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t- 
 
 CIVIL ENGINEERING STUDIES 
 
 SANITARY ENGINEERING SERIES NO. 28 
 
 THE STIMULATORY EFFECTS OF CATTLE RUMEN 
 
 LIQUOR AND DIGESTED SLUDGE SUPERNATANT 
 
 ON THE METHANE FERMENTATION 
 
 By 
 V. KOTHANDARAMAN 
 
 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, 1965 
 
THE STIMULATORY EFFECTS OF 
 CATTLE RUMEN LIQUOR AND DIGESTED SLUDGE SUPERNATANT 
 ON THE METHANE FERMENTATION 
 
 by 
 V . Kothanda raman 
 
 Supported by 
 
 Division of Water Supply and Pollution Control 
 
 U.S. Public Health Service 
 
 Research Project WP-000394 
 
ENGINEERING LIBIMT 
 
 ii 
 
 ACKNOWLEDGEMENT 
 
 The author wishes to gratefully acknowledge with sincere 
 gratitude the valuable advice and encouragement of Dr. R. E„ Speece, 
 who suggested this problem and under whose able guidance the investi- 
 gation was carried out. 
 
 The author is also thankful to Dr. R. S. Engelbrecht, and to 
 all colleagues and personnel of the Sanitary Engineering Laboratory, 
 who helped during the performance of the investigation. 
 
 This research was supported by research grant WP-0039^ from 
 the Division of Water Supply and Pollution Control of the United States 
 Public Health Service and was carried out in the Sanitary Engineering 
 Laboratory of the University of Illinois. 
 
I I I 
 
 ABSTRACT 
 
 THE STIMULATORY EFFECTS OF CATTLE RUMEN 
 LIQUOR AND DIGESTED SLUDGE SUPERNATANT ON THE 
 METHANE FERMENTATION 
 
 In the effective utilization of low molecular weight fatty acids 
 in anaerobic digestion, the most significant environmental factor deter- 
 mined was the need of methane organisms for some trace nutrients. These 
 requirements were found to be met by the addition of municipal digested 
 sludge supernatant to assay digesters. Also, the cattle rumen liquor 
 which abounds in anaerobic microbial population notably Anaerob ic lacto - 
 bacter ia , Methanobacter ium ruminantium , etc., supplements the needs of 
 bacteria with unknown nutritional requirements. The purpose of this study 
 was, to isolate the fraction in the digested sludge supernatant causing 
 stimulation, to verify the adaptability of the rumen liquor anaerobic or- 
 ganisms and to verify the effect of the growth factors in rumen liquor 
 on the anaerobic digestion of volatile fatty acids. 
 
 Activated carbon adsorption, solvent extraction, cation and 
 anion exchange, acid hydrolysis, ashing, and dialysis were the fractionation 
 procedures used either alone or in combination to more clearly identify 
 the stimulatory factor or factors. From this study it was found that the 
 addition of rumen liquor by an amount more than 20 per cent of the di- 
 gester contents inhibited methane fermentation due to volatile acid 
 toxicity. Addition of lesser quantities than 20 per cent had no beneficial 
 effect on the digestion. Addition of increased quantities of centrifuged 
 and evaporated digested sludge supernatant caused rapid deterioration in 
 the anaerobic digestion. It was also found that most of the fractions 
 added lowered the activity of methane bacteria and the remaining fractions 
 
I V 
 
 had very little or no beneficial effect though the corresponding quantity 
 of centrifuged supernatant caused stimulation. The ionic species of the 
 dialysable inorganics in the supernatant at pH 7-0 were found to inhibit 
 appreciably the volatile acid utilization by anaerobic bacteria. One or 
 several of the biochemical metabolic control mechanisms namely negative 
 feed back, enzyme repression, etc., were believed to be responsible for 
 the reduced activity of the assay digesters upon the addition of fractions 
 which caused a change in physical and chemical environmental conditions 
 within the assay digester. 
 
TABLE OF CONTENTS 
 
 page 
 
 ACKNOWLEDGEMENT i i 
 
 ABSTRACT i i i 
 
 LIST OF TABLES vi i 
 
 LIST OF FIGURES ix 
 
 I. INTRODUCTION 1 
 
 PURPOSE OF STUDY 3 
 
 II. LITERATURE REVIEW 5 
 
 III. EXPERIMENTAL PROCEDURE 11 
 
 EXPERIMENTAL EQUIPMENT 11 
 
 INITIATION OF DIGESTERS 11 
 
 GAS MEASUREMENT AND EXPULSION OF DIGESTER SAMPLE 12 
 
 VOLATILE ACID DETERMINATION 12 
 
 FEEDING 13 
 
 FRACTIONATION PROCEDURE 15 
 
 Centr i f uged Supernatant 15 
 
 Supernatant Filtered Through Activated Carbon 15 
 
 Recovery of Organics Adsorbed on Activated Carbon 15 
 
 Deionized Fraction of Supernatant 16 
 
 Dialysis of Supernatant 17 
 
 Solvent Extraction 17 
 
 Acid Hydrolysis of Supernatant 18 
 
 IV, RESULTS AND DISCUSSION 19 
 
 V. CONCLUSIONS 59 
 
V I 
 
 page 
 
 VI. REFERENCES 60 
 
 APPENDIX A TABLES OF DATA FOR VOLATILE ACID UTILIZATION 
 AND GAS PRODUCTION FOR DIGESTERS IN FIGURES 
 1 TO k2 62 
 
 APPENDIX B EFFECT OF ALTERING THE PHYSICAL, CHEMICAL AND 
 
 BIOLOGICAL EQUILIBRIUM IN ANAEROBIC DIGESTION 77 
 
 APPENDIX C FREE AMINO ACIDS IN DIGESTED SLUDGE SUPERNATANT 83 
 
V I I 
 
 LIST OF TABLES 
 
 Table page 
 
 1 FEED SOLUTION "A" 13 
 
 2 FEED SOLUTION "B" 13 
 
 3 FEED SOLUTION "C" 14 
 
 4 FEED SOLUTION "D" 14 
 A VOLATILE ACID UTILIZATION RATES AND GAS PRODUCTION 63 
 
 DATA FOR FIGURE 1 63 
 
 DATA FOR FIGURE 2 63 
 
 DATA FOR FIGURE 3 63 
 
 DATA FOR FIGURE 4 64 
 
 DATA FOR FIGURE 5 64 
 
 DATA FOR FIGURE 6 64 
 
 DATA FOR FIGURE 7 65 
 
 DATA FOR FIGURE 8 65 
 
 DATA FOR FIGURE 9 65 
 
 DATA FOR FIGURE 10 66 
 
 DATA FOR FIGURE 1 1 66 
 
 DATA FOR FIGURE 12 66 
 
 DATA FOR FIGURE 13 67 
 
 DATA FOR FIGURE 14 67 
 
 DATA FOR FIGURE 15 67 
 
 DATA FOR FIGURE 16 68 
 
 DATA FOR FIGURE 17 68 
 
 DATA FOR FIGURE 18 68 
 
 DATA FOR FIGURE 19 69 
 
V I I I 
 
 Table page 
 A (Continued) 
 
