobic Digestion of Raw Sewage Sludge," Sewage and Indu s trial Wastes , Vol. 30, 10; 1958 6. Jeris, J. S., and McCarty, P. L. , "The Biochemistry of Methane Fermentation Using Cl4 Traces," Journal Water Pollution Control Federation , Vol 3_7_; 1965. 7. Jeris, J. S. and McCarty, P. L., "Significance of Individual Volatile Acids in Anaerobic Treatment," Proceedings of 17th Industrial Waste Conference , Purdue University; 1962 „ 8. Lawrence, A. and McCarty, P. L „ , "Kinetics of Methane Fermentation" Water Pollution Control Federation, Atlantic City, October; 1965. 9. McCarty, P. L. and Vath, C. A., "Volatile Acid Digestion at High Loading Rate," International Journal of Air and Water Pollution , Vol 6^, 65; 1962. 10. Smith, P., Private Communication 11. Speece, R. E. and McCarty, P. L. , "Nutrient Requirements and Biological Solids Accumulation in Anaerobic Digestion," 1st International Conference on Water Pollution Research, pp. 305-333, Pergamon Press, New York, 1964. 12. Todd, H. R. , "A Study of the Acid Intermediates in the Methane Fermentation," Ph.D. Thesis, University of Illinois; 1936. ■50- APPENDIX -51 TABLE 1 Average Activity and Average HAc Feed Rate Constant Detention and Varied Looding Days > of Average Substrate and Loading Acid Forma- Methane tion Formation (COD mg/l/day) (COD mg/l/day) Gas Reduction (ml /I /day) 19-27 R 0.005 0.05 0.1 0.2 96 675 1530 2590 1340 1830 2470 3640 895 1200 1490 2240 Acetic Acid Fed (mg/l/day) 1.280 1200 930 1050 Residual Vol. Acid (mg/1 ) 20-27 A 0.005 0.05 0.1 0.2 254 560 717 1380 1660 2200 2280 2580 1115 1480 1570 1620 1410 1700 1490 1130 18-26 18-22 " M 0.005 0.05 0.1 0.2 267 855 1307 2840 1490 2207 2680 2680 1060 1560 1930 1540 1180 1320 1370 708 651 628 1824 18-26 21-27 G 0.005 0.05 0.1 0.2 267 1050 2060 1270 615 2380 3130 1310 358 1620 2140 1190 413 1320 1120 1533 682 863 2288 20-26 20-27 Set Sew Control 127 338 815 1698 465 1055 760 1510 1280 830 * R: raw sludge A: activated sludge Ms Metrecal G: Glucose •52- TABLE 2 Volatile Mat te r Destruction and Ga s Produ ction Efficiency Constant Detention and Varied Loading NO Substrate _ ^ #/cf/dy) Loading Complex Org. Dest. (mg/l/day) Tot. Vol. Matter Dest. (mg/l/day) Gas Tot (c Prod.. Per .Vm„ De-st,, f/lb/Vm) Methane Per Tot. Vm. Dest, (cf/lb/Vm) Gas Tot Prod. Per . Vm„ fed (cf/lb/Vm) 1 R 0.005 27 1430 12.2 6.6 11.7 2 0.05 1560 2740 7.1 4.3 9.7 3 0.1 1230 2240 10.6 7.0 8.9 4 0.2 1800 2790 12.5 8.1 8.0 5 • A 0.005 101 1620 12.5 7.4 12.7 6 0.05 298 1960 11.7 7.0 9.5 7 0.1 553 2130 12.2 7.0 8.5 8 0.2 1075 2180 11.9 7.6 6.4 9 M 0.005 967 17.0 9.9 13.0 .0 0.05 322 1650 15.2 8.7 13.1 .1 0.1 770 2160 14.0 7.8 12.1 2 0.2 1720 14.9 10.4 11.6 G 0.005 0.05 0.1 0.2 Set Sew. Control 560 1190 1740 206 1810 2150 1610 27.8 14.2 16 . 2 13.1 443 1300 1.4.2 12.5 21.0 8.3 9.4 6.4 9.3 8.3 11.8 12.5 13.7 10.5 9.5 10.7 ■53- DESTRUCTION OF THE PROTEIN AND LIPID COMPONENTS OF RAW SLUDGE DURING ANAEROBIC DIGESTION 54 DESTRUCTION OF THE PROTEIN AND LIPID COMPONENTS OF RAW SLUDGE DURING ANAEROBIC DIGESTION Introduction The objective of this study was to observe the degradation of the protein and lipid fraction of raw sludge in anaerobic digestion. This was observed under natural conditions prevailing when only raw sludge was added as well as under a condition in which supplemental additions of acetate were made to in- crease the volatile acid concentration to 2000 mg/1 daily. These observations were made while varying the detention time and tempera- ture. The studies were made under constant as well as varied loading conditions. Constant loading rates were achieved by adding the same quantity of sludge each day to