OHWEHB* UW*"" LI B RAHY OF THE, U N IVLR.SITY or ILLI NOIS 628 lJL65c ENGINEERING COHf . R00* 3 The person charging this material is re- sponsible for its return on or before the Latest Date stamped below. Theft, mutilation, and underlining of books are reasons for disciplinary action and may result in dismissal from the University. University of Illinois Library Digitized by the Internet Archive in 2013 http://archive.org/details/biologicalremova29bane >-stem. under Aerobic Conditions. F/M 1,40, Temperature 20- 107 Removal Characteristics of Potato Starch in a Bovine Serum Albumin-Acclimated System under Aerobic Conditions, F/M 1.9, Temperature 20°C " L08 4-> Change in Total-COD Removal with Acclimation of Glucose- Sludge to Potato Starch at 20°C 110 Change in Starch-COD Removal with Acclimation of Glucose- Sludge to Potato Starch at 20°C 111 46 Change in Starch-COD Removal with Acclimation of Glucose- Sludge to Potato Starch at 20°C 112 -/ Removal Characteristics of Starch in an Acclimated Aerobic System by Cells and their Components at 20°C 114 ^6 Removal Characteristics of Starch-COD in an Acclimated Aerobic System by Cells and their Components at 20°C 115 Rate Constant of Total-COD Removal at Different F/M and Temperatures for an Acclimated System 119 50 Rate Constant of Total-COD Removal at Different Initial MLSS and Temperatures for an Acclimated System 120 51 Rate Constant of Starch-COD Removal at Different F/M and Temperatures for an Acclimated System L23 52 Rate Constant of Starch-COD Removal at Different Initial MLSS and Temperatures for an Acclimated System 53 Relationship between Rate Constants of Starch-COD Removal and Total-COD Removal in Acclimated Systems 130 54 Michaelis-Menten Plot for Starch-COD Removal Rates 132 55 Effect of Temperature on Total-COD Removal Rate Constants 135 56 Effect of Temperature on 5tarch-C0D Removal Rate Constants 136 57 Effect of Temperature on the Constants 'b' S 'c' 137 5S Adsorption of Starch by the Acclimated Sludge Systems . . 59 Adsorption of Starch by Acclimated Sludge Systems w: Different Sludge Concentrations at 5°C 152 XIV Figure Page 60 Change in Starch Adsorption by Glucose Sludge with Accli- mation to Starch 154 61 Standard Curve for Potato Starch-Iodine Test 176 62 Acclimation of Metrecal Sludge to Potato Starch Substrate - MLSS Balance 182 63 Acclimation of Metrecal Sludge to Potato Starch Substrate - Change in Substrate Removal Rates 183 64 Acclimation of Metrecal Sludge to Glucose Substrate - MLSS Balance 184 65 Acclimation of Metrecal Sludge to Maltose Substrate - MLSS Balance 185 66 Acclimation of Metrecal Sludge to Bovine Serum Albumin Substrate - MLSS Balance 186 INTRODUCTION ■. ... : waste treatment is biological treatment , Since its inception in the late nineteenth century and early twentieth century, biological waste treatment has made tremendous strides „ Various industrial wastes which were con- sidered to be toxic to the microorganisms only 10 to 15 years ago have been successfully treated by the use of the proper type of microflora , Although there are still some unanswered questions relating to the treat- ment process, significant advances have been made towards explaining the biochemical and physical nature of the process for simple substrates, especially in the activated sludge process. The contributions from the fields of microbiology, biochemistry, and physiology have proved invaluable in the understanding of the complex activities of microorganisms in the waste treatment works. This clearer understanding of the biological treatment process (especially with soluble wastes) provides a sounder basis for the design of the treatment facilities and has reduced the pollutional loads on streams by thousands of pounds „ The understanding of the removal of colloidal and particulate wastes in biological waste treatment is still very scanty (1)„ The colloidal fraction of a waste may vary according to the character and origin of the waste. In domestic sewage, Balmat (2) estimated that colloids represent as much as 52 percent of the biological oxygen demand (BOD) and 54 percent by weight of the suspended solids of the sewage. Some industrial wastes may have a higher content of colloidal organic load. For example, wastes from the textile desizing process contain large quantities of starch (3); waste from paper and pulp factories and food processing industries have a high colloidal content also Casein in dairy wastes, protein water in wastes from the potato starch manufacture, and blood in the slaughter house wastes are other examples of colloidal macromolecules in industrial waste „ Despite the fact that various chemical precipitation methods have been developed for the treatment of colloidal wastes, the ultimate disposal of the sludge produced is often accomplished by biological methods . Existing methods of biological waste treatment which successfully remove colloidal matter either alter the hydration and electric forces, resulting in agglomeration and settlement, or produce exoenzymatic degradation of the colloidal mole- cules to sizes small enough to permit utilization by the microorganisms „ There is a dearth of literature on the treatment of colloidal wastes by biological means, although it is established that colloids in waste constitute a significant parto One reason for the lack of research on this aspect of the problem could be the difficulties of analytical procedures for determining colloids in the presence of microorganisms , which themselves are of colloidal size. The present work was undertaken to determine the basic physico- chemical and biological factors responsible for the removal of a purely colloidal substrate in an activated sludge system„ 2. LITERATURE REVIEW The colloidal fraction of sewage has attracted the interest of many investigators. The early workers were mainly interested in separating and estimating the amount of colloids present in the sewage (4)(5). They assumed an arbitrary distinction between the suspended particulate and colloidal matter a and made no attempts to critically study the method of differentiation adopted in their investigations. It was Mills (6) in 1932 9 who first systematically investigated the sewage colloids and classified them according to their particle size,, This work was primarily concerned with the occurrence and stability of the colloids as affected by factors like pH, ionic strength, and other cations, Balmat (2) in 1955, using improved techniques, classified sewage colloids on the basis of particle size,, He was mainly interested in separating various particulate and colloidal fractions, chemically charac- terizing them and studying the BOD removal rate of these fractions. He concluded that colloids (one micron to one millimicron size) and supra- colloids (nonsettleable solids greater than one micron size), represented as much as 52 percent of the total BOD and 54 percent by weight of the suspended solids in the sewage, Heukelekian and Balmat (7) reported the mineral and organic composition of the different fractions of sewage , indicating that colloids have high calcium and phosphorus, and supracoiloids have high phosphorus, silicon, and calcium contents. They also attempted to separate the fractions by solvent extraction into total grease (soluble in petroleum ether), alcohol-soluble (exclusive of grease and containing amino acids) and alcohol-insoluble (containing polysaccharides) fractions, Balmat (8), using a Warburg respirometer , reported the BOD removal kinetics of the colloidal fraction of the sewage., The rate constant, k (common logarithm), for supracolloids , was found to be o 09 and that of the col- loids was o 22„ He indicated that comminution of some wastes to one micron size or smaller would greatly increase their rate of biochemical oxidation,, No report has been published characterizing the components of sewage which are soluble but of colloidal size,, This category includes various poly- saccharides like starch, dextrins 9 glycogen, etc, some proteins like albumins, hemoglobin, etc, and other macromolecules soluble in water „ The first study involving purely colloidal substrates, i e , starch and dextrin, in an activated sludge system in the laboratory was reported in 1947 by Placak and Ruchhoft (9)„ It was observed that very high initial removals, 30 to 80 percent, were obtained when activated sludge systems were fed these substrates, followed by a continuous removal over the 22- to 2U-hour aeration period, so that the