 DATA FOR FIGURE 20 69 
 
 DATA FOR FIGURE 21 69 
 
 DATA FOR FIGURE 22 70 
 
 DATA FOR FIGURE 23 70 
 
 DATA FOR FIGURE 2k JO 
 
 DATA FOR FIGURE 25 71 
 
 DATA FOR FIGURE 26 71 
 
 DATA FOR FIGURE 27 71 
 
 DATA FOR FIGURE 28 72 
 
 DATA FOR FIGURE 29 72 
 
 DATA. FOR FIGURE 30 72 
 
 DATA FOR FIGURE 31 73 
 
 DATA FOR FIGURE 32 73 
 
 DATA FOR FIGURE 33 73 
 
 DATA FOR FIGURE 3k Ik 
 
 DATA FOR FIGURE 35 7k 
 
 DATA FOR FIGURE 36 Ik 
 
 DATA FOR FIGURE 37 75 
 
 DATA FOR FIGURE 38 75 
 
 DATA FOR FIGURE 39 75 
 
 DATA FOR FIGURE kO 76 
 
 DATA FOR FIGURE k] 76 
 
 DATA FOR FIGURE k2 76 
 
 C R VALUES OF AMINO ACIDS 88 
 
IX 
 
 LIST OF FIGURES 
 
 Figure Page 
 
 1 CONTROL DIGESTER FOR COMPARING PERFORMANCES OF 
 DIGESTERS IN FIGURES 2 TO 10 21 
 
 2 EFFECT OF ADDING ASH OBTAINED FROM 1.7 LITERS 
 CENTRIFUGED AND CARBON FILTERED SUPERNATANT 21 
 
 3 EFFECT OF ADDING ASH OBTAINED FROM 1.7 LITERS 
 
 OF CENTRIFUGED SUPERNATANT 22 
 
 k EFFECT OF ADDING ASH OBTAINED FROM DIALYSIS TUBE 
 
 CONTENTS AFTER DIALYSIS OF 1 . 7 LITERS OF 
 CENTRIFUGED SUPERNATANT 22 
 
 5 EFFECT OF ADDING DIALYSIS TUBE CONTENTS AFTER 
 DIALYSIS OF 1.7 LITERS OF CENTRIFUGED 
 
 SUPERNATANT 23 
 
 6 EFFECT OF ADDING 1.7 LITERS OF CENTRIFUGED AND 
 DEIONISED SUPERNATANT 23 
 
 7 EFFECT OF ADDING DIALYSED AND CARBON FILTERED 
 FRACTION OBTAINED FROM 1.7 LITERS OF CENTRI- 
 FUGED SUPERNATANT 2k 
 
 8 EFFECT OF ADDING DIALYSED AND DEIONISED FRACTION 
 OBTAINED FROM 1.7 LITERS OF CENTRIFUGED 
 
 SUPERNATANT 2k 
 
 9 EFFECT OF ADDING CARBON FILTERED AND DEIONISED 
 FRACTION OBTAINED FROM 1.7 LITERS OF CENTRI- 
 FUGED SUPERNATANT 25 
 
 10 EFFECT OF ADDING CARBON FILTERED FRACTION OF 
 
 1.7 LITERS OF CENTRIFUGED SUPERNATANT 25 
 
 11 CONTROL DIGESTER FOR COMPARING PERFORMANCE OF 
 
 DIGESTER IN FIGURE 12 26 
 
 12 EFFECT OF ADDING 1.7 LITERS OF CENTRIFUGED 
 SUPERNATANT 26 
 
 13 CONTROL DIGESTER FOR COMPARING PERFORMANCES OF 
 DIGESTERS IN FIGURES }k, 15 AND 32 27 
 
 }k EFFECT OF ADDING 4.0 LITERS OF CENTRIFUGED 
 
 SUPERNATANT 27 
 
F i gure Page 
 
 15 EFFECT OF ADDING 6.0 LITERS OF CENTFUFUGED 
 SUPERNATANT 28 
 
 16 CONTROL DIGESTER FOR COMPARING PERFORMANCES OF 
 DIGESTERS IN FIGURES 1 7 TO 25 AND 34 28 
 
 17 EFFECT OF ADDING BUTANOL SOLUBLE FRACTION OB- 
 TAINED FROM 1.7 LITERS OF CENTRIFUGED SUPERNATANT 38 
 
 18 EFFECT OF ADDING BUTANOL INSOLUBLE FRACTION OB- 
 TAINED FROM 1.7 LITERS OF CENTRIFUGED SUPERNATANT 38 
 
 19 EFFECT OF ADDING PETROLEUM ETHER SOLUBLE FRACTION 
 OBTAINED FROM 1.7 LITERS OF CENTRIFUGED SUPERNATANT 39 
 
 20 EFFECT OF ADDING PETROLEUM ETHER INSOLUBLE FRACTION 
 OBTAINED FROM 1.7 LITERS OF CENTRIFUGED SUPERNATANT 39 
 
 21 EFFECT OF ADDING DIALYSABLE FRACTION OBTAINED FROM 
 
 1.7 LITERS OF CENTRIFUGED SUPERNATANT 40 
 
 22 EFFECT OF ADDING THE FRACTION OBTAINED BY PASSING 
 1 7 LITERS OF CENTRIFUGED SUPERNATANT THROUGH 
 
 ANION EXCHANGER 40 
 
 23 EFFECT OF ADDING 89O ml OF SETTLED SUPERNATANT 41 
 
 24 EFFECT OF ADDING THE FRACTION OBTAINED BY PASSING 
 1.7 LITERS OF CENTRIFUGED SUPERNATANT THROUGH 
 
 CATION EXCHANGER 41 
 
 25 EFFECT OF ADDING 1 50 ml OF RUMEN FLUID 46 
 
 26 CONTROL DIGESTER FOR COMPARING PERFORMANCES OF 
 DIGESTERS IN FIGURES 27 TO 31 46 
 
 27 EFFECT OF ADDING 1.0 LITER RUMEN FLUID 47 
 
 28 EFFECT OF ADDING 1.0 LITER CENTRIFUGED RUMEN 
 
 FLUID 47 
 
 29 EFFECT OF ADDING 1.0 LITER CENTRIFUGED AND DE- 
 IONISED RUMEN FLUID 48 
 
 30 EFFECT OF ADDING CENTRIFUGED AND CARBON FILTERED 
 FRACTION OBTAINED FROM 1.0 LITER OF RUMEN FLUID 48 
 
 31 EFFECT OF ADDING SOLIDS OBTAINED BY CENTRIFUGING 
 
 1 .0 LITER RUMEN FLUID 49 
 
XI 
 
 Figure Page 
 
 32 EFFECT OF ADDING 25 ml RUMEN FLUID kS 
 
 33 EFFECT OF ADDING DEIONISED ACID HYDROLYSATE 
 
 OBTAINED FROM 0.5 LITER OF ACTIVATED SLUDGE 51 
 
 3k EFFECT OF ADDING 200 ml RUMEN FLUID 51 
 
 35 EFFECT OF ADDING ETHER SOLUBLE FRACTION OB- 
 TAINED FROM 1.0 LITER OF SUPERNATANT AT pH 1.0 52 
 
 36 EFFECT OF ADDING ETHER INSOLUBLE FRACTION OB- 
 TAINED ^ROM 1.0 LITER OF SUPERNATANT AT pH 1.0 52 
 
 37 EFFECT OF ADDING NEUTRALISED HYDROLYSATE OB- 
 TAINED BY ACID HYDROLYSIS OF 0.5 LITER OF 
 
 ACTIVATED SLUDGE 53 
 
 38 EFFECT OF ADDING DEIONISED ACID HYDROLYSATE 
 
 OBTAINED FROM 0.5 LITER OF SUPERNATANT 53 
 
 39 EFFECT OF ADDING NEUTRALISED AND DEIONISED 
 HYDROLYSATE OBTAINED FROM THE ACID HYDROLYSIS 
 
 OF 0.5 LITER OF SUPERNATANT 5^ 
 
 UO CONTROL DIGESTER FOR COMPARING PERFORMANCES OF 
 
 DIGESTERS IN FIGURES 33 s AND 35 TO 39 5^ 
 
 M EFFECT OF 100 PER CENT DILUTION WITH MAKE UP 
 
 SOLUTIONS AND WATER 79 
 
 U2 EFFECT OF HIGHER NITROGEN CONCENTRATION IN 
 
 DIGESTER ATMOSPHERE 80 
 
I. INTRODUCTION 
 
 The processes employed in primary treatment of waste waters are 
 designed to separate a portion of the suspended particulate materials from 
 colloidal and dissolved matter. In secondary treatment processes, the 
 colloidal and dissolved substances are converted microbial ly into sus- 
 pended matter which are subsequently separated by gravity sedimentation. 
 The solids separated, which constitute a major fraction of the BOD in the 
 waste handled, require additional treatment. In many treatment works the 
 solids are decomposed by a heterogeneous anaerobic microbial population 
 in a sludge digestion tank. 
 
 The anaerobic digestion of complex organic wastes is commonly 
 considered to go through two basic phases: liquefaction and gasification 
 (5, 8, 18). During the liquefaction stage, also known as the acid fermenta- 
 tion stage, the organic materials, contained mostly in the solid fraction 
 of the sludge, are slowly hydrolyzed and brought into solution by saprophytic 
 organisms, designated as acid formers, through extra cellular enzymes, 
 Under normal conditions of digester operation, the insoluble organic matter 
 is broken down into volatile, low molecular weight organic acids and carbon 
 dioxide. During the subsequent gasifaction stage, the volatile acids pro- 
 duced in the liquefaction stage are decomposed into carbon dioxide, and 
 methane by a second group of bacteria which are commonly termed methane 
 formers . 
 
 As a result of the activity of methane formers, the quantity of 
 organic matter actually in solution, in a well operating digester, normally 
 remains low. There is, however, a build up of certain salts in solution 
 
such as ammonium, calcium, and magnesium bicarbonates resulting from the 
 breakdown of proteins, carbohydrates and fats. These constitute the natural 
 buffers, which enable the maintenance of a favorable pH range of 6.5 to 
 7.4 in the digester all the time. 
 
 The bacteria responsible for the stabilization of sludge re- 
 quire a source of carbon, energy, nitrogen, inorganic nutrients, and growth 
 factors as well as proper physical and chemical environments. The major 
 constituents of domestic sewage are carbohydrates, lipids, proteins and 
 other organic compounds which are decomposed during anaerobic digestion. 
 Such wastes provide a nutritionally well balanced environment, energy and 
 organic growth requirements for the anaerobic microorganisms. In the case 
 of specific industrial wastes, it may be necessary to supplement the es- 
 sential nutrients. 
 
 When unbalanced digestion conditions exist, the methane pro- 
 ducing organisms cannot remove the volatile acids as quickly as they are 
 formed and consequently an increase in the volatile acids concentration 
 results. This increase can take place in a few days and lower the pH of 
 the digester contents significantly. It has been demonstrated that under 
 adverse conditions, the most prevalent species of volatile acids in solution 
 are acetic acid, propionic acid and butyric acid (14, 22). This indicates 
 that the organisms responsible for the degradation of these compounds are 
 perhaps the most sensitive to environmental changes, and so are largely 
 responsible for the digester upsets. 
 
 The end products resulting from the biochemical reactions in- 
 volved in the degradation of organic wastes are primarily carbon dioxide 
 and methane. Small amounts of ammonia, hydrogen sulfide, hydrogen, and 
 
nitrogen are also evolved. Therefore, the process continually gasifies the 
 organic wastes, provided a favorable environment is maintained for the micro- 
 organisms responsible for the transformation. 
 
 PURPOSE OF THE STUDY 
 
 Although anaerobic digestion has become widely adopted, relatively 
 little is known about the metabolic processes which occur, and the re- 
 lationships between many factors which affect its efficiency. Since most 
 of the biologically degradable organic matter in the digester is eventually 
 converted to methane and carbon dioxide by methane bacteria, the activity 
 of this group of bacteria can govern the overall performance of the digester. 
 A knowledge of the specific factors which stimulate the activity of methane 
 bacteria would prove to be of great help in process design, especially in 
 the treatment of industrial wastes, and in the daily maintenance of digesters 
 treating domestic wastes. 
 
 Earlier studies in the identification of stimulants for methane 
 bacteria had revealed the potentiality of the domestic digested sludge 
 supernatant, as a stimulant in the anaerobic degradation of low molecular 
 weight fatty acids, e.g. acetic, propionic, and butyric acids. Thus, the 
 primary purpose of this study was to identify that fraction in the digested 
 sludge supernatant which was responsible for the stimulation of volatile 
 acids utilization. Various fractionation procedures, e.g. solvent extraction, 
 activated carbon adsorption, acid hydrolysis, dialysis, ionic separation, 
 etc., were used either alone or in combinations. A study of the presence of 
 free amino acids in the digested sludge supernatant was also carried out by 
 two dimensional paper chromatography. The stimulatory potential of cattle 
 
rumen liquor was also investigated, using these same fractionation procedures 
 
 All variables, other than the different fractionated additives 
 to the digesters were controlled by maintaining similar conditions in the 
 digesters. Comparisons of the digestion rates were made by determining 
 the amount of substrate utilized per day and the amount of gas produced each 
 day . 
 
II. LITERATURE REVIEW 
 
 In the past, raw sewage sludges were employed as substrate in most 
 laboratory studies (k, 5, 14). Such sludges permit satisfactory performance 
 of both groups of microorganisms, as long as the environment is optimum. 
 However, several investigators (18, 23, 2k) have attempted to use nutritionally 
 balanced synthetic substrates. These often resulted in unsuccessful degradation 
 of the organics fed. Apparently there was some critical material that was 
 not present in the feed used. 
 
 Stander (26) in a study of the digestion of winery and compressed 
 yeast wastes found that, the maintenance of a specially prepared inoculum 
 and the practice of re inocu lat ion with this inoculum as an integral part of 
 the process, greatly enhanced the efficiency of purification of the fermenta- 
 tion effluents. This was hypothesized to be because the waste liquors were 
 usually sterile or contained the specific organisms of the manufacturing 
 process and therefore received no fresh supply of the bacteria necessary 
 for the anaerobic digestion. In these investigations, satisfactory digestion 
 was maintained, not by reducing the rates of feed as soon as the volatile 
 acids concentrations increased, but rather by the addition of an inoculum 
 to remedy the state of bacterial unbalance. 
 
 It is reported by Pfeffer and White (2k) that Gulp was not able 
 to obtain satisfactory digestion of dilute organic wastes when employing 
 Metrecal or acetic acid as a synthetic substrate. With an initial seed of 
 digested sludge, satisfactory digestion was obtained until the original 
 seed was purged from the system. Then the gas production decreased and the 
 volatile acids concentration began increasing. It was possible to 
 
re-establish good digestion by adding more digested sludge or dried super- 
 natant sol ids . 
 
 Also Neibel (23) found it necessary to use digested sludge super- 
 natant to maintain satisfactory digestion of acetate. This was presumably 
 because the growth medium usually contains extracts from the natural en- 
 vironment of the organisms and assures the presence of the necessary nutri- 
 ents for growth. 
 
 Further it is reported by Speece and McCarty (25) that. Leary 
 found the addition of small amounts of powdered Milorganite to a carbohydrate 
 containing mash accelerated the fermentation rates. Reductions in fermenta- 
 tion time of 10 to 20 per cent and higher yields were noted. 
 