digesters operating at the various detention times and adding sufficient dilution water to achieve the desired detention time. This had the obvious ad- vantage of being able to elucidate the effect of detention time alone without having to consider the effect of varied loading rate which results when deten- tion time is controlled by feeding varied quantities of sludge. The total solids, volatile solids, protein and lipid components of the raw sludge feed were determined. The degradation* of these components during anaerobic digestion was ascertained by analyzing the effluent samples and comparing with the feedo An attempt was then made to fit the results to a model developed by Herbert, Elsworth, and Telling (1). Procedure Laboratory digesters containing 750 ml of actively digesting sludge were used in this study. Raw sludge from the Urbana-Champaign Sewage Treatment Plant was used as the feed. In some instances it was concentrated by allowing natural flotation to occur overnight and draining off the solids-free water. -55- Digesters were set up according to the following schedule for the first phase; 35 °C 25 °C Constant Loading Varied Loading Constant Loading Varied Loading Loading Rate „ (#volatiles/ft /day 0.15 0.09-0.35 0.15 0.038-0.23 Detention Time (Days) 6-25 6-25 6-25 10-60 During the second phase of the study, the volatile acid concentration was supplemented by adding acetate in order to increase the concentration to 2000 mg/1 at the start of each day. The detention times studies were from 10-30 days. Discussion of Results The first phase of this study was conducted for 51 days with those digesters which operated well and for 81 days with those digesters which proved difficult to operate at the shorter detention times. The former, stable digesters were used for the second phase of this study. During this second phase, the raw sludge was supplemented with acetate to continually maintain a high volatile acids con- centration. The operation of these digesters is indicated in Figure 1, 2, 3, and 4. The three-day average gas production of the respective digesters is plotted vs days of operation. In order to achieve satisfactory digestion in some of the shorter detention time digesters, it was necessary to lengthen the detention time for the periods indicated in the figures. Difficulty was encountered in achieving satisfactory operation of the follow- ing digesters: ■56- Temp Type of Loading Rate Detention Time (C°) Loading constant (#/ft 3 /day) .15 (Days) 35 6 35 constant .15 7.5 35 varied .35 6-6.25 25 constant .15 7.5 25 varied .23 10 The highest methane production was obtained from the following digesters: Temp. (C°) Type of Loading Constant Loading Rate (#/ft 3 /day) .15 Detention (Days) Time CH 4 (Vol Production ../vol, /day) 35 °C 15 1.31 35 °C Varied .30 7.5 2.18 25 °C Constant .15 15 1.17 25 °C Varied .20-. 15 11.5-5 1.17 Figure 5 shows the total gas production and gas production per pound of volatile matter added under varied loading conditions. It is to be expected that the total gas production decreased with increasing detention time in the varied loading rate study, since less volatile matter is added to achieve the longer detention time. However , at detention times of 15 days or more, total gas production was essentially the same at both 25 °C and 35 °C. The same was true for the amount of gas produced per pound of volatile matter added at these temperatures. Figure 6 reinforces the above observations, since a constant loading rate was used at all detention times. Figure 6 also indicates that gas production is relatively independent of detention time and