total removal was over 90 percent « The high initial removal was attributed to adsorption of the substrate by the sludge „ However, they did not pursue this finding under different conditions and neither did they try to correlate the initial uptake with the overall removal kinetics of the substrate „ Thus, their results were of a more qualitative nature than quantitative,, Gaudy (10) t using a glucose acclimated sludge, obtained a high initial removal of glycogen substrate „ Although the increased sludge did indicated ad- sorption, he found on analysis that the sludge did not have an equivalent high carbohydrate content » He concluded that the adsorbed carbohydrate was probably used immediately for synthesis of protein in the cello With soluble starch, he did not obtain any initial removal and concluded that adsorption was not prevalent in this case This discrepancy between the behavior of glycogen and starch was attributed to the fact that the glycogen substrate was not completely soluble and had the appearance of a colloidal suspension, whereas the starch was completely soluble , This explanation does not seem reasonable , since even soluble macromolecules can be adsorbed on surfaces f and the adsorption of soluble proteins on soils is an excel- lent example of this (11). Gaudy (10) was investigating the effect of a qualitative shock load of starch and dextrins to glucose acclimated cells s and so he did not study the kinetics of the removal of these substrates in greater detail,, nor did he further investigate the high initial adsorption phenomenon observed with glycogen. In the industrial waste field, there are numerous references to the biological treatment of colloidal waste; for example, dairy wastes (12) (13), textile wastes (14)(15), potato starch industry waste (16)(17), and citrus waste (18). No unified approach to the study of the decomposition rate or mechanism for the colloidal components has been reported. Usually the colloid, be it protein, carbohydrate, or lipid, is present together with various other organic matter, some smaller (soluble) and some larger (particulate) matter. Therefore, any conclusions drawn from studies on these mixed wastes would not be entirely representative of removal of the colloids. Chemical treatment (19) (20) has also been used for the removal of colloidal components of industrial wastes as mentioned earlier. With chemical treatment, precipitated colloids are difficult to dewater, some- times become a waste disposal problem, and often require anaerobic diges- tion. There have been reports of recovery of proteins after chemical precipitation in potato starch industry waste (21), but the economics of this process often do not justify the recovery. It will be apparent from the literature discussed above that no coordinated and unified efforts have been made to ascertain the removal characteristics of pure macromolecules of colloidal sizes in activated sludge systems. The works reported by Ruchhoft et al. (22), Placak and Ruchhoft (9) and Gaudy (10) were fragmentary, and they did not study the colloidal substrate removal thoroughly, since this did not constitute the primary purpose of their research. Further, the mechanism of removal of colloidal components of waste in activated sludge systems has not been clarified. It has been suggested by Eckenf elder and Gloyna (23) that colloidal matter in waste is removed on contact by a physico-chemical process like adsorption on the activated sludge surface, Smallwood (24) experimentally showed that adsorption was the predominant phenomenon in the removal of a colloid, C, u «tagged Chlorella algae, in an activated sludge system. However, the substrate used was quite unusual and perhaps not representative of colloidal wastes. Further, the presence of 40 percent of the radioactivity in the supernatant liquid after six hours of aeration does not necessarily mean that the balance of the radioactivity was adsorbed on the cells in the activated sludge system. There is a good possibility that the balance of the radioactivity was incorporated into the cells. In this context, mention must be made of the modified activated sludge processes like "Biosorption" (25) , "Contact Stabilization" (26), and "Bioflocculation" (27), which are largely dependent on the high initial removal characteristics of the particular waste and the sludge. The mech- anisms proposed for these processes are essentially the same as those mentioned earlier in the case of the conventional activated sludge process. These processes are applicable only in cases where a very high percentage (70-80 percent) of the incoming organic matter is sorbed or complexed with the recycled and reactivated sludge in a short contact period. It has been reported by Weston (26) that with filtered and unfiltered sewage, 76 per- cent and 68 percent respectively of the BOD fed was removed on contact with stabilized activated sludge, thereby indicating a high removal of soluble and colloidal organic matter in sewage « The "Contact Stabilization" process has been successfully applied to textile waste (28) and canning waste (29), both of which contain high proportions of colloidal matter „ The large size of the colloid molecules prohibits their direct entry into the microbial cells where the biochemical oxidation of the organic matter actually occurs „ Extracellular enzymes are necessary to degrade these molecules to sizes small enough to cross the cytoplasmic membrane o No reports have been published to indicate the presence or im- portance of these exoenzymes in degrading the macromolecules in activated sludge systems „ Thus the mechanism of the removal of colloidal substrates in the activated sludge system is still in the theoretical stage without much experimental evidence <> ,■ 3, SCOPE OF THE INVESTIGATION From the preceding discussion, it will be evident that there is a great need to obtain precise information about the removal characteristics of colloidal components of a waste in the biological treatment process , This knowledge would prove invaluable in designing treatment plants for industrial waste with a high colloid content (e.g., textiles, food, paper and pulp)„ The use of the "Contact Stabilization" process may be appropriate for wastes containing solely colloidal components, if there has been a high initial sorption of colloids on the sludge „ The present investigation was initiated to determine the removal kinetics and mechanisms responsible for removal of a colloidal substrate in an activated sludge system under different operating conditions „ The vari- ables which affect the removal kinetics of the substrate in an activated sludge system and which were studied were temperature, food-to-microorganism ratio (F/M), acclimation to various substrates, and anaerobiosis The operating variables for the activated sludge system chosen were selected in order to obtain a comprehensive idea about the removal characteristics of the colloidal substrate „ The colloid chosen for study was soluble potato starch,, a lyophilic molecular linear colloid „ The use of starch in industry is very widespread „ Some wastes from the food, paper and pulp, textile and fermentation indus= tries contain either starch or its degradation products,, The quantity of starch in domestic sewage is probably negligible, but some other polysac- charides may be present. Starch is an ideal colloid to study in an activated sludge system since it can form a colloidal solution on heating and can be separated easily from the microorganisms by membrane filtration or centrifugation. Moreover, simple quantitative tests specific for starch are available. s 10 4. THEORETICAL CONCEPTS 4.1 Colloids Colloids are particles which are so small that they behave in some respects like molecules, and sometimes molecules so large that they behave in some respects like particles. Although colloids generally vary in size from 2 my to 100 my (0.1 y), particles larger than 100 m may also _7 behave as colloids (30). (One my is equal to 10 angstrom or 10 centi- meters.) Particles having large extensions in one or two dimensions will also exhibit colloidal phenomena. Thus, a particle of 1 my thickness and 1 y length would be considered as a colloid. Balmat (2) defined sewage colloids as particles between 1 my to 1 y, and used the term supracolloidal for those particles ranging in size from 1 y to 100 y. For the purpose of this investigation, the upper size limit of the colloid was defined to include