I I III I I III I IIII III 401.4 ‘ «INN III (0-400 I SOME BIOCHEMICAL AND BACTERIDLDGICAL STUDIES OF ACTIVATED SLUDGE THESIS FOR THE DEGREE 01’ II 8. George Harold Robinson 1933 SOME BIOCHMICAL AND BACTERIOLOGICAL STUDIES OF ACTIVATED SLU DGE SOME BIOCHEMICAL AND EACTERIOLOGICAL §Zfl2l§§ pg ACTIVATED SLUDGE lb I§E§l§ Submitted to the Graduate Faculty at the Michigan State College - of Agriculture and Applied Science in Partial Fulfillment of the Requirements for the Degree Master of Science by (‘ !’- a f \ 'n‘ or George Hid fighineon 1933 THESIS I. II. III. IV. VI. VII. VIII. IX. INDEX Acknowledgement Introduction Scope of Problem Review of Literature Experime ntal A. Description of Plants B. Methods of Laboratory Plant Operation C. Chemical Analyses and Methods D. Bacteriological Methods Results A. Chemical considerations B. Bacteriological considerations Discussion Summary Literature C ited '1‘ 5'5 (j. r—' 0- 1 l i 1 :u' b L} K.) Page gcxnowyrnong The author wishes to express his sincere appreciation to the following: Mr. E. F. Eldridge, for his guidance and aid in the chemical work; Dr. W. L. Mallmann, for his advice on the bacteriological considerations; the Engineering Experiment Station, for their financial aid; to the Simplex Ejector and Aerator Corporation of Chicago, for the use of an experimental aerator unit; and to various members of the Departments of Bacteriology and Chemistry for their excellent cooperation in making this work possible. .‘h.v I It’d LNTRODUCM During the past eighteen years considerable work has been done on the disposal of wastes by aeration-a method commonly called the "Activated Sludge" process. This means of waste disposal is a distinct improvement over the anaerobic and aerobic prooess-Imhoff system using filters for secondary treatment. Without doubt the two major improvements over the anaerobic processes are (1) the almost complete absence of noxious odors and, (2) the retention and synthesis of the nitrogen, otherwise lost, in a form which increases the fertilizer value of the dried sludge. Although many studies of both chemical and bacteriological nature have been made on the process, no correlation studies stressing the relationship of these two proceedures have been conducted. Naturally, studies undertaken separately by chemists and bacteriologists would offer a wide variance in results as well as an expression of great divergence of opinions regarding the most intricate of the biological methods of sewage purification. .fp“ The mechanism is not clearly understood either chemically or biologically, or surely someone would have discovered most of the important factors govern- ing that most troublesome feature of the process- 'bulking'. It is very true that many theories have been advanced along with much data bearing out the contentions, but 95 per cent of the data has been collected at.a plant in operation which for one reason or another has gone through a period of bulking. It has been the author's contention, that the laboratory was the best place to carry out eXperimental procedures. Here, one could vary the influencing factors and once getting consistent results could later apply them to a large scale plant in an effort to secure the practical effects. This conception of research has been ably proven after watching the increase in numbers of research laboratories in large industrial plants, together with the increase of small commercial testing laboratories in the larger communities throughout the United States during the past fifteen or twenty years. The work embodied in this paper was undertaken with these views in mind: (1) to combine chemical and bacteriological studies, (2) to use laboratory facilities to the greatest extent possible, (3) to apply them to a small sized plant in order to get practical results and, (4) to visualize any practical application to a full size plant. This work will, in many cases, duplicate previouse findings. However, in some instances different results were obtained, also different methods of arriving at the same conclusions were used, and a few new factors were determined which have influence on bulking. soon: Or mam . This problem is a study on some mechanics of the activated sludge process with particular emphasis on bulking. Bulking is a situation encountered when the sludge remains suspended and does not settle. All of the methods used revolve around food require- ments and overloading. The methods of creating and later correcting a bulking activated sludge were governed by food types, their utilization, and effect upon pH. The hydrogenion concentration is very important in the maintenance of a rapidly settling sludge as well as being an important factor in the efficiency of the purification process by the sludge. Variations in conditions affecting pH and load studies were accomplished by carbohydrate and protein additions, separately at first, and then together. A brief review of the progress of experimentation follows, listing the treatments in chronological sequence: 1. For carbohydrate utilization lactose was selected, due primarily to the fact that dairy wastes were being studied at the time. The exact amounts used and reasons for such concentrations are given under the ”Experimental". The carbohydrate effect on pH, suspended solids, settleable solids, carbonate and bicarbonate production, were considered and also utilization as food by sludge. 