temperature within the limits of this study. These appears to be a slightly greater total gas production at 15 days detention times as compared to shorter or longer detention times. The somewhat reduced gas production at shorter detention times is probably due to incomplete degradation of the raw sludge. At the longer detention -57- times, endogenous metabolism may consume a greater amount of intermediate degradation products, leaving less available for gas production. Figures 7 and 8 reveal the fact that when the protein and lipid concentra- tion was plotted versus detention time, each had a minimum value in the effluent sludge at a short detention time followed by an increase. It has been anticipated that there would be a consistent decrease. In the series of varied loading rate studies, the 35 °C digesters showed a minimum value of organics in the effluent at 7.5 days detention time and an increased value at 10 days detention time. With the 25 °C digesters, comparable values were noted at 15 days detention time and 20 to 25 days detention time respectively. In the latter studies, the reduction of the volatile matter was determined only with the digesters of de- tention times longer than ten days. Therefore, the volatile matter in the effluent vs detention time curve showed a consistent decrease with increasing detention time. The first trend has some similarity to the steady-state relationship between detention time and bacterial concentration developed theoretically and examined (1) experimentally by Herbert, Elsworth and Telling, although their data was col- lected with a pure culture. Their theory is based on the material balance in a completely-mixed type of continuous culture vessel: Change in bacterial concentration = growth - out put dx dt = u x - Dx (1) Change in substrate concentration = input - output - consumption growth = input - output - a , .. , yield constant f - ds r - » s - ¥- (2) ■58- x = bacterial concentration in vessel S = substrate concentration in vessel S = feeding substrate concentration is. - ,.-. _. , inflow rat e , number of complete volume changes D = dilution rate ( — r r r & , volume of vesseL or reciprocal of detention time) u = specific growth rate . - , .weight of bacteria formed. y = yield constant=( r 2 : : — — ) weight of substrate used By using the Michaelis-Mentern equation: u = m S u = Maximum growth rate ■ m K o + S T7 . S K = substrate concentration when growth rate is half of maximum they derived: dx u , on Tt " * [ ™ K 1-5 - D ] < 3 > f - D(S R - S) - JUL ^-h- ) (4) At steady state: dx dS_ dt dt s - k / 5 b ( u - D } (5) m T-Y(S R - S) 'Y[S R -K S ( - B_ ) j m The plot of this relation is shown in Figure 15. In these results it could be assumed that the protein concentration as well as lipid and volatile matter concentration shown in Figure 7 (but not in Figure 8) is proportional to the total concentration of undegraded substrate and the cell mass in the effluent, that is s in the vessel. The summation of S and x of Figure 15 will yield the curve shown -59- in Figure 16. This plot of the summation of x + S resembles the mirror image of the curves in Figure 7, since the inverse of detention time is plotted. However the difference lies in the fact that the summation of x and S in Figure 16 does not have the minima and maxima shown in Figure 7. This latter difference might be explained by considering endogenous respiration,, The derivation of equation (5) and (6) has neglected the endogenous consump- (2) tion of cell masses. Although Speece and McCarty have indicated that the en- dogenous consumption rate in anaerobic digestion in terms of