 McCarty and Vath (18) reported that the digestion of acetic acid 
 could not proceed at high rates if only the normal inorganic nutrient salts 
 were included in the feed. They observed that the digesters after attaining 
 a maximum rate of acetic acid utilization of about 3 g/1 of digester capacity 
 per day, dropped below 1 g/l/day on continued operations of the digesters 
 under similar conditions as obtained in the earlier period. This led to 
 their conclusion that some material contained in the seed sludge and re- 
 quired for bacterial growth was depleted by continued operation. The addition 
 of dried solids, obtained by evaporating one liter of digester supernatant 
 obtained from a municipal sewage treatment plant, immediately increased the 
 rate of utilization from about 500 mg/l/day to about 1^400 mg/l/day. They 
 also obtained the phenomenal rate of acetate utilization and gas production 
 of 21.9 g/l/day and over 100 liters of gas per day per 6 liters of digester 
 contents respectively by the increased and continued additions of digester 
 supernatant liquor solids at periodic intervals. These rates correspond to 
 
the destruction of organic wastes at unit rates of 1.37 lb organic matter/ 
 (day) (cu ft) and over 16 cubic feet of gas per cubic foot of digester capacity 
 per day. The acetic acid utilization rate began to drop when the addition 
 of the solids was discontinued. 
 
 In their attempt to determine the nature of material in the super- 
 natant solids causing stimulation of the methane production, they found that 
 the stimulant was soluble in boiling water and ethanol, but insoluble in 
 petroleum ether. The stimulant was not found to be affected by autoclaving. 
 They also noted that ashing of the solids at 600°C destroyed the stimulatory 
 effect. Acid hydrolysis of the supernatant solids over a steam bath in 6 N 
 hydrochloric acid for 2-j hours, destroyed all of the stimulatory effect. 
 This led them to believe the stimulatory effect was due to complex proteins 
 or other trace organic nutrients which are usually destroyed by acid hydrolysis 
 The addition of several organics, known to be required in trace amounts by 
 different anaerobic bacteria, namely tryptophan, methionine, thiamine, ribo- 
 flavin, adenine, etc., gave no stimulation. 
 
 From the beneficial effects of adding digester supernatant to 
 the experimental digesters in the study of McCarty and Vath, (18) there 
 appears to be no practical limit to the possible volatile acid fermentation 
 rate when optimum environmental conditions, viz., physical, chemical, and 
 nutritional, are maintained within the digesters. Probably the rates 
 obtained in their study are beyond a practical limit for full scale operation 
 since any slight decrease in the methane production rate at these high 
 levels would result in a rapid increase in acid concentration in the digesters 
 with a resultant drop in pH within a short time. Yet this has clearly 
 demonstrated the ability of methane formers to degrade volatile acids at 
 rates far higher than normally observed. 
 
8 
 
 Barker (2) reported that the methane organisms appeared to require 
 only inorganic nutrient salts and substrate for growth. However, several 
 other workers (12, 18, 23,25) found in their studies with synthetic sewages, 
 that inorganic nutrients alone were not sufficient to maintain high digestion 
 rates and the need for some additional material was consistently noted. The 
 beneficial addition of materials other than the digester supernatant solids 
 has been indicated by others. Heukelekian and Heinemann (12) investigated 
 methods for the enumeration of methane organisms and reported that more rapid 
 growth occurred when yeast extract was added than when using inorganic nu- 
 trients alone. 
 
 Speece and McCarty (25) reported in their studies of acetate 
 utilization by methane bacteria that inclusion of ferric chloride in the 
 nutrient salts enabled digestion rates of 1 g/l/day continuously, without 
 inclusion of any organic stimulants. Thus, the methane bacteria could 
 maintain normal low digestion rates with only inorganic nutrients in the 
 feed. They further observed that stimulation of the rates of acetate utili- 
 zation by methane bacteria was found to be produced by many different pure 
 compounds. Acetate utilization rates of 63OO mg/l/day were observed after 
 the addition of iron, cobalt, thiamine, vitamin B._, glycine and proline. 
 
 Pfeffer and White (2k) also demonstrated the stimulatory effect 
 of iron in the fermentation of volatile acids and observed that the most 
 effective digestion was obtained with the addition of iron in sufficient 
 concentration to keep the soluble phosphates in the digesters below 50 mg/1 . 
 Addition of aluminum and calcium to the digesters produced the same effect 
 
 as iron 
 
Various claims have been made regarding the beneficial effects in 
 anaerobic digestion by the addition of bacterial cultures and b iocata lysts . 
 (A biocatalyst is a substance that activates or stimulates a biochemical 
 reaction). Foremost among biocatalysts are the enzymes that are produced 
 by bacteria to break down complex substances into forms that are available 
 for energy and synthesis of protoplasm. Heukelekian and Berger (ll) and 
 McKinney (20) have discussed the theoretical and practical considerations 
 which are expected to result from the addition of pure bacterial cultures 
 and specific enzymes, as well as those resulting from commercial preparations. 
 Heukelekian and Berger (]]) found that the addition of enzymes and bacterial 
 preparations did not increase the liquefaction of non-sterile fresh sewage 
 solids as measured by the BOD of the supernatant liquor. McKinney discussed 
 some of the theoretical aspects of the biochemistry of anaerobic digestion 
 with the specific purpose of evaluating the addition of hydrolytic enzymes 
 and concluded that if the plant design is such that the biological system 
 can function properly, maximum efficiency could be attained without the 
 addition of organic catalysts. 
 
 Sufficient enzymes are produced during the digestion of domestic 
 wastes, so that the addition of different enzymes does not increase the ef- 
 ficiency of the process. This was also the conclusion of a special committee 
 on enzymes and biocatalysts of the California Sewage and Industrial Waste 
 Association ( 1 9) . But the beneficial effects of these additives in industrial 
 waste treatments were not altogether ruled out. 
 
 The work of McCarty and Vath and several others (18, 23, 27) 
 have clearly established the stimulatory effect of the addition of digester 
 supernatant solids. The identification of that fraction in the digester 
 supernatant which enables the fermentation of acetic and other low molecular 
 
weight fatty acids will greatly aid our knowledge of the biochemistry of 
 
 methane bacteria. Also, the addition of such fractions may help increase 
 
 the possible rates of digestion, and thus enhance the process efficiency, 
 
 particularly in the case of industrial waste treatment units. 
 
 In summarizing the effects of various additives designed to promote 
 
 the activity of methane fermenting organisms, it is worthwhile quoting the 
 
 remarks of Helmers et a]_. (lO) on nutritional requirements in the biological 
 
 stabilization of industrial wastes. 
 
 "A property of several important industrial wastes which 
 limits the amount that can be treated successfully in 
 combination with domestic sewage is a deficiency of certain 
 nutritional elements essential for bacterial growth notably 
 nitrogen and phosphorous. 
 
 "Because of the highly variable conditions that must be met 
 by waste treatment plants and the differences in the degree 
 of control these plants have over treatment process variables, 
 the critical BOD loading or critical nutritional requirement 
 will vary from plant to plant., even though they may be treating 
 the same waste. Thus, the critical requirements may be equal 
 to maximum requirements in some instances and they may be 
 somewhat less than the maximum requirements in others. In any 
 event, no advantage is to be gained by supplying nitrogen or 
 phosphorous in excess of sludge growth requirements." 
 
11 
 
 III. EXPERIMENTAL PROCEDURES 
 
 EXPERIMENTAL EQUIPMENT 
 
 One liter narrow mouth pyrex bottles fitted with three holed rubber 
 stoppers and glass tubings served as the digester units in this study. These 
 were connected to gas collection tubes which were in turn connected through 
 a manifold of tygon tubing to two 20 liter reservoirs containing a saturated 
 sodium chloride solution acidified with 15 per cent sulfuric acid. These 
 reservoirs were connected through a pressure reducing valve to the compressed 
 air delivery line. With the manipulation of a three way control valve inter- 
 posed between the reservoirs and the pressure reducing valve, pressure of 
 about 3 feet of water could be maintained in the reservoir and consequently 
 within the digesters. By applying pressure to the reservoir, gas or liquid 
 could be expelled from the digesters. Releasing the pressure caused a vacuum 
 of about two to three feet of water within the digesters, which facilitated 
 the da i ly feed i ng . 
 
 These digesters were kept in an incubation room in which a tempera- 
 ture of 35.0°C — 1.0°C was maintained, except during periods pertaining to 
 the experimental run for which the data are presented in Figures 26 to 31 
 when marked daily temperature variations were noted. The contents were kept 
 continuously mixed by placing the digesters in a shaking rack. 
 
 INITIATION OF DIGESTERS 
 
 The effects of the fractions added were studied with digesters 
 started with initial seed obtained from the primary sludge digester of the 
 Urbana -Champa ign sewage treatment plant. The experimental digesters were 
 purged of air with nitrogen by water displacement method, after which 750 ml 
 
seed was transferred anaerob i ca 1 ly to each digester and the contents were 
 flushed again with nitrogen for five minutes to expel any trace of oxygen 
 in the digesters. All the experimental digesters were operated on a 15-day 
 hydraulic detention time. 
 
 GAS MEASUREMENT AND EXPULSION OF DIGESTER SAMPLE 
 
 Before withdrawing samples for analysis, gas production volumes 
 at atmospheric pressure were recorded. 
 
 After placing the system under pressure, the contents were 
 thoroughly mixed manually, and samples of 50 ml were withdrawn in a graduated 
 cylinder. Excess gas was then expelled and the system placed under vacuum. 
 About two milliliters of the digester contents trapped in the sample delivery 
 tubes were put back in the digester by gently opening the stop cock. This 
 insured that the entire digester contents were kept continuously mixed and 
 representative samples were withdrawn every time. 
 
 VOLATILE ACIDS DETERMINATION 
 
 The tentative distillation method as given in the 11th edition of 
 Standard Methods for the Examination of Water and Wastewater (26) was used 
 throughout this study. The exceptions to the procedure were: the distillation 
 was made with only 50 ml sample instead of 100 ml; the sample was not centri- 
 fuged prior to the analysis for volatile acids; the titration of the dis- 
 tillate was performed with 0.117 N sodium hydroxide instead of 0.1 N sodium 
 hydroxide . 
 
 Analysis for pH and alkalinity was carried out at intervals of 
 three to four days on the samples, prior to the volatile acids determination, 
 in order to monitor the chemical environment in the digester and to take 
 
corrective steps whenever found necessary. 
 
 FEEDING 
 
 The daily feed volume of 50 ml comprised the organic and in- 
 organic feed solutions at concentrations indicated in Tables 1, 2, 3 and k, 
 acetic acid and make up water consisting of 75 per cent distilled water and 
 25 per cent tap water. Since it is assumed that tap water contains most of 
 the trace elements required for bacterial growth, it was blended with dis- 
 tilled water. This practice helped to minimize the formation of sulfides 
 i n the d i gesters . 
 