2. Protein utilization was determined by the use of peptone additions in measured amounts. Peptone was used because of its solubility in water, also because of the absence of other impurities or constituents which would tend to influence results. The effect of protein matter upon pH, suspended solids, settle- able solids, carbonate and bicarbonate production, and in addition the conversion of N33 through nitrite formation to nitrates was carefully considered. 3. Mixtures of lactose and peptone were also used and the same observations made as on these substances when used singly. This addition of carbohydrate and protein mixture should theoretically maintain the sludge characteristics more or less uniformly. A condition which was not maintained when they were used singly. h. rat content determinations were made at periods on sludges from the large plant along with settling rates to determine the effect of various fat concentrations upon the settling property of the sludge. 5. Lactic acid was used to determine its availability for sludge organisms. An increase in pH was taken as a measure of this utilization. 6. On all of these aforementioned studies bacteriological samples were taken for total counts on plain and milk powder agar, the number of acid and alkali producers, nitrate reducers, and liquefiers were made. In addition the relationship between plankton and bacteria were determined. Later, some data were obtained stressing possible carbonate and bicarbonate production in various media by organisms isolated from the sludges. This work was undertaken in an effort to secure, if possible, an explanation of the behavior of pH results during the periods of sludge treatment with synthetic food wastes. From a survey of literature it was found that no previous work had been done on alkali production in association with the aerobic treatment of sewage. This phase of the problem seemed quite important. m 9;; LITERATURE About 1883 Angus Smith (8) found that jars of sewage, in which considerable algae growths were found, failed to putrefy as readily as jars of sewage contain- ing no algae. He inferred that oxygen was produced by the growths. Following his first observations, he placed variable amounts of algae in large jugs into which he later placed raw sewage. Similar jugs were set up using no algae. These were used for controls. He was able by this set-up to prove that the presence of oxygen aided in sewage decomposition and that less odor was produced, than when no oxygen was present. Other workers as Arden, Lockett, Fowler, Clark, Bartow, and others, (8) took up the study of oxygen as an agent for rapid decomposition of sewage. The bottle tests were not in use to any extent after 1905. Small aeration plants were installed and at Lawrence, Hassachussetts H. W. Clark (5) installed the first activated sludge system in this country. The studies at Lawrence were started during 1911. Dr. Clark's experiments were carried out in carboys and gallon bottles using a afl hour aeration period. He found that algal growths produced oxygen in bottles containing weak sewage. In this point he substantiates 90 the work done previously by Smith. The sewage was purified in 2% hours in both open and sealed bottles. Shorter periods of aeration gave good clarification but nitrates were not given a chance to develop, where as with 24 hours aeration about 15p.p.m. nitrates were produced. He also found that the settled material, sludge remaining in the carboys and bottles, contained considerable nitrogen. This observation indicates the increased agricultural value of the sludge as fertilizer. Arden and Lockett (6) confirmed Clark's work at Manchester; and by drawing off the supernatent liquor leaving the settled sludge in the bottom of the tank as a starter, were able to completely purify raw sewage with 6 to 9 hours simple aeration. A large accumulation of sludge could be obtained by allowing the sludge to settle, drawing off the supernatant, and again filling the bottle with raw sewage and aerating for 6 to 9 hours. By repeating this process a number of times a large quantity of activated sludge was produced. This was the first work reported on the ”starter" idea, e. g. using a developed activated sludge as a starter for future plant operations. Clark did not use a (starter in his work and as a result six weeks were required to develop an activated sludge. Cramer (7) 10. points out that sludge protozoa occur commonly in soil and in ground water, but not in human and animal wastes. This might account for the long period of time required by Clark for sludge development. The work of Ardern, Lockett, and Clark was, without doubt, the most important when considering the origin and development of the activated