specific maintenance -1 -1 "a" is 0.005 (day ) or 0.0002 (hr ) under starved condition, several researchers have reported that the "a" value varies depending on the stage of the growth as (3) well as on the temperature. Marr, Nilson and Clark have reported that the value of "a" with E. coli is 0.028 (hr ) under glucose limited growth and is almost zero at unrestricted growth. Therefore, it could be reasonable to assume that the endogenous consumption of the material in the vicinity of the washing-out detention time is almost zero and at the detention time where the TS- (3) substrate- limited growth governs, "a" is 0.02 to 0.03 (hr ) for an aerobic sys- tem with glucose substrate. As for the effect of temperature on "a ,r , Marr et.al. reported the following with E. coli under aerobic conditions: at 15°C a = 0.005 hr" 1 at 28°C a = 0.028 hr" 1 Then if we take endogenous respiration into account, equations (1) through (6) could be replaced by equations (7) through (10); dx | = u x ■ Dx - ax (7) f ■ ds r - DS - ¥ - « ■ < 8 > By the same procedure: K Q (a + D) S = u - (a + D) (9) m ■60- a 4- D Y [S - K_ u - (a + D) " = — R s E__ i + a(1 p Y) (10) The magnitude of the endogenous respiration rate may vary from nil. at unrestricted growth to as high as 107 o of the specific growth rate under starvation conditions. In order to check the adaptability of the protein concentration to the Herbert type curve the protein curve of Figure 7 was replotted in Figure 17 using dilution rate on the abscissa. The hypothesized lines of bacterial concentration and de= gradable substrate concentration were also added. Since some of the protein is undegradable a there will never be complete degradation. However s the zero scale could be shifted upward to coincide with the level of undegraded protein at in- finite detention times and then the Herbert- type curve would more nearly describe the results of this study. The hump in the curve cannot be explained., The protein and lipid curves must be examined in conjunction with the gas production curves. By comparing Figure 7 with Figure 5 9 it can be noticed that the gas production rate at 35 °C is highest at the detention time where the pro- tein and lipid curves show a minimum. The protein and lipid curves are represen- tative of bacterial population as well as raw sludge substrate concentration. The protein, lipid, volatile matter and total solids analyses would measure the quantity of these materials in the raw sludge substrate as well as that found in the bacteria which are synthesized during degradation of the raw sludge. The great majority of protein, lipid, volatile matter and total solids would be attributed to the raw sludge, while only a minor fraction would be contributed by the bacteria which are synthesized during the d egradation process. In anaerobic digestion, the fraction of synthesis is quite low. In the above discussion the case of constant loading was omitted. This is because the experiment of constant loading seems to be a study of quite a different scheme of investigation from the view point of the steady-state relation such as the Herbert-type curves shown in Figure 15. With the varied loading rate study, application of Herbert's curves -61- was not as successful;, but with the results from the constant loading study , it provided a good basis of explanation. In the case of constant loading rates , the concentration of the substrate decreases proportionately to the reciprocal of the detention time (proportionately to the dilution rate "D") . Therefore, if the experimental condition of this scheme is expressed in terms of the substrate