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SEP 2 b 1968 iijiiS a L161— O-1096 Digitized by the Internet Archive in 2013 http://archive.org/details/uptakeofsolublep44hall k 7 TOTAL SOLUBLE PHOSPHORUS, SOLUBLE ORTHOPHOSPHATE, AND TOTAL GROSS PHOSPHORUS DURING AERATION OF ACTIVATED SLUDGE FROM STOCK UNIT k\ 8 SOLUBLE PHOSPHORUS UPTAKE IN ACTIVATED SLUDGE AND BLANK SYSTEMS DURING AERATION kk 9 SOLUBLE PHOSPHORUS UPTAKE, CELL FRACTIONATION STUDIES k3 10 VARIATION IN SOLUBLE COD DURING AERATION OF ACTIVATED SLUDGE: MLSS STUDIES 56 11 VARIATION IN MLSS DURING AERATION OF ACTIVATED SLUDGE: MLSS STUDIES 57 12 SOLUBLE PHOSPHORUS UPTAKE AS A FUNCTION OF MLSS CONCENTRATION 59 13 INFLUENCE OF INITIAL SUBSTRATE CONCENTRATION ON UPTAKE OF SOLUBLE PHOSPHORUS 66 }k PHOSPHORUS UPTAKE AS A FUNCTION OF INITIAL SUBSTRATE CONCENTRATION 67 VI 1 1 F igure Page 15 SOLUBLE PHOSPHORUS UPTAKE AS A FUNCTION OF COD UPTAKE 69 16 SOLUBLE PHOSPHORUS UPTAKE AS A FUNCTION OF MLSS SYNTHESIS 71 17 UPTAKE OF SOLUBLE PHOSPHORUS AT VARIOUS INITIAL SUBSTRATE CONCENTRATION - CONSTANT INITIAL SOLUBLE PHOSPHORUS CONCENTRATION, k5 mg/1 as P 0, 74 18 SOLUBLE PHOSPHORUS UPTAKE, SYSTEM RESPONSE TO CON- TINUOUS AERATION, MIXING WITH N 2 AND QUIET SETTLING 77 19 SOLUBLE PHOSPHORUS UPTAKE, SYSTEM RESPONSE TO AERATION RATE 79 20 PHOSPHORUS UPTAKE AS INFLUENCED BY DISSOLVED OXYGEN 81 21 SOLUBLE PHOSPHORUS UPTAKE AS A FUNCTION OF DIS- SOLVED OXYGEN CONCENTRATION 83 22 SOLUBLE PHOSPHORUS UPTAKE AS A FUNCTION OF pH . . 86 23 SOLUBLE PHOSPHORUS UPTAKE AS A FUNCTION OF INITIAL SOLUBLE PHOSPHORUS CONCENTRATION .... 88 2k VARIATION IN SOLUBLE COD AND MLSS CONCENTRATION DURING AERATION OF ACTIVATED SLUDGE: INITIAL SOLUBLE PHOSPHORUS CONCENTRATION STUDY 90 25 INFLUENCE OF INITIAL SOLUBLE PHOSPHORUS CONCEN- TRATION ON TOTAL 6 HOUR UPTAKE OF SOLUBLE PHOS- PHORUS BY ACTIVATED SLUDGE SYSTEMS OPERATED AT DIFFERENT LOADING RATES 92 26 PHOSPHORUS CONCENTRATION OF NUCLEIC ACIDS AND METABOLIC INTERMEDIATES -- RESPONSE TO INITIAL SOLUBLE PHOSPHORUS CONCENTRATION 95 I. INTRODUCTION During recent years an increased concern has developed with regard to the excessive enrichment of surface waters by inorganic nu- trients contained in the effluents from municipal and industrial waste- water treatment facilities. The traditional objectives of sewage treatment, i.e., the reduction of the biochemical oxygen demand, and the removal of particulate matter, are no longer always adequate to protect surface water supplies from pollution. The biological oxidation of sewage liberates the mineral constituents of the organic matter being treated. These newly released minerals, along with the inorganic nutri- ents already present in the influent sewage, are, for the most part, discharged in the effluents from treatment facilities. As the volume of wastewater has grown, the effect of these dissolved minerals in establishing an undesirable proliferation of algae and aquatic plants within receiving streams has become increasingly evident. A number of investigators (1-*+) have reported that the dis- solved inorganic nutrients of greatest significance in this respect are the compounds of nitrogen and phosphorus, particularly phosphorus. At the same time that the growth in wastewater volume was bringing about the intensification of this problem, the expanded use of phosphates in agricultural fertilizers, in synthetic detergents, and in a variety of industrial processes, greatly increased the concentration of phosphorus compounds in the aqueous environment (5). This has had the effect of further aggravating an already serious problem. The importance of phosphorus as a limiting factor in con- trolling obnoxious aquatic growth has been emphasized by the investi- gations of Fogg and Wolfe (6), Buljan (7), and others (8, 9)- These investigators have demonstrated that certain forms of blue-green algae, among them Anabaena , Anacystis , Aphanizomenon , Calothrix , and Nostoc , are capable of fixing atmospheric nitrogen, thereby augmenting the supply of usable nitrogen in their environment. In many instances, therefore, algae may be able to utilize all available phosphorus, making phosphorus the limiting factor in the development of algae blooms. For this reason, it is generally held that the removal of phosphorus from wastewaters should be helpful in the control of obnoxious algae blooms. Because of the foregoing considerations, interest is currently running very high in methods for reducing the phosphorus concentration of secondary effluent. A Presidential Advisory Committee, formed in 1 965 » has considered this problem. Department of the Interior Secretary, Stewart L. Udal 1 , has recently indicated that the cities of Cleveland, Ohio and Detroit, Michigan will be required to remove phosphate from their sewage effluents to protect Lake Erie from excessive algae growths (10). The State of Virginia currently requires phosphate removal from sewage effluents discharged into the Potomac River in the vicinity of metropol itan Washington, D. C. (10). In response to this interest, numerous treatment processes have been proposed as means of dealing with the problem. All of these pro- posals have involved tertiary treatment of one form or another. For a variety of reasons, principally economics, none of these proposed methods has been widely accepted. This suggests, therefore, that a different approach to this problem is needed. 3 It seems rather strange that so little interest has been shown in evaluating the phosphorus removing capabilities of existing wastewater treatment facilities. As mentioned earlier, most published data indicate that very little phosphorus is being removed in conventionally operated secondary treatment plants. Certain investigators, however, have demon- strated that activated sludge treatment systems can, when operated under certain conditions, remove significant amounts of phosphorus from the waste stream. The Sewage Commission of the City of Milwaukee, for in- stance, reports phosphorus removals of 60 percent or better in their two plants (11). Levin and Shapiro (12), investigating phosphorus removal in the District of Columbia Sewage Treatment Plant, reported that a significant increase in phosphorus removal could be effected by the simple expedient of increasing the aeration rate. More recently, it has been reported that operational changes in two activated sludge plants in San Antonio, Texas have increased phosphorus removal from "typically low" values to "over 90 percent" (13). The nature of the operational modifications which are responsible for the improved phosphorus removal efficiency has not yet been reported. These recent findings point to the possibility of significant reductions in secondary effluent phosphorus concentration through the proper utilization of existing waste treatment facilities, and indicate the need for continued research in this area. The investigation reported herein was undertaken to delineate some of the factors which contribute to the uptake of soluble phosphorus by activated sludge treatment systems. It is believed that the information obtained from this study will be useful in improving the phosphorus removal efficiencies of activated sludge systems, and will contribute fundamental knowledge regarding the uptake of phosphorus by activated sludge mixed liquor, II. LITERATURE REVIEW The role of phosphorus as a key element in man-induced eutrophi- cation of surface waters, and the numerous proposals advanced as means for reducing the concentration of this element in the aqueous environment have been reviewed in great detail by several authors (14-18). It is felt that nothing is to be gained from a reiteration of their views in these pages. Consequently, the purpose of the following review is to cover only those past efforts that are in keeping with the purpose and scope of this investigation. Specifically, only those references that contain pertinent information related to the utilization of the activated sludge process for reducing the phosphorus concentration of secondary effluents have been selected for review here. Because of economic considerations, sewage treatment has always relied heavily upon naturally occurring biological processes. It seems reasonable, therefore, to investigate the possibility of using a biological method for removing phosphorus from sewage effluents. Surprisingly, very little attention has been directed towards this possibility. Wuhrmann (19) investigated the elimination of phosphorus, calcium, and nitrogen from sewage effluent through metabolic uptake by heterotrophic microorganisms. He explored the use of the bacteria present in sewage and sludge, and concluded that the carbon: nitrogen: phosphorus (C:N:P) ratio in domestic sewage demonstrates a large deficit in organic carbon. He con- cluded that insufficient usable carbon was present in domestic sewage to permit significant reduction in dissolved phosphorus. The activated sludge treatment plant of the City of Zurich was cited as achieving a 25 percent reduction in dissolved phosphate, and the implication was that additional phosphorus uptake was limited by carbohydrate deficiencies. 5 Bogan (3) also studied the possibility of using the microorganisms which constitute activated sludge to take up phosphate, and dismissed it on the grounds that, "Ordinary domestic sewage does not provide a balanced diet for activated sludge, being deficient in carbon and nitrogen with respect to the amount of phosphorus normally present" (3) • Stumm and Morgan (20) compared the relative composition of domestic sewage with the mean stoichiometric relation between carbon, nitrogen, and phosphorus in bacterial organisms. They concluded that present day aerobic biological treatment is not capable of eliminating more than 20 to 50 percent of the nitrogenous and phosphatic constituents of domestic sewage, because of the limited availability of organic carbon in the sewage. They also stated that the C:N:P ratio of the waste is a decisive factor in the elimination efficiency of inorganic nutrients by biological treatment. Thus, the treatment of wastes with high ratios of assimilable carbon to inorganic nutrients, such as sugar wastes, gen- erally leads to higher nutrient removal efficiencies. This view was presented earlier by Sawyer (21), who demonstrated that the periodical addition of glucose to activated sludge, over a period of several days, would finally produce an effluent free of inorganic phos- phorus. He stated that such a process might find limited application in the treatment of certain wastes, but pointed out the adverse economics involved in such a scheme. Although all of these researchers offer evidence opposing the possibility of significant reduction in effluent phosphorus concentration through the use of the activated sludge process, other investigators have reported data which do not necessarily fit the carbohydrate, i.e., avail- able carbon, limitation theory. Alarcon (22), studying phosphate fractions present in the various stages of the Baltimore, Maryland sewage treatment plant, found that the dissolved phosphate content of the effluent from the activated sludge process varied widely from time to time. At one sampling, the effluent from the activated sludge process contained onl y one-tenth the dissolved phosphate present in the effluent from the trickling filter. At this plant a portion of the flow goes to the activated sludge system, with the remainder being diverted to the trickling filters, so that the character of the raw waste entering both systems would have been essentially the same. A second sampling failed to show any significant difference between the respective dissolved phosphate concentration of the two ef- fluents. Alarcon suspected that insufficient aeration in the activated sludge process might have been the cause of the high phosphate content in the aeration tank effluent of the second sampling. Rudolfs (23) had earlier reported that phosphorus was released from sludge which was allowed to become anaerobic. Accordingly, Alarcon (22) took samples of the activated sludge mixed liquor into the laboratory for closer observation under conditions of vigorous aeration. Aliquots of the vigorously aerated samples were withdrawn periodically, and filtered through a membrane filter to remove all particulates, including bacteria. The orthophosphate content of the filtrate was then determined. The results of this experiment are shown in Figure 1. Initial uptake was quite pronounced, and was followed by uptake at a decreasing rate which resulted in almost total removal of the dissolved orthophosphate by the sixth hour of aeration. Alarcon con- cluded that the decrease in the orthophosphate content of the filtrate CO S-i O cu e •H H C O •H 4J CU Od sp