sludge practice. Others have had important places in the actual mechanical and other engineering improvements, but to those three must go the honor of being the real founders of the new biological method of sewage treatment. A. J. Martin (8) in his fine book covering the development of the process up to the year 1927, gives credit to many who have introduced improvements and also to those who have contributed materially to a plausible explanation of the finer features of the system. A complete historical resume has been given by him and at present is the finest record ever compiled covering the complete activated sludge process. It is needless to give a complete historical summary when such an excellent reference is at hand. Suffice it to give credit only to those men who have really built up the system, either by research or discovery. 11. Copeland (9) of the Milwaukee Sewage Commission gives the finest definition of activated sludge. He states, "The sludge embodied in sewage and consisting of suspended organic solids, including those of colloidal nature, when agitated with air for a sufficient period, assumes a flocculent appearance very similar to small pieces of sponge. Aerobic and facultative aerobic bacteria are present to the extent of 12 to lfl million per cc. some having been strained from the sewage and others by natural growth. Among the latter are those which possess power to decompose organic matter especially of an albuminoid nature-others (meaning bacteria) absorbing this free nitrogen liberated from the decomposition, convert it to nitrites and nitrates. This takes time, air, and favorable environment such as suitable temperature, food supply, and sufficient agitation to distribute them to all parts of the sewage. “The process preserves the aerobic bacteria by keeping the sludge, which is their natural food in intimate contact with air at all times and keeps them thoroughly supplied at all times with frest food from the raw sewage, throughout the whole body of which 12. they are in intimate contact.” He also mentions that the process should not be confused.with the practices in which air alone was used in attempts to purify sewage, but which made no use of sludge as an aid to oxidation. The activated sludge process was defined in 1916 by a committee appointed by the American Society Civil Engineers as ”A biochemical process by which the purification of sewage is accomplished by passing it through tanks in which the sewage sludge is artificially agitated and intimately mixed.with sewage, and is supplied with the requisite oxygen for the Optimum development of countless numbers of nitrifying organisms incorporated in and adherring to the sludge, the final settling of which causes a distinct clarification of the oxidized sewage“. As to some of the finer points of the process, air, agitation, type of sewage treated, per cent sludge return, and load per day are very important factors to be taken into consideration when best operating conditions are to be obtained. Streander (10) listed common methods which are in general use today. They are listed as occurring under two main divisions, (A) aeration and agitation using compressed air, and (B) aeration and agitation by mechanical means. Ridge 13. and Furrow aerators, Spiral flow aerators, and the air lift aerators make use of diffused air. The Link- Belt aerator, Simplex aerator, and an experimental aerator now being used by the American Well Water Works Association make use of the mechanical system. The Dorrco aerator is a combination of both mechanical and compressed air agitation. Surface aeration using agitation currents for air bubble distribution is the basis for all mechanical methods. These methods were the result of observations made from the economic side of sewage treatment; also from the findings of chemists and bacteriologists relative to the possibility of bulking conditions being caused by too great an agitation by mechanical means or by too much air being forced through the sludge suspension. Haworth (11) in 1919 described mechanical aerators, the designs of which are only being improved upon today. Aeration in contact beds and trickling filters was probably the_first use of air in plant operation. These observations were made by Fort (12) in 1915, and previously by Clark (5) and Fowler (8) at the same place (Lawrence, Mass.) 1H. Buswell, Shire, and Neave (13) in 1928 theorized that air bubbles were surrounded by a shell which hinders aeration. By opposing the water flow the shell was disrupted and increased diffusion was noted. They found that simple mechanical aeration caused fairly rapid absorption to take place and only 0.01 cu. ft. of air actually was required for one gallon of sewage. Lockett (1h) as early as 1917, found no advantage in quantites of air above that required for circulation. Good effluents in 3 hours with 20 per cent sludge content and 1.5 hours with M0 per cent sludge content were obtained. He used 1.5 cu. ft. of air per gallon with 20 per cent sludge. A given quantity of air introduced intermittently produced consistently better results than continuous aeration. This latter treatment caused a removal of 99 per cent of the bacteria. Buckworth (15) in 1915, found 2 hour agitation satisfactory. At Milwaukee during 191A and 1915, Fuller (16) reporting on the year's Operation at the first full scale plant in the United States, observed that 2 to 3 hour aeration removed 96 to 99 per cent of the bacteria present; also the nitrites were completely removed. In 1916 after another year's experience T. C. Hatton (17) reported that 15. 