concentration "S _" and the deten- R tion time (or dilution rate "D") , each detention time at a constant loading refers to a point on a different curve in Figure 15, since S is different in each di- gester. The protein concentration was treated like a bacterial concentration and replotted on Figure 18 using Table 4 in the Appendix. The broken lines are hy- pothetical qualitative Herbert-type curves. Each plot is equal to the sum of the substrate concentration and the bacterial concentration in terms of protein. For example, the digester of 7.5 days detention was fed with a substrate having 7 5 a concentration Sr> = —— G = 1/2 C . Thus the plotted value should be equal to * 15 o o n the sum of the substrate concentration and the bacterial concentration at the 7.5 day detention period. (1) - Herbert et.al. have defined the output as "(D) (x) which means the production of cells per unit volume of the vessel per unit time (t-t) x ( ^) = gm/l/hr). They examined the theoretical equation expressing the relation between the output and detention time experimentally and gave such an output curve as shown in Figure 16. If we assume that the gas production is proportional to this "output", the gas production vs detention time at constant loading could be expressed as a tieline connecting the corresponding point of each Herbert-type curve as shown in Figure 19 and 20. By changing the dilution rate, scale to a detention time at the larger values of "D" s the scale will be contracted and at the smaller values of "D' the scale will be expanded as can be seen in the scale of Figure 17. It can be seen from Figures 5 and 6 that the gas production curve at constant loading is much flatter than that at the varied loading. -62- An interesting comparison is shown in Figure 21„ The gas production rates at equilibrium operation were plotted as Curve I for the study of varied loading rate and varied detention time. After this study was completed, the digesters continued to receive raw sludge at the same rate and at the same detention time. However, acetic acid (HAc) was added daily in sufficient amounts so that the volatile acid concentration was maintained in excess of 1000 mg/1. Thus, the rate of gas production was not limited by low volatile acid concentrations as is normally the case when only raw sludge is added. The gas production values under conditions of excess volatile acids are plotted as Curve II in Figure 21. Curve I describes the rate of activity of the acid-forming stage of digestion at the respective detention times, because the rate-limiting step in the over all transformation was acid formation under these conditions. However, Curve II can be said to more nearly describe the rate of activity of the me thane -forming stage of digestion, because the substrate for the methane-forming microorganisms was not limited under the conditions in which this data was obtained. Summary 1. A reaction kinetic scheme developed by Herbert, et.al. for continuous pure culture in the steady state may be applicable with some modifica- tions to domestic wastewater sludge digestion. 2. There is a narrow range of short detention times at which the effect of protein and lipid reduction is nearly equal to that reduction which occurred at much longer detention times. 