l/§ui 'a^qdsoqdoLpao 9 T c l n T S 8 was the result of the uptake of dissolved orthophosphate by the activated sludge. His data also indicate that orthophosphate was apparently se- creted by, or leaked out of, the sludge solids after the sixth hour of aeration. By the 26th hour of aeration the orthophosphate content of the filtrate had returned to about 60 percent of its original value. Attempting to demonstrate the biological nature of this "uptake" of dissolved orthophosphate, Alarcon added mercuric chloride to aerating mixed liquor, and followed the dissolved orthophosphate content of the filtrate. The uptake of orthophosphate in the presence of mercuric chloride was only 10 percent of the initial dissolved orthophosphate content of the system. Similar investigations, presumably unknown to Alarcon, had been performed in 1959 by Srinath e_t a_l_. (2k) in India, where they were at- tempting to grow rice in "hanging gardens" in the effluent end of an activated sludge tank which provided a four-hour aeration period. Srinath and his co-workers observed that, although the influent raw sewage con- tained "a considerable amount" of water-soluble phosphorus, the rice plants suffered from a serious phosphorus deficiency. These investigators exam- ined the process in much the same manner as Alarcon, by aerating volumes of mixed liquor and analyzing for dissolved phosphorus in the settled supernatant. Figure 2 presents the results obtained. As in Alarcon's later work, these results showed a rapid initial decrease in supernatant dissolved phosphorus during the first kS minutes of aeration, followed by a declining rate of disappearance which resulted in almost complete re- moval of the dissolved phosphorus by the sixth hour. U o X! e H C o •H ■U 00 ^O CM d sb l/§ui •' snaoqdsoqj siq^ios ^aiBft w D O M fa 10 Srinath and his co-workers also determined the three minute permanganate value of the raw sewage and settled supernatants , a measure of the readily oxidizable carbonaceous material present in the waste. They reported that a reduction in the dissolved phosphorus content of the waste occurred in conjunction with a reduction in the three minute permanganate value. This seemed to suggest that the observed reduction in soluble phosphorus might somehow be associated with microbial metabol i sm. These investigators attempted to demonstrate the biological nature of the phosphorus uptake by the addition of mercuric chloride, as well as by heating the sludge to 40°C for ten minutes prior to the mixing of the sludge with the raw waste. In both cases, phosphorus uptake was adversely affected. Srinath e_t^ aj_. also deomstrated the effect of the solids content of the mixed liquor on phosphorus uptake. They reported that maximum uptake was produced by "20 to 30 percent sludge," i.e., 20 to 30 ml of return sludge per 100 ml of mixed liquor, which achieved water soluble phosphorus reduction, in the settled supernatant, in the 90 percent range after six hours aeration. Aerated raw sewage, i.e., zero percent sludge, took up practically no phosphorus during the same period. In 1962, Feng (25) reported on the results of investigations into the usefulness of the soluble orthophosphate concentration of acti- vated sludge mixed liquor as an index of the biological activity of the process. He demonstrated the ability of activated sludge to remove much of the orthophosphate from solution. His results indicated that this removal was dependent upon temperature, aeration rate, and the mixed liquor suspended solids (MLSS) concentration of the system. 11 Levin (1^+), in 1963, reported on further studies of this phenomenon, which he had conducted at the District of Columbia Sewage Treatment Plant. In laboratory scale experiments, Levin found that a small amount of return sludge in raw sewage dramatically improved soluble orthophosphate uptake over that obtainable in raw sewage alone. He was able to show that as much as 80 percent of the dissolved orthophos- phate contained in a mixed liquor solution composed of 30 percent return sludge and 70 percent raw sewage, by volume, was "...taken up by the sludge organisms with no addition of substrate." He also announced the demon- stration of a "luxury" uptake of soluble orthophosphate, i.e., "...uptake in the absence of growth." He concluded that "...rate of aeration, pH, and return sludge..." are highly important variables affecting "metabolic uptake" of orthophosphate by activated sludge mixed liquors during aeration, Rudolfs (23), in 1 9^7 > reported a release of soluble phosphorus from sludge which had been permitted to become anaerobic. This has been mentioned previously in connection with Alarcon's (22) hypothesis concern- ing the difference in orthophosphate content of effluents from trickling filter and activated sludge systems. The study made by Levin (14) con- firmed that orthophosphate leaked from the particulate matter in the mixed liquor when the activated sludge was permitted to settle with no aeration. Levin also demonstrated that a similar leakage, or secretion, of ortho- phosphate occurred at pH ' s of 5-0 and 6.0 even though the mixed liquor was being aerated. Campbell (26), reported that return sludge, kept under anaerobic conditions at room temperature for four hours leaked phosphate from the sludge solids into the liquid. 12 In addition to the loss of orthophosphate from the particulate matter back into the liquid environment under adverse conditions of dis- solved oxygen and pH , another type of orthophosphate leakage, or possibly secretion, has been reported. Data presented by Sekikawa e_t a]_. (27), as well as that reported by other investigators (14, 22), show that soluble orthophosphate is released from MLSS during extended aeration. Sekikawa and his group also reported that the aerobic digestion of solids resulted in the release of soluble orthophosphate. Irgens and Halvorson (28), on the other hand, while studying the aerobic stabilization of a mixture of activated and raw sewage sludges, found that there was a significant uptake and retention of orthophosphate by the solids in the system. It was concluded that phosphate was being utilized for the synthesis of new microbial protoplasm and retained in the sol ids. Vacker, Connell, and Wells (29) recently reported on studies carried out at three activated sludge plants in San Antonio, Texas. They showed up to 90 percent removal of total phosphate in an activated sludge plant where digester liquors were not returned to the system. They con- cluded that plant loading rate, dissolved oxygen concentration in the aeration tanks, and the degree of return of digester liquors were important parameters in the operation of an activated sludge plant for maximum phos- phate removal. Their work also indicated that leaking of phosphate from the solids back into the liquid under anaerobic conditions was not sig- nificant once the phosphate uptake was complete and a high dissolved oxygen level had been sustained throughout the end half of the aeration tank. According to other sources (13, 30) wastes at several other plants 13 around the country have since been treated experimentally in the same manner and, without exception, very high phosphate removal has been achieved. Levin (31, 32) has obtained a patent on a system for reducing the amount of phosphate in activated sludge sewage treatment plant ef- fluent- The system is based on the ability of activated sludge micro- organisms to take up more phosphate than they need, when the aeration rate is three to five times greater than normal. To recover the phos- phate, the organisms are removed from the mixed liquor by froth flotation. The sludge is then allowed to stand in an anaerobic tank, where the organisms give up their phosphate. The organism-containing sludge is then recycled to the aeration tank and the phosphate-rich supernatant fluid is treated with lime for the precipitation of tricalcium phosphate. Alarcon (22), Srinath et a\_. (2k), and others (\k, 27) have clearly demonstrated the changing rate of phosphate uptake. Their work suggests that phosphate can be taken up in excess of the limitation that would be imposed by a high C:N:P ratio in cellular tissue. In addition to these suggestive results, there is evidence that bacteria are able, under certain conditions, to store phosphorus. Harold (33) states that metachromatic granules, sometimes called "volutin," have been observed in microorganisms since 1888. These granules have been found to correspond to deposits of inorganic polyphosphate. They occur in fungi, algae, and bacteria. The function of these deposits is not yet understood (33, 3M • However, it is apparent that, if phosphorus can be stored within the cells comprising activated sludge, the C:N:P ratio }k of the cells would be variable, and would no. longer be a significant theoretical barrier to the possibility of utilizing these cells for reducing the phosphorus content of secondary effluents. In this con- nection, it is interesting to note that as early as 1955. Greenberg, Klein and Kaufman (35) reported that activated sludge organisms were able to take up more phosphorus than that actually required for satis- factory BOD removal. 15 III. PURPOSE AND SCOPE OF THE INVESTIGATION The investigations cited in the previous chapter have con- tributed a great deal of information with regard to reducing the phos- phorus concentration of activated sludge effluents. However, it is evident that a number of questions regarding the process or processes involved in this reduction remain unresolved. Present knowledge con- cerning the uptake and release of soluble phosphates by activated sludge mixed liquor appears to be insufficient to permit the application of this process as a practical means for controlling the phosphorus content of effluents from existing treatment facilities. Consequently, there is still a definite need to obtain additional information concerning the removal of soluble phosphorus by the activated sludge treatment process. The present investigation was undertaken to study the removal kinetics and mechanisms responsible for the removal of soluble phosphates in laboratory scale activated sludge systems, under various operating conditions. The variables which reportedly affect the removal kinetics of soluble phosphates in an activated sludge system and which were studied were: MLSS concentration, substrate concentration, dissolved oxygen con- centration, pH, degree of mixing, and soluble phosphate concentration. These parameters were selected following an appraisal of the existing literature on the subject. 16 IV. MATERIALS AND METHODS A. Experimental Equipment The activated sludge used in the various phases of this investi- gation was obtained from laboratory scale activated sludge units of the fill and draw type, which received a synthetic substrate consisting of glucose, yeast extract, and appropriate minerals. These units consisted of 65 by 500 mm Pyrex brand glass test tubes, having a total volume of approximately 1400 ml. The culture volume in each unit was one liter. Each cylinder was covered with a rubber stopper to prevent the loss of liquid through spraying. Air, nitrogen gas, and carbon dioxide were supplied as needed through fritted glass diffusers suspended from rubber tubes passed through holes in the stoppers. The compressed air which supplied these units was first passed through an oil trap, and then diffused through distilled water so as to saturate it with water vapor. This procedure insured a clean air supply, and also considerably reduced evaporation losses in the units. Throughout this investigation, these units were operated in a constant temperature water bath at 25°C B. Bacterial Cultures To initiate this investigation, a single activated sludge unit was started, using seed obtained from the waste treatment plant operated by the Urbana-Champa ign Sanitary District, Urbana, Illinois. This and all subsequent units were operated as follows. After 23-5 hours of aeration, solid material adhering to the inner surfaces of the unit was resuspended and distilled water was added to compensate for evaporative losses. After mixing, the required amount of mixed liquor was wasted, and aeration was 17 discontinued for one-half hour to allow the sludge to settle. After settling, the supernatant liquid was drawn down to the 250 ml mark, and the nutrients and substrates were added. The unit was then refilled to the operating level with distilled water and aeration was resumed. When filled to the one-liter operating level, the units had a freeboard of approximately five inches. The original unit, hereafter designated as the stock unit, was operated so as to contain, immediately after feeding, an MLSS concentra- tion of 1500 mg/1, and a COD concentration