those particles which could be filtered through a 1.20 y membrane filter. The definition based on size is arbitrary since it entirely neglects the important organization that colloidal matter is known to possess. But from the engineering point of view,, it affords a way of distinguishing a colloidal particle from smaller molecules. According to this definition, a bacterium is also a colloidal particle. Colloids have been classified in various ways. Depending on the stability of their dispersed state in a medium (which is often called sol in colloid chemistry),, colloids have been defined as lyophobic or lyophilic (31). Lyophobic sols are those in which no affinity exists between the particle and the solvent. If the solvent is water, they are said to be "hydrophobic," The stability of these sols is mainly dependent upon the charge of the particles. Lyophobic sols are not very stable. In lyophilic 11 colloids, there is an interaction between the particle and the solvent, one combining with the other and becoming solvated. Most organic colloids are lyophilico Groups which have a tendency to bind water are called hydro- philic groups,, The -OH group in hydroxides and polysaccharides, as well as the -COOH and -NK groups in proteins are examples of hydrophilic groups,, What are the properties which make the colloidal particle different from other particles? Firstly, colloids pass through simple filter paper, but may be retained on ultrafilters (10-200 my), whereas coarse particulate matter is retained on the former and small molecules pass through the latter. Thus, particle size is a very important difference between colloidal and non- colloidal systems „ Viscosity is one of the most important properties of a colloidal system. While the viscosity of lyophobic systems differs slightly, if at all, from that of the dispersing medium, the viscosity of lyophilic systems is much greater than that of the dispersing medium. The reason for this behavior becomes apparent when the nature of the particles com- posing the internal phase of lyophobic and lyophilic systems is compared,. The lyophilic colloids are, without exception , substances which become solvated with the dispersing medium and this property accounts for their viscosity. The viscosity of lyophilic sols depends on their particle shape. Linear colloids have viscosities 10-100 times higher than spherocolloids. The diffusion coefficient of a colloidal particle is low because of its large size^ and only the smallest particles can be dialyzed at a very low rate. Colloidal solutions exert a very low osmotic pressure. This peculiar property of colloids is easy to understand. The colloidal particles cannot penetrate any membrane, so the solvent will diffuse in to dilute the colloidal solution resulting in the osmotic pressure. Moreover, 12 according to Van't Hoff's Law s the larger a molecule, the lower will be its osmotic pressure for any given weight concentration, as osmotic pre- sure is a colligative property. Interfacial reactions and events are of paramount importance in colloidal systems. A colloidal system has two kinds of interfaces — firstly, the macroscopic interface which the solution shares with air (gas/liquid), and with the walls of the container (liquid/solid), and secondly, the submicroscopic interface in a colloidal solution between the colloidal particles and the solvent. Since organic colloidal solutions are mostly hydrophilic sols, they have a low surface tension. When a protein is dissolved in water, it lowers the surface tension moderately. A much greater effect is achieved by surface active substances such as soaps, saponins and detergent substances; on the other hand, substances like com- mercial starches have very little influence on the surface tension of the solution. It is a \■ < in X o Q LU _l < > Si 2 o _J > o LU o OC < in LU Z> cT/6lu *N0llVaiN3DN0D 59 2 • - < CO X LU 2 < 2 1 > O LU CJ . ^^--^() Vo Nl x / \o ^ \ o >^ TIME, hours (URE 10 : STARCH -COD REMOVAL IN AN AEROBIC ACCLIMATED SYSTEM WITH DIFFERENT F/M RATIOS AT 5° C 12 65 a o m E-« < CO W CO >- CO W CD Q ►J CO o W H < J" > W J CQ < E- CJ < Q W E- < h-i CJ o < CQ < > o s: o <*; < E- CO d) +J ■H O o -t-> H c •H o C o CD Q +J a> O (fl 0) u &-' r^ Ph 1 x: U, rC rH Ns^ 1 o TO H rH & > ""v. rH m o M P B 6 o to CD ro lO CO rd •H CO rH o O LT> o O P CO \ m :* ro rj- cn •H J bO o c C^ ID CO G S E CN rH 1— 1 Q rH O T3S 03 o -f o 4-> H fc "V CO m CO co CM ■H ro o M CO rH ro rH rH c H P o w < E Q O CJ H H 1 to 0> 1 xz > P r-N o o rd U u 6 oz xz ro cu *^s P pi; CO in co cm o m rH r^ h m cm CM rH rH O O c o o o o O rH O C£ Eh d) P rH r0 \ 0) PS hO E o o m o o O CD CM -}■ CD "I" CM CO CM GO s E m CM CM ^^ \ t> lO rH -f CD Em bO o o o o o E O rH CM CM CO j/Ow *sanos Q3QN3dsns uonon CBxm 66 o o m O o o o O o o IT) o ro m o o If) ro tf/6w 'NOIlVdlNBDNOD */&w 'sanos a3QN3dsns aonon oixm 67 o O o o o O o lO o ro CM CJ o o m o o o < *Y 2T z° oo X CJ < IS H o? CO ()H < o OCT % muj o2" 2 or 2s UJ UJ U_ =35 t- UJ 5^ UJ CJ < co > CO X Q o UJ _l < % 5 o _J ^ CJ UJ CJ b < o occ £ fiuj o2- £ UJ LJ U. z 32 t- UJ si UJ U < a: < CO >- CO X Q o UJ 1— _J < $ 5 o _J 2> UJ O 70 2 < z . CO X o° u — o cr H O < 5 w \- z co o o £ < y^ h- CD cr O O LU Q. cr 0. Ll 2 2 O < LU hours UICS i NDER 2.5, T U) => 5 uT cr \ SLU 5 u- ^S LU < CO cr >- < CO X o Q LU 5 5 o _i 2 o UJ CJ (Z < m LU cr tf/6uj 4 NOIlVdlN30NO0 71 f/6iu 'N0I1VU1N3DN0D 72 tf/6iu 'NOI1VU1N3DNO0 73 J/&w 'N0llVdlN30N0D 74 Here also there was an immediate uptake of starch by the sludge at the start of the experiment which was indicated not only by the substrate loss in the filtrate but also by the increase of the initial mixed liquor suspended sol ids „ The total-COD removal followed a linear rate of removal after an initial lag or slower removal rate. This lag was particularly noticeable at higher F/M ratios. The starch-COD removal rate was logarithmic for all the test runs, at least for the first two hours of aeration as indicated in Figure 19 „ However, in some cases there was a divergence from the logarithmic removal rate when the concentration of starch in the mixed liquor was low,, The cell-free starch-COD degradation rate was linear and much lower than the starch-COD degradation in the activated sludge. This lower cell-free starch-COD degradation occurred at a lower starch concentration also (Fig- ure 12), The intermediate degradation products of starch are also shown in Figures 11 through 18, There was a build-up of these products till all the starch-COD in the system was removed. Figure 20 shows the starch removal with acclimated sludge in a Warburg respirometer at 20°C and an F/M ratio of 0,92, The characteristics of total-COD and starch-COD removal were very similar to those observed with activated sludge aeration units. There was a characteristic lag initially followed by linear removal of total-COD and total-carbohydrate, A study of the total-carbohydrate curve and total-COD curve indicates that there was no accumulation of intermediate degradation products other than carbohydrate during degradation of starch in a Warburg teste This is also indicated in an acclimated activated sludge experiment (Figure 13), 75 2000 1000 700 500 300 200 100 70 50 30 20 10 /\/*tinntnr* Sludge N^F/M = 12.5 \ \f/M: 6.2 I2ll\\ \ \ I \ \ \ V \f/M = 5.6 ^ /M \ = 2.5 \ 0.6 \ \ \ \ ( A \ } F/M = l.83 \ 1 o n\ 1 \ F/ 'M=l.33 \ O TIME, hours GURE 19 : STARCH -COD REMOVAL IN AN AEROBIC ACCLIMATED SYSTEM WITH DIFFERENT F/M RATIOS AT 20° C 76 y a> £ O ID 5; co co 0) 3 o o o V) 0) ■D Q O c o ~° E ^ o a) O r n — ' -r-i ' o o JZ I C £ CO o — — » c 0) o CO O O 1 i- T3 >< CD o >- O O *T *- +- O O » CO (- »- O £ CL Hill A \ c ; 0) o> o ■o c UJ »/&w 'sanos a3QN3dsns uonon a3Xiu\i \ cj\ cc LU Q UJ I- (f) >■ a o o O CVJ LU CC z> cc LU Q_ LU o < CM cn d cc LU x : to o o 3 £ cc 2? 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CO cc u. < 0£ CO < o »- z CO cc CO LU Z 1- o < 1- cc Q < Z X o CJ o < o > CD o o 2 o j/6w 4 NOI1VU1N30NOD 77 Table 5 demonstrates the rate of total-COD removal , rate of starch-COD removal , initial adsorption 8 initial MLSS and initial starch concentrations o There was an increase in the rate of total- and starch- COD as the F/M was decreased „ 6„2olo3 Substrate Removal at 30°C The characteristics of removal of starch in an accli- mated activated sludge system at 30°C was very similar to that reported for 20°C, except that the removal rates for starch-COD and total-COD were higher at 30°C than at 20°C The lag observed in the removal rate of total- COD in some cases at 20°C was quite obvious with most F/M ratios at 30°C<, Figures 21 through 26 present the data of starch removal at 30°C at different F/M ratios , The starch-COD removals were replotted in a semi- log plot in Figure 27,, The rate of total- and starch-COD removal, initial adsorption, and initial solids are reported in Table 6„ The cell-free starch degradation after a 10-minute contact period of substrate and cells in the activated sludge unit at 30°C was higher than at 20°C (Tables 5 and 6). 