1.75 cubic feet of air per gallon with 20 per cent sludge using a h hour contact period was sufficient to treat sewage. Burn (18) reported that sufficient agitation is ample to keep a developed activated sludge in excellent condition. This supposition, which has been proven many times in the laboratory, also suggests the possibility that excessive air being blown through the sludge mixture might cause a break- ing up of the clumps to cause them to form colloidal particles with markedly reduced specific gravity. This causes bulking. He found that when air was turned off for A hours and then turned on again bacterial conditions in the sludge returned to normal after 6 hours aeration. He also found that free ammonia, nitrite, and nitrate conditions were normal after 7 hours aeration while only 3 hours aeration was required to cause normal albuminoid ammonia content. While these determinations demonstrated that normal dissolved oxygen conditions existed after only two hours aeration. Air evidently had considerable effect on the nitrogen status in the sludge. Buckworth (19) reported 90 per cent purification on oxygen absorption and 76 per cent purification on albuminoid ammonia. III III.II.. l6. Bartow and Mohlman (20) found 15.0 P.P.M. nitrates were formed in 15 days by blowing #830 cu. ft. of air through fresh sewage. The sludge was accumulated and after the 3lst treatment 3 cu. ft. of air per gallon for 5 hours caused complete nitrification, i. e. complete conversion of nitrogen to nitrates. These studies indicated that one hour aeration appeared to be ample for practical treatment. The worm life was active during these studies. There were many Vorticella and Rotifera. The nitrogen curves during the first blowing show that nitrates are formed from nitrites almost quantitatively. In all cases the removal of the supernatant caused decreased time in the purification on the succeeding batch. Bartow and Mohlman also observed during these nitrification studies that the dried sludge from this experiment had a fertilizer value of 829.00 per ton. The growths of flowers in pots gave better results when this dried sludge was used than when an equivalent amount of nitrogen was furnished from dried blood. In 1916, Bartow and Molhman (21) used the free ammonia and nitrite content as indicators for aeration. H. P. Eddy (22) in 1916, observed that different types of wastes necessitated variations in amounts of air used. 17. Tannery wastes required 10 cu. ft. of air per gallon with a 12 hour aeration period. In tannery waste treatment it was found that over $8 per cent of the fate were digested. The action of activated sludge on the nitrogen compounds was found to be marked by Carel (23) in 1920. He found that the ammonia content of sewage, when aerated, remained constant, but when activated sludge was added the ammonia was converted to nitrates within 2h hours. These results show the necessity of bacteria plus air rather than air alone in the nitrogen conversions. This study was extended by Dienert and Girault (2“) who experimented with allonges in a water bath whose temperature was held at 25°C. A #50 cc. quantity of activated sludge mixture containing about 12 gm. dry material was added to 1200 and 1500 cc. of sewage respectively and to ordinary water. The air was bubbled through until ammonia disappeared, then the time was noted, nitrites and nitrates were estimated. This work was continued for 9 months. They concluded that the free ammonia conversion is practically the same in all sewage concentrations studied as well as in ordinary water to which ammonia had been added. The same men (25) then attempted to find the effect of various concentrations of sludge upon the rate of nitrification. One liter samples 18. of Seine river was used and to these 50, 100, 200, and #00 cc. sludge mixtures were added. These sludge mixtures corresponded to 1.5, 3.0, 6.0, and 12.0 gms. dry matter. The air was bubbled through at the rate of 50 liters per hour. The free ammonia, nitrite, and nitrate nitrogen determinations were made. Observations indicated that all sludge mixtures readily gave an effluent free of ammonia and non-putrescible. Due to the great variation in bacteria in the sludge and in the different samples of water, the reduction in bacteria was very inconsistent. McVea and Fugate (26) reporting on the Houston, Texas plants found that as long as 1 p.p.m. nitrate was present about 99 per cent relative stability was experienced. Ammonia content of over 8 p.p.m. correlated with.poor plant operation. Ardern (27) in 