3. The optimum sludge detention time for methane formers again came out to be 15 days when the volatile acid concentration was kept over 500 mg/1 by feeding acetic acid with sludge. However, it was 7.5 days when only raw sludge was fed and at this detention time the concentration of pro- teins and lipids was at a minimum. -63- 4. The washing-out time appears to become longer by 3 to 4 days when the temperature is lowered from 35 °C to 25°Co -64- I. 1 o o UJ < > o z < o z o < O o E 2 Ul cc t o z o p o z> Q O ■ O < r « i « t i t i 3 D in N" > 1 > < - o o I • vO NO UMT\ — — < lo O □ ' 1/1 >- m o 00 LA vO ca sO LA O a. 2 O ae o o or c J a. LA J- t SAM O^ x fA CM ca uj 2 CA o ? O -J s a. -I 2 < r-^ «/> CM ""*— * < t- x v> o o mm z z CM CM < o> (j9)S96{p (UJ osi . At?p/|) s»9 -63- (jajs»6!p (in o5Z • **P/i) «»9 o CM UJ o % » \ 1 IA =1 1 1 4j 5 An V0I8OM3 VNV ) 03XIK < \ ■ 1 L 1 IA T ° / 9 ' 1 (A O J - 1 < D 1 > • ■ 1/1 o CD la r-- en ("A LA LA -3- C 2 i 1 ^ • »- 5 a to ■■■ UJ _, •1 ^ ' 1 JTsv, 1 (A >- l 3 u. J ii JtJ ! H L- •0 u\ -^ " ii vfl NO UM/\ — — i o • O □ < D t> ' (/I TO o 00 v0 l_T\ -J" - LA ON (j»js»6!P tut Q5Z • *»P/l) s»o -•7- I s • a u 00 I *r MV 9NK3WO" \£> | ■ *G fc. * Z - -. . ^ u> 8 o C ■ ^ \ f s l ITS X • ^ 1 . s ON 5 * > K 2««S D < D > • < » ' J^^k m li 3 8 * (M ^fjf. (T\ G ,' 1 C 1 o (j9)«96tp |w oS^ • Atp/i) t#9 •^ 3.0 *— 2 2.0 __ 1.0 _ — FIGURE 5 EFFECT OF VARIED LOADING RATE AND VARIED — A- DETENTION TIME ON DIGESTION OF RAW SLUDGE — 1 • — «^^ / 25° C i till 1 5.0 ESS? I I 1 ! 35° C 25° C v ! ! 10 20 30 DAYS DETENTION TIME 40 50 60 69 3.0 2.0 -_ 1.0 FIGURE 6 EFFECT OF OETENTION TIME ON DIGESTION OF RAW SLUDGE WITH CONSTANT LOADING RATE :' j 35° C • - t ^-- 25 # C _L 5.0 1 N ! B I A 10 55* C 25* C 20 DAYS DETENTION TIME $51 I I 30 •70* UJ <-> 5 ° = in I- « Z o £ z\° UJ l- < 8 3 1- z UJ 1- Z o H Z UJ 6 PETENTION DETENTION 5 !j!! CVJ i to 1 O 1 i m t 1 t 1 1 t 1 , 1 I 1 1 1 1 B < X o o o to o 10 >» i o O 1 2 I- i Z ! U» 1 I- I g| 80 indino 1— o CO z o LU h- o: z ID LU 1- O LU LU Q LU h- < a: a> 2 LJ O a: H- 3 _J O indino 81 FIGURE 21 EFFECT OF ACETATE SUPPLEMENTATION ON GAS PRODUCTION 4000 _ o ■o - 3000 O o o E z o t- u 2000 Q o CC 0. 1000 30 20 DETENTION TIME (days) 82 References 1. Herbert, D., Elsworth, R. , and Telling, R.C., "The Continuous Culture of Bacteria; A Theoretical and Experimental Study" J. Gen, Microbiol . 14, 601=622, (1956). 2. Speece, R. E. and McCarty, P. L. s "Nutrient Requirements and Biological Solids Accumulation in Anaerobic Digestion" , Proc. First International Confer - ence on Water Pollution Research , 1962. Pergamon Press, London (1964). 3. Marr, A. G. and Clark, D.J. and Nilson, E.H., "The Maintenance Requirement of E. Coli " , Annals New York Academy of Sciences , 536=548, ("1963). 4. Orme-Johnson, W. H., and Woods, C. E., "Colorimetric Determination of Pro- teins and Free Amino Acids", Water and Sewage Works , R=339 Reference Number, (1964). 5. Loehr, R. C. and Rohlich, G.A., "A Wet Method for Grease Analysis", 17th Purdue Industrial Waste Conference , (1962). 6. Loehr, R. C. and Higgins, G. C, "Comparison of Lipid Extraction Methods" Int. J. Air Wat. Poll . , Pergamon Press, Vol. 9, pp 55-67, (1965). 7. Koch, A. L. and Levy, H. R. , 1965, "Protein Turnover in Growing Cultures of E. coli.", J- Biol.. Chem., 217:947-957, (1965). 83- a UJ o Q 3 o ID Z * 2 O Q O f V) < o a Z < >- > o < Ul <9 O UJ UJ u. u < I a z < z o h- o o o oc a. to < (9 cr UJ CC O u. X o (/> or uj q: e o Aop/|/|ui rtop/i/Buj qoo •cwd sv9 aoi A1IAI10V «0J -76- 0) Ul o I ^-' 1- Q. z I Z UJ o H H < < OC -J h- UJ rr 00 D UJ u. o c/> i m z > o Q Id Si < UJ oc 1- 3 H (/) z UJ UJ u z UJ ° I C/5 li~ > Q O Ul ^ U. ^ • o o UJ u. u_ Ul o z o u. 00 % X d/6) £ onoo aivuisans 2 «o T d UJ I- < cc Z o 3 00 U> * CM H/6) x onoo "iviaaiova 77 (i/6) s -onoo 3ivdisans X 5 or UJ o z o o UJ !i c/> GO CO CO Q - z < LU I « U. yj I- O < m LU I u. o z o id a: o LU LU h- X LU I- o U LU § i -I LU 5 t * * CM (!