6,2,2 Constant MLSS; Variable F/M Ratio 9 Aerobic Systems This experiment was performed in order to find out if the low activity of cell-free starch-COD degradation reported earlier was due to substrate inhibition of the enzyme systems „ Further , it would be interest- ing to see if the rate constants (starch-COD and total-COD) were affected by variation of F/M or MLSS or both 78 to U 03 e CO Pi /— V X> o p a) a) a ■H a CN p p o X) co ^ o m a) P co • H r-N o +J •— \ • H '— > CO f» > u c P P o (h c o o o Uh •H +J m o3 05 -H a) 03 CO •H •H -H cm tC P -H +-> h ^ ,Q < p CO XI Mh XI 3 ft O »H in p +-> o V_-Z W .-3 co o PQ < P-, C w c u c s fc Q k o X5 co co bO o P o 1 CD ! o O 03 H CN rH H u E K \ H Ifl a> bO CU p Pi e O CO h o -a 03 O CO rH U 03 O p w o xi E- < rH l rt 1 •H CO Hi o O O O o in m O M CO \l CN o J- co en [-» d- CN •H J bOI lo CN rH CO LO CM CN H c ^ 61 CN rH •-{ rH co en cr> j- CN LO -J" LO -f CN CN rH rH CO CJ> CD U) J" CM O LOOOO OO O CO H CO CO CN r-co CO -]- co r-- co co ID H CD rH H CN CO rH O CD CN LO LO LO J" H CO J" CO CN CN CN CN CN o o o o o O O O o o co i> CN CO CO CD 3- o H ■-{ 3- CO CN CN CN H LO H rH rH rH rH <-{ lo co r- cm i 1 CN o rH CO LO LO H p o CJ rH rH i fC CI) I x > p s-** o 03 u fc £ Pi x rd CO *w* +-> Pi 03 LO rH cn CD 00 LO O CN t-* CO J" o O CO co en o rH j- co en f< CN LO LO CN rH LO O LO J" CN rH o c-» f- CO LO -j- CO lO CD o o o o o o a o rH o 03 f-i I > CO J3 o o O LO o LO o t- H o p *\ CO CO co lO J- CT; H t^ 03 e 03 •-{ CD ro * CN C-J H CM -M CO Pi "N^ Pi bO H e bO H CO CO o s e CD CM CO CO LO ^D CM LO 'v. "v. O a o a o o o o Lm bO o rH rH H CN UT CD CN rH (Q <-{ O O rH O O O CO f> LO rj- CJ) CN CO CO CO CN CN H CN CN J" cn CN ID CD CN CD LO 00 CO O +- CN CN J" O O rH H rH CN c o o CJ 1 rC CJ !m rd p Q CO O CJ c B o X V X) U CU rg M p Ifl co co j/Guj 'sanos a3QN3dsns uonon oixm 79 o o in rO O O O fO O O ID o O o CM o o m 1 1 1 c o <»< o V. x» o> p E O en 00 ii CO CO _J 5 1 LSS arch-COD >tal-C0D ter mediate De roduct-COD 1 — S U=>£ E Q. - o c i*ft TD 1 1 1 1 a> o 3 a -r O o <_> \ - \ / T3 a) \ / jO \ y o \ ^s' - a) ■o < a Q O o o "^ z < z . CO X 3" o — o rr 1- O < n f° H- z CO O < y& i— od ac o O LU Q. or a_ u_ y 5 O < UJ ~ H- »» CO o ■ — UJ (J> Q 1^ CO z o TIME CTER UJ < CO cr >- < CO i o Q LU < > !5 o _j 2> u UJ o QL < o o <\J o o o CVJ UJ 3 O tf/6w 'NOIlVdlNlDNOD tf/6w 'sanos a3QN3dsns donon G3xm 80 < X a: < 00 o h- <-> z z 5k o2 X pro o < IS H o? CO <~>b < O UK & 5£ £ o:5 LU LU U. <»- <20 or . IE, hou STICS UNDEi M2.75 si LU 1— < < to >- CO X Q o LU _J 5 5 5 o _J 5 o LU o cr < ^> OJ LU cr 3 O J/6uj 4 NOIlVdlN3DNOD 83 £ < o uc % O^ 2 cc5 LULU U- <»~ to q: - LUCO *4\» z 35 is LU O < q: < co >- CO X Q o LU -J < 5 5 o _j 5 CJ LU CJ cr < CD CM LU or 3 O J/6uj 'N0llVdlN3DN0D 85 iUUU 1000 700 500 \ F/M = 5.62 Iw/MV 300 200 100 \ \\-l 61 S \ \f/m\ \ \ 3 7 \ F/M=0.79\ \\ F/M \ \ = 126 \ \ 70 50 \ F/M =2.75 30 20 in 1 TIME, hours GURE27 : STARCH -COD REMOVAL IN AN AEROBIC ACCLIMATED SYSTEM WITH DIFFERENT F/M RATIOS AT 30° C 86 w CQ < O O O CO H < S w CO >- CO H U O D CO Q W H < > M E-> CJ < o w t- < M _3 O O < P~ CQ < > o O < H CO +J 03 U +J rH •H Cm H (0 •H 4-> •H c rd •H ■P ■H a a o »H ■p in u p G CD o c C ) Q O o o o o o cm en oo in cm rH cm ro en co rH H rH O O O CM o p 0) c rd a> c_> a-: Ph 1 [n x: ■H 1 o fl H U > i— 1 m o a) ■p 6 u CO a; r>; x: CM en o cm -j- H H J- CM en o m O in CO m CD r^ ro rf o CO co H CD Lf) J CM n H o 13 ro CJ (U "■H XI H CO CO o H C^ CO p rH fc \ O a* H H O CO •H ft O bO CM CM CM CM CM ■H c P w E H o T3) < Q O o II H itJ H CD i CM x: > +J — N en d- in CM 00 CM CJ O m p. H oo r^ m -J- CO fc E CC A o o o a o a rO rH O rO E P 0) O Pi <-H Ml E o m o in CO rH Cn (D ID CO CO CM Cm h0 El en id ^h m cm •vJ i> cm co r- t>- co g| o rH rH cm co in 87 In this set of experiments, the acclimated mixed liquor suspended solids were kept at about 800 mg/1, and the starch substrate concentration was varied to give different F/M ratios „ These experiments were carried out at 20°Co Figures 28 through 31 represent the starch removal characteristics at different F/M ratios but fairly constant MLSS in the acclimated activated sludge systems o The characteristics of starch removal under constant MLSS were very similar to those with constant substrate concentrations The initial lag in the total-COD removal, especially at higher F/M values, was noticeable followed by a linear removal„ The starch-COD removal rate was logarithmic (Figure 32), and the rates were higher at low F/M ratios The cell-free starch-COD removal rate decreased as the substrate concentration decreased „ Thus, the inhibition of the enzymes did not appear to be responsible for starch degradation in the cell-free system by the substrate „ The starch-COD and total-COD removal rates are reported in Table 5 6„2„3 Oxygen Deficient Systems In this system starch was added to acclimated sludge just like the other system at 20°C, but instead of compressed air diffusion through the diff users, compressed nitrogen was used. Figures 33 and 34 show the starch-COD removal with a nitrogen- blown, anaerobic activated sludge system. The starch-COD was removed in a manner similar to that reported earlier with aerobic conditions (Figure 32) „ The MLSS and total-COD of the system were practically unchanged during the experiment. However, there was a slight decrease in the total-carbohydrates after six hours of nitrogen diffusion„ Apparently, the enzymes responsible 88 ar/6uj 'sanos a3QN3dsns yonon cgxiin Q UJ 2= CO _l CO O -I 2 5 << ?£ £ u 2fE Q.HU »JL. °^ o ^ q u o ^ ^ o ^ H S => CO GQ |_ ft a: or H UJ LU o < CL 5 or ^ •^ c\j Jr/6uu 'N0IJLVU1N30N0D < > « O UJ O UJ (/) ^ tr > > co u. CO CJ LU a: Ll 89 jv&w 'sanos Q3aN3dsns uonon Q3xm o Q £ O m to ■o a.- 3 o o o o Q c o o ■D O i_ o> - v. CO u_ 0> CVJ UJ cc z> o Jy6ui 'N0I1VU1N3DN0D 90 ar/6w 'sanos a3QN3dsns donon Q3xm t/buj 'NOIlVdlN30NO0 Q LU H- < ^ CO _l CO U -I ^ 2 < 2 b — CO ^ 2 ?; O y o cr ^ x b i- co ^ ^ £ o b <^ «/) 2 z c52 u u o sz - <0 <-> m UJ 2 ^O* I— — 3 h- CO CD |— E g < tfi ft a: h LU UJ CJ < CL CHAR NDER TEM O LU 5 ■- "5 LU CO S a: >- > CO Lu o ro LU or 3 e> u_ 91 2 h-. < £ 2 CO X o o o or K o < Q CJ h- z co O o LU or 3 h- < u < J— O GO O or LU Q_ or Q_ O LU < 2 LU Xirs ICS LU Q CD 00 CO wor Z) 2 2 LU 5 lE jZ»~ LU K o h- < CO CO LK >- CO < CO -J X 2 o Q LU H _l < > 5 5 < o _j CO 2 o z UJ CJ o QC < CJ rO UJ or 3 O tf/6w 'N0!lVaiN3DN0D 92 2000 1000 TIME, hours FGURE 32 : STARCH -COD REMOVAL IN AN ACCLIMATED SYSTEM UNDER VARIOUS CONDITIONS, AT 20° C 93 J/Buj 'N0llVdlN30N0D 94 tf/6w , NOIlVaiN30N0D 95 for the degradation of starch were active even under anaerobic conditions „ An initial starch adsorption on the sludge occurred as it had under aerobic conditions o Table 5 also reports the starch-COD removal rate and the initial starch adsorption under oxygen deficient systems,, 6o2 4 Constant Substrate; Acetate Buffer Aerobic System The low activity of cell-free starch degradation with accli- mated sludge has been reported earlier„ In all of these experiments phosphate buffer was used It has been reported (73) that calcium ions are needed to activate amylases of some organisms and phosphate buffer was detrimental to the amylases of such systems » Therefore , the possibility existed that because of removal of calcium by phosphate buffer, the cell- free starch degradation was low. Acetate buffer (pH 7„2) was used in an experiment at 20°C with acclimated activated sludge and the starch-COD fed was 1U45 mg/1, although the total-COD was much higher (about 3000 mg/1) because of the acetate-buffer added„ Figure 35 represents the starch-COD degradation of aerobic acclimated activated sludge at 20°C with an acetate buffer system „ It will be evident that starch-COD removal was very similar to that obtained with phosphate buffer earlier (Table 5) The cell-free starch-COD removal rate was higher than that obtained under similar condi- tions with phosphate buffer but was still much lower than that obtained with whole-cell systems. 