 TABLE 1 
 FEED SOLUTION "A" 
 
 NH^HCO 1 .32 g/1 
 
 KHCO 1 .00 g/1 
 
 NaHCO 1 .67 g/1 
 
 (NH /+ ) 2 HP0 z+ 50.00 mg/1 
 
 NH^MO £2+ 30.00 mg/1 
 
 
 
 TABLE 
 
 2 
 
 
 
 
 FEED 
 
 SOLUTION 
 
 "B" 
 
 
 CaCl 2 
 
 
 
 
 1 Ml g/1 
 
 
 MgCl 2 6H 2 
 
 
 
 
 1 .02 g/1 
 
 
 MnCl 2 4H 2 
 
 
 
 
 9.00 mg/1 
 
 
 CoCl 2 
 
 
 
 
 0.25 mg/1 
 
 as cobalt 
 
 AlCl 
 
 
 
 
 10.00 mg/1 
 
 
\k 
 
 TABLE 3 
 
 FEED SOLUTION "C" 
 
 Thiamine 10.00 mg/1 
 
 Prol ine 10.00 mg/1 
 
 Glycine 20.00 mg/1 
 
 Benz imidazole 10.00 mg/1 
 
 TABLE k 
 
 FEED SOLUTION "D" 
 
 FeCl« 4H 2 71 .20 mg/1 
 
 The amount of acetic acid included in the feed to each digester 
 was determined by the volatile acid concentration in that digester. The 
 quantity was calculated from the volatile acid analysis. An example of this 
 calculation is given below. 
 
 a = Volatile acids remaining on the previous day. 
 
 b = Quantity of acetic acid fed on the previous day. 
 
 c = Quantity of acetic acid remaining at the end of the day. 
 
 a + b - c = Quantity of acetic acid used during the day. 
 
 Throughout the experiments, the concentration of volatile acids 
 remaining at the end of each day was maintained between 500 mg/1 and 1700 
 mg/1. When the amount of acetic acid to be fed was high, it was added in 
 two or three stages during the day. This intermittent feeding and the main- 
 tenance of an excess acetic acid eliminated the danger of overtaxing the 
 alkalinity in the digester and minimized the variation in utilization rate 
 due to substrate concentration. 
 
FRACTIONATION PROCEDURES 
 Centr i fuqed Supernatant 
 
 The digester supernatant used in this study was obtained from the 
 Urbana -Champa i gn sewage treatment plant secondary digesters, in batches of 
 20 liters and stored at room temperature. The supernatant was centrifuged in 
 Servall superspeed centrifuge at 12,100 x G for ten minutes and the clarified 
 supernatant was used for further fractionations, discarding the solids. Two 
 liters of the supernatant was found to yield 1.7 liters of clarified super- 
 natant on centr i fuging. In order to study the effect of the centrifuged 
 supernatant before being subjected to further fractionations, 1.7 liters of 
 the clarified supernatant representing two liters of plant supernatant was 
 evaporated over a steam bath to a volume of about 25 to 50 ml and added to 
 the experimental digester. 
 
 Supernatant Filtered Through Activated Carbon 
 
 A volume of 1.7 liters of centrifuged supernatant was allowed to 
 flow by gravity through a column containing 700 ml of activated carbon, 30 
 mesh size, at a rate of 75 ml per hour so as to allow ample time for the 
 adsorption of organics in the supernatant on the activated carbon. At the 
 outlet of the column, a syphon connection was provided to avoid short cir- 
 cuiting of the supernatant within the column. The filtrate was evaporated 
 to about 25 to 50 ml over a steam bath and was added to the experimental 
 d igester . 
 
 Recovery of Organics Adsorbed on Activated Carbon 
 
 The organics adsorbed on the carbon column were recovered using 
 chloroform in a Soxhlet apparatus. A minimum of three passes of chloroform 
 through the activated carbon held in the cylinder of the Soxhlet apparatus 
 
16 
 
 were insured in order to completely desorb the organics from the activated 
 ca rbon , 
 
 The chloroform was then separated in a distillation apparatus. 
 When the contents of the distillation flask were about 20 to 25 ml, the 
 distillation was stopped and the contents were transferred to a beaker. 
 The distillation flask was rinsed with 25 ml of distilled water which was 
 then transferred to the beaker. This was to ensure the maximum recovery of 
 the organics from chloroform. 
 
 The beaker with the organics recovered and traces of chloroform 
 was heated in a boiling water bath for 30 minutes and at the same time sub- 
 jected to a vacuum of about 20 inches of mercury. This ensured the complete 
 removal of the chloroform. The organics which remained suspended in water 
 were refrigerated for subsequent addition to the experimental digester. 
 
 Deionized Fraction of Supernatant 
 
 Ion exchange resins IR-4B RNH + 0H _ form, IRC-50 RC00~ H + form 
 were used for partial deionization and Amberlite MB-3 mixed bed H and OH 
 forms were used for total deionization. Columns of size k cm diameter and 
 27 cm long were used with glass wool plug. Fifty ml resin was mixed with an 
 equal volume of deionized water and the resultant slurry was poured into the 
 column in such a manner as to exclude air pockets from the settled resin. 
 Then the resin column was again washed with 50 ml of deionized water. The 
 excess water was drained off until the fluid surface just covered the top of 
 the resin bed. The column was normally filled to two thirds the height with 
 resin and the surface was covered with a layer of glass wool. 
 
 After preparing the ion exchange column in the above manner, 1.7 
 liters of centrifuged supernatant was passed through the column at the rate 
 
17 
 
 of 0.60 ml/hr/ml of resin. The effluent was either dried over steam bath and 
 refrigerated for subsequent use, or subjected to additional fractionation 
 procedures, evaporated and added to the experimental digesters. 
 
 Dialysis of Supernatant 
 
 A volume of 1.7 liters of centrifuged supernatant was evaporated 
 to 50 ml over a steam bath. The evaporated supernatant was then placed in 
 a bag made of cellophane whose pores measured less than a few millimicrons, 
 according to Karlson (15), and served as a selective membrane permitting the 
 passage of inorganic ions and organic coloring matters, but not proteins, 
 lipids, carbohydrates and other macromolecu les „ The ends of the bag were 
 tied with double knots and placed in two liters of distilled water which was 
 continuously stirred with a magnetic stirrer. The contents of the bag were 
 allowed to dialyse and to reach equilibrium with the distilled water over a 
 period of 48 hours. The dialysed matter was then either evaporated over a 
 steam bath or subjected to other fractionation procedures and evaporated as 
 the case may be before being used. 
 
 Solvent Extraction 
 
 The stimulatory effects of butanol and petroleum ether soluble 
 fractions in centrifuged supernatant were studied. A volume of 1.7 liters 
 of centrifuged supernatant was added to an equal volume of solvent in a 
 four liter bottle, and tightly stoppered, and shaken for 48 hours during 
 which time the two liquid phases reached equilibrium. The solvent was then 
 separated from the supernatant using a separator/ funnel and the organic 
 fraction was recovered from the solvent by distillation. The solvent in 
 the fractionated organics were removed by vacuum distillation. 
 
18 
 
 Acid Hydrolysis of Supernatant 
 
 The pH of two liters of plant supernatant was brought to 1.0, 
 using 6 N hydrochloric acid and was allowed to hydrolyze over steam bath 
 for 2k hours. The hydrolyzed matter was resuspended in distilled water, 
 stirred thoroughly and centrifuged to remove the inert fraction. The super- 
 natant was then deionized, evaporated and added to the digesters. 
 
19 
 IV. RESULTS AND DISCUSSION 
 
 The results of the study of McCarty and Vath (18) led them to con- 
 clude that the ability of methane organisms to ferment volatile acids at high 
 rates is limited only when proper environmental conditions for growth are 
 not maintained Also, a very favorable environment is created in the digester 
 by the addition of digested sludge supernatant. It was found in this present 
 study that the digested sludge supernatant obtained from the Urbana -Champa i gn 
 Sanitary District, and the fractions derived from it, failed to produce ap- 
 preciable stimulation of the methane fermentation of low molecular weight 
 fatty acids. 
 
 Figure 1 shows the utilization rates of propionic acid in mg/l/day 
 and the gas produced in ml/l/day for a control digester started with 100 per cent 
 active seed obtained from the primary digester of the Urbana -Champa i gn Sanitary 
 District, Before mixing was started, the utilization rate remained at an 
 average value of 1100 mg/l/day and increased to a maximum value of 2460 mg/l/day 
 when the shaker was in operation. There was a corresponding increase in gas 
 production also. The digester, after being operated for a period of fifteen 
 days, with only the organic and inorganic nutrients shown in Tables 1, 2, 3 
 and k and make up water used, to dilute it to the initial volume after wasting 
 50 ml daily for the purposes of analysis and to maintain a hydraulic and 
 solids detention time of 15 days, started showing a decreasing trend in the 
 rate of utilization. This trend clearly demonstrates that some factor initially 
 present in the seed sludge became limiting in the metabolic transformation 
 of propionic acid into carbon dioxide and methane, as the initial digester con- 
 tents were wasted daily. Apparently this factor was not being supplemented 
 
20 
 
 LEGEND 
 
 Volatile Acid Utilized mg/l/day 
 Volatile Acid in the Digester mg/ 
 Gas ml /l /day 
 
 SUBSTRATE USED 
 Figures 1 to 12 and 26 to 31 -- Propionic Acid 
 Figures 13 to 25 and 32 to hi — Acetic Acid 
 
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29 
 
 in the daily addition of nutrients. A very similar trend was exhibited by 
 all the control digesters of this study shown in Figures 11, 13, 16, 26 and kO 
 
 Figure 12 shows the effect of adding 1.7 liters of centrifuged 
 supernatant. Soon after the addition, the rate of utilization dropped from 
 1690 mg/l/day to 78O mg/l/day due to changes in chemical equilibrium, but 
 the digester recovered quickly and showed a utilization rate of 1330 mg/l/day 
 on the fourth day after the addition and a rate of 1 500 mg/l/day on the 
 ninth day after addition. During these periods the utilization rates of 
 the control digester (Figure 11) showed rates decreasing from 1180 mg/l/day 
 to 950 mg/l/day. From the fifteenth day onwards both the digesters showed 
 an increase in utilization rates owing to the fact that the substrate con- 
 centration in the digesters was maintained above 500 mg/l/day at all times. 
 The control digester and the assay digester under discussion after reaching 
 peak rates of 2010 mg/l/day and 2310 mg/l/day respectively exhibited de- 
 clining tendencies, but the rates of decrease in substrate utilization were 
 100 mg/l/day and 15 mg/l/day respectively. These figures indicate to a 
 certain extent the stimulatory effect of the addition of 1.7 liters of 
 centrifuged supernatant. Though the effect is not very pronounced, probably 
 due to the increased salt concentration which resulted from the addition, 
 stimulation resulted from the addition. The difference in the rates of 
 decrease in utilization is of special significance. In spite of the daily 
 wasting of the digester contents, the concentrations of the critical factor 
 or factors required for methane fermentation were held at a higher level in 
 the assay digester as compared to the control digester and these were respon- 
 sible for the increased rate of utilization of the assay digester. There 
 appears to be a critical concentration requirement of these growth factors 
 
30 
 
 for optimal utilization rates, below which the rates begin to decline. 
 
 In the event of the identification of these growth factors, it will not be 
 
 a formidable task to determine these optimum concentrations. 
 