1917, discussed the relationship of temperature and nitrification. The question of temperature is not serious in considering the extent of nitrification. They also concluded that the maintenance of activity of sludge was not dependent on the stage to which nitrification was carried. Copeland (2S) and Ledener (29) found temperature to have but little effect upon bacterial removal and clarification. The work done from 1920 to 1928 covers only the necessary research incidental to improved plant design. This work has nothing in common 19. with this particular paper so no effort was made to record the literature. Buswell (3n), Heukelekian (31), Rudolfs (32), and Levine (3) and (h) as well as Slaughter (33) and Eldridge (35)(36) and (37) have done considerable work on aerobic and anaerobic processes of sewage purification, particularly on dairy wastes. Their findings have been used to a considerable extent in the general discussions appearing in this thesis. After this time, research work on the activated sludge process is progressing rapidly at present with the advent of many small installations in some of the smaller towns and cities throughout the country. 20. EXPERIMENTAL Description of Plsggg In this work the mechanical aeration plant was the one use by Eldridge in his studies on the application of the activated sludge process to milk wastes. This plant consisted of a large cylindrical tank of approximately 2300 gallons capacity to the water line, as aerator unit furnished by the courtesy of the Simplex Ejector and Aerator Corporation of Chicago, a settling tank of 600 gallons capacity, and two centrifugal pumps. The necessary piping, orifice box, and electrical equip- ment was furnished by the Michigan Engineering Experiment Station. The plant, as set up for the treatment of the wastes from the college dairy, is shown in figure 1. The des- criptive points are indicated. A sewer line from the dairy building was accessible through a manhole located about midway between the Kedzie Chemical Laboratory and the Red Cedar river. Here a 3 ft. sump was constructed some 8 feet below the ground surface. It was of concrete construction and afforded an excellent location from which to obtain dairy waste. The waste was taken from the sump by means of a pipe (A) and pumped by the first of the centrifugal pumps (B) to the orifice box (a) from where the flow to the large aeration tank (D) could be governed. The photograph shows the overflow line from the orifice box returning to the sump basin. The flow of raw waste to the aeration tank entered behind a baffle plate some 3 feet in depth, thus reducing to a minimum, the possibility of any untreated sewage reaching the effluent. This effluent pipe (E) lead from the large cylindrical tank on the opposite side (shown in figure 3) and can be seen emptying into the hopper-bottomed settling tank (F). There was also a baffle plate so constructed in this tank, that the aerated waste entered in the enclosed portion, thereby eliminating the churning action on the settling sludge in the large central portion of the tank necessarily caused by the continuous flow from the aeration tank. A weir was constructed in the outflow end of the settling tank to decrease the possibility of floating sludge making its way to the final effluent pipe (K). This tank was also provided with a 2 inch drain pipe (G). This pipe was tapped by another smaller pipe, fitted with a valve (H), which furnished a means of returning certain amounts of settled sludge to the aeration tank for seeding purposes. The second centrifugal pump (J) served to pump the 22. sludge from the settling tank over into the aeration tank. This pump was not operated continuously as was the first one, but periodically according to the amount of return sludge desired. These periods were varied throughout the work and are given in the paper. At Mason, Michigan the plant set-up was the same as that used at East Lansing, (fig. 2) except that an automatic time switch was installed to operate the return sludge pump. By this means an accurate amount of sludge could be returned each hour. Also, the necessity of personal attention each hour was eliminated. A small laboratory plant was used to aerate the aeration tank contents of the larger plant after the development of a good sludge. This small set-up enabled the writer to study mixtures of sludge and raw waste during various periods in the operation of the larger plant. The laboratory set-up was extremely simple in construction as is seen in Fig. A. It consisted of two galvanized iron tanks, cylindrical in shape, of about twenty-five liters capacity. Each tank was fitted with 6 inch filtrose plate, in turn connected to a compressed air line. Light in construction and easy to operate, they formed a very useful piece of apparatus for the study of sludge mixtures as carried out in this work. It can readily be seen that in using a laboratory set-up of this design, a synthetic waste can be added and the steps in its utilization could Figure I. ' ‘ VII-I .? . ,.33 ‘~ I 43-:an Pipe frum sump basin. First