of 3000 mg/1 . This corresponds to a loading rate (F:M) of 2, i.e., 2 gm COD/gm of MLSS - day. A mixture of glucose and yeast extract in a ratio of 4:1 (as COD) was used as the organic substrate for this and all subsequent units. Additional nitrogen, in the form of ammonium ion, was supplied to maintain a C0D:N ratio of approximately 15« Phosphorus, in the form of monobasic potassium phos- phate (KrLPO.) and dibasic sodium phosphate (Na HOP, *7rL0) , was supplied to maintain a COD: P ratio of approximately 75:1- Table 1 lists the sub- strates, nutrients, and other operating parameters used in the maintenance of the stock unit. In order to insure that this and all other units were function- ing properly, measurements of pH and MLSS concentration were made daily. As they were needed, other units were started, using seed ob- tained from the stock unit, and acclimated to the various conditions being studied . As soon as the MLSS concentration stabilized, indicating com- pleted acclimation, experiments were begun. The experimental procedure TABLE 1 SUBSTRATE AND NUTRIENT CONCENTRATION AND OTHER OPERATING PARAMETERS OF STOCK FILL AND DRAW ACTIVATED SLUDGE UNIT 18 Compound Initial Concentration (mg/1 except as indicated) Gl ucose Yeast extract NH^Cl MgS0 4 -7H 2 FeS0 /f -7H 2 MnS0 /+ -H 2 CaCl 2 -2H 2 KH 2 P0 4 Na 2 HP0 4 -7H 2 Na 2 C0 Tap water 2400 (as COD) 600 (as COD) 600 250 10 10 10 68 134 2650 100 ml/1 iter PH Waste schedule Initial loading rate (F:M) Air flow rate 7-5 50% of mixed liquor/ day 4 ftV(hr)(l) 19 was initiated by harvesting the waste sludge from the acclimated unit under study. The harvested sludge was allowed to settle, and the super- natant then decanted. Substrate, nutrients, and water were then added so as to prepare an experimental unit, identical in all respects to the acclimated unit, which was sacrificed during the course of the experiment. In this way, the acclimated unit was protected, and could later be used for replicate studies. C. Analytical Procedures 1. Mixed Liquor Suspended Solids The determination of mixed liquor suspended solids concentration was made by the membrane filtration method described by Winneberger et al . (36). Membrane filter disks having a pore size of 0.4-5 microns and a diameter of kj mm were used for all determinations. The stainless steel membrane filtration apparatus and the filter disks were supplied by the Millipore Filter Corporation, Bedford, Massachusetts. 2. Chemical Oxygen Demand Chemical oxygen demand (COD) was determined in accordance with Standard Methods (37)- The following quantities of reagents and sample were used in this investigation: 20 ml of sample of aliquot diluted to 20 ml 10 ml of 0.25 N potassium dichromate 30 ml of concentrated sulfuric acid containing 22 gm of silver sulfate per nine-pound bottle of acid 0.10 N ferrous ammonium sulfate 0.4 gm mercuric sulfate per sample The mercuric sulfate addition is recommended for the elimination of chloride interference. 20 3- Dissolved Oxygen Measurements of dissolved oxygen concentration were made using a YSI Model 51 Oxygen Meter, supplied by the Yellow Springs Instrument Com- pany, Incorporated, Yellow Springs, Ohio. This instrument was calibrated in air-saturated distilled water at 25°C each time it was used. 4. pH Practically all of the studies performed to date by other re- searchers, using laboratory scale activated sludge systems, have utilized phosphate buffer systems for pH control. However, because of the extremely high phosphorus concentration required of such systems, it was felt that they would be unsuitable for use in the present investigation. Consequent- ly, a carbonate buffer system was employed for pH control in this study. This buffer system was prepared as follows. Initially a small volume of a 1.0M solution of sodium carbonate was added to each activated sludge unit. A small quantity of gaseous carbon dioxide was then diffused into the system until an equilibrium pH was established. The pH was then ad- justed up or down to its desired value by changing either the amount of sodium carbonate added to the system or by adjusting the flow of carbon dioxide entering the system, or by a combination of these changes. For several reasons, chiefly economics and ease of operation, the units used in this investigation were operated so as to use a minimum amount of carbon dioxide for a given pH. In other words, pH control was exercised as much as possible by controlling the sodium carbonate con- centration of the systems. Additional fine control was obtained, as necessary, by adjustment of the amount of carbon dioxide entering the system. 21 It was found that a buffer system such as this, though easy to generate, requires somewhat more attention than buffer systems commonly employed for this type of work. However, with a little practice it is not difficult to control the pH to within + 0.1 unit of the desired level. Measurements of pH were made using a Beckman Model 76 Expanded Scale pH Meter with a Beckman 39183 Combination Electrode. This equipment was manufactured by Beckman Instruments, Incorporated, Fullerton, California 5. Phosphorus Concentration Phosphorus concentrations were measured as soluble orthophos- phate, soluble total inorganic phosphate, total soluble phosphorus, and total gross phosphorus, i.e., total phosphorus in an unfiltered sample. The procedures used for orthophosphate and total inorganic phosphate are modifications of the methods outlined in Standard Methods (37)- These modifications were suggested by Dr. David I. Jenkins (38). The procedure used for total phosphorus, both soluble and gross, is a modification of the procedure suggested by Harris and Popat (39) • These procedures are outlined below. All reagents except those indicated by an asterisk were prepared according to the 12th Edition of Standard Methods (37)- Directions for preparing the noted reagents are given in Appendix A. The phosphorus data presented in this report are expressed as P-OV > except where indicated. This choice was made rather arbitrarily, and is simply a reflection of the author's past experience in presenting such data. In an earlier work (ko) , dealing with the occurrence and per- sistence of phosphate compounds in activated sludge, phosphate concentra- tions were determined as recommended in a report prepared by the Association of American Soap S- Glycerine Producers (4l). This latter report, for some 22 obscure reason, recommended that phosphate data be expressed as P . It became then, the author's habit to express all phosphorus data in this manner. If one delves into the literature on the subject of phos- phorus compounds in aqueous systems, it soon becomes obvious that phosphorus concentrations may be, and are, expressed in a variety of ways. Chief among these methods of expression are: as P ; as PO, ; and, as P. This lack of uniformity in the presentation of data often leads to significant difficulties in the correlation and interpretation of data from various sources. Perhaps this would be the proper place to suggest that one of the things which would do much to promote under- standing, information exchange, and future research in this area would be the adoption of a standard method for the expression of phosphate data. In retrospect, it seems, for a number of reasons, that the proper mode of expressing phosphorus data, at least in studies dealing with biological systems, would be as elemental phosphorus, i.e., as P. However, the adoption of a standard method for the expression of phosphorus data will probably be a long time in arriving. Therefore, for convenience, the following conversions are given (42): 1 mg/1 P 2 = 0.43 mg/1 P = 1-34 mg/1 P0^ a. Soluble Orthophosphate i. Filter the sample through a 0.45 micron membrane filter, ii. Place the filtered sample in a 50 ml volumetric flask and dilute to about 40 ml, mix by swirling. 23 1 1 1 IV VI VI 1 Include a reagent blank with each five samples. Start the determination by adding 3 ml strong acid molybdate solution to the flask containing the reagent blank. Mix by swirling. After 30 seconds, add k ml aminonapthol sulfonic acid (ANS) reducing agent, dilute to 50 ml and mix. At 1 minute, proceed with the next flask, and so on. Read each flask 6 minutes after the introduction of the ANS reducing agent. Read the percent transmission of the samples using a Spectronic "20" colorimeter (Bausch £■ Lomb, Rochester, New York), at a wavelength of 630 mu.. Use a red filter and red sensitive phototube, and 1/2 inch square cuvettes, Be sure to include at least on standard with each series of tests. A calibration curve may be developed, as shown in Figure 3, by reading the percent transmission of a series of standards . b. Soluble Total Inorganic Phosphate i. Filter the sample through a 0.^5 micron membrane filter, ii. Place the filtered sample in a 250 ml Erlenmeyer flask, dilute to 40 ml and add 8 ml of 8 N sulfuric acid, iii. Add 2 boiling chips to each flask, cover with a watch glass, and boil gently for kO minutes. 2k 4J c O U and others (*+l), and con- sisted of digesting the sample in the presence of perchloric and nitric acids. Perchloric acid is particularly suited for the digestion of organic matter because of its high oxygen content and its speed of reaction. It has been noted, however (39) > that the addition of a small amount of nitric acid to the perchloric acid-sample mixture will cause the oxidation reaction to begin 26 4J c o u o C cd 4J 4J •H e w C 5-i H 500 Phosphorus Content, micrograms as P 9 Cv FIGURE 4 CALIBRATION CURVE FOR THE DETERMINATION OF TOTAL INORGANIC PHOSPHATE . 27 at a lower temperature, and will help to insure a more complete oxidation than is possible with the use of perchloric acid alone. It has also been reported (39) that the ANS reducing agent, used in the soluble orthophosphate and soluble total inorganic phosphate procedures outlined earlier, does not produce satisfactory results in the presence of perchloric acid. For this reason elon (para-methyl -amino-phenol sulfate), a more suitable reducing agent (39) > is used in the total phosphorus procedure outlined below. i. Place the sample, either filtered or gross, in a 22 x 175 nnm Pyrex brand glass test tube, ii. Add 2 boiling chips, 1 ml of 70-72 percent perchloric acid", and 2 drops of concentrated nitric acid-' to each tube. Mix immediately by gentle swirling. iii. Heat the samples gently for about 15 minutes, or, until the intense oxidation reaction, characterized by a vigor- ous foaming action, subsides. Add two more drops of nitric acid to each tube and continue to heat untul the digestion is complete, e.g., dense white fumes of per- chloric acid appear in the tube. The resulting solution should be clear; if not, allow to cool slightly, add 1 or 2 ml deionized water and 2 drops of nitric acid and continue to heat until clarity results. Be sure to carry out this operation in a_ fume hood behind a protective screen See Appendix A. 28 iv. Allow the clarified samples to cool and transfer the contents of the tubes to 50 ml volumetric flasks. Di lute to about kO ml . v. Include a reagent blank and at least one standard with each set of samples. The reagent blank is prepared by adding 1 ml of 70-72 percent perchloric acid to approxi- mately kO ml of water in a 50 ml volumetric flask. Standards are prepared by adding known amounts of ortho- phosphate to 50 ml volumetric flasks, and adding 1 ml of 70-72 percent perchloric acid to each flask, vi. Start the colorimetric analysis by adding 2 ml of 5 percent ammonium molybdate solution* to the flask con- taining the reagent blank. Mix by swirling. After 30 seconds add k ml of elon reducing agent", dilute to 50 ml and mix. vii. At 1 minute, proceed with the next flask, and so on. Read each flask 15 minutes after the introduction of the elon reducing agent, viii. Read the percent transmission of the samples using a Spectronic "20" colorimeter, at a wavelength of 820 mu,. Use a red filter and red sensitive phototube and 1/2 inch square cuvettes, ix. A calibration curve for this method is shown in Figure 5 See Appendix A. 29 c o u 0) CD a c 03 •U 4J •H 6 100 200 300 400 500 Phosphorus Content, micrograms as Py^5 FIGURE 5 CALIBRATION CURVE FOR THE DETERMINATION OF TOTAL PHOSPHORUS. 