 Figures \k and 15 show respectively the effects of adding 4.0 
 liters and 6.0 liters of centrifuged supernatant solids. These digesters, 
 when compared with their corresponding control digester in Figure 13, 
 reveal that the addition of k.O liters had no stimulatory effect. Whereas 
 the addition of 6.0 liters caused a marked deterioration in the digestion, 
 the rate decreasing sharply from 9^+0 mg/l/day to 320 mg/l/day. However, 
 the digester showed signs of recovery on the sixth day after the addition. 
 This may have been because the salt concentration was reduced below the 
 toxic level due to daily wasting of the digester contents or because of 
 acclimation. This digester reached a maximum rate of 800 mg/l/day as 
 compared to 1500 mg/l/day for the control digester before the experimental 
 run was terminated. 
 
 McCarty and McKinney (17) found that toxicity which may be present 
 at the higher volatile acid concentrations in the case of stuck digesters, 
 is associated with salt toxicity and is dependent for the most part on the 
 cations in solution rather than the anions. According to them, the cations 
 studied in order of increasing toxicity for methane producing organisms 
 based on equivalent concentrations are Ca , Mg , Na , K and NH, . These 
 cations are much more toxic if added on a slug basis than when added over a 
 period of time. They also concluded that ammonium toxicity appeared similar 
 in nature to the other ions at low pH but appeared related to the concentration 
 of free ammonia in solution at high pH . Pfeffer and White (2k) concluded 
 from their studies that effective anaerobic digestion proceeded when the 
 
31 
 
 soluble phosphate concentration was 50 to 60 mg/1. Excessive concentrations 
 of Fe , Al , and Ca resulted in reducing the soluble phosphate to such 
 a low concentration that the microorganisms were not able to metabolize the 
 substrate because phosphate was limiting. Burgess (k) reported that concen- 
 trations of 200 mg/1 of zinc, 70 mg/1 of chromium, 90 mg/1 of lead, 7 mg/1 
 of nickel slowed the anaerobic digestion rate considerably. 
 
 With the addition of supernatant and the fractions derived there- 
 from, a change in chemical characteristics within the digester is anticipated 
 Mechanisms of bacterial metabolism and the ability of the microorganisms to 
 grow in an altered environment are important factors. Unstable conditions 
 will exist until the microorganisms have fully become adapted to their new 
 environment. Such conditions may bring an environmental selection of the 
 more compatible microorganisms. These and other causes may be responsible 
 for the reduced activity of the digester when 6.0 liters of centrifuged 
 supernatant was added. 
 
 As the slug addition of k.O liters and 6.0 liters of centrifuged 
 supernatant failed to show any stimulation and since only the addition of 
 1.7 liters of centrifuged supernatant showed some stimulation, this quantity 
 of centrifuged supernatant was adopted for further fractionation studies. 
 
 Speece and McCarty (25), and Pfeffer and White (2k) in their 
 studies established the beneficial effects of the addition in trace 
 quantities of iron, cobalt and aluminum in anaerobic digestion of acetic 
 acid. Also, White, Bryant and Caldwell (28) have indicated that Bacteroides 
 rumi naco la , an anaerobic ruminal bacterium, which ferments glucose with the 
 production of succinic, acetic and formic acids, and most other strains re- 
 quire heme which contains covalently bonded iron. Thus it could be seen that 
 
32 
 
 there is a possibility of the need for some trace cations obtainable from 
 digested sludge supernatant. 
 
 In order to assess the importance of the role of inorganic elements 
 present in the supernatant, ash obtained from 1.7 liters of centrifuged super- 
 natant was added to a digester and the results are presented in Figure 3- 
 The addition did not prove beneficial as is apparent when compared with the 
 rates obtained in the control digester in Figure 1. These cKgesters registered 
 maximum utilization rates of 15 10 mg/l/day and 2460 mg/l/day respectively. 
 This is probably due to the slug addition of inorganic salts in the absence 
 of any other organic volatile growth factors. It is to be recalled here that 
 the addition of 1.7 liters of supernatant solids did show a slightly higher 
 rate of utilization than the control. 
 
 It is interesting to note the results obtained from the digesters 
 to which ash obtained from dialysis tube contents after the removal of 
 dialysable organic and inorganic substances and the ash obtained from carbon 
 filtered supernatant were added. These results are presented in Figures h 
 and 2 respectively. In the former case, the digester attained a maximum 
 rate of 2130 mg/l/day which is 620 mg/l/day over and above the rate obtained 
 when total fixed solids were added (Figure 3) • From this it could be hypoth- 
 esized that a fraction of the inorganic salts present in the supernatant , 
 which could be removed by dialysis, is one of the factors contributing to 
 the reduced activity of the methane organisms. Referring to Figure 2, 
 there was neither apparent stimulation nor inhibition due to the addition. 
 The course of the utilization rates for this digester followed closely the 
 rates obtainable with the control digester. As the activated carbon adsorbs 
 most of the organics and their chelates, the lack of inhibitory effect of 
 
33 
 
 this additive suggests the possibility that the inorganic compounds with 
 molecular size less than a few millimicrons are responsible for the re- 
 pression of bacterial activity and are dialysable and exist as organic 
 chelates in solution which could be eliminated by adsorption on activated 
 carbon column. 
 
 From the experimental results so far discussed, it becomes ap- 
 parent that by the fractionation procedures, the stimulatory factors in- 
 itially present in the supernatant are either modified in character, or the 
 synergistic beneficial effects of some components in the fraction separated 
 and the unknown stimulatory factors are lost. This is probably the reason 
 why the stimulatory factors do not appear in one or the other fractions 
 obtained from the digested supernatant. It could also possibly be happening 
 that the fraction obtained is neither beneficial nor detrimental to bio- 
 logical activity in which case, the addition in a concentrated form of the 
 fraction does not alter the biological process. The results presented in 
 Figure 2 fall under this category. 
 
 As the metabolic pathways involved in the conversion of low 
 molecular weight fatty acids into methane and carbon dioxide and the pro- 
 ducts of intermediary metabolism and enzyme systems in anaerobic digestion 
 have not been well delineated so far, it is difficult to pinpoint the 
 actual mechanism of inhibition taking place, by the addition of various 
 fractions. In the course of this study it was found that the fractions 
 added as indicated in Figures 7, 3, 10, 20 and several others exerted a 
 partial or total inhibitory effect on methane bacteria even though the un- 
 modified centrifuged supernatant of equal volume showed stimulation. It 
 is possible that the reduced rates of metabolism may be partly due to the 
 
34 
 
 decreased concentrations of certain enzymes resulting from the blocking of 
 
 active sites by inhibiting agents. The glycolytic scheme in the anaerobic 
 
 conversion of glucose into ethanol and carbon dioxide or lactic acid is 
 
 either totally inhibited or modified, by the addition of sulfhydryl reagents 
 
 like iodoacetate , p-mercury benzoate, etc. which react with free "SH" 
 
 groups on enzyme t r iosphospha te dehydrogenase and by the addition of 
 
 fluoride ion which inhibits the activity of the enzyme enolase. The enzyme 
 
 chymotrypsin one of the important proteinases, active in small intestines 
 
 of warm blooded animals, is inhibited by the compound di-isopropyl fluoro 
 
 phosphate by forming an inactive DIP-chymotrys i n complex. 
 
 The possibilities of metabolic control processes namely positive 
 
 feed back, negative feed back, enzyme induction and enzyme repression 
 
 playing a part in modifying the rate of utilization of acetic and propionic 
 
 acids used as substrates cannot be ruled out. It is worthwhile quoting 
 
 Conn and Stumpf (7) on metabolic regulation. 
 
 "The kinetics of a single reaction is governed by the 
 concentration of the enzyme, substrates, co-enzymes, 
 cations and anions; by the temperature and pH and where 
 applicable, by the -SS-/-SH ratio, the NADPH/NADP + and 
 NADH/NAD"** ratios, and the occurrence of the activators or 
 inhibitors. Indirect factors include the rate of con- 
 version of enzymes or pro-enzymes to the fully active 
 enzymes, the rate of synthesis and the breakdown of the 
 enzyme protein and co-enzymes. The action of hormones 
 is superimposed on the more direct kinetic factors. 
 A small change of any of these factors by the physical 
 environment, disease or hormonal effects may have a 
 rather profound effect on the overall performance of 
 the metabolic machinery." 
 
 Metabolic repression involves a control mechanism by which the 
 
 concentrations of certain cellular enzymes are regulated by small molecules- 
 
 frequently end products -by inhibiting the formation of an enzyme which acts 
 
35 
 
 on an early stage of the metabolism of the repressors. This constitutes a 
 situation where the synthesis of a particular metabolite is controlled by 
 the need for it. Considering a system, where there may be more than one 
 metabolic pathways operating, the end product of the first being utilized 
 by the second--In the conversion of a substrate into its final products-- 
 unless there is a continuous and uninterrupted demand for the final product 
 of the first pathway by the second, there will be an accumulation of this 
 final product provided the metabolic repression control mechanism is not 
 b -ought into play. This is analogous to the activities of acid and methane 
 formers in the degradation of complex organic wastes into methane, carbon 
 dioxide, etc., in an anaerobic digester. Thus a thorough understanding of 
 the individual biochemical processes involved in the anaerobic utilization 
 of low molecular weight fatty acids is essential. Though Heukelekian and 
 Berger (11), McKinney and others (19? 20) came to the conclusion that the 
 addition of enzyme preparations are not beneficial in the anaerobic digestion 
 of domestic wastes, such an idea cannot be altogether ruled out. If the 
 rate limiting step or steps in the overall biochemical processes and the 
 factors responsible for decreased reaction rates in those rate limiting 
 steps could be identified, the enzymes involved in the limiting steps or 
 the required growth factors, activators, etc., could be supplemented from 
 an external source. 
 
 Figure 5 shows the effect of adding dialysis tube contents after 
 separating the dialysable organic and inorganic matter from the digested 
 sludge supernatant. The digester showed an immediate drop from 1200 mg/1/ 
 day to 960 mg/1 /day compared to a drop of 11 80 mg/1 /day to 1060 mg/1 /day in 
 the control digester (Figure l) due to changes in temperature during the 
 
36 
 
 corresponding period and recovered after four days from the day of addition 
 When the contents of the digester were mixed continuously from the fifth 
 day after addition, the rate increased to 2040 mg/l/day as compared to the 
 control digester rate of 2460 mg/l/day. The behavior of the digester was 
 similar to that of the control except for the maximum rates attained soon 
 after the continuous and complete mixing of the digester contents. Though 
 the initial increase in rate of utilization immediately after restoring the 
 mixing operation was k05 mg/l/day in the assay digester as compared to the 
 rate of increase of 400 mg/l/day in the control digester, this trend was 
 not maintained longer than two days as against a period of three days ex- 
 hibited by the control before leveling off. Also the rate of decrease in 
 utilization after reaching a maximum was higher in the assay digester. 
 These phenomena are probably due to some physical, chemical or biological 
 transformations, which the fraction added underwent, in a period of two 
 days. They were accelerated by mixing when thoroughly brought in contact 
 with the total cell mass, and resulted in not only curtailing the rate of 
 increase in utilization but caused a more rapid decrease in the overall 
 rate of ut i 1 izat ion. 
 
 Figure 6 shows the effect of adding centrifuged and deionized 
 supernatant. As the supernatant was passed at pH 7-0 through a mixed 
 bed ion exchanger of H and OH form, the effluent from the ion exchanger 
 is devoid of the prevalent ionic species at pH 7-0. Since the rate of 
 utilization of the assay digester was less than that of the control at the 
 time of making the addition, due to unknown causes which limited the 
 activity of the digester, the effect of the addition could not be evalu- 
 ated ful ly. 
 