6„2o5 Cell-Free Starch Degradation Tables h 9 5 and 6 have reported the rate of cell-free starch-COD degradation with acclimated sludges at 5°, 20° and 30°C o It will be 96 tf/6w *N0llVdlN3DN0D 97 observed that the rate increased with an increase in temperatures and was not inhibited by substrate concentration,, The use of acetate buffer at 20°C increased the rate about three-fold indicating possibly some calcium removal effect of the phosphate buffer It has been reported by Nomura et_ al„ (74) that a-amylase of B a subtilis was liberated in the stationary and endogenous growth phase and very little extracellular a-amylase was obtained in the log growth phase „ This hypothesis was tested in the systems „ All the cell-free starch-COD degradation tests reported earlier were obtained with sludges at or near the log growth phase „ An experiment was performed to see if the filtrate from acclimated activated sludges in the endogenous phase would have a higher cell-free starch-COD degradation rate Figure 36 shows the cell-free starch degradation with the filtrate from an acclimated activated sludge system in the endogenous phase. The cell-free starch degradation rate in the log growth phase obtained from Figure 12 is also plotted in Figure 36 for comparison „ The rate of cell~free starch degradation is re- ported in Table 5 and was found to be much higher than the cell-free systems described earlier „ However , the rate of starch-COD removal with the fil- trate from acclimated potato starch sludge in the endogenous phase was still lower than the removal rate of whole cells „ 6„3 Starch Substrate Removal in Non-Acclimated Aerobic Systems These experiments were conducted to determine whether the enzyme system responsible for starch degradation in a non-acclimated activated sludge system was inducible, and also to study the nature of starch removal kinetics with these non-acclimated systems,, 98 o o o CM u o < CO 3 o 2 UJ o o Q Z o ? X h- O uT £ C\J * * < > o CO 2 UJ UJ cc St Q h O O u_ 1 X o UJ CD o: o < 1 CO CO CD ro UJ o: CD JT/DW l NOIlVaiN30NO0 99 6o3ol Glucose Systems In this experiment the. sludge had been acclimated previously to glucose „ Potato starch was added to this sludge and its removal charac- teristic at 20°C plotted as indicated in Figures 37 and 38 „ There was a marked lag in the total -COD and starch-COD removal rates at F/M ratio 2 54 9 after which the total-COD removal followed a linear rate and the starch-COD followed a logarithmic rate„ At the lower F/M ratio (lo24) 9 there was no lag and the rate of total-COD removal was linear j just as it had been in the acclimated sludge system,, In this experiment, the starch-COD removal was logarithmic up to two hours „ There was a change in the starch-COD removal rate after two hours in some cases 8 as shown in Figure 39 „ Table 7 presents the starch removal rates, total-COD adsorbed, initial MLSS concentration and initial filtrate total-COD concentration with glucose acclimated sludge at 20°C„ On comparing the data of starch removal by glucose acclimated sludge in Table 7 with that in Table 5 (for acclimated sludge) it becomes evident that starch-COD and total-COD rates were much lower with glucose acclimated sludge „ 6 3o2 Maltose Systems The sludge used in this system was acclimated to maltose „ The starch removing characteristics of maltose-acclimated sludge are shown in Figures 39 9 40 and 41 „ There was no lag observed in starch removal rate in the experiments „ However, the total-COD removal characteristics in Figure 40 (F/M 1„20) were different from that obtained in the previous experiments mentioned earlier „ After two hours during which the removal rate was linear, there was a break, and the removal rate was retarded from "5" c m N CP £ o 8 if) o o o O o o 100 »/&w c sanos Q3QN3dsns uonon aaxii/\i c o o T3 O k_ o> a> a CM O O o u Q O U I o 3 O o — E o o CD CO CO "T" O O o -Q — i «/6uj 4 sanos a3QN3dsns uonon oixm 101 UJ c o o T3 O i_ o> §81? I 0) o 1 E 3 co " o j- "o i o *- ® o CO t- .£ Q. CO o y/6w 'N0IJLVU1N3DN0 102 2000 1000 700 500 300 200 ^ At If e F/M l.2> 100 70 50 30 20 10 A Glucose System □ Maltose System • Metrecal System O BSA System TIME, hours IGURE 39: STARCH -COD REMOVAL IN NO N - ACCLIMATED AEROBIC SYSTEMS AT 20° C 103 cq < O o o CM < CO w H CO CO w o Q CO Q W E-" < > i— ( C_> < Q W < C_) o <: s o s; CO < > o o < co , >^ >> >! X> X) to to fO (0 x> Xf 3 3 X> D Q o a X) 43 43 03 TO to w q 4_, 4J w E e X) E H CM J" CD ^ •H •H rH 0) »H fc H H O 4-> H « f 1) to O U o a H •p < 3 H to Q) CO H o x: y CO CJ a) +j (0 C J-. O ♦ Q H .— i O i-> °H CJ to — 1 o o O o o O o o o o £m 11 ■m -^ CD o rH o CO O ai d- CM J- H (- bO CO _r -t d- CO _t to rH rH CO H T3 G B H -H H H rH H H H -H rH t0 j- a> •H b O «-> H a •H C o o o O O O o O to o o O O ID O o CO o CD CM CO CM CO CM LO rH r- CM CM J" CM rH rH H rH rH H rH O rH C X) H! O (!) •H ! XI H o o O O o o o o CN +>r| i\ o o c-» cm U3 r-> o r> o -H 10 O W) H rH rH H co J- C +J W E n O X) H < Q O O i 43 CJ & (0 CO O CD CM CO rH O _r r- CM O O O O CM O CM CM O o o CM rH cd r~- lo rH CO rH O O H Q O rH c_> to 1 > H O tO E P (!) Pi M o C£ o m CD CD rH C CD _r o CO rH o CO o ir> 00 LO CD r-~ J CO rH CM CO CO W 6 :* J- O If r» t- .=1- CM un CM CD 3T CD CM rH O H M o o o a o a V o a o E H CM rH CM H rH rH H rH rH */&w 'sanos a3QN3dsns uonon aixm 104 o O o o If) o (VI <\J co u o 59 OJ X J— .— cm a: < IS H o? co ob < o oa: £ o9- £ UJ UJ u_ - CO a UJ U, 1 UJ a: o LU OT 3 O o u < I UJ CO o < 2 J/6LU l N0llVdlN3DN0D 105 E o m CO CO 3 O o o o o o £/6iu 'NOIlVdlNBDNOO 106 then on. The total~COD and starch-COD removal rates (Table 7) were lower than the rates of acclimated systems (Table 5), but were higher than those obtained for glucose acclimated sludge „ 6.3.3 Metrecal System Figure 42 represents the starch removal characteristics of the Metrecal acclimated sludge. There was a significant lag in the starch removal characteristics of the system, although after 23 hours of aeration 9 97 percent of the fed substrate had been removed. The removal rate of starch-COD (as plotted on semi-log paper, Figure 39) was much lower than most of the systems at the same F/M ratio (Table 7). 6.3.4 Bovine Serum Albumin System In this experiment the sludge had been acclimated to bovine serum albumin. The starch removal characteristics of this sludge are presented in Figure 43. There was a significant lag in both the starch-COD and total-COD removal rates, as in the case of Metrecal sludge. However, after a seven-hour lag total-COD removal occurred linearly. The starch- COD removal rate, Figure 39, was initially lower than the Metrecal sludge, but after the lag period, the rate improved and finally it was higher than that of the Metrecal system. 6.3.5 Acclimation of Glucose Sludge to Starch It i-rill be observed from Table 7 that, in general, non-acclimated sludges adsorbed less starch than acclimated sludges. In this experiment, E O O o c ■D o 3 O o o ■o d) jO l_ o v> -a o ? O « o * t: o v. c/) W i I /' // // 107 LU I- C/) >■ C/) Q UJ / // I / / \ \ / \ / \ / \ * \ U u < I o i- < i- o O Q 3 _1 CO I LU CO O CJ 3 _l LU 3 *uaojad 'ONINMAGU QOO-HDdVlS 112 i N» l ^ ^^ 1st Day fl 2nd Dav\ \\4th Day _\6th Day • ' >\ TIME, hours 10 12 IGURE 46: CHANGE IN STARCH-COD REMOVAL WITH ACCLIMATION OF GLUCOSE -SLUDGE TO POTATO STARCH AT 20° C 113 All these observations could be explained if it were assumed that the enzyme system responsible for starch degradation was confined to the outer cell surface, i.e., cell wall. During the endogenous phase, it is likely that there was an excretion of these enzymes into the medium, and therefore, the activity of the starch degrading enzyme was much higher. The isolated cell wall and soluble components of the cell were tested separately for starch-COD removal characteristics, as depicted in Figures 47 and 48. The starch-COD removal rate of a whole cell sludge, a part of which was used for cell wall preparation, has also been presented „ Table 8 gives the removal rates and amount of starch adsorbed by the cell and its component s. The starch-COD removal rates were expressed per gram per liter of protein in the system in order to compare the various cell fractions on an equal basis. The rate of removal of starch-COD by the cell wall and soluble component fractions was much higher than that obtained for whole cells. The higher starch-COD removal rate of the cell wall sus- pension as compared to whole cell does indicate a cell surface bound enzyme systenio The adsorption of starch on the cell wall suspension was also observed as with the whole cells. 