centrifugal pump. Orifice box. Aeration tank. Effluent pipe from aeration tank (Shown in Fig.3) Hopper bottomed settling tank. Drain pipe from settling tank. valve in pipe allowing a return of settled sludge. Second centrifugal pump. Overflow pipe from orifice box. Figure 11. Plant set—up at Mason, Michigan. Figure III. Rear view of plant set—up at East Lansing, Michigan E. Effluent pipe from aeration tank Figure IV. A. View of tanks used for the laboratory plant. B. View of diffuser unit as used in the laboratory plant. be easily observed along with the correlated effect on the tank contents. The amount of air used in the laboratory set-up was not measured; only enough was allowed to bubble through to cause a boiling appearance on the surface. In the larger plant two types of waste were studied. The wastes from the College dairy consisted almost entirely of common dairy waste as can washings,bottle washings, floor washings, churn scourings, and, at times, old cottage cheese. There was only one toilet connected with the sewer line. The wastes did not have a constant pH because of the cleaning and scouring powders used. Some were very caustic while otheres were slightly acid because of the sulphuric acid which at times was used for cleansing purposes. In general, the wastes were quite milky in appearance and had the distinct odor of slightly soured milk. The sump had to be cleaned out from time to time. The materials removed, consisted largely of settled milk solids smelling very strongly of butyric acid and of protein cleavage products characteristic of anaerobic decomposition. The wastes encountered at Mason, consisted primarily '64-. of the final effluent of the Mason Sewage Treatment plant. The Mason plant used primary sedimentation and separate sludge digestion. Link-Belt equipment is used on the sedimentation tanks. The final effluent had an average biochemical oxygen demand of 120 to 130 p.p.m. The pH value ranged from pH 7.” to pH 7.6. The raw waste encountered at Mason was relatively strong in the B. 0. D. and solids content. It was composed of about 50 per cent domestic sewage and about 50 per cent waste from a large commercial milk products plant. Such sewage is fairly characteristic of a number of smaller cities and towns throughout the country. The milk waste in question consisted of washings from cans, large storage tanks, coolers, vacuum pans, and from casein and casein vats. As a great percentage of the work at the milk plant consisted of powdered milk preparations, the solids content of the raw milk waste was very high. Eldridge (36) in 1929 found them to be 1160.0 p.p.m. The average taken at the time of opening operations at the Mason plant showed about 1030.0 p.p.m. These results showed hardly any variation and can be considered as a normal average. Aside from the powered milk product, the plant manufactures condensed milk, casein, and milk sugar. The plant normally handles approximately 120,000 lbs. of milk per day, but during the time of Operation of the mechanical aeration system at Mason a decrease in production occurred so an accurate report cannot be given as to the number of pounds of milk handled per day. It probably did not fall below 75,000 pounds per day at any time. The volume of waste from the milk plant was about 225,000 gallons per day. In addition, the domestic wastes of the town which has about 1500 inhabitants connected with the sewage system, increased the volume to a combined total of 450,000 gallons per day with an average oxygen demand of 600 p.p.m. Methods offiLaboratory Plant Operation In operating the laboratory plant it was not necessary to keep check upon the time periods as much as when dealing with the larger set-up. The aeration was continuous and no settling tank was used. Continuous reaeration conditions existed enabling a study of the action of various sludges under continuous activation. The samples of about 35 liters were taken from the larger tank, placed into the small tanks and the air turned on. Initial determinations were made in all cases. It was not considered necessary to record the analysis in a few cases especially if no abnormal condition of the sludge existed. The food material (synthetic waste) was added in measured amounts i.e., lactose in sufficient quantity to make 400 p.p.m. concentration, while peptone was added in amounts necessary to form a concentration of 300 p.p.m. In all cases amounts were added which corre- sponded as closely as possible to amounts found naturally in wastes of the types studied. Woodman's ”Food Analysis" was used as the reference text in this respect. Chemical Analyses and Methdds As far as possible the methods outlined in the "Standard Methods of Water Analysis" published in 1925 by the American Public Health Association were followed. At times modifications of these methods were made to conform with the heavier concentrations of the various compounds sought in strong wastes and sludges. These are listed in full as follows: 1. Total Solids One hundred cubic centimeters of the sludge was placed in a taree porcelain dish and evaporated to dryness over a water bath. It was then placed in an oven at 103°C and dried for one hour, cooled in a desiccator for 15 minutes and then weighed. The weight in milligrams multiplied by 10 gives parts per million total solids. 