37 
 
 Figure 7 shows the effect of adding the dialysed and carbon fil- 
 tered fraction representing the dialysable inorganics of the digester super- 
 natant. By dialysed fraction it is meant the fraction of the digested sludge 
 supernatant which permeates out of the dialysis tube and is dispersed in 
 the aqueous medium maintained outside the dialysis tube. Though the rate of 
 utilization of this assay digester was higher than that of the control at 
 the time of addition, the rate dropped appreciably from 1220 mg/l/day to 
 890 mg/l/day, and reached a maximum of 1 440 mg/l/day upon complete mixing 
 of the digester contents. This fraction was not stimulatory, but on the 
 other hand, was inhibitory. 
 
 Figure 8 shows the effect of adding the dialysed and deionised 
 fraction. The results of this digester when compared with the results of 
 the digester in Figure J, make it apparent that the factor responsible for 
 the reduced activity is largely due to the ionic species of the dialysable 
 inorganics which are eliminated in the former case by de ionizat ion . Even 
 still, the rate of utilization of the digester under discussion was less 
 than that of the control. At the termination of the run, the digester was 
 showing an upward trend in the utilization rates. 
 
 Figure 9 shows the effect of adding the carbon filtered and de- 
 ionised fraction and Figure 10 indicates the effect of the addition of cen- 
 trifuged supernatant passed only through activated carbon column. The 
 maximum rates of utilization reached by these digesters were 1850 mg/l/day 
 and 1530 mg/l/day as compared to the rate of 2460 mg/l/day for the control. 
 These again point to the significant inhibitory effect of the inorganic 
 ionic species in the digested supernatant at pH about 7.0. The overall 
 inhibition is probably due to the added fraction forming a complex with the 
 
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 digester contents rendering some essential factor unavailable for the 
 methane bacteria or due to changes in physical and chemical equilibrium 
 wi th i n the di gester . 
 
 Figures 17 and 18 show the effects of adding butanol soluble and 
 the butanol insoluble fractions. The control digester in Figure 16, after 
 reaching a maximum rate of 1460 mg/l/day declined to a value of 970 mg/l/day 
 on the fifteenth day after initiating the digester. The digester in Figure 
 17 touched a minimum value of 920 mg/l/day upon making the addition, but 
 soon recovered to yield the highest value of 1 460 mg/l/day on the sixth day 
 from the day of addition. The digester thereafter maintained a uniform 
 -■ate of 1200 mg/l/day till the termination of the experimental run. Though 
 the maximum rate did not exceed that of the control, the rate of utilization 
 was comparable to that of the control except for a short period of accli- 
 mation needed soon after making the addition. On the other hand, the 
 addition of the butanol insoluble fraction appeared to be slightly beneficial 
 There was no lag exhibited by the digester upon the addition of the insoluble 
 fraction and the maximum rate obtained in this digester was 1510 mg/l/day. 
 The addition of insoluble fraction showed greater utilization rates than the 
 rates obtainable either with the control digester or with the addition of 
 butanol soluble fraction. Hence it is to be concluded that a greater 
 fraction of the stimulatory factors in the supernatant are insoluble in 
 butanol. It is significant to note that the stimulatory character of the 
 supernatant was not very much altered by its treatment with butanol which 
 was not the case in other fractionation procedures used in this study, 
 
 Figures 19 and 20 depict the effect of adding petroleum ether 
 soluble and the petroleum ether insoluble fractions, It is recognized that 
 simple lipids, steroids, gl ycerophosphat i des among fats except sph i ngo 1 i p i ds 
 
43 
 are soluble in petroleum ether. 
 
 Other polar substances like proteins, nucleic acids, etc., are 
 insoluble in petroleum ether. By these additions, the effect of simple 
 lipids and sterols present in the supernatant could be assessed. From 
 Figure 19 it could be seen that the rate of utilization increased from a 
 value of 7^0 mg/l/day to ]kk0 mg/l/day gradually in a period of ten days 
 after the addition. However, the rate did not exceed that of the control. 
 The addition of the ether insoluble fraction caused a rapid decrease in 
 the rate from 1400 mg/l/day to 350 mg/l/day, before it begdn to recover 
 again. This fraction exerted a marked inhibition on the methane fermentation, 
 The digester could recover only after proper dilution or acclimation or due 
 to other causes . 
 
 Figure 21 shows the effect of adding the dialysable organic 
 and inorganic fraction obtained from the centrifuged supernatant. The 
 digester reached a maximum rate of 1 580 mg/l/day before making the addition. 
 The rate continually decreased to a low value of 700 mg/l/day on the 
 seventh day from the day of addition, after which the digester recovered. 
 Referring to Figure 5, where dialysis tube contents were added to the di- 
 gester, the rate of utilization was not far from that of the control. 
 Hence it is to be concluded that the dialysable matter in the supernatant, 
 probably inorganic in nature as could be deduced by comparing with the 
 performances of the digesters in Figures 7 and 8, is responsible for the 
 reduced activity in the methane bacteria. 
 
 Figure 22 shows the effect of adding centrifuged supernatant 
 passed through anion exchangers of OH form, in order to study the effect 
 of anions present in the supernatant. The pH of the digester was 7-10 
 
after the addition which was within the normal limits for digester oper- 
 ations. There was no lag due to the addition indicating that the bacterial 
 population readily adapted to the new environment. The rate of utilization 
 reached a maximum value of 1590 mg/l/day on the fourteenth day from the 
 day of addition and then started to decline. The control digester could 
 reach a maximum of only 1460 mg/l/day and dropped to 970 mg/l/day on the 
 fifteenth day from the initiation of the digesters. The assay digester 
 not only showed a higher rate of utilization, but also sustained this trend 
 for a longer period than the control, before starting to decline in 
 utilization rate. As the stimulatory potential exhibited by this fraction 
 is not far different from that shown by the whole centrifuged supernatant, 
 the results are suggestive of the fact that the prevalent anions at pH 7-0 
 are not responsible for the stimulation and that there are factors other 
 than the concentration of the stimulant, involved in controlling the 
 kinetics of utilization of these stimulatory factors. If the concentration 
 of the stimulants was only the controlling factor, the rate of utilization 
 should shoot up to the maximum value soon after the lag period instead of 
 showing a gradual increase. 
 
 Figure 23 shows the effect of adding settled supernatant. This 
 was added to study the effect of particulates which were otherwise eliminated 
 in centri fugi ng. Though there was an immediate drop in the rate soon after 
 the addition, the digester recovered after a lag of three days and showed 
 continuously increasing rate of utilization attaining a maximum of 1670 
 mg/l/day before the termination of the run. The effect of the addition 
 was to cause a higher rate of utilization in the assay digester and was to 
 sustain this trend for a longer period than was possible in the control. 
 
Figure 2k depicts the effect of adding centrifuged supernatant 
 passed through cation exchanger of H form. The pH of the digester was 7-0 
 after the addition. The rate of utilization increased from 1280 mg/l/day 
 to 1 450 mg/l/day after the addition, but started decreasing from the third 
 day after the addition, to the lowest value of 5^0 mg/l/day on the seventh 
 day after the addition. Thereafter it recovered to reach a value of 1260 
 mg/l/day before the termination of the experimental run. The anions re- 
 maining in the fraction were not found to be beneficial in the anaerobic 
 digestion process. 
 
 The ruminant fluid in cattle is known to contain an abundant 
 anaerobic microbial population, among which most prevalent species are 
 Anaerob ic lactobac i 1 1 i , Methanobacter ium rumi nant ium , Lachnospi ra mu 1 1 i parus , 
 Bacteroides , etc. Small numbers of yeasts of the genera, Candida and 
 Trichosporon have been isolated from rumens of normal cattle (13). Many 
 research workers in the field of animal nutrition recognize the importance 
 of the addition of rumen fluid to culture media for the primary isolation 
 of bacteria of unknown nutritional requirements, because it meets with most 
 of the nutritional needs of the bacteria. In order to study the adaptability 
 of these and other ruminant anaerobes in the digestion of synthetic sewage 
 and to study the effect of growth factors in rumen fluid on anaerobic di- 
 gestion, quantities of rumen liquor varying from 25 ml to 1.0 liter were 
 added to assay digesters. 
 
 Figures 32, 25, 3^, and 27 show respectively the effects of 
 adding 25 ml, 150 ml, 200 ml and 1.0 liter of rumen fluid to the digesters. 
 Figure 32 when compared with the corresponding control digester performance 
 shown in Figure 13, reveals that the rates of utilization remained more or 
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 the digesters reached maximum rates of 1990 mg/l/day and 19&0 mg/l/day at 
 the same time. There was no beneficial effect revealed by the addition of 
 25 ml rumen liquor, nor did it inhibit methane fermentation, On the other 
 hand, the addition of 150 ml and 200 ml rumen fluid depressed the rate of 
 utilization appreciably upon making the addition. Only after about six days 
 of acclimation did the digesters recover. Still, they failed to reach 
 even the maximum rate exhibited by the corresponding control digester 
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 ac'd concentration in the digester to increase to a maximum of 9^00 mg/1 
 and apparently the activity of the methane bacteria was inhibited by high 
 volatile acid concentrations. 
 
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 carbon filtered rumen liquor the maximum volatile acid reached in the 
 digester was 4400 mg/1 as against the high values of about 8000 mg/1 in the 
 other two digesters under discussion. This is due to the adsorption of the 
 organic fraction on the activated carbon. 
 
 Figure 31 shows the effect of adding the centrifuged solids ob- 
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 by the addition, but reached a maximum rate of 1 1 90 mg/l/day as against 1040 
 mg/l/day for the control digester. None of the fractions added, except the 
 centrifuged rumen liquor solids, were beneficial in anaerobic digestion of 
 fatty acids, 
 
 Figure 33 shows the effect of adding deionised acid hydrolysate 
 
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55 
 
 obtained from 0.5 liter of activated sludge. This when compared with the 
 corresponding control digester in Figure kO , reveals that there was no effect 
 on methane fermentation due to the addition, The rates of utilization of 
 the assay digester closely followed that of the control digester. 
 
 Figure 35 shows the effect of adding ether soluble fraction ob- 
 tained from 1.0 liter supernatant at pH 1.0. This was done to render all 
 the organic and inorganic compounds with carboxyl functional groups and 
 primary, secondary phosphates of phospho proteins, phospholipids and other 
 functional groups protonated. Thus, they become nonpolar and are dissolved 
 in the nonpolar petroleum ether solvent. As the digester was not showing 
 rates 'comparable to the control digester (Figure kO) , even before making 
 the addition, the relative effect due to the addition could not be fully 
 assessed. Though the digester was showing a rate of 670 mg/1/day at the 
 time of addition, the rate reduced to zero thereafter, indicating the complete 
 inhibition of the methane fermentation. The pH and alkalinity in the digester 
 were 7.05 and 36OO mg/ 1 as CaCO at the time of the termination of the run. 
 
 Figure 36 indicates the effect of adding the ether insoluble 
 fraction obtained from 1.0 liter of supernatant at pH 1.0. The fraction 
 under consideration largely represents the supernatant devoid of nonpolar 
 organic or inorganic matter at pH 1.0. This did not prove to be stimulatory. 
 The rate of utilization started decreasing gradually after the addition 
 and did not recover within the test period. 
 