/•) x onoo ivmaiova o— -| 78« 50 FIGURE 7 EFFECTS OF SOLIDS DETENTION TIME ON EFFLUENT CHARACTERISTICS VARIED LOADING AND VARIED DETENTlOt PROTEINS 25° C LIPIDS 25° C < o UJ o z 3 u. UJ 5 8 MX a. 50 10 -71- V.M. 25 # C i. 20 30 DAYS KTrMTinN TIMF 40 50 60 so g < o UJ o EFFECT OF SOLIDS DETENTION TIME ON EFFLUENi CHARACTERIS CS WITH CONSTANT LOADING AND VARIED DETENTION PROTEINS 35 # C PROTEINS 25 # C LIPIDS 25 # C u. o u L z UJ o UJ a. T. S. 35* C SO T. S. 25 # C V.M. 25 # C ■ ' mni i 10 -7ti 20 30 DAYS DETENTION TIME I * o z a < o o UJ 2£ > UJ o - ,7 * I UJ o o -I < or u. O or UJ 01 or UJ go * 9 2 £$o< o o o o o @®@ o z £ >, o 5 > Ui o h £ u O < UJ < 5 u. < o o o o o m oo m < or UJ o. O U. o V) o CM o o o o o o o o o eg o o o *dp/i/6uj qoo & o m to 5 o K UI ar w * 8 i W T 5 X H D *- s %! o ? O Q >• -J > O < < or > < o or or iu UJ 2 2 o p u. u. ♦ 9 £8S3 £X§> o o - z O § or UJ m CM ft o o o o m o o o o o s 8 o • < Q If) (VJ Wp; Q; LU UJ S. or p u. <*9 ox o fOO < > o < UJ o ^ UJ o E w 5 u- h o z o < If) O <\J I- < o o o If) o o o o o o o o o CM Aop/i/ftui Q00 - z o UJ ° h o o o >- > o < o UJ UJ u. o < I X o z < UJ o Q D J 00 If) C\J < >- < o o o 00 to (\J or UJ o. O 00 >- < o *Dp/|/oui QOD u. o ro or O UJ a _J < O in to Q 2 S I- O o 5 < Q X O < < o o z < b > o < 2 :> CD wmmmmw#m%mm in m mmmsmmtmmmfm •n m o UJ o z < o z o < o z v////////mwMW/y////m&* mmmmmm CM O *g o o o o o o CO Aop/ |/|UI Aop/|/ftui qoo o (VI Odd SV9 dOJ A1IAI10V dOd -73- z g H O o o CC 0. (/) < cc UJ 2 or o u. X o or UJ 1 o u. Q O < APPENDIX ■84- Analytical Procedures The Folin reaction was applied to the analysis of proteins in sewage (4) sludge by procedure reported by Orme-Johnson and Woods . A modification was made in replacing centrifugation with filtration using glass fiber filterpaper ™ r ! 11. . r -n -. - m i . . /Standard deviation. 30 minutes after the addition of Folin reagent. The precision ( — — — — — — — ) b r \ average was 10-20%. The lipid analyses was carried out according to the method reported by Loehr and Rohlich , and Loehr and Higgins. The reproducibility was such that the lipids of a thickened raw sludge came out to be .99% and .98% on two successive analyses. Sample Preparation The total solids of fresh, raw sludge taken from Urbana-Champaign Sani- tary District Plant was usually 3.0 to 4.07 o . By standing overnight, a thickened layer of sludge floated. By siphoning off the supernatant, a condensed sludge of 5 - 6% total solids was obtained. All of the raw sludge in this experiment was thickened by this method. -85- TABLE 1 Properties of Raw Sludge 1st Raw Sludge 2nd Raw Sludge 3rd Raw Sludge Items Sept. 25, 1965 Oct. 23, 1965 Nov. 25, 1965 Total Solids % 5.10 5.63 4.94 Volatile Solids % 3.25 4.07 3.59 Proteins (mg/1) 7400 7240 7450 Lipids (mg/1) 9784 8486 10,100 ■86- TABLE 2 Properties of Effluent and Gas Production with Varied Loading and Varied Detention Time at 35 C Items Period of Exper. Raw Sludge No. Ldg. Det. 1 0.35 6.25 2 0.30 7.5 3 0.23 10 4 0.15 15 5 0.09 25 teins g/D 1 2 3 7400 7240 7450 3810(53) 6195(83) 4100(56) 6180(86) 4560(61) 3060(41) 5460(75) 3780(51) 4590(63) 4050(55) 3939(54) ids ID 1 2 3 9784 8486 10,100 5806(60) 5942(70) 7734(77) 4544(47) 4920(58) 4030(40) 4406(45) 5972(70) __ _.. 