114 E z o < rr h- Z UJ o z o o 1500 i_ co 1000 500 COD Fed • COD Adsorbed V J \ ^* FT*-*- Initial V Solids V o 1500 COD Fed \ \ \ Ii 1500 V COD Fed '000 500 A. Whole Cells _.*_ MLSS _..« — Starch-COD — * — Protein 2000 1500 E a) _i - 1000 4 B. Soluble Cell Components * Starch-COD -A — - Protein 4- C. Cell Walls — MLSS -.- Starch-COD — Protein TIME, hours FIGURE 47: REMOVAL CHARACTERISTICS OF STARCH IN AN ACCLIMATED AEROBIC SYSTEM BY CELLS AND THEIR COMPONENTS AT. 20° C 115 2000 1000 700 500 300 200 100 70 50 30 20 !• _4„~4 0_ll ^^— ^^ * JUIUUII. V^CII > lUtllUM ^ f«ll \A/„II TIME, hours IIGURE 48 : REMOVAL CHARACTERISTICS OF STARCH-COD IN AN ACCLIMATED AEROBIC SYSTEM BY CELLS AND THEIR COMPONENTS AT 20° C 116 w CQ < O o o CM < CO H IS w O CU o o CO H Q < CO w o w o Q D -J CO Q U H < S rH C_) o < < > o u Pi a o oi < CO co r* u (0 E 0) Pi cu o o c -H O H -H fO CO S C Q> O rH H £ o a) o woo co o c *;c o •;c •H rH CU Q p to +J O f0 •rH to o rH H o o O +J ^ i P — „ o CN H •H 4-> Xi c bO (N rH IT) c rH Fil 'tare cu o o E rH r-\ H rH f0 •H P •H c rH o CD o o LO en o TJ H o a) n3 a X) H i & P ,c o H o co c & t3 — 1 to < CO o o CO o o CN c 1 a •H *-> o H U cu rH o m CX O faO rH < CU o o o l> H a> d- J- co Q O CJ rH rH i to cu I J" CD rC > P ^■N IT) CD <7> O O f0 rH ID CD CD rH E Pi rC « c o to cu ^-^ O rH rH p Pi CO bO S E CM en \ J" CO u. bO o o E rH rH o p cu CU E ■H P rC P T3 CU bO C to rC o p •H CU o c »H to p c cu E •H rH CU ex X Q) CU rC P cp O P rH f0 P W CU rC P P fO co fO p c cu p c o o c •H (1) P o rH CX CU rC P co T3 cu c •H E rH cu p cu (0 fO a o a i rC O rH fO p CO >1 H C o cu c •H E rH CU p cu T> P O c rH ra CU » fO p CU 5 Q rH O O O rH O rC bO I £ H rH r0 rH rH +J O CU O 117 7. DISCUSSION OF RESULTS It will be evident from the experimental results and the scope of the investigation that the characteristics of starch removal in an activated sludge system are dependent on many variables and the most impor- tant of these, it is believed, have been dealt with here in detail. How- ever , there are some variables, like pH, presence of other substrates, nutrient deficiency, etc., which may also affect the removal of starch in an activated sludge system and have not been studied. 7.1 Starch Removal in Acclimated Systems 7.1.1 Kinetics of Starch Removal The total-COD removal kinetics in all the systems studied with acclimated sludge followed a linear rate until a low COD level was reached similar to the results obtained by Wuhrmann (37). Eckenf elder and O'Connor (34) have also stated that at high substrate concentrations, linear kinetics were obtained for a few industrial wastes. The total-COD removal represents the amount of starch degradation products assimilated by the sludge at any time, although total-COD itself represents the amount of undegraded starch- and starch degradation product-COD, In the present study there was an initial lag in some cases, especially with high F/M ratios, which was fol- lowed by the linear removal kinetics. This lag was due to a quantitative shock of high substrate concentration per cell. These cells had been acclimated to potato starch at an F/M ratio of about 1.13 and any change of this ratio produced this lag. There was no lag in Figures 12, 13, 14 and 22, where the F/M ratio was very close to that of the acclimation units (Appendix B). 118 The rate of total-COD removal is plotted for various values of F/M and initial solids concentration at different temperatures in Figures 49 and 50 respectively. The total-COD removal rate was dependent on the initial solids concentration and not on F/M ratio „ This was quite evident when the removal rates for constant MLSS system at 20°C were plotted „ In Figure 49, the rate of removal of total-COD remained practically constant in the case of constant MLSS and variable substrate system with a change in F/M. If total-COD removal rate was dependent on the F/M then there should have been an increase in the removal rate with an increase of (F/M) , Since this rate was dependent on initial mixed liquor suspended solids , in Figure 50, there was little variation of the rate at a constant sludge concentration system. The removal of total-COD represents direct assimilation of the starch degradation products by the cell. This total- COD removal rate was found to be independent of substrate concentration, because of the linear substrate removal kinetics observed in most of the experiments. Therefore, the proportional variation of the total-COD removal rate due to change in (F/M) would be expected to be dependent upon the initial mixed liquor suspended solids. This would perhaps mean that the permease systems responsible for uptake of starch degradation products were constant for a particular sludge concentration, and were not dependent on the amount of substrate present. At 1200 mg/1 sludge concen- tration the rate of removal of total-COD was about 400 mg/l/hr at 20°C regardless of the concentration of starch (Figure 50). If the substrate concentration was 200 mg/1, it would take only half an hour for the 1200 mg/1 sludge concentration to remove it from the system, and if the substrate concentration was 1200 mg/1, it would take about 3 hours to remove it from the system. 119 JM/J/6W '1NV1SN00 31VH IVAOIAGU OOO-lViOl 120 1 I i 1 1 h- 2 8 w 5 £ o: lu 10 LU H- U- CO 5 ^ — 1 - 2800 mg/G L AT ATED \ *M o \ ' - CO ^ < ol 10 l 4 W Q 5? Q ^ *3 \ o \ ► o^ O - \ cm y — 8i1j o a: WO. | O « \ _ x o \ CO li_ \ ' - 1600 QUOR SU TOTAL TURES J o. \ ° Ij < - O 0) \ ^ Ll OC § o \ P O LU 1 : Accl Jubstr MLSS \ • - 1200 MIXE NT EMP «. \ I -i < h- 1 Aerot irburg nstant nstan t> N. \ r < H P CO Q Z 5^ Z <-» o o o $ o o o i 8" ° < ° O • D CM w X. \ □ T CO LU CO 1 H- _l < S o o: ^V \ 1 * - < \ - m - * 1 1 1 1 1 1 P § Q O o o o c o o o CD O 00 CO s CO TJ r z> o JD - 5 W I- Q < LU H; _J < < 2 MOV CCLI £ < 01 s< o O o q: I o E O CO 0> (T LU e < 0) •*— — E o en o m E o o < to CO CO _) 2 in r> 3 CO o 10 >> CO 0) r Xj o c o c c o c n < 5 o 0) < to c c xr CD O o o o i c> o £ o CJ O ^ o CM O • a ■ < Ml- o ,_( J M) 'JLNV1SN00 31VU HVA0IAI3d QO3-H0UV1S 128 liquor suspended solids systems (Figures 28 through 32) indicated that starch-COD removal was not dependent on initial solids but on the F/M, as the rate of starch-COD removal at constant sludge concentration in Figure 52 was quite independent of the sludge concentration. The dependence of starch-COD removal rate on the F/M ratio rather than the initial sludge concentration would indicate that the enzymatic action responsible for the starch degradation involved both the sludge and substrate concentrations . The initial removal rate of starch-COD has been found to be dependent on the substrate concentration because of the logarithmic removal characteris- tics in most of the experiments . Also, the starch-COD removal rate changed with the alteration in the initial solids concentration in the experiments with constant substrate level. Therefore, it would be expected that the starch-COD removal rate would be dependent on both the substrate and sludge concentrations or on the F/M ratio. At high (F/M) values there was a divergence from the linear plots for 20°C and 30°C in Figure 51, as also observed in Figures 49 and 50 with total-COD removal rates. The plausible explanation for this decrease in the starch-COD degrading rate at high (F/M) values was that the enzymes of the system were not saturated with substrate and so the overall rate was lower. This divergence from linear relationship in Figure 51 for 20°C was at (F/M)" of 0.95 and of 0,65 for 30°C. The dependence of starch-COD removal rate on F/M rather than on initial solids was different from that observed in the case of total-COD removal rate. However , the basic difference between the two removal phe- nomena must be emphasized in order to understand the variation, Total-COD can only be removed by assimilative processes of the cell, whereas the starch-COD removal need not be due to assimilation, but simply to degrada- tion into smaller fragments giving negative starch-iodine test. Although 129 both processes are enzymatic, the starch-COD removal rate is perhaps inde- pendent of total-COD removal rate. This conclusion is based on the anaerobic experiments discussed later. The starch-COD removal rate constant k is, therefore, dependent on the inverse of F/M ratio at constant temperatures. So, k = cCF/M)" 1 18 s The values