 Figure 37 shows the effect of adding neutralized hydrolysate 
 obtained by acid hydrolysis of 0.5 liter of activated sludge, The maximum 
 rate of utilization was 1250 mg/l/day as compared to the rate of 1220 
 mg/l/day for the control (Figure kO) . This, when compared with Figure 33, 
 

56 
 
 showing the effect of adding the hydrolysate obtained by acid hydrolysis of 
 0.5 liter of activated sludge, reveals that the results are not materially 
 affected by neutralizing the hydrolysate, before making the addition, as the 
 maximum rate of utilization obtained in the latter case was also 1270 mg/l/day 
 
 In order to study the effect of different ionic species in the 
 acid hydrolysates obtained from supernatant at phi 1.0 and 7-0, portions of 
 the acid hydrolysate were passed through deionizing columns at pH 1.0 and at 
 pH 7.0 separately and the resulting fractions were added to assay digesters. 
 The digester to which deionized hydrolysate at pH 1.0 was added (Figure 38), 
 showed a slightly higher rate of utilization compared to the control, i.e., 
 1290 mg/l/day as against 1220 mg/l/day for the control. The rate of utiliza- 
 tion for this digester at the termination of the experimental run was 900 
 mg/l/day as against 640 mg/l/day for the control digester. Though the in- 
 creases in rates were not very significant, the addition did have some 
 beneficial effect on anaerobic digestion. On the other hand, the digester 
 to which deionized hydrolysate at pH 7-0 (Figure 39) showed only a maxi- 
 mum rate of 1160 mg/l/day which was less than that of the control. Thus 
 it could be concluded that some component in the supernatant with acid or 
 phosphate functional group on it. is causing a slightly increased rate of 
 utilization in anaerobic digestion. 
 
 The mixed bacterial population and the complexity of the 
 mechanisms of anaerobic digestions obscured the evaluation of the effects 
 of adding the fractions obtained from the digested supernatant and activated 
 sludge. Though earlier workers notably McCarty and Vath (18) did show that 
 the digested supernatant stimulated anaerobic digestion, the supernatant 
 obtained from U rbana -Champa i gn Sanitary District did not prove to be 
 
57 
 
 stimulatory, A marked difference in the characters of the two supernatants 
 is that in the case of McCarty and Vath's study, addition of increased 
 quantities of supernatant solids resulted in increased acetic acid utilization 
 rates, whereas in the case of this study addition of increased quantities 
 of supernatant solids drastically reduced the fatty acid utilization rates. 
 
 Among the 3k fractions of digested sludge supernatant and rumen 
 liquor studied in all, only seven of them showed positive stimulation in 
 anaerobic digestion, but the beneficial effects were not very significant. 
 The seven fractions are as given below: 
 
 1. 1.7 liters centrifuged digested sludge supernatant 
 (Figure 12) . 
 
 2. Butanol soluble fraction of the centrifuged digested 
 sludge supernatant (Figure 17). 
 
 3. Butanol insoluble fraction of the centrifuged digested 
 sludge supernatant (Figure 18). 
 
 k. Centrifuged supernatant passed through anion exchanger 
 
 on ly (Fi gure 22) . 
 5- Settled digested sludge supernatant (Figure 23). 
 
 6. Deionized acid hydrolysate of digested sludge 
 supernatant at pH 1.0 (Figure 38). 
 
 7. Solids obtained by centrifuging 1.0 liter rumen 
 fluid (Fi gure 31). 
 
 The addition of settled supernatant caused stimulation to the 
 extent of 15 per cent over the control and the addition of centrifuged 
 supernatant also resulted in a stimulation of 15 per cent over the control. 
 Thus it is logical to conclude that the stimulatory factors exist in either 
 

 
 
58 
 
 soluble or colloidal forms which are not eliminated by centr i fugi ng at 
 12,100 x G and not as settleable suspended solid particulates. The treatment 
 of centrifuged supernatant with butanol resulted in the distribution of the 
 growth factors in both the butanol soluble and insoluble phases. The stim- 
 ulatory character of the supernatant is not altered by its treatment with 
 butanol. The addition of centrifuged supernatant passed through anion ex- 
 changer showed stimulatory effect on methane fermentation, and hence it 
 could be hypothesized that the anions do not play a prominent role in stim- 
 ulation as there is no significant difference in the stimulations caused by 
 this fraction and the whole supernatant. The acid hydrolysate of the di- 
 gested sludge supernatant had stimulatory potential. These stimulatory 
 factors were observed to exist in ionic form at pH 7-0. When the hydrolysate 
 was neutralized and deionized, the fraction lost all its stimulatory effect. 
 T hus a component of the hydrolysate with carboxyl or phosphate functional 
 groups should be responsible for the stimulation. These functional groups 
 are ionized and retained on ion exchange column at pH 7-0. Due to the 
 heterogeneous nature of the rumen liquor solids, it is not possible to 
 ascertain the nature of stimulant present in such solids without additional 
 fractionation studies on the solids. 
 
59 
 V. CONCLUSIONS 
 
 1. Mixing of the digester contents and substrate concentration 
 in the digester has a profound effect on the rate of anaerobic digestion of 
 volat i le fatty acids. 
 
 2. There is a limiting concentration for the addition of digested 
 sludge supernatant, beyond which the addition proves inhibitory. In this 
 study the limiting concentration was found to be 4.0 liters of evaporated 
 supernatant per 750 ml digester contents. 
 
 3. The stimulatory factors in supernatant are in soluble or 
 colloidal forms which are not eliminated by centrifuging at 12,100 x G, 
 
 k. Treatment of the digested sludge supernatant with butanol 
 does not alter its stimulatory characteristics. A greater fraction of 
 the stimulant is insoluble in butanol. 
 
 5. Salts responsible for reducing the activity of anaerobic 
 bacteria are dialysable with a molecular size less than a few millimicrons 
 and exist as organic chelates. 
 
 6. The ionic species of the dialysable inorganics in the super- 
 natant inhibit methane fermentation. 
 
 7. The anions present in the centrifuged supernatant at pH 7 
 are not responsible for the stimulation of methane fermentation, 
 
 8. Some component in the supernatant with carboxyl or phosphate 
 functional group causes a slightly increased rate of utilization in methane 
 fermentation . 
 
 9. Rumen liquor and the different fractions derived therefrom, 
 except the centrifuged rumen liquor solids, have inhibitory effects on 
 methane fermentation. 
 
60 
 VI. REFERENCES 
 
 1. "Amberlite Ion Exchange Resins Laboratory Guide." Rohm &■ Haas Company, 
 
 Phi ladelphia, Pa. 19105, 39 p. 
 
 2. Barker, H.A., "Biological Formation of Methane." Industrial and 
 
 Engineering Chemistry , 48, 1438-1442 (1956). 
 
 3. Block, R.J., Durrum, E.L., and Gunter Zweig, A Manual of Paper Chroma - 
 
 tography and Paper Electrophoresis . Academic Press, Inc., New 
 York, 484 p. (1955). 
 
 h. Burgess, S.G., "Anaerobic Digestion of Sewage Sludge." Institute of 
 Sewage Purification , Journal and Proceedings , Part 5 , 411- 
 423 (1962). 
 
 5. Buzzel, J.C., Jr., and Sawyer, C.N., "Biochemical Versus Physical Factors 
 in Digester Failures." Journal Water Pollution Control Federation , 
 35, 2, 205-221 (1963). 
 
 6 Clark, J.M., Jr., Experimental Biochemistry . W.H. Freeman and Company, 
 
 San Francisco, Calif., 229 P- (1964). 
 
 7 Conn, E.E., and Stumpf, P.K., Outlines of Biochemistry . John Wiley and 
 
 Sons, Inc., New York, 391 p. (1963)- 
 
 8 Eckenfelder, W.W., Jr., and O'Connor, D.J., Biological Waste Treatment . 
 
 Pergamon Press, New York, 299 P- (1964). 
 
 9. Fruton, S.J., and Simmonds, S., General Biochemistry . John Wiley and 
 Sons, Inc., New York, 1077 p. (1958). 
 
 10. Helmers, E.N., Frame, J.D., Greenberg, A.E., and Sawyer, C.N,, "Nutri- 
 
 tional Reguirements in the Biological Stabilization of Industrial 
 Wastes. II. Treatment with Domestic Sewage." Sewage and In - 
 dustrial Wastes , 23_, 7, 884-899 (1951). 
 
 11. Heukelekian, H., and Berger, M. s "Value ofCulture and Enzyme Additions 
 
 in Promoting Digestion." Sewage and Industrial Wastes , 2_5_, 11, 
 1259-1267 (1953). 
 
 12. Heukelekian, H., and Heinemann, B., "Studies on the Methane Producing 
 
 Bacteria." Sewage Works Journal , V\_, 6, 965-970 (November 1939) . 
 
 13- Hungate, R.E., Bryant, M.P., and Mah , R.A., "The Rumen Bacteria and 
 Protozoa." Annual Review of Microbiology , ]8_, 131-166 (1964). 
 
 14. Kaplovsky, A.J., "Volatile Acids Production During the Digestion of 
 
 Seeded, Unseeded, and Lined Fresh Solids." Sewage and Industrial 
 Wastes , 23_, 6, 713-721 (1951). 
 
61 
 
 15 Karlson, P., Introduction to Modern Biochemistry , Academic Press, New 
 York, 433 p. (1963). 
 
 16. Lederer, E., and Lederer, M., Chromatography--A Review of Principles and 
 
 Appl icat ions . Elsevier Publishing Company, New York, 711 p. (1957). 
 
 McCarty, P.L., and McKinney, R.E., "Salt Toxicity in Anaerobic Digestion." 
 Journal Water Pollution Control Federation , 33., 4, 399-415 (1961). 
 
 McCarty, P.L., and Vath, C.A., "Volatile Acid Digestion at High Loading 
 Rates." International Journal of Air and Water Pollution , 6, 
 65-73 (January 1962) ! 
 
 McKee, J.E., Chmn . , "B ioca ta lyt i c Additives in Waste Treatment." 
 Sewage and Industrial Wastes , 26, 9, 1162-1172 (195*0. 
 
 McKinney, R„E„, "B iocata lysts and Waste Disposal. II. Fundamental 
 
 Biochemistry of Waste Disposal." Sewage and Industrial Wastes , 
 25, 10, 1129-1135 (1953). 
 
 McKinney, R.E., Microbiology for Sanitary Engineers . McGraw-Hill Book 
 Company, Inc., New York, 293 p. (1962). 
 
 Mueller, L.E., Hindin, E., Lunsford, J.V., and Dunstan, G., "Some 
 
 Characteristics of Anaerobic Sludge Digestion. I. Effect of 
 Loading." Sewage and Industrial Wastes , 3J_ S 6, 669-677 (1959). 
 
 Neibel, D.W., "The Effect of Temperature on Acetate Fermentation in 
 
 Waste Treatment." Unpublished Master of Science Thesis, Univer- 
 sity of II 1 inois (1963) . 
 
 Pfeffer, J.T., and White, J.E., "The Role of Iron in Anaerobic Digestion." 
 Presented at the Nineteenth Annual Industrial Waste Conference, 
 Purdue University, Lafayette, Indiana. 
 