30 6. Fractionation Procedure Experiments were performed during the course of this investigation in which it was desirable to follow the soluble phosphorus taken up by the mixed liquor microorganisms and to identify particular compounds formed by the microorganisms. This was accomplished by observing the distribution of the phosphorus among various broad classes of compounds. It was there- fore necessary to carry out a chemical fractionation of the cells. In such a fractionation, it is desirable to use a procedure which gives "clean" fractions, i.e., a procedure which isolates all of a given class of compounds in one fraction or another. However, if large numbers of samples are to be fractionated, the procedure must also be relatively simple and rapid. The procedure described below is a modifi- cation of the procedure outlined by Roberts et aj_. (43) which was developed from the investigations of Schmidt and Thannhauser (44), Friedkin and Lehninger (45), Schneider (46). and Davidson et_ a_l_. (47). This procedure is designed to produce relatively clean fractions with a minimum of manipulation. It has been used extensively in the laboratory of Roberts e_t aj_. (43), for the fractionation of phosphorus compounds from cells of Escherichia col i . i. Centrifuge a suitable sample of mixed liquor at 17,300 g for 20 minutes at a temperature of 0°C. The centrifuge used during this investigation was a Sorvall RC-2 Refriger- ated Centrifuge, manufactured by Ivan Sorvall, Incorporated, Norwalk, Connecticut. 31 ii. Carefully pour off the supernatant fluid. Rinse the pellet by filling the centrifuge tube with 0.85 percent sodium chloride (NaCl) solution and pouring off im- mediately without disturbing the pellet, iii. Wash the cells by resuspending them in 10 ml of 0.85 percent NaCl solution and centrifuging again. Save the supernatant from this operation. This is known as the wash water fraction, and contains only that phosphorus which is associated with the cell but which is on or outside the eel 1 wal 1 . iv. Resuspend the washed cells in 7 ml of a 5 percent solution of trichloractic acid (TCA) and transfer the suspension to a small 18 x 100 mm glass centrifuge tube. Allow the suspension to stand 30 minutes at to 5°C, then centri- fuge and save the supernatant fluid. This supernatant is the col d - TCA - sol uble fraction, and contains most of the inorganic cations present within the cell. In addition most of the small phosphory 1 ated molecules, such as ATP, ADP, etc., are recovered in this fraction, v. Remove any residual fluid remaining from the cold-TCA operation from the walls of the tube with a sterile cotton swab and resuspend the precipitate in 7 ml of a 75 percent ethyl alcohol (ET0H) solution. Keep this suspension at 40-50°C in a water bath or over for 30 minutes and then centrifuge. Save the supernatant fluid. This fraction contains most of the lipids and phospho- lipids of the cells, and some small amount of protein. 32 vi. Resuspend the precipitate from the above operation in 7 ml of 5 percent TCA solution. Place the suspension in a boiling water bath for 30 minutes and then centri- fuge. A clean glass marble is useful to cover the top of the tube during the boiling, so as to prevent evapor- ation of the contents. The supernatant fluid from this operation is known as the hot - TCA - sol uble fraction. This fraction is composed almost exclusively of nucleic acids, with the possibility of slight amounts of a polysaccharide material . vii. Save the precipitate remaining from the previous operation, This is termed the residual fraction, and contains the principal protein fraction of the cells. The total phosphate content of each of these fractions was determined according to the procedure outlined earlier in this report. Investigations revealed that the NaCl, TCA, and ETOH that were present in the various fractions did not interfere with the determination of total phosphate by this method. 33 V. RESULTS AND DISCUSSION The experimental results obtained during this investigation are presented below. In order to facilitate the presentation of these data the results of all experiments are presented in graphical or tabular form. A discussion of results is included with the presentation of each phase of the experimental work. Because of the considerable controversy that surrounds the subject of phosphorus uptake by activated sludge systems, all of the experiments reported in this study were re- peated, so as to provide confirmation of results. The inclusion of all these data would only serve to make this report more voluminous than it has any right to be, and would not increase its substance to any ap- preciable degree. Thus, for the most part, only typical results are shown and discussed. A. Prel iminary Typical data obtained during this investigation are shown in Figure 6. These particular data were developed using sludge from the stock unit, as described in Table 1. These results show a reduction in the concentration of soluble phosphorus of approximately 90 percent during the 24-hour aeration period. More important, however, is the observation that only 50 percent of the total reduction is accomplished during the first 6 hours of aeration. This implies that most existing activated sludge plants, with aeration periods restricted to 6 hours or less, are severely limited in their phosphate removal capability. 34 4000 &0 6 a> •p cd o •H C M C O •H 4J 03 c •H e 0) a CO 2 o £> a CO O i^i ,c o CL, CM cu CU rH CO J2 CTJ 3 i-H rH o \ {/) W) 1 6 CTJ u o H r 1 I ii MLSS 3000 2000 - 1000 Soluble COD / c 80 Ao - 60 - 40 - 20 - 1 1 1 1 1 4 8 12 16 20 Aeration Time, Hours 24 FIGURE 6 VARIATION IN SOLUBLE PHOSPHORUS, SOLUBLE COD AND MLSS DURING AERATION OF ACTIVATED SLUDGE FROM STOCK UNITc 35 The data of Figure 6 also show that the uptake of soluble phos- phorus is not dependent upon either the rate of soluble COD removal or the rate of change in MLSS concentration. The uptake of soluble COD and the increase in MLSS concentration are essentially complete after 2 hours aeration, whereas the uptake of soluble phosphorus continues throughout most of the aeration period. This observation has implications which will be elucidated later. One further point needs to be made with regard to the data of Figure 6. Several investigators (12, 22, 2k) have reported a "leakage" or secretion of soluble phosphorus from the sludge solids back into solution after 8 to 10 hours aeration. This secretion purportedly re- sulted in an increase in the soluble phosphorus concentration of the system after prolonged aeration. However, the data of Figure 6 fail to show any evidence of such an occurrence in this system. This observation applies to all of the experiments conducted during this study, as secretion of phosphorus back into solution was never encountered during the course of this investigation. B. Phosphorus Balance in Activated Sludge As was previously discussed in Chapter 2, several investigators (12, }k, 22, 2k, 25, 27) have reported the uptake of soluble phosphorus by activated sludge. These reports have indicated that with proper operation- al control the activated sludge process can be utilized to attain sig- nificant reductions in the soluble phosphorus content of biologically treatable wastewaters. However, none of these investigations has ade- quately described the role of the microorganisms present in activated 36 sludge in the observed disappearance of soluble phosphorus from activated sludge mixed liquor. In other words, what fraction of the observed dis- appearance of soluble phosphorus from activated sludge mixed liquor can be attributed to the metabolic activity of the microorganisms present in the mixed liquor? The answer to this question would seem to be essential to the complete elucidation of the mechanism of phosphorus removal in activated sludge systems. Admittedly, Alarcon (22), Sr i nath et_ a]_. (24), and others (12, 14), did consider this question. Their attempts to clarify this point led to their use of mercuric chloride (22), heat (24), and 2,4-dinit rophenol (12, 14) as means of investigating the nature of the phosphate uptake. The results of these investigations indicate that most of this uptake is of a biological nature, i.e., associated with the metabolic activities of the sludge organisms. There are, however, several means, aside from metabolic uptake, by which soluble phosphorus may be removed from a biological system such as activated sludge mixed liquor. These include adsorption onto the surface of the microorganisms (48, 49, 50), adsorption onto inorganic solids present in the mixture (51) , adsorption onto the walls of the container (52), the formation of insoluble precipitates through interaction between phosphate ions and other ions present in the system (17> 53) > as well as the ad- sorption of phosphate ions onto air bubbles and their subsequent removal by entrapment in either the foam generated on the surface of the system, or in aerosols leaving the system (54). In addition, these previous inves- tigations (12, 14, 22, 24) were, for the most part, concerned only with the uptake of soluble orthophosphate. However, it is reasonable to assume 37 that the orthophosphate originally present in the system may be converted, through microbial action, into other forms of soluble phosphorus. In such a case, because of the nature of the orthophosphate determination, soluble orthophosphate will appear to have been removed, even though there is actually no net change in the total soluble phosphorus content of the system. In like manner, the possible conversion of other forms of soluble phosphorus to the ortho form must also be considered when performing a soluble phosphorus balance on such a system. None of the investigations cited previously (12, }k, 22, 2k, 25, 27) reported any consideration of these possibilities. There are, in short, several mechanisms which must be considered and evaluated in order to determine the relationship between the disappear- ance of soluble phosphorus from a microbial system and the metabolic activity of the system. The methods, i.e., mercuric chloride, heat, and 2,k- dini trophenol , used by other investigators (12, 14, 22, 2k) to evalu- ate this relationship are subject to criticism because although they are known to produce an effect on the metabolic activity of the cells, they may also produce an effect on other possible mechanisms by which soluble phosphorus may be removed from the system. It therefore seemed necessary to consider, by some other means, the role that microorganisms play in the observed disappearance of soluble phosphorus from activated sludge mixed liquor. To this end a series of experiments were performed with a view toward the systematic evaluation of the efficacy of these proposed mechanisms in the removal of soluble phosphorus from microbial systems. 38 1. Evaluation of Suggested Mechanisms of Soluble Phosphorus Uptake by Activated Sludge Four different experimental systems were employed for this phase of the study. These systems were designed so as to permit an evaluation of the influence of the various components of activated sludge mixed liquor, i.e., biological solids, organic substrate, and inorganic nu- trients, on the soluble phosphorus removal capacity of the total system. One of these systems was an experimental laboratory scale acti- vated sludge unit of the fill and draw type, composed of biological solids, organic and inorganic nutrients, and water, as described earlier in Table 1. The other three systems were modifications of this total system in that they were "blanks," i.e., void of one or more of the ingredients con- sidered to be essential to the proper functioning of the activated sludge process. These "blanks" were prepared as follows. One of the blanks consisted of all the above mentioned components except biological solids. A second blank was devoid of both biological solids and organic substrates, and thus contained only inorganic nutrients and water. The third blank contained only soluble phosphorus, buffering material, and water. All systems were operated in an identical manner with regard to pH, tempera- ture, and air flow rate. A description of these systems is given in Table 2. Using these experimental systems it was possible to evaluate the effect of the biological solids, i.e., microorganisms, on the disap- pearance of soluble phosphorus from activated sludge mixed liquor, without the use of materials that are foreign to the system, such as poisons or TABLE 2 DESCRIPTION OF EXPERIMENTAL UNITS USED FOR PHOSPHORUS BALANCE IN ACTIVATED SLUDGE UNITS 39 Initial Concentration (mg/1 except as indicated) Ingredients Activated Sludge System Blank No. 1 Blank No. 2 Blank No. 3 Biological solids 1500 Glucose, as COD 2400 2400 Yeast extract, as COD 600 600 NH^Cl 600 600 600 MgSO^-7 H 2 250 250 250 FeSO^-7 H 2 10 10 10 MnSO if -H 2 10 10 10 Ca CI 2 - 2 H 2 10 10 10 KH 2 P0 i+ 68 68 68 68 Na 2 HP0 4 '7 H 2 134 134 134 134 Na CO., 2650 2650 2650 2650 Tap water 100 100 100 Dist i 1 led water, make one 1 iter of sol ut ion to yes yes yes yes PH 7-5 7-5 7-5 7-5 Waste schedule pe of mixed 1 iquor p rcent er day 50 - - - Initial loading rate (F:M) 2 - - - Air flow rate ft 3 /(hr)(l) 4 4 4 4 inhibitors. This approach also permitted an evaluation of the extent to which several other proposed phosphorus uptake mechanisms contribute to the total soluble phosphorus uptake of activated sludge systems. Typical data obtained during this phase of the investigation are shown in Figure 7- These particular data were developed using the experimental activated sludge unit as described in Table 2. These results show a reduction in the concentration of soluble orthophosphate of approximately 90 percent during the 24-hour aeration period. These data also show that the total soluble phosphorus concen- tration is reduced by approximately the same amount, i.e., 90 percent, during the same period of aeration. Inspection of these data reveals that the rate of uptake of total soluble phosphorus is almost identical to the rate of soluble orthophosphate uptake, except during the first hour of aeration. The difference in these two uptake rates during the early stages of aeration is attributed to the rapid conversion of condensed and/or organic phosphates, originally present in the yeast extract feed solution, to the ortho form. Such a conversion is known to occur in aqueous solutions, and the rate of conversion is reportedly influenced by bacterial action, being higher in sewage than in pure water (55). The data of Figure 7 indicate that this conversion is essentially complete in this system by the end of the first hour of aeration, and that there- after the rate of uptake of total soluble phosphorus is identical to the rate of soluble orthophosphate uptake. From these data it may be concluded that the disappearance of total soluble phosphorus from solu- tion is caused by the uptake of soluble orthophosphate. 