of constant of proportionality "c" at different tem- peratures are presented in Table 9, and were obtained directly from the linear portion of curves in Figure 51. The relation between starch-COD removal rate of the acclimated activated sludge and the total-COD removal rate has been presented in Figure 53 for different temperatures. At a particular temperature there was a more or less linear relationship between the two provided the substrate level was maintained constant. If the mixed liquor suspended solids were kept constant instead, then the starch-COD removal rate was practically independent of total-COD removal rate. This has been explained earlier. The total-COD removal rate was dependent on initial solids whereas starch-COD removal was dependent on F/M. In the constant MLSS system the total-COD removal rate was constant but starch- COD removal rate varied with changed F/M ratios. Attempts were also made to plot the starch-COD removal rates in the Michaelis-Menten relationship at 20°C, with a constant sludge concen- tration, Equation 19. The linear form of the Equation 9 was used, which is i . £ . i ' + i 19 v V L V 130 I i i 1 i i 1 i < 1 1 — - < < °o> CO E CD ■^- CO >> If) f ■ < - ■o 0* 0) \\ * o ^— E o £$ o - o o O u. CD < o _l s LU LU or CC ro io LU ac z> e> u. 131 The initial velocities v were obtained from the initial slope of the starch- COD removal curves in Figures 28 through 31. Figure 54 shows the Michaelis- Menten plot of the data with constant sludge concentration system. These data (full circle in the Figure 54) did not produce the predicted straight line. This would perhaps indicate that the assumption involved in the Michaelis-Menten relationship (low enzyme concentration, rate limiting step, etc.) was not applicable in this case. Further, as has been pointed out the starch breakdown by the bacterial cell would be a very complex reaction and a single Michaelis-Menten relationship not capable of representing the kinetics. There was indication of inhibition of the initial starch-COD removal rate at the higher substrate concentrations. The starch-COD removal rate, as has been pointed out earlier, was dependent on F/M ratio, and so at higher substrate concentrations the amounts of enzymes present were in line with the relationship as shown in Figure 51. The values of initial velocity v for constant substrate level but different sludge concentrations are also plotted on Figure 54. The varia- tion in — was due to the change in sludge concentration, since in Equa- tion 8 -TT- depends also on sludge concentration. With the help of these constant substrate velocity of starch-COD removal data and the previously plotted constant sludge starch-COD removal rates, a straight line plot was made in Figure 54. The calculated V s the maximum initial velocity was 2100 mg/l/hr at the sludge concentration of about 800 mg/1. The L,,, ob» tained from the slope of the straight line, was 122 mg/1, which is the sub- strate concentration at half maximum velocity. However, the usefulness of this L v and V value is doubtful since it was evident earlier that the data on the starch removal rate at constant sludge concentrations did not follow the Michaelis-Menten relationship. The straight line plotted in 132 o c o CO o to 0) _l o S w. 0) U1 X) XJ 3 o CO "C X) o .«-— . 5 3 E I E o o 1 0) E iT> 1 O I 1 co o 1 00 I 01 l^ 01 o j £ CO CO tf) .o 1 o o _l 3 1 ^ CO 1 < . 1 o c o c o I \a ^~ 1 o 1 k. c c 1 0> o o 1 < o o 1 O • < \ 1 o 1 00 1 e id E If) ex £ E 1 E 1 • o o 1 o o i- 1 a) t\i ~ l <\j 00 10 Z,< \ • <<< 1 (\j < < < J * 1 \ I o> 1 E I o \ ro 1 1 I 1 °° |\ o OJ 10 ©< C7> cr Ld o o o - ro LU or t- co CD CO if> b CO LU I- < < > o Q O o I X o q: < i- co tr o o _j Q_ I CO < X u LO LU - 2 — '(A1I0CTI3A IVIIINI) 133 Figure 54 was perhaps not representative of the true situation „ 7,1.2 Effect of Temperature The effect of temperature on the starch-COD and total-COD rate constants has been shown in Figures 49 through 52, Table 10 represents the change in rate constants with temperature obtained from Figures 50 and 51 for the linear portion of the curves. If the temperature variation of the rate constants followed Van't Hoff-Arrhenius equation law, then a plot of log k vs. tt ( k = rate constant, T = absolute temperature) would give a straight line. Figures 55 and 56 show the relationship between — and the rate constants of total- and starch-COD respectively. The AE value in Equation 11 was calculated to be 21,000 cal per mole and 13,950 cal per mole for total-COD and starch-COD removal rates respectively. The AE value calculated for starch-COD removal rate was close to that reported for activated sludge systems of 14,400 cal per mole (34). The effect of tem- perature on "b" and "o" values, which were reported in Table 9 and which perhaps are more representative of the effect of temperature on the reac- tion rates since they eliminate a variable on which the rate constants depend, the initial solids in the case of total-COD removal and (F/M) for starch-COD removal, was determined. Figure 57 shoves the relationship of temperature and these constants "b" and "c." The AE values calculated from Figure 57 were 20,400 and 16,500 cal per mole for the total-COD removal rate constant and the starch-COD removal rate constant. These values are slightly different from those obtained earlier. However, these values would be more representative of the actual situations since "b" and "c" are true constants at a fixed temperature, as compared to k values 134 o H M CQ < CO w E- 00 >- 00 w e> Q D 00 Q W H < > rH Eh o < Q W H < S H cj CJ < o oo w E- < Pi < > o s g cj < 00 Eh < « W w E-> Pm O H O w (0 > o e (X o o o i o u +J 00 CJ o o CO CJ I O '-> O U cm x: CJ o in in cj o o n j m H t\ h- in to t-^ co o o o o o O O O ID rH n j m in id a • • a a o o o o o CM l> CM O m io t-~ en o o O O O H rH C o o o o o o o o M C m o \ n d- in m o bO o o o e » E O O O O o w a) & +-> r-^ 03 CJ •^ « H o ~\. H CM bO ffl £ > 6 (U PS Q O CJ CJ 1 O m rH ro 4-> o F-H in in o o in m o co ht o to H c~- oo cm oo zf J- m m in o o CM CM cm =t j- id en cm in co H J- rH H CM CM CM CO CO cm in en h co id en rH rH H CM CM CM CM •r{ 00 rH o o c c o o c )J 00 \ o o o o o o o •H J WJ H" ITi uo c~- CO en o C s c H C7» E in h- z en z o o LU < q: _j o UJ q: o o o o 200 135 0003 >-l .0 004 (ABSOLUTE TEMP)"' (°K) FIGURE 55: EFFECT OF TEMPERATURE OKl T0TAL-C0D REMOVAL RATE CONSTANTS 136 00030 00035 0.0040 (ABSOLUTE TEMPERATURE), (°K) -I 0.0045 FIGURE 56: EFFECT OF TEMPERATURE ON STARCH-COD REMOVAL RATE CONSTANTS 700 400 - 200 0) CO Q O O o> E CO o (J 100 70 40 20 10 0.003 FIGURE 57 : EFFECT OF ■ • \ \ • A \ \ \ A -A o. o \ c 1 I \ ° \ 2- \ 2> \ \3 \ \° \ 5» \ o \ \ \ V \ 70 137 40 2 1.0 0.7 E v. o> E o < co O o 0.4 02 \-l 0.0035 (ABSOLUTE TEMPT,' ( TEMPERATURE ON •K)' 1 THE o.i 004 CONSTANTS 'b'a'c' 138 which were shown to be dependent on initial solids concentration of the system in the case of the total-COD removal rate and (F/M) for the starch-COD removal rate. The calculated Q values between 20° and 30°C obtained from Figure 57 were 2,57 and 3,19 for starch-COD removal rate coefficient "c" and total-COD removal rate coefficient "b" respectively . 7.1.3 Effect of Oxygen Deficiency The oxygen deficient studies involved blowing nitrogen gas in place of air through the glass diffuser in the acclimated activated sludge system at 20°C. The oxygen deficient system has been referred to as an- aerobic from time to time, on the basis of the absence of aeration. Figures 33 and 34 indicate that the starch-COD was removed quite efficiently with the nitrogen-blown system, but total-COD remained practically constant. There was a slight decrease in the total carbohydrate content, indicating conversion of carbohydrate degradation product of starch to some other un- identified forms. The sludge concentration was also quite unchanged during the period of the test. These results would mean that the enzyme systems responsible for starch degradation were active under anaerobic conditions as long as there was good mixing of the substrate with the cells. The initial adsorption of starch by the sludge was also not affected by anaero- bic conditions. The total-COD of the system remained constant at the level after adsorption even after all the starch was degraded. Thus, the amount of starch adsorbed was somehow utilized by the sludge, because the increased sludge mass obtained initially was maintained even after all the starch was degraded in the system. If the