 Speece, R„E„, and McCarty, P.L., "Nutrient Requirements and Biological 
 Solids Accumulation in Anaerobic Digestion." In "Advances in 
 Water Pollution Research." Proceedings of the First International 
 Conference on Water Pollution Research , 2, 231, Pergamon Press, 
 New York (1964) . 
 
 Standard Methods for the Examination of Water and Wastewater . Eleventh 
 Edition. American Public Health Association, Inc., 1790 Broadway, 
 New York 1-9, N.Y., 626 p. (I960). 
 
 Stander, G.J., "Part V--Re i nocu lat ion as as Integral Part of the An- 
 aerobic Digestion Method of Purification of Fermentation Ef- 
 fluents." Institute of Sewage Purification , Journal and Pro - 
 ceedings , Part 4 , 447-458 (1950). 
 
 White, D.C., Bryant, M.P., and Caldwell, D.R., "Cytachrome Linked 
 
 Fermentation in Bacteroides Ruminacola." Journal of Bacteriology , 
 84, 4, 822-828 (October 1962). 
 
62 
 
 APPENDIX A 
 
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 IN FIGURES 1 TO 42 
 
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APPENDIX B 
 
 EFFECT OF ALTERING THE PHYSICAL, 
 CHEMICAL AND BIOLOGICAL EQUILIBRIUM 
 IN ANAEROBIC DIGESTION 
 
 77 
 
78 
 
 Study of the Effect of Altering the Physical, Chemical and Biological 
 Equilibrium in Anaerobic Digestion 
 
 Two four liter aspirator bottles with three holed rubber stoppers 
 and glass tubings to facilitate feeding, gas collection and withdrawal of 
 samples, were inter-connected by a rubber tubing which was kept clamped. 
 Anaerobic digestion of acetic acid was initiated in one of the bottles 
 with 3-0 liters of digested sludge seed using the technique already des- 
 cribed in the earlier chapter "Experimental Procedures." On the fifth day 
 after the initiation of the digester, the second aspirator bottle was 
 thoroughly purged with 100 per cent nitrogen. Half of the contents of 
 Digester #1 , which was initiated first, were transferred anaerobica 1 ly to 
 the second aspirator bottle by releasing the clamp after which the tubing 
 was again clamped. The contents of Digester #1, were brought to its 
 original volume of 3-0 liters by using the nutrient solutions and make 
 up water. The contents of Digester #2 remained at 1.5 liters. Both the 
 digesters were operated on 15 days hydraulic and solids detention basis. 
 
 The utilization and gas production rates of Digester #1 are shown 
 in Figure 4l and those of Digester #2 are shown in Figure k2 . Digester #1 
 reached a maximum utilization rate of 1 96O mg/l/day on the fifth day, 
 when half the contents were transferred to Digester #2. Immediately after 
 the transfer and dilution back to the original volume of 3 = liters in 
 Digester #1, the utilization rate dropped to 280 mg/l/day. As against 
 this change, Digester #2, which represents the original contents of the 
 Digester #1, showed a rate of 920 mg/l/day. These organisms, before being 
 transferred to Digester #2, were using acetic acid at the rate of i960 mg/1/ 
 day but showed a reduced rate of utilization on being transferred. The only 
 
79 
 
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 change in environmental conditions anticipated here was the increased 
 concentration of nitrogen in the digester atmosphere. 
 
 Neglecting the effect of the difference in the rates of synthesis 
 in the digesters due to the difference in utilization rates, the total viable 
 organisms were the same in both the digesters, though the concentration of 
 the organisms in Digester #2 was nearly double that in Digester #1. On the 
 third day after making the transfer, Digester #1 showed a rate of utilization 
 cf 630 mg/l/day, i.e., 1 .89 grams of acetic acid per day and Digester #2 
 showed a rate of 1070 mg/l/day which is 1.60 grams of acetic acid per day. 
 Digester #1 showed gradual recovery in utilization rates reaching a maximum 
 rate of 1070 mg/l/day before starting to decline. Digester #2 steadily 
 decreased to reach a minimum value of 390 mg/l/day on the ninth day and 
 then recovered to give a maximum rate of utilization of 920 mg/l/day before 
 starting to decline again. 
 
 The contents of Digester #1 showed a pH of 7-0 and alkalinity of 
 3350 mg/1 before the transfer. On being transferred to Digester #2, the 
 corresponding values were found to be ~J .h and 3050 mg/1 respectively. 
 The changes in pH and alkalinity were probably due to the release of carbon 
 dioxide from the sludge mass to the nitrogen atmosphere of the digester. 
 Though the pH and alkalinity were in the desirable ranges for digester 
 operation, the upset in bicarbonate, carbonic acid and carbon dioxide 
 equilibrium had greatly reduced the utilization rate of acetic acid. As 
 the digester gas produced daily was expelled, the concentration of nitrogen 
 in the digester atmosphere was reduced and the carbon dioxide and methane 
 concentrations in the digester atmosphere might have attained a stable 
 value. This is probably the reason why the digester recovered after the 
 
82 
 
 ninth day after the transfer of contents and began to show higher rates 
 of ut i 1 izat ion . 
 
 In either case, the utilization rates were far less than the 
 rates obtainable without the physical, chemical and biological equilibrium 
 being changed. The methane bacteria are therefore found to be very sen- 
 sitive to these changes and their activity is very much reduced by a change 
 in any one of these factors. Digester #2 recorded a lesser utilization rate 
 compared to Digester #1 probably due to the following and other unknown 
 reasons: 
 
 1. The increased nitrogen concentration in the digester 
 atmosphere appeared to exert a deleterious effect on 
 anaerobic digestion by altering the carbon dioxide, 
 carbonic acid and bicarbonate equilibrium. 
 
 2. The concentration of metabolic end products, some of 
 which may have been toxic, was higher in Digester #2 
 than in Digester #1 in which the contents were diluted 
 
 by 100 per cent with nutrient solutions and make up water. 
 
APPENDIX C 
 
 FREE AMINO ACIDS IN DIGESTED 
 SLUDGE SUPERNATANT 
 
 Int roduct ion 
 Experimental Procedure 
 Resul ts 
 
 83 
 
8k 
 
 Introduction 
 
 The major constituents of domestic sewage include proteins, 
 carbohydrates, lipids and other organic compounds of which protein content 
 is usually between 30 and 50 per cent (20). Proteins are polymers of 
 amino acids and the hydrolysis of proteins results in a mixture of amino 
 acids and ammonia. The amino acids are essential for microbial metabolism. 
 Most bacteria are not capable of synthesizing all of the amino acids that 
 are required for their metabolism. Such amino acids must be derived from 
 the environment. As proteins are macromolecu les , which are too large to 
 pass through cell membrane, they are hydrolysed by extra cellular enzymes 
 secreted by microorganisms, into free amino acids, peptides, and polypeptides 
 These may then permeate through the cell membrane and become available for 
 utilization by the microorganisms. 
 
 Also, the enzymatic breakdown of these proteins is the main source 
 of ammonium ions which constitute the bulk of the bicarbonate alkalinity 
 found in the digester. It was felt that a knowledge of the free amino acids 
 in the digested sludge supernatant will be useful in evaluating the stim- 
 ulatory potential of the digested sludge supernatant being investigated. 
 
 Experimental Procedure 
 
 Two dimensional ascending paper chromatography using Whatman 
 
 paper No. 1 was adopted for better resolution of the individual amino 
 
 acids. The digested sludge supernatant was centrifuged at 12,100 x G, 
 
 and then passed successively through ion exchange columns of Amberlite 
 
 IR-4B of OH" form and Amberlite IRC -50 of H form. This procedure yields 
 
 an eluate containing all the amino acids eliminating the ionic salts 
 
85 
 
 which interfere with the chromatographic resolution of amino acids (16). 
 The supernatant was then evaporated over a steam bath to achieve a volume 
 reduction of about 20 in order to increase the concentration of amino 
 acids present in the digested sludge supernatant. 
 
 A single spot of the concentrated sample was applied 2.5 cm 
 from the lower corner of the Whatman paper using a capillary tube. In 
 order to keep the spot size to less than 3/8" in diameter and at the same 
 time deliver sufficient quantity of the sample on to the paper, the sample 
 was dried as it flowed on the paper by blowing air. After the spot dried 
 completely, the sheet was formed into a cylinder by stapling the edges 
 together so that they did not touch. The paper cylinder was then placed 
 in an upright position into a closed pyrex cylinder containing the first 
 solvent phase. 
 
 The first solvent phase consisting of butanol, acetic acid, 
 and water was prepared by taking 500 ml of freshly shaken mixture of equal 
 volumes of water and normal butanol and then adding and mixing 60 ml of 
 glacial acetic acid. After the layers separate, the upper layer was used 
 as the moving phase. An aliquot (50-100 ml) of the lower layer contained 
 in a beaker was placed in the chromatogram chamber (3). The solvent passes 
 the spot where the sample is applied and each constituent in the sample 
 moves along the solvent at a unique rate, so that after some time all the 
 components of the mixture will occupy a distinct position somewhere along 
 the path of the flow of the solvent. After the solvent front had reached 
 within an inch from the top edge of the paper, the paper was removed and 
 the solvent front was marked with a pencil. The paper was dried and 
 rerun in a second solvent whose front was at right angles to that of the 
 
86 
 
 first solvent. The second solvent in this case was a mixture of 55 m 1 of 
 2, 6 lutidine, 25 ml of ethanol, 20 ml of water and 2 ml of diethylamine (3) 
 
 The paper after being run on both the solvents, was dried and 
 sprayed with ninhydrin under a hood, to develop purple colored amino acid 
 spots. In certain cases it was necessary to expose the paper to 103°C 
 for three minutes before the amino acid spots could be fully developed by 
 the ninhydrin reaction. The purple spots were marked with pencil and the 
 ■'R " values, which are the ratios of the movement of amino acid spots to 
 the movement of the solvent front were calculated for both the solvent 
 phases used 
 
 The procedure was repeated with standard amino acids on separate 
 Whatman papers and the corresponding "R " values were computed. 
 
 Results 
 
 The results obtained from the paper chromatographic runs are 
 presented in Table C. From the results tabulated, it is apparent that only 
 four amino acids could be resolved from the digested sludge supernatant. 
 The "R " values of the supernatant spots 1, 2, and 3 closely tallied with 
 those of lysine, histidine and threonine respectively. The "R " values of 
 the spot No. h did not tally with any of the other standards used Though 
 one could expect to detect all the naturally occurring amino acids in the 
 digested sludge supernatant, it was not so in this case, due probably to the 
 following or other unknown reasons: 
 
 1. The varying degrees of susceptibility of the different amino 
 acids to microbial degradation may have caused some of the 
 amino acids to undergo complete decomposition. 
 
87 
 
 2. Due to the counter ion pair formation of ammonium, guanidium, 
 imidazolium and other ions present in different amino acids, 
 
 which are responsible for the specific ninhydrin reaction 
 in the development of purple color, the amino acids might 
 have gone undetected by the ninhydrin test. 
 
 3. The complex nature of the digested sludge supernatant may 
 result in incomplete resolution of the amino acids and the 
 consequent overlapping of one another. 
 
 k. There may have been preferential uptake of one or the 
 other of the amino acids resulting from the enzymatic 
 hydrolysis of proteins, by the microorganisms. 
 
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