41 1 _ .., , 1 1 CO pj *-l o X! a co £1 fa co co o U O rH — aJ ii _ •u / / -< i r/ 3 — o / / "-' - CO / / o CO rH CX P o // - C ] - $ : , Ar3 i CN ND TOTAL E FROM O CN OSPHATE, A AT ED SLUDG vO CO X > rH J-l fa M 3 o PS G PC S < n O fa cu O B •H a z CN H PQ O rH P3 M C r-J H O •H CO 2 ■P W a} ^ H < o M •H u W 5* < C M P H CO 2 o £g CO M O oi X & Oh Q w co ^ s pq Jj CO O JH CO CO §ututbui9^ ^uaoaaj f sruioL[dsond a^qn^os T^^oj, co 2 "om 45 it should be pointed out that the activated sludge units used in this study were maintained on a synthetic, soluble substrate. Thus, these systems received only small, carefully limited amounts of the cations which are known to participate in the precipitation of phosphorus fr< aqueous solutions, i.e., iron, calcium, magnesium, manganese, and aluminum. In addition, these systems were essentially devoid of inorganic suspended solids, such as clays and silts. In the more general case of an activated sludge plant treating a domestic or industrial waste, it is reasonable to assume that at least two of the aforementioned mechanisms of phosphorus removal, i.e., sorption onto inroganic solids, and the formation of in- soluble precipitates of phosphorus, might indeed play a significant role in the overall removal of soluble phosphorus from the waste. The magnitude of this contribution would of course depend upon many factors, such as the concentration and characteristics of the suspended inorganic solids, and the concentration of the various metallic ions present in the waste, as well as the pH of the system, and the solubilities of the metal phosphate precipitates, to name a few. All that is being claimed here is that, probably because of the characteristics of the synthetic medium, these mechanisms are not significant in the removal of soluble phosphorus ob- served in the activated sludge units used in this study. 2. Cellular Phosphorus Balance The evidence presented thus far in this report has demonstrated the predominant role that microorganisms play in the removal of soluble phosphorus from activated sludge mixed liquor. However, a question per- sists as to the type of microbial activity responsible for this removal. 46 Does this uptake result from the storage of phosphorus on or within the cells, or is it the result of the metabolic assimilation of phosphorus into new cellular material? In other words, is it a "luxury 11 uptake and storage, or is it actually controlled by the metabolic needs of the cells? In order to answer this question it was necessary to trace the soluble phosphorus taken up by the mixed liquor microorganisms, and to observe its distribution among certain broad classes of phosphorylated compounds formed by the microorganisms. In this way it was possible to determine any change in the phosphorus content associated with these com- pounds that occurred during the uptake of soluble phosphorus. The fraction- ation procedure used during this phase of the investigation was described in Chapter k. These experiments were performed using sludge from the stock unit, as described in Table 1. Typical results obtained during this phase of study are pre- sented in Table 3> which shows the fractions studied and the phosphorus concentration of each fraction after various periods of aeration. These data show a predictable decrease in the system's soluble phosphorus con- centration throughout the aeration period, along with a corresponding increase in the phosphorus concentration of the various cellular fractions. Inspection of these data reveals that the phosphorus concentration of each cellular fraction was approximately doubled during the aeration period. Although not shown here, a doubling of the system's MLSS con- centration was also recorded during this period. These two facts in- dicate that the uptake of soluble phosphorus in this system is associated with the synthesis of new cellular material, i.e., associated with a metabol ic uptake. kl CO < LU LU < LU o < CD CO OL O Q_ CO O X 0_ CO LU ID CO z o o 1— I I- o LU O en E s_ c 0) u c o o l/l 3 o JZ O- i/> O a) c E «h — 1 E c ro .a +-> 3 O — 1- o CO . — l/l f0 l/l ■M O O s- I- cu c • rH o W i- cd Q_ cc; i/) T3 • ^H ,, — ^ u < 1- o •^H 4-1 a.) o i — nz o ■* — - 3 1/1 D.O Cl o l/l X O •» — -C Q- l/l 0) U ■M •IH CO < ■— • c-H o o ■a h- -O cu A-cf t^J- CT\CO r^-oo cri r-~ ■ — OvOLAOOOCsjCM LALA-^- 1^-00 ooo — Lr\CTvLTiLAO > »0'— vD — — J--J" CO CM ro 0O 00 -j" OO LnLAOMALnMroO CTv^OOO^O-d- OvO- o ■— CM-— C\J CM OA CM -d" -3" \£> — CO CT\LnCT\LAO00 OroO — ■ — O O ■ — O r^. — r — o o n^d mn cx> ex) r~- md -d- cn vO -j" vO po rvvDCO o r~- OO 1^ J" vO CO CO v£> OO r-^ (v-iOOvDOOLAOO oovO rsj-JvD' — cniJ" i — cni co vD r 3" 48 Figure 9 is a graphical presentation of some of the data given previously in Table 3. These results show the soluble phosphorus uptake plotted along with the total insoluble phosphorus, i.e., the sum of the phosphorus content of all cellular fractions, including the wash solution. It is apparent from this figure that the reduction in the soluble phos- phorus content of the system, i.e., soluble phosphorus uptake, is closely paralleled by an increase in the system's cellularly associated phosphorus. Indeed, it can be seen that essentially all of the soluble phosphorus removed from the system is recoverable in the system's various cellular fractions. This is strong evidence of the association between the cells' biosynthetic activity, i.e., synthesis of cellular material, and the up- take of soluble phosphorus. The phosphorus content of the nucleic acid fraction is also shown in Figure 3, and demonstrates that the uptake of soluble phosphorus in these systems results in a corresponding increase in the phosphorus con- tent of the cells' nucleic acid fraction. Again, this is a strong in- dication that the soluble phosphorus uptake observed in these systems is the result of the cells' biosynthetic activity. For convenience, selected data from Table 3 are presented in Table h, showing the distribution of insoluble phosphorus in the solution used for washing the cells, as well as in the various cellular fractions. This distribution was computed as percent of the total insoluble phos- phorus, i.e., total gross phosphorus minus total soluble phosphorus, and was based on the average TGP of the system. **9 200 m O P-i CO ff 4J c d) 4-1 i-< 1-1 O 0) M O a CO o 160 _ 120 Total Insoluble Nucleic Acids e Uptake 1 — c 24 Aeration Time, Hours FIGURE 9 SOLUBLE PHOSPHORUS UPTAKE, CELL FRACTIONATION STUDIES. 50 00 < to X> cC o X Q_ oo o x a. LU _l CD O o zz o 1—1 V- ZD CO I— I a: \~ to i— i o co LU I— t Q X> \- CO o I— I < o LU O 10 !_ O X o_ i/> o X 4-J Q. C — C XI O D o — o 10 10 ZS c !_ 1— I O X — cl ro i/i +j O O X h- o. c o !_ Q) 0) C E •- c 0) o o s_ u QJ L. 10 o x 4-> Q- O l/l X O *— ' Ifl u o CD c~i (T\ O LACT» cricr\i^cricricr>cr\cr\cr> en CTi LA00 vD vDOO ITHANN -d - -d" f^ravfi LA-d" -d - la ■4- CMCMOvOLAOvOO — LAOO 00 J" -d" — -4- — 00 v£> LA -d" vX> vO r~- v£> vO v£< 00 CM |S(M\D O N 000 04 ■ — LA-d"cACMcM-d"LAcM -d- 00 LA CTi v£> -d" — r^— CVJ CM0000 rOOO O LA-d" ro CM CM ■— CM ■ — CM — CM CM r^-cMOO r^ — vo -d" vo la O OA O ' — ■ — OOOO CAOOMDOOLAOO CA v£> CM -J v£> — CM-d" i — (M M\fl Nd ZJ !_ o X CL 10 o CT) <0 l_ CD > (0 C o T3 0) 10 OJ X 51 These data show that essentially all of the phosphorus removed from solution during aeration can be traced to the cells composing the activated sludge. Table k shows that an average of 93-9 percent of the total soluble phosphorus removed from the system was recovered in the various cellular fractions. Only small amounts of the soluble phosphorus taken up by the cells could be removed by washing with isotonic saline solution. There is some doubt, however, whether or not washing with saline solution will effectively remove phosphorus that might be adhering to the surface of the cells. For this reason it should be pointed out that additional research (57) in this area has been conducted in the Sanitary Engineering Laboratories of the University of Maine, to determine if phosphorus can be removed from the cells by washing with slightly acid solutions. These studies have shown that washing with solutions having pH values as low as 5.0, for 15 minutes, does not produce a significant reduction in the amount of phosphorus associated with the cells. It was found that pH values on the order of 2 to 3 were necessary to achieve significant release of phosphorus from the cells. All of this information seems to indicate either that only very small quantities of phosphorus are held on the cell surfaces, or that the bonds holding the phosphorus molecules to the cell surfaces are quite strong. Table k also shows that most of the phosphorus removed from solution was distributed in various fractions within the cell, principally in the form of nucleic acids and low molecular weight intermediates. These two fractions contain approximately 60 percent and 20 percent, respectively, of the cells' total phosphorus. 52 These data also show that there was no significant change in the distribution of phosphorus within the cells during the aeration period. Apparently the phosphorus that was removed from solution was used for the synthesis of new cellular material having approximately the same phos- phorus distribution as the older cells. In other words, there appeared to be a smooth transfer of phosphorus from the solution into the various cell fractions, with no discernable increase in any fraction's contribution to the total insoluble phosphorus content of the system. Thus it may be concluded that the uptake of soluble phosphorus by these systems is not the result of an accumulation or storage of phosphorus within a particular cellular fraction but rather results from the cells' use of phosphorus in the synthesis of new cellular materials. C. Influence of Mixed Liquor Suspended Solids Concentration Several investigators have referred to the role of MLSS in the uptake of soluble phosphorus by activated sludge systems. However, for several reasons, the influence of MLSS concentration on this uptake has remained in question. Srinath e_t a]_. {2k) were apparently the first to investigate the effect of the solids content of the mixed liquor on phosphorus up- take. As mentioned earlier, they reported that maximum uptake was pro- duced by a mixed liquor containing 20 to 30 percent return sludge on a volume basis. This liquor was reported to have effected a 90 percent reduction in the soluble phosphorus concentration of the settled super- natant after six hours aeration. On the other hand, aerated raw sewage, 53 containing no return sludge, took up practically no soluble phosphorus during the same period. Unfortunately, no data were presented as to the MLSS concentration of these sludges. In 1962, using return activated sludge and feeding effluent from a primary settling basin, Feng (25) evaluated the effect of five different MLSS concentrations and two different aeration rates on the uptake of soluble orthophosphate by activated sludge. With the higher rate of aeration there was, in general, greater orthophosphate removal in systems having a low MLSS concentration. There are, however, certain inconsistencies in Feng's data. Moreover, at the lower aeration rate, the different MLSS concentrations had no discernable effect on ortho- phosphate reduction. Sekikawa e_t a_l_. (27) using an aeration rate almost twice as great as Feng's (25) highest rate, found the concentration of MLSS to have little effect on orthophosphate removal. Levin (14), and Levin and Shapiro (12) found that a small amount of return sludge in raw sewage dramatically improved orthophosphate up- take over that obtainable in raw sewage alone. However, they reported a lack of proportionality between the percent of return sludge used in their systems and the observed orthophosphate uptake of the system. Data concerning the actual concentration of MLSS in these sludges were not included in their reports. It should also be pointed out that, insofar as can be determined, none of these investigators (12, 14, 2k, 25, 27) used acclimated systems in their studies. There seemed to be then, adequate reasons for investi- gating the effect of MLSS concentration on the uptake of soluble phosphorus by activated sludge. 