adsorbed starch was degraded and not uti- lized then we would have obtained a decrease in sludge mass, and an increase of the total-COD of the system to the value originally fed. 139 It is proposed that in the mixed population of the activated sludge system, a small number of microorganisms was capable of utilizing the starch substrate under anaerobic conditions „ If the period of test was continued for longer periods, perhaps these microorganisms after multi- plication and growth would have utilized all the total-COD remaining in the medium. This would be a typical case of selection or acclimation of the sludge to new anaerobic conditions. The starch-COD removal rate constant under anaerobic conditions is plotted in Figure 51. The rate constant was higher than under aerobic conditions at F/M 1.28, but was of the same order of magnitude as in the aerobic system at F/M 2.2. The reason for the high starch-COD removal rate constant at F/M 1.28 for anaerobic system as com- pared to aerobic system is not evident. There was a possibility that active cell mass in the former system was higher, but this explanation can only be hypothesized, since no measurements of active cell mass were made. 7.1.4 Cell-Free Starch Removal The degradation of starch in the acclimated activated sludge membrane filtrate (referred to here as cell-free starch degradation), after a short period of contact between starch and sludge at different temperatures and growth conditions, has been presented in Figures 5, 8 and 9 for 5°C, Figures 11, 12, 28, 29 , 30 and 31 for 20°C, and Figures 22, 23, 24, 25 and 26 for 30°C. The cell-free starch-COD removal rates were, in each case s much lower than the rates obtained with whole cells, and for the period of test followed linear removal kinetics. The cell-free starch-COD removal rates have been reported in Tables 4, 5 and 6 for the three different tem- peratures. There was some variation of the cell-free starch-COD removal rate with different F/M ratios at a particular temperature, but on the 140 whole, the rates increased with an increase in temperature. The cell-free starch-COD removal rates were only 0.2 to 9 percent of the initial whole- cell starch-COD removal rate, (The average value was about 2 percent.) Thus, it would seem that starch degradation by acclimated sludge filtrates (cell-free) was negligible as compared to whole-cell starch degradation. It would be concluded from the above that the amount of exoenzymes secreted by the cells in the medium was insignificant. It has been reported by various investigators (74) (77) (78) that amylases of most microorganisms were liberated in the medium (extracellular) and in many cases had been isolated from culture filtrates (79) (80), These reports were contrary to the findings here. It may be argued that this discrepancy may be due to three reasons, (i) there was inhibition of amylase by high substrate (starch) concentrations, (ii) the use of phosphate buffer removed the cal- cium ions necessary for amylase activation, or (iii) the enzyme system responsible for starch degradation was situated on the cell wall and not liberated in the medium under conditions of the test. In the studies, starch concentration had been about 1500 mg/1. In Figure 12 the cell-free starch-COD degradation was determined at dif- ferent times of contact between starch and acclimated sludge; i.e., 5 minutes and 20 minutes. At the start of each of the cell-free starch-COD degradation tests, the starch concentration was different. At 5 minutes the starch concentration in the medium was around 1000 mg/1 and at 20 min- utes it was about 600 mg/1. Even so s the rate of degradation of starch was the same for both cases (16 mg/l/hr). In another study the concentration of sludge was maintained constant and the concentration of starch was varied from 200 mg/1 to 1525 mg/1 (Figures 28 through 31), There was hardly any inhibitory effect 141 of high starch concentration noted in the cell-free starch-COD degradation rate. As a matter of fact, there was an increased cell-free starch-COD degradation activity at higher substrate concentration, as reported in Table 5, although still the cell-free rates were much lower than the whole- cell starch degradation rates, Nomura et al. (81) found that increased starch concentrations produced higher amylase yields in B. subtilis „ Thus, it was unlikely that high starch concentrations were inhibitory for enzymes in sludge filtrates. Further, there was no indication of inhibition by high substrate concentration in the whole-cell starch-COD degradation, so the high substrate inhibition of amylase in sludge filtrate was ruled out. The second possibility of low cell-free amylase activity due to the calcium ion removal by phosphate buffer used, has been reported for Malt-a-amylase (73). However, in bacterial amylases the calcium ion is so thoroughly complexed with the enzyme that extra addition of calcium ion for activation is not necessary (76). Further, Fukumoto et al. (82) re- ported enhanced amylase production by B. subtilis with phosphates, espe- cially when carbohydrates were used as carbon source. The experiment with acetate buffer, Figure 35, shows that the starch-COD removal rate with whole-cell system was very similar to that obtained with phosphate buffer at comparable F/M and temperature. However, there was a stimulatory effect of acetate buffer on cell-free starch degradation (Table 5) as compared to systems with phosphate buffer. The cell-free starch-COD degradation rate with acetate buffer was still only about 4,5 percent of the initial starch- COD removal rate with whole cells. Thus, although the results of acetate buffer system did not answer the question of low starch degradation activity of cell-free sludge filtrates, there was a higher activity of amylase in the 142 filtrate compared to phosphate systems, It was pointed out by Fukumoto et_ al_o (82) that the earlier the phosphate was added to B. subtilis system the larger was the amount of amylase produced, but the amount of the pro- tein secreted to xhe medium was lower. So the decreased amylase activity in cell-free sludge filtrates with phosphate buffer compared to acetate buffer systems could be due to lower enzyme secretion from cells to the medium in the phosphate system, although the total enzyme activity with whole cells was similar in both cases. Thus, the stimulatory effect of acetate buffer compared to phosphate buffer for higher amylase activity in sludge filtrates was perhaps not due to calcium ion removal in case of phosphate buffer but due to reduced amylase secretion into the medium by the cells in a phosphate buffer system. The third possibility for the low amylolytic enzyme activity in sludge filtrate was the presence of a large fraction of the starch degrading enzyme system in association with the cell walls. These enzymes are at- tached to cells and degrade starch on the cell surface rather than in the medium. Only a small fraction of the total amylase is secreted in the medium producing low starch degrading activity of the cell-free sludge filtrates. In order to determine if the cell walls had high amylase activity, acclimated sludge cells were disrupted. The isolated crude cell wall frac~ tions were separated from the soluble cell components and its starch-COD degrading activity plotted in Figures 47 and 48. It will be evident from Figure 47 and Table 8 that the starch-COD degrading rate for the cell-wall fraction was much higher than even whole cells when expressed on the basis of total protein of the system. Although the techniques for isolation of cell walls were not very refined, the results show very definite indications 143 that the amylase system for the sludge was located in association with the cell walls o The higher rate of starch-COD degradation with cell wall preparation was perhaps due to higher enzyme amount per gram of cell wall as compared to whole-cell. In Aspergillus oryzae , a-amylase has been known to be bound to the cell wall depending on the pH of the medium (83), Thus, it would be possible to have a system in which the microenvironment of the cell has a pH different from the medium which allowed retention of a-amylase on the cell surface. Most of the reports (77) (78) indicate that in bacterial systems the location of amylase is extracellular. Recently there have been some reports of bacterial intracellular amylases (84) (85), which could be the precursors of the extracellular amylases. However, the