3. Ash (Fixed residue) This determination was made on the same sludge sample as was used in the total solids determination. 27. After weighing for total solids, the dish was ignited for one hour in a muffle furnace at 700°C. The dish was then placed in the 103°C. oven for 15 minutes, cooled in the desiccator and then weighed. The weight in milligrams multiplied by 10 gives parts per million ash. 3. Volatile Matter (Loss on ignition) This result was obtained by the difference between the total solids in p.p.m. and the ash content in p.p.m. 4. Suspended Solids A 25 cc sample was taken for this determination. It was passed through a weighed Gooch crucible containing a matrix of asbestos. After filtration the mass was washed with two 10 cc portions of distilled water. The increase. in weight multiplied by 40 gives the suspended solids in p.p.m. 5. Settleable Solids and Settling Rage Use of 1 liter graduates instead of Imhoff cones was made in.this determination. A liter of aeration tank contents was placed in the cylinder. At 15, 30, 45 and 60 minute intervals readings were taken of the settled sludge in cubic centimeters. The 60 minute interval was considered as final in this determination and was recorded as the total quantity of settleable solids. The readings were made in cubic centimeters rather than in parts per million. Graduates were used because of the decreased friction caused by the slanting sides of an Imhoff cone. One hour settling periods were selected, although for general work 2 or 3 hours should be taken as the standard. 6. EH3 determination The new Hellige' Aqua Tester was used for this determination. Amounts were taken according to the “Standard Methods" and clarified with CuSO4 and KOH. Aliquots were then taken and the comparisons made using thecmmparator. Calculations were made accordingly. 7. £93 determinations The Hellige' apparatus was used in this determination and also in the nitrate estimation. The samples were taken (aliquots) and diluted to the mark. One cc.sulphanilic acid and 1 cc. alpha-naththylamine reagent were added and the comparisons were made at the end of 10 minutes. Calculations were made accordingly. 29. 8. £93 - Nitrate determination. The supernatant liquid was used for this analysis. Amounts from 1 cc. to 25 cc. were used during the course of the studies. These were evaporated to dryness over the water bath. After cooling,1 cc. of phenoldisulphonic acid was used to moisten the residue. Stirring with a glass rod insured intimate contact of the acid with the residue. The material was diluted with nitrate-free water and strong KOH added until a dark yellow color was produced. The solution was filtered through a filter paper and diluted to the mark in the comparison tube. Comparisons were made using the comparator. Calculations were made according to the amount of sample evaporated. 9. Total Nitrogen The Kjeldahl method was used and the final filtrate titrated with standard HCl solution. Results were reported in p.p.m. rather than in per cent. The results for the total nitrogen determination do not include nitrite and nitrate nitrogen. It includes only that nitrogen which is decomposed by H3804. Therefore, as the organic nitrogen not including the ammonia nitrogen is assumed to be combined in the protein molecule, the protein is calculated as (tdal nitrogen in p.p.m.-ammonia nitrogen in p.p.m.) x 6.38 : protein in p.p.m. 10. 30; Carbohydrate Determination A modification of the method used by Munson and Walker as outlined in Woodman's "Food Analysis" was used in this estimation. The modification is given in detail by Eldridge (38) and is repeated here, in part: 8.. A Gooch crucible was prepared and dried to constant weight. A 450 cc. sample of the material studied was ' placed in a 500 cc. volumetric flask. 10 cc. of CuSO4 solution (69.28 gms./ 1 liter) and 35 cc. of 0.1N NaOH or its equivalent were added. The contents were diluted to the mark, mixed well, and filtered through a dry filter. 25 cc. Fehling‘s solution B(l73 gms. potassium sodium tartrate - 50 gms. sodium hydrate in 500 cc. solution) were added to a 400 cc. pyrex beaker. Then 50 cc. of the filtered sample from (d) were added to the mixture and the beaker covered with a watch glass. This solution was boiled exactly 2 minutes over a burner so regulated as to bring 100 cc. of solution to boiling in exactly 4 minutes. The mixture was then filtered hot through the Gooch crucible. Residue of Cugo was washed with 31. i. The precipitate was then washed with 10 cc. of alcohol and by 10 cc. of ether. J. The Gooch filter was then removed to the 103°C. oven and dried for 30 minutes, cooled in a desic- cator and weighed. k. The weight of Cuzo in milligrams x the factor 14.44 gives the amount of reducing sugar present, in p.p.m. 11. pH determination In the chemical studies a