5^ Five different activated sludge units were initiated for this study. The feeding and maintenance of these units were exactly as des- cribed earlier for the stock unit. Substrate and nutrient concentrations, and other operating parameters, were identical with those described in Table I, except for the wasting schedule. By wasting different amounts of mixed liquor from each unit, different MLSS concentrations could be maintained in the various units, even though they received the same feed. Table 5 shows the wasting schedule, loading rate and other pertinent data for each of these units. As soon as these units had become acclimated to the desired conditions, data collection was begun. The data on COD removal obtained from these units are summarized in Figure 10. From these data it can be seen that the initial rate of COD uptake varied from unit to unit, increasing as the F:M values became smaller. However, in each unit, the soluble COD remaining eventually reached an equilibrium value of approximately 100 mg/1 . Thus, each unit was successful in taking up approximately 2900 mg/1 COD during the aeration period. Figure 11 presents the data on the increase in MLSS concen- tration obtained from the units used in this portion of the study. These data show a situation similar to that observed in the data on COD uptake, i.e., the initial rate of MLSS increase became larger as the F:M values were decreased. However, it can also be seen that, as aeration was con- tinued, the MLSS concentration of each unit eventually reached an equilib- rium value, which was, in each case, approximately 1500 mg/1 higher than the initial MLSS value. This net increase in MLSS concentration is repre- sentative of the net metabolic synethsis of the system. Thus, in each 55 TABLE 5 OPERATING PARAMETERS: MLSS CONCENTRATION STUDIES Unit Number Wasting Schedule (% of mixed liquor/day) 16 20 33 50 67 Initial F:M 0.38 0.50 1.00 2.00 4.00 Initial COD (mg/l) 3000 3000 3000 3000 3000 Initial MLSS (mg/l) 8000 6000 3000 1500 750 56 r r r- Tp-T— T" 1 -T=c5 w> CO o CM II o ii oo rH c-l o 33 0) R w M Q P & CO s r P. cm 2 VH •H H CO rJ • ■ 2 Cn L C o 4J crj • • X-N. ^-/~\~-£ J * nCT J \o cu < rJ CO Q w r\* LJT^ /*rv L/V 1 5 o O O o o o o > o o o o o o HI o o o o o o h en CM en CM x/§ui 'aoo ^iq^ios o o 3 *3 — r co • o II X • • o o o CO o o o CM fT*T\ II • • fa oo CM vO O O O o o o o CO o o o •» CM o o o o o o en o o o n CM o o o CO u o 33 a c o •H u u CD T/8m 6,000 ^Vi>^ 4,000 2,000 2 r^ F:M = 0.5 ■ — o — F:M = 1.0 F:M = 2.0 J L 12 18 24 Aeration Time, Hours FIGURE 11 VARIATION IN MLSS DURING AERATION OF ACTIVATED SLUDGE: MLSS STUDIES, 58 system, approximately 2900 mg/1 of COD were taken up, and approximately 1500 mg/1 of MLSS were synthesized, during the aeration period. In other words, each unit was able to use about 50 percent of its available substrate for the synthesis of new cellular material, over the 2k hour aeration period. This value compared favorably with synthesis values given by other sources (58, 59). Moreover, this value is supported by data on initial COD, and initial and final MLSS concentration, collected on these units over a period of several months. Figure 12 presents a summary of the data on phosphorus uptake obtained from these units. It may be seen from this plot that there was no discernable difference in either the rate or extent of soluble phosphorus uptake by these different systems. These data indicate that a change in the concentration of MLSS in the aeration basin of an actual treatment plant would have little effect on that plant's ability to take up soluble phosphorus. The data presented in Figures 10, 11, and 12, also show that the rate of soluble phosphorus uptake in these systems, being essentially constant in each unit, is not related to either the rate of soluble COD uptake, or the rate of increase in MLSS concentration. However, when the rate of uptake or increase in these parameters is ignored, and comparisons are made on the basis of total uptake or increase during the 2k hour aera- tion period, it becomes apparent that these new parameters, i.e., total soluble phosphorus uptake, total soluble COD uptake, and net increase in MLSS increase, are somehow related. 59 C3 o •H 4J cd u u C OJ o c o u CO CO o CM o o o o o o o o o m o o o lo f^ 00 vO CO H I I I I OODO« 3 O 33 CD 6 •H H C o •H 4J ctf 0) o oo ^0^d sp l/Sm f a^P^dn snaoqdsoqj aiqivfos co <: CO 33 Pi O CO o w 1-1 PQ 3 O CO CM w erf P Pn 60 For example, Figure 12 shows that the uptake of soluble phos- phorus in all five units was essentially completed by the end of the 2k hour aeration period. Furthermore, each of these units took up essentially the same total amount of soluble phosphorus during this period. Table 6 is a further summarization of the data presented in Figures 10, 11, and 12, showing the total amount of soluble phosphorus, and soluble COD taken up by each unit, and the net increase in MLSS concentration in each unit during the 2k hour aeration period. It may be noted from these data that the ratio of net MLSS increase to total phosphorus uptake (MLSS increase: P uptake) was essentially the same for each unit, the average value being approximately k2-.]. This fact will be elaborated upon later. For now it is sufficient to say that, in all five units, regardless of F:M ratio, approximately 1 unit of soluble phosphorus was taken up for each k2 units of MLSS that was synthesized during the 2k hour aeration period. There is one further feature of these data which needs to be discussed. The data of Table 6 also show that the ratio of total soluble COD uptake to total soluble phosphorus uptake (C0D:P) was approximately the same for all units, and averages 81:1. It should be pointed out here, that in these systems that the portion of the soluble COD that is removed from solution is representative of the BOD of the substrate, i.e., that fraction of the total COD that the microorganisms present in the system are able to degrade biologically. Thus it may be stated that in these systems the ratio of BOD uptake to phosphorus uptake (B0D:P) is essentially constant, and averages 81:1. This observation has a definite bearing on the question of whether or not activated sludge systems are able to effect "luxury" uptake of soluble phosphorus. 61 TABLE 6 UPTAKE OF SOLUBLE PHOSPHORUS AND COD: MLSS CONCENTRATION STUDIES Unit Number Initial MLSS (mg/1) 8000 6000 3000 1500 750 Total Uptake of Soluble Phosphorus (mg/1 as P^) 82.0 82.5 84.5 82.2 84.5 (mg/1 as P) 35.2 35.4 36.3 35-3 36.3 Total Uptake of COD (mg/1) 2925 2850 2825 2860 2875 COD Uptake:P Uptake 83 81 78 81 79 Net Increase i n MLSS (mg/1) 1400 1450 1625 1500 1550 MLSS IncreasetP Uptake 39-8 40.9 44.6 42.5 42.6 w 62 A number of investigators have reported on the BOD: P ratio necessary for the satisfactory operation of activated sludge systems (35, 58, 60, 61, 62). A summary of these findings is shown in Table 7. In general, these reported ratios are based on the minimum amount of phosphorus needed for satisfactory stabilization of the BOD. Thus, these ratios may be thought of as defining the critical amount of phosphorus required for stabilization of the BOD. When the B0D:P ratio of 81:1 obtained in this study is compared ith the values reported by these other investigators, it appears that the activated sludge systems used in the present investigation may have been taking up more phosphorus than was required for the most efficient stabilization of the substrate. If such a comparison is valid, it would tend to support the existence of a "luxury" uptake of phosphorus by activated sludge, i.e., phosphorus uptake greater than that actually required for satisfactory activated sludge operation, as has been reported by others (12, 14) . D. Influence of Initial Substrate Concentration Previous investigators (12, 14, 21) have reported that the intro- duction of carbohydrates to activated sludge mixed liquor stimulates the uptake of soluble phosphorus. In addition, the results presented earlier in this report have indicated that connection between the biosynthetic activity of the activated sludge microorganisms, and their uptake of solu- ble phosphorus. In the light of this information, it seemed desirable to study the influence of initial substrate concentration (COD.) on the uptake of soluble phosphorus by mixed liquor. 63 CD < .. t— t <=> H- O <_> 00 < Q >- LU CO a z co LU LU SI h" 21 CO o < o 5 LU CH Ll. o Li_ O 1- ■z. >- LU cc 2: < I- €25 ■=> cc CO h- s_ CO E 0) en -* C > '- 1 c 3 O >> 3 O CT .1-1 ■O 1_ O" —H CD 4-> CD O 0) 4J 1_ ro TD 4-1 i_ ro N C N 1 — •t-H 0) (D »— -i-t 03 . E 4- (0 — E • f-H E in E •-« XI O .rt •rt _D c ro 4-> c ro • ^-1 4-1 0) ro •■H 4-1 2: in a: in 2; o LA ^_ CM 1 4- 03 4-1 in CO 4J (0 c 2 aj E 4- 53 O i_ r-H c 3 O cr •r-H (U 4J L. 00 N •r— t ro 1 E •-— ( .,— 1 X3 c n3 o o CNJ 1 1 0) 4-> Ifl 03 s_ 2 4- 4- O C c 3 | 4-1 03 03 N • <— 1 tD . E •l-i ■r-l _Q c ID CA LT\ OA *~~"* ^~v ' S CM 0) . , — , s —' ' — — 1 CO 1_ ro| LA • 3 '^—^ *— ■ 1 4->| ro| ro CO a)| !_ ,--~N CD O H +J CD TJ mO aj >- E E 0) .^ 2 *— '— i_ u ro 0) a) C_3 LU co 3; x 64 Four activated sludge units were initiated for this study, and acclimated to separate initial substrate concentrations. These units were operated in the same manner described earlier for the stock unit, except that each unit received a different amount of substrate (as COD), and the mixed liquor in each unit was wasted differently so as to maintain the same initial MLSS concentration in each unit. Table 8 gives the substrate concentration, wasting schedule, and other pertinent parameters for each of these units. Table 8 also shows that as the COD. was varied, the phosphorus concentration was also varied, so as to maintain a COD: P ratio of approximately 75:1 in the feed solution. All other nutrient concentrations and operating parameters for these units were the same as those given in Table 1. Typical data obtained during this phase of the study are pre- sented in Figure 13- It is apparent from these data that increasing the initial substrate concentration has a marked effect on the uptake of soluble phosphorus by activated sludge. Figure 14 is a presentation of some of the data given in Figure 13, showing the ultimate, i.e., 24-hour, soluble phosphorus uptake plotted against the initial substrate concentration. From inspection of these data, it is apparent that soluble phosphorus uptake is directly propor- tional to organic loading, provided that aeration is continued until phosphorus uptake ceases. It was assumed that the soluble phosphorus uptake observed in these systems might more properly be a function of substrate utilization rather than substrate loading. For this reason, the 24-hour soluble phos- phorus uptake was plotted against the 24-hour uptake of soluble COD. These TABLE 8 SUBSTRATE AND NUTRIENT CONCENTRATION AND OTHER OPERATING PARAMETERS: INITIAL SUBSTRATE CONCENTRATION STUDIES Unit Number 65 Initial Substrate Concentration (mg/1 as COD) a) glucose b) yeast extract c) total 400 600 1200 2400 100 150 300 600 500 750 1500 3000 KH 2 P0 4 (mg/1) 11 17 34 68 la 2 HP0 4 -7 H 2 (mg/1) 22 33 67 134 Wasting Schedule (% of mixed liquor/day) 14 20 33 50 Initial MLSS Concentration 1500 1500 1500 1500 Initial F:M 0.33 0.50 1.00 2.00 Initial C0D:P Ratio 75:1 75:1 75:1 75:1 66 7T~ 1 y 1 TT "-S J— r-1 \ 50 rH a ^ o a o o — rH rH ' o II 6 n Q O o ' Q CJ o o — rH — n3 1 II ' rH •H cd •U 1 Q •H •H L o 1 *-» C P <-> 1 *H «-A !H 1 c SA 1 <-< 1 M toO \ \ ^ 1 6 W I ,H o \ o \ CO \ II \ Q O 1 aj | I 'i-i \ S X O i-l •H •U •H c H 1 ' 1 L Csl O CM - v£> CN - 00 O 00 O O O CM tn o w 3 e & 53 O Z o M H 2 w Q M O 3 25 O O SC CJ •\ W 3 M H l— 1 o CNI o M •H 4J En o c O C O u Z O M 6 55 0) 4J it appears that the activated sludges used in this investigation were taking up phosphorus in greater amounts than was necessary for successful stabilization of the substrate. Thus, these data also support the "luxury" uptake theory advanced by other investigators (12, 14). Another interesting feature of the ratio of COD uptake to phosphorus uptake obtained in this investigation is its constancy. Regardless of the F:M ratio, and regardless of whether the F:M ratio was varied by holding the substrate concentration constant and varying the MLSS concentration, or vice versa, these systems took up a constant amount of soluble phosphorus for each unit of COD that was utilized. This observation leads to the conclusion that within the limits of this investigation, the uptake of soluble phosphorus in activated sludge systems is directly proportional to the utilization of substrate, i.e., the anabolic activity of the microorganisms present in the system. 