2900(30) 4080(48) --- 2616(27) 2176(26) fi) 1 2 3 5.10 5.63 4.94 4.16(82) 4.08(83) 3.93(77) 4.55(81) 3.38(69) 4.44(87) 4.17(74) 3.89(76) 3.53(63) 3.89(76) 3.52(63) ) 1 2 3 3.25 4.07 3.59 2.46(76) 2.81(78) 2.13(66) 2.80(69) 2.22(62) 2.48(76) 2.67(66) 2.77(85) 2.31(57) 1.79(55) 2.08(51) 1-d) 1 2 3 .99 2.18 1.73 1.31 .80 3 /LB) 1 2 3 2.75 7.30 6.82 7.70 7.85 le 2 through 4: le 2 through 4; Figures in parenthesis are percentages left undegraded. Figures underlined were used for graphs plotted. ■87- TABLE 3 Properties of Effluent and Gas Production Varied Loading and Varied Detention Time at 25 C Items Period of Exper. Raw Sludge No. Ldg. Det. 7 .23- .20 10-11.5 8 0.15 15 9 0.09 25 10 0.075 30 6 0.038 60 roteins (mg/D 1 2 3 7400 7240 7450 4230(57) 6360(88) 4440(60) 5160(71) 4530(61) 3270(44) 4890(68) 2850(39) 3270(44) 3120(43) 4530(61) ipids Bg/D 1 2 3 9784 8486 10100 5930(61) 6284(74) 3760(38) 4270(50) 3462(34) 2480(25) 3790(45) 2500(26) 3548(42) 1620(16) 2632(31) 3010(30) . s. (%) 1 2 3 5.10 5.63 4.94 3.82(75) 3.38(66) 4.00(71) 3.40(69) 2.60(51) 3.76(67) 2.46(48) 3.62(64) 1.87(37) 3.63(65) 3.07(62) . s. (X) 1 2 3 3.25 4.07 3.59 1 2.57(78) 1.70(52) 2.45(60) 2.20(61) 1.62(50) 2.32(57) 1.58(49) 2.28(56) 1.19(37) 2.29(56) 1.04(54) as 1/1- d) 1 2 3 1.15 1.17 .69 .54 .32 l as / ft J /LB) 1 2 3 5.1 7.8 6.8 6.3 8.6 ■88- Properties of Constant Loading (0.15 TABLE 4 Effluent and Ga and Varied Det #Volatile Solid s Production with ention Time, at 35 C s/ft 3 /day) 1 1 "1 Items Period of Exper . Raw Sludge No. Det 6 12 7.5 13 10 14 15 J 15 ' 25 3 roteins (mg. with- drawn/day) 1 2 3 370 362 373 210(57) 192(52) 258(69) 155(42) 280(77) 313(84) 189(51) 230(64) 81(22;U 178(49) ] jipids (mg. with- drawn/day) 1 2 3 490 425 505 229(47) 182(37) 251(50) 153(31) 215(51) 357(71) 145(30) 204(48) 94(i9;H ; 128(30/ J 1 r. s. (gm with- drawn/day) 1 I 2.55 2.82 2.47 202(79) 1.87(73) 1.92(78) 1,74(68) 2.55(80) 2.12(86) 1.95(76) 1.77(63) 1.38(54A 1.62(5* T. S. (gm with- drawn/day) 1 2 3 1.63 2.04 1.80 1.13(70) 1.01(62) 1.22(68) 0.92(56) 1.41(69) 1.39(77) 1.39(85) 1.16(57) .68(42) .882(4?A las 1/1- d) 1 2 3 1.17 1.2 1.31 J 1.07 J -as/ |:ft 3 /LB) 1 2 3 7.8 8.0 7.7 1 6.27 -H ll ■89- I ) J r r L_ I j >N 1 | -d I 4-1 | rd I d | cu fi cu j 4-) d O O O vo oo o 1^. <* #* m en <|- 00 rd i ^ En CM ^ VO r-l r-l| i— 1 i—l 00 ON m vo cu j 4-1 4-1 ! d j, CU CU o ol o o o> en r^ i-i -O d oo on O 00 oo m r^ en i— i r^- in on o • o « • o 4-1 > cd -O 4-J 00 -d | d 1 d a ^ cu cd •Hen | d o olo O CM vO CM o en cm oo m o T) 4J i-4 r~~. cm r^ N o ~^ J 4-1 cm cn|— ' j !S Oi en <± r-- cm m CO CU cu o 0) M ■u CU •H 1 d. ^ 4-> T3 | cu O o CM O ^ CU ! d 00 en vO ON T3 CU t-I j i— i vO 00 • o 0) P- M I 4-1 i—l i—i r-l O d O cd ■ o mi •H M > i-H " w i—l Ph H KD CU •d ■ •d o o ,-H S-2 i-l O *~s w U ^^ •h o e o o ^ O CO H Ph HW ■! > -90- I TABLE 6 Properties of Effluent and Gas Production with Constant Loading and Varied Detention Time at 25°C (0.15# Volatile Solids/f t 3 /day) Items Period ; of Raw Exper. ; Sludge No . 16 Det. 1 7.5 17 18 19 10 | 15 | 25 20 30 J oteins lg.with- ■awn/day) 1 2 3 370 362 373 350(95) 272(74) 268(74) 222(60) 258(71) 226(61) 220(60) 156(43) 248(67 174(4sJ pids lg.with- •awn/day) 1 2 3 490 425 505 290(59) 220(45) 296(70) 188(38) 214(50) 173(34) 138(28) 148(35) 95(201 357(?) | S. ;m with- •awn/day) 1 2 3 2.55 2.82 2.47 1.54(60) 1.34(53) 2.44(86) 1.69(66) 2.00(71) 1.70(69) 1.24(49) 1.42(50) 0.94(37) 1.28(45X _] S. ;m with- ■ awn/ day) 1 2 3 1.63 2.04 1.80 1.02(63) .75(46) 1.50(74) 0.85(52) 1.23(60) 1.10(61) 0.76(47) 0.85(42) 0.58(36j = 0.79(39) s /1-d) 1 2 3 1.0 1.17 1.0 1.0 t J /LB) 1 2 ; 3 5.9 7.8 5.9 5.9 j -91- >> cd X CO X 4-> d H-| cd ~-~. co M X Pi •H ■H i—l X O cd C/3 o i-i CO 4-) -H d jj t« cd 4-1 i-l CO O d > o m 4-1 w •H O -i £ ^ w o o cni ml 13 > CO cd co pd (xi i—l CM o o O ^D ON C\ o o LO LO co co "3 cd co Pd Pn o o CO £> r-~ O vO LO CT\ CM CM CT\ vO >^> o CO r-l O CM v-i r^ £> LO O COsf i— I v£> CT\ LO LO 00 CN| -ct r^ co co co LO CM C^ 00 X o u\ •H M— I CO) U O CO fid. 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