Ydden.electometric device was used for the determination of pH values. The apparatus used was manufactured by the William Welch Company. In bacteriological studies, however, a different apparatus was used. This apparatus used the colorimetric principle (Hellige' Comparator) and was manufactured by the Fischer scientific Company of Pittsburgh. Fairly close similarity was obtained on standard solutions of known pH when run on both pieces of apparatus. The alkalinity produced by some bacteria was too great to be determined by the electrometric method, otherwise that method would have been used throughout the course of the studies. The colorimetric method was not used universally because of the error introduced by the sludge particles particularly while studying the chemical behavior. 13. Alkalinity Determination Alkalinity was determined according to ”Standard Jill! I I Methods" with the exception of a few cases where 0.2N acid was used in titrating instead of 0.0211. This occurred in the bacteriological studies and facilitated end point readings in presence of minute quantities of phosphates, which had considerable effect on them. The calculations were made on 50 cc. samples and results are given in p.p.m. bicarbonate and p.p.m. carbonate. l3. Bacteriological Methods The methods varied during the course of the determinations so they are described as they are dis- cussed. 33. RESULTS It was assumed in this work that a multiplicity of factors were responsible for any and all bulking character- istics of activated sludge. Morgan and Beck (1) in 1928, observed that filamentous organisms increased in number in sludges which were treated with carbohydrate wastes. Scott (2) in 1928, observed that the types of waste had considerable effect on bulking of activated dludge, particularly milk, starch, and brewery wastes. These are naturally very high in carbohydrate content. Scott also pointed out that the damage done to activated sludge by milk wastes was irreparable. Buswell, (39) in 1931, emphasized the appearance of filamentous organisms of the Sphaerotilus type in wastes of high starch content. Bach, (39) also concluded that “Carbohydrates were the chief causes of bulking“ in that they promote growth of organisms of the Sphaerotilus genus. All of the Cladothrix organisms, however, have a dense interwoven structure and therefore are very inducive to bulking. Smit (40), in 1932, working independently corroborates considerable work covered in this paper. He attacked the problem from a different angle however using a "Continuous Operating” experimental set—up, while the small laboratory set-up used by the author was primarily one of reaeration. The cylinders used by Smit in the determination of settling rates and of settleable solids were of 250 cc. capacity, 34. while 1 liter capacity containers were'used in this work. The time periods of 190 minutes duration were used while 60 minute periods were used here. However, most of the results obtained at Amsterdam, were compatible with those we obtained. The exceptions will be discussed more completely in the "Discussion". With these views in mind, the author undertook the work from the food and load standpoint. Dairy wastes have been studied at length by Levine (3) a (4), Slaughter (33), and Eldridge (35), (36), and (37). Because of the work done here by the latter two men and also because of the interest still shown at this institution regarding dairy waste disposal the primary studies in this paper were made on wastes from the College dairy, which has been stated previously. By filling the large cylindrical tank with the raw waste and aerating from Q to 6 days a fairly good sludge was develOped. Within 65 hours from the start of aeration a decrease of 64 per cent in B.O.D. in theésration tank was observed. At this time new waste was admitted for 8 hours and then a 48 hour period in which no new waste was admitted ensued. On April 16 the actual operation of the plant was begun. After continued operation until May 4, 1932 the first samples were removed from the aeration tank. 35. A dark brown, rapidly settling sludge had developed by this time and operation of the plant had been progressing in a most satisfactory manner. In Table IA are recorded the results of a preliminary study to determine the relationship of the amounts of carbohydrates, fats, and proteins in the raw influent, aeration tank effluent, and final effluent. The results of 6 samplings from the orifice box overflow, aeration tank effluent, and final effluent are averaged. The averages showed 100 per cent utilization of carbohydrate in the aeration tank and an increase of 294 per cent in the protein content. The protein content then decreased markedly in the final effluent. The fat content slowly diminished throughout the plant showing a gradual utilization. As these samples were collected over different periods of the day variations in the raw waste were to be expected. This may be due to the hourly changes encountered during the course of their operating day. 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