69 o o o CO a o o En o r-i O o o ^ 2 O M CM a H 0) CJ ■a 4J P*4 <: Q O CO < O w t-H % o o ,J0 rH o o co CO § o Oh CO o w CO m ( S Z d SB l/Sui) snaoqdsond 3iqrr[os jo a^adfl satiny. :ji ft D 70 The data reported by Jenkins (63) may also be used to support this conclusion. Working with continuous flow, pilot scale activated sludge units operated at loading rates varying from 0.1 to 3.0 pounds of COD removed per pound of volatile suspended solids per day (lb COD removed/lb vss-day) , Jenkins (63) was able to show that the ratio of phosphorus removed to COD removed, at all loading rates, was a constant value. He reported this value to be approximately 0.0086 pounds of phosphorus removed per pound of COD removed. It is obvious that this value cannot be compared directly with the ratio of phosphorus uptake to COD uptake obtained from this investigation, because of the differences in the type of operation and in the units of expression. Nevertheless, Jenkins 1 data do support the conclusion that this ratio is constant over a wide range of activated sludge loading rates, and that the uptake of soluble phosphorus in activated sludge systems is related to the system's anabolic utilization of substrate. Additional data obtained during this phase of the study are presented in Figure 16, which shows the total 24-hour uptake of soluble phosphorus plotted versus the increase in MLSS concentration that occurred during this period. The slope of the curve shown in Figure 16 can be used to compute the ratio of net MLSS increase to total phosphorus up- take (MLSS increase: P uptake) in these systems. As computed from these data, this ratio has a value of 42.5:1, a value almost identical to that which was obtained from the MLSS concentration studies discussed earlier (Chapter V, Section C) . Thus, within the limits of this investigation, it was concluded that regardless of the F:M ratio, and regardless of the 71 o o m r-i CO M CO W CO CO co 2 rH fc O \ o o M o e l*ii r-H O •\ M CD CO B CD § >-i b o C < M CO CO • •H CO H C w o •H 3 e P D 01 < CO D 8 sc fa CO o £ a m 3 o CO CT> 5.3 d sf t/3ui f snaoqdsonJ aiqnxos T^ioi fa 80 Inspection of the data given in Figure 19 reveals no significant difference in either the rate or extent of soluble phosphorus uptake in the two systems. Within the limits studied, therefore, aeration rate does not appear to be an important parameter in the uptake of soluble phosphorus by these systems. The data of Figure 19 also show that, although the dissolved oxygen concentration was lowest in the unit operated at the lower aeration rate, it was apparently high enough to enable the unit to function in a normal manner. In other words, DO concentrations of approximately 1.5 mg/1 , or greater, do not appear to restrict these systems' uptake of soluble phosphorus. This conclusion will be expanded upon later. Results of additional experiments performed during this phase of the investigation are shown in Figure 20. In these studies an experimental activated sludge unit was started as described previously. After 6 hours of aeration, the mixed liquor content of the unit was split into two aliquots. One of these aliquots continued to be aerated as before, and the second aliquot was allowed to settle quietly. After 6 hours of quiescent settling, the second aliquot was gently but thoroughly mixed and split into two portions. One of these portions was allowed to settle quietly as before, and the second was aerated at the same rate as origina 1 ly . The data presented in Figure 20 have several features of interest to this discussion. For the continuously aerated activated sludge, it may be seen that the removal of COD and the increase in MLSS were quite typical, and were essentially completed by the end of the second hour X/Sm 'SSTW Jo Q00 81 o o o m o o o O o o en o o o CM o o o 00 o o o o o o CN "T ^ f T T T ■ i-l 6 1 li. Q - co o • O CO • r^ CO ^ 1 o II o CU o C 4J iH Q O •H O 3 •> 4J o rH 4J ctj r^ O c a CO cu •H 3 D Reaer /\ CO _ - «* ' rH ^ s 1 en ous ion 5.6 to 7. 1 •H M Q C < o o CN o CN v£> CN CO u O ^^^^ pH = 6.0 - 60 s, >v pH = 6.5 40 - \ >v PH - 7.5 pH = 9 o T^^\^ - 20 - 1 1 1 I i Aeration Time, Hours FIGURE 22 SOLUBLE PHOSPHORUS UPTAKE AS A FUNCTION OF pH, 87 These studies were carried out using activated sludges that were originally developed for the studies on the influence of initial substrate concentration, which are described in Table 8. However, for this phase of the investigation the experimental units were modified si ightly, as fol lows. The experimental units were prepared in the normal manner, using biological solids, feed solution and water as described earlier. However, before aeration was begun, the contents of the units were split into three aliquots. One of these aliquots remained unaltered, and thus had an initial C0D:P ratio of approximately 75:1. The remaining two units each received different additional amounts of the stock phosphorus solution. The effect of this operation was to establish three experi- mental units, identical in ail respects except initial soluble phosphorus (SOP.) concentration. Aeration was then begun in all three aliquots, and sampling was started. It should be pointed out that the stock phosphorus solution was of sufficient strength to insure against sig- nificant dilution of the three systems. Figure 23 is representative of the results obtained from these studies. These particular data were obtained from systems containing an initial MLSS concentration of 1 500 mg/1 , which received an initial COD loading of 750 mg/1, giving them an initial F:M of 0.5- The initial C0D:P ratio in the three systems was approximately 75:1, 39:1, and 23:1, for the low, medium, and high phosphorus systems, respectively. From these data it can be seen that the initial rate of soluble phosphorus uptake was the same in all three aliquots. The total magnitude of up- take, however, was increased in the systems having higher SOP. concentra- tions. This was rather surprising in light of the fact that, in all 88 CO > CN cu a en 2 O Cu CO O £ CU i-H o CO 25 20 - 15 _ 10 - 1 1 1— 1 1 Symbol SOP. Cone. — (mg/1 as P 2 5 ) . 75 A - 45 - - D 23 -» - - 1 1 1 1 |_ Aeration Time. Hours FIGURE 23 SOLUBLE PHOSPHORUS UPTAKE AS A FUNCTION OF INITIAL SOLUBLE PHOSPHORUS CONCENTRATION. 89 cases, phosphorus was apparently not limiting, there being significant amounts remaining in solution in each of the systems at the end of the aeration period. These data also reveal that the incremental increase in the 6- hour uptake value was not proportional to the incremental increase in the SOP. concentration. This fact suggests that the uptake mechanism can be saturated at high SOP. values. 1 Figure Ik presents the data on soluble COD uptake and the in- crease in MLSS concentration obtained from these three systems. Inspection of these data reveals that the uptake of COD and the increase in MLSS was essentially the same in all three aliquots. These observations are quite puzzling when considered in conjunction with results presented earlier in this report. These data, however, do support the contention that, under certain conditions, activated sludge systems may take up soluble phosphorus in amounts greater than that needed for normal cell growth. These data also indicate that the relationships between COD uptake and phosphorus uptake, and between MLSS increase and phosphorus uptake, which were presented earlier in this discussion, were probably influenced by the amount of soluble phosphorus available to the systems. Therefore, the ratios that were derived and presented earlier, i.e.: COD uptake: P uptake^" 80: 1 MLSS increased uptake = 42: 1 are correct only for those systems having an initial COD: P ratio of ap- proximately 75:1. Most probably, these values would be different for systems having initial COD: P ratios other than this. 90 T D O O en CM § uh. 1 ^T 6 o o 0\ CO • • Q O O o o O o O O O O O o O o o O O O O O o O O o o o LO o LO O m O m O m o m o CM CM rH rH CM CM rH rH CM CM rH rH t/Sui 'uoT^^j^uaouoo SSTW i ^ en CM tu I w en Q O O -±* "TO m 0- Q O O ^k v£> CO U o X 6 •H H C O •H 4J CTJ U 0) m _ CM o O o o o o o o o o o m O m m o m m o m r^ m CM r>» m CM r^ m CM l/3m 'QO0 aiqnios % CM 91 Figure 25 presents further data relative to the influence of initial soluble phosphorus concentration on the uptake of phosphorus by activated sludges. These data show the effect of initial soluble phos- phorus concentration on the uptake of soluble phosphorus in activated sludge systems operated at different substrate loading rates. The initial MLSS concentration was 1500 mg/1 in all cases. The initial COD concen- tration is shown on each curve. It may be seen that for each of the loading rates studied an increase in the system's initial soluble phos- phorus concentration resulted in an increase in the system's uptake of soluble phosphorus. These data indicate that both substrate loading and initial soluble phosphorus concentration greatly influence the total amount of soluble phosphorus that can be taken up by activated sludge systems. In order to gain a better understanding of this phenomenon, fractionation studies were performed on the microorganisms composing the activated sludge. For this study only two aliquots were used, one con- taining a normal initial concentration of soluble phosphorus (initial C0D:P = 75:1), and one containing a high initial concentration of soluble phosphorus (initial C0D:P = 40) . The purpose of this study was to compare the various cellular fractions under conditions of high and low SOP. con- centration, in the hope of discovering the cellular fraction or fractions which accounted for the excess phosphorus. Data from this study are presented in Table 3. Inspection of these data shows that the system containing the higher SOP. concentration was able to take up considerably more soluble phosphorus (approximately 60 92 m O Hi CO 03 60 6 4-1 a en O a w o £ 0) r-J XI 3 tH O CO 5-1 O PC vO 4J O H 100 80- 60- 40 _ 20 _ 1 l 1 C0D i = 3000 mg/1 ^^-— - / C0D i = 1500 mg/1 - — / g _ ir CODi = 750 mg/1 C0D i = 500 mg/1 1 1 1 - 40 80 120 160 200 Initial Total Soluble Phosphorus Concentration^, mg/1 (as P 2 5 ) FIGURE 25 INFLUENCE OF INITIAL SOLUBLE PHOSPHORUS CONCENTRATION ON TOTAL 6 HOUR UPTAKE OF SOLUBLE PHOSPHORUS BY ACTIVATED SLUDGE SYSTEMS OPERATED AT DIFFERENT LOADING RATES. 93 CO UJ 3 Xl -1 < O h- CO 0. Z) _J < CO i-h 3 I— a: HH o Z IT hh a. CO u. o O I o_ LU <_> LU Z -J LU CO 3 3 _1 _1 en L_ O z. co LU t-H _1 z CD « o < CO 1- LU Z HH O O HH =3 H- H- < co oc: h- z. z. O LU HH (_) 1- Z < o z. o o HH CO \- Z> o a: < o cc -x. Ll_ Q_ co —1 o _l X LU Q_ O CO s_ *- ■M LA C O 0) CM U Q_ C O irt <_> 05 in en E CL o a; c E — •■h E -Q "o c CO D. „ — ,. "S C Q) o "H 3 _i a) td -M "rt O i/) .c i_ co en a. a: .^ o < < o I— o ~H +J a) o — :c u — ' 13 1/1 -o Q-x" — I- O LU -C a. •»-> m o O :r in ? O .c s: ■M ' en jr "? in o &5 co _l LA IS OO • • — .c o o en ro • iH en \D po- la -3- r~-.r--.CTi CNJ- LA-3 - -Cf LAIAN (TiCT\-4- CT> r-. ro CO LA CMCMCACM-3"LALAO J-O-J-OLAOOO OOOr-^v£)LA— LACXD -3" LA LA LA r-. O — — OOOOOOOO LA LA CA vX) vO — CM -Cf -3" LALA0O — CA vO LA • — ■ — CA O O CM [ — csj ca -3- r^ oo -d- -^ v£> CM CM r--. CM I — v£) — ca CJN ca CTv — CM LA v£> OO 00 OLAOOLALAOO OCA-J-CMCMLAOOCTv nj- r^oo -J - cAOOOvDOOO CA vD CM -4" vO vO -3" — CM CA vD -4" LA O CM o_ o CM o_ CD in CO — — cn E E CT\ o o_ 0_ o O CO CO II II _c cn ■? • rH o 94 mg/1) than the low SOP. system. A close examination of these data shows that the only significant difference in the phosphorus content of the various fractions at the high and low SOP. concentrations is found in the two acid-soluble fractions, i.e., the metabolic intermediates and other low molecular weight compounds, and the nucleic acids. The data for these two fractions are shown in Figure 26. The data of Table 9 and Figure 26 show that these two fractions account for more than 90 percent of the additional phosphorus taken up by the high SOP. system, with ap- proximately three-fourths of this going into the nucleic acid fraction. These data seem to indicate that this uptake of excess phosphorus was also closely related to the cells' anabolic activity. However, the reason for this excess or "luxury" uptake is not understood at the present t ime. Table 10 is a summary of selected data from Table 9> showing the phosphorus content of the various cellular fractions as a percent of each system's total insoluble phosphorus. These data show that es- sentially all of the phosphorus removed from solution by both the high and the low SOP. system was recoverable in the various cellular fractions, l These figures show that an average of 108.9 and 97-8 percent of the soluble phosphorus taken up by the high and low SOP. systems, respectively, was recoverable in the cellular fractions. This observation again points to the dominant role that the microorganisms play in the uptake of soluble phosphorus by these systems. Although not shown here, other data obtained during the course of this experiment revealed that the uptake of soluble COD, and the net 95 O On CO i G O •H ■U ctJ M ■U c Q) O fl o o CO O a. CO o 160 120 High SOP. Metabolic Intermediates 6 12 18 Aeration Time, Hours 24 FIGURE 26 PHOSPHORUS CONCENTRATION OF NUCLEIC ACIDS AND METABOLIC INTERMEDIATES — RESPONSE TO INITIAL SOLUBLE PHOSPHORUS CONCENTRATION. 96 o o to 00 < >- _J cc to < 2Z _1 LU X> \- _J to _1 >- LU tO O Li_ Q- O O to z o ^ l-H O 1- -J o ^ 1 — CO Q en Z LU QC < _l 1- CO to X < 1—4 CJ3 h- O. H- 1 x *\ to -z. LU l-H l-H Q tO => O 1- DC to o X z o_ o to l-H O 1- X < Q_ LU Q. i/i O x 4-> Q_ C — C XI O 3 o — o I/) l/l ZJ c U I— I O x ■— Q_ u o L. O CU 0) Q- CC c cu CD X> 4-> — H O l/l L. CD QL c€. l/l ■a u <" < <-> I— u •■H 4-> CU O — x u — ' 3 — 1 x O x a. c/i : o - x 0_ o XI CO l/l CU -— N 4-« < CD O .rH |_ T3 cu "a E — L- O CU o +J — c CU c E — •- E LA CO O O en en x en en en x C\J ■ — 0A CX) CM 1^. moo O vD >X> PA vO LACTi NNOO\C?\000 PA Psl 0O -d" -Cf CTi v£> 0O PA LA LA -d" r^cv^roj- O r^. ■ — CTvvD I — pa ■ — LA -J- -d" M rorarOLA OOCM- OOvD — CM O LA-Cf CO CT\ — — >x»vD vo la la vo r^-r^ OOvOOOLAOJ" r^rv~»-cj-vx> r^-r^cTioo r^-r^vr) LrvLAi^^t^r^. cm r--»r^o — r^vD o c^if0 4- r^vO CM cm -d" 0OOO |-^ CM -d - LACT\-Cf pAv£>LAO > t-d"cMcMCM cr»-d" — cm rwflco CO CM O I 0O LA CX\ O CM PA pa _ _ _ fsj CM vO 0O \£> CO CTvCALA vX>cMOvX>r-»— CTVCM PA -d" PA CM • — CM — CM COCOLALAPAPACMCM OO — O — OOO N-NMOCOLAN — ^ pa — — OOO PAOOOvOOOO PA v£> CM -d" M3 v£> -d~ — CM PA v£> -d" 00 cr\ oo o CA J- O -d" CTi CA vD r-- CM -d"' CA CM CM o • • X LA LA a. O O CM CM ai o_ CL i/i O l/l (/) !_ CD CD en i — ■— ■— • *\ "^v^ CD en en +J E E O 4-1 en r~- O CU f— cr\ en CD 4- M- s_ o o CU > •r* • i-H CD 0_ Cu o O C to to o II II -o it was shown that the uptake of soluble phosphorus by activated sludge was proportional to the uptake of soluble COD. For these systems, this relationship may be expressed as follows: Soluble Phosphorus Removal Rate = 0.0129 lbs P/(lb COD) (day) The corresponding ratio of COD uptake to P uptake is approximately 79:1- 106 Studies on these same systems also revealed that the ratio between MLSS increase and phosphorus uptake was approximately 42:1, or, approximately 0.23 lb P/(lbMLSS increase) (day) . It was also found that the uptake of soluble phosphorus by the activated sludge used in this investigation is affected not only by COD uptake, but also by the system's initial soluble phosphorus con- centration. Thus it is emphasized that the values given here apply only to the special case where the initial C0D:P ratio was approximately 75:1- 6. The initial soluble phosphorus content of the mixed liquor exerts a marked effect on the uptake of soluble phosphorus by activated sludge. In identical systems operated at different initial soluble phos- phorus concentrations the initial rate of uptake was the same, but the magnitude of the uptake was increased in the systems having higher initial concentrations of soluble phosphorus. It was concluded that under certain conditions, i.e., low initial C0D:P ratios, soluble phosphorus may be taken up in amounts greater than that needed for normal cell growth. 7- Soluble phosphorus reduction in activated sludge mixed liquor is dependent upon the DO concentration of the mixed liquor. DO levels of approximately 2 mg/1 appear to be necessary for the optimum uptake of soluble phosphorus. 8. Soluble phosphorus uptake in acclimated activated sludge systems was shown to be influenced by the pH of the system. Units oper- ated at pH 6-5 did not take up soluble phosphorus as readily as those operated at pH 7-5- However, there was little further reduction in the 107 uptake capacity of systems operated at pH 6.0. On the other hand, systems operated at pH 9-0 appeared to be as effective in the removal of soluble phosphorus as those operated at pH 7-5- It was concluded that slightly alkaline systems were most efficient in removing soluble phosphorus. 108 VIII. SUGGESTIONS FOR FUTURE WORK In view of the conclusions arrived at from the present study, it is suggested that this investigation should be extended to include the f ol 1 owi ng: 1. Similar studies, employing other than purely soluble substrates, to determine the influence of colloidal and suspended organic matter on the uptake of phosphorus by the activated sludge process. 2. An investigation of the effect of temperature on the up- take of soluble phosphorus by activated sludge mixed liquor. 3. Studies with lower initial C0D:P ratios to determine if the phosphorus uptake mechanism in activated sludge can be saturated. k. Studies to determine if the relationships observed in these batch studies can be duplicated in continuous flow systems. 5. Investigations to establish the role of cellular energy storage products in the uptake of soluble phosphorus by activated sludge. 6. Studies to determine if the uptake of soluble phosphorus by activated sludge microorganisms is related to their uptake of dissolved oxygen. If such a relationship could be demonstrated, it would offer further proof of the biological nature of phosphorus uptake by activated sludge. 109 REFERENCES 1. Chu, S. P., "The Influence of the Mineral Composition of the Medium on the Growth of Planktonic Algae," The Journal of Ecology, 31, 2, (1943). — — — 2. Sawyer, C. 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N., Fraser, S. C, and Hutchison, W. C, "Phosphorus Compounds in the Cell, I. Protein-Bound Phosphorus Fractions Studied With the Aid of Radioactive Phosphorus," Biochemical Journal , 49, (1951). 48. Varma, M. M., and Reid, G. W. , "Present Status of the Coliform Test, 1 Water & Sewage Works Journal , 109 , 7, (1962). 32 49. Varma, M. M., "The Relationship Between Absorbed p and pH of the Substrate," Water & Sewage Works , 1 10 , 10, (1963). 50. Varma, M. M., and Reid, G. W., "Comparison of Respiration and Metabolism of Biological Slimes Using Radiophosphorus ," Journal Water Pollution Control Federation , 36 , 2, (1964). 51. Carritt, D. E., and Goodgal, S., "Sorption Reactions and Some Ecological Implications," Deep-Sea Research , J_, 4, (1954). 52. Hassenteufel , W. , Jagitsch, R., and Koczy, F. F., "Impregnation of Glass Surface Against Sorption of Phosphate Traces," Limnology and Oceanology , 8, 2, (1963) • 53. Lea, W. L., Rohlich, G. A., and Katz, W. J., "Removal of Phosphates From Treated Sewage," Sewage and Industrial Wastes , 26, 3, (1954). 113 54. Baylor, E. R., Sutcliffe, W. H. , and Hi rschf iel d, D. S., "Adsorption of Phosphates onto Bubbles," Deep-Sea Research , 9_, 2, (1962). 55- Sawyer, C. N., Chemistry for Sanitary Engineers , McGraw-Hill Book Company, Incorporated, New York, New York, (i960). 56. Shapiro, J., "Phosphorus and Phytopl ankton," Science , 130 , 3371, (1959). 57- Barnes, B. B., and Hall, M. W., Unpublished Research, Department of Civil Engineering, University of Maine, Orono, Maine, (I967). 58. Eckenfelder, W. W. , Jr., and O'Connor, D. J., Biological Waste Treat - ment , Pergamon Press, New York, New York, (1961). 59- Eckenfelder, W. W., Jr., Industrial Water Pollution Control , McGraw- Hill Book Company, Incorporated, New York, New York, (1966). 60. Sawyer, C. N., "Bacterial Nutrition and Synthesis," Biological Treatment of Sewage and Industrial Wastes , Volume I , Aerobic Oxidation , Edited by J. McCabe, and W. W. Eckenfelder, Jr., Reinhold Publishing Corporation, New York, New York, (1956). 61. Helmers, E. N., Frame, J. D. , Greenberg, A. E., and Sawyer, C. N., "Nutritional Requirements in the Biological Stabilization of Industrie Wastes, II. Treatment With Domestic Sewage," Sewage and Industrial Wastes , 23, 7, (1951). 62. Helmers, E. N., Frame, J. D., Greenberg, A. E., and Sawyer, C. N., "Nutritional Requirements in the Biological Stabilization of Industria Wastes, III. Treatment With Supplementary Nutrients," Sewage and Industrial Wastes , 2k, k, (1952). 63. Jenkins, D. I., "Phosphorus Removal by Sewage Treatment Processes," University of California News Quarterly , \J_, 2, ( 1 967) - 6k. Shapiro, J., "Induced Rapid Release and Uptake of Phosphate by Micro- organisms," Science , 155 ? 1269, (1967) • 65. Sawyer, C. N. , and McCarty, P. L., Chemistry for Sanitary Engineers , McGraw-Hill Book Company, Incorporated, New York, New York, ( 1 967) • 114 APPENDIX A Reagents used in the determination of total phosphate (39)- i. Perchloric acid - 70-72 percent, ACS reagent grade. This may be purchased at this strength, ii. Nitric acid - concentrated, ACS reagent grade, iii. 5 percent ammonium molybdate solution - dissolve 5 gm ammonium molybdate, ACS reagent grade, in 100 ml dis- tilled water, iv. Elon reducing agent - dissolve 1.25 gm P-methy laminophenol sulfate (also known as elon, graphol, or metol), 31.25 gm sodium bisulfite, and 6.0 gm sodium sulfite, all ACS re- agent grade, in 250 ml distilled water. Store solution in dark bottle in refrigerator. 115 VITA Millard Wayne Hall was born at Edison, Georgia, on May 30, 1936. After graduating from Griffin High School, in Griffin, Georgia, he en- rolled in the Vanderbi 1 t University College of Engineering. He left Vanderbilt University temporarily in March, 1956, and in July of that year entered the U. S. Army Corps of Engineers. After serving in Germany, he was honorably discharged in 1959> and returned to Vanderbilt University to pursue studies in Civil Engineering. He was graduated from Vanderbilt University in June 1962, with a B. E. in Civil Engineering. In September 1962, he entered the Graduate College of the Uni- versity of Illinois, as a recipient of a U. S. Public Health Service Traineeship award. He received an M. S. in Sanitary Engineering from the University of Illinois in October 1963- As part of the requirements for his Master's degree, he prepared a report of a Special Problem en- titled, "Inorganic Compounds If Phosphorus in the Activated Sludge Process." From September 1963 to the present he has been a doctoral can- didate in the Sanitary Engineering program at the University of Illinois. During the first three years of this period he received financial support from a U. S. Public Health Service Research Fellowship, and from the Ford Foundation Load Program. In September 1966, he was appointed as Instructor in the Depart- ment of Civil Engineering, University of Illinois. In January of 1967. he resigned this position to become Assistant Professor of Civil Engi- neering at the University of Maine, where he is presently located. 116 He is married to the former Miss Marlene Murphy of Griffin, Georgia, and they have two children, Crystal Dianne, age nine, and Michael Wayne, age k. He is a member of Phi Kappa Sigma social fraternity, and the Fellowship of Christian Athletes. He is also an ordained Deacon in the Southern Baptist Church. His professional associations include the American Water Works Association, the Water Pollution Control Federation, the American Society of Civil Engineers, and the Society of Sigma Xi. His professional publications are listed below: 1. Hall, M. W., and Sproul, 0. J., "The Role of the University in Water Utility Research and Development," Maine Water Utilities Association Journal , 4 3 , 5> ( 1 967) - 2. Hall, M. W., and Engelbrecht, R. S., "Uptake of Soluble Phosphate by Activated Sludge: Parameters of Influence," Proceedings, Seventh Industrial Water and Waste Confer - ence , University of Texas, Austin, Texas, (June, 1967) • 3- Sproul, 0. J., Keshavan, K. , Hall, M. W. , and Barnes, B. B., Potato Processing Wastewater Treatment , 14th Ontario Industrial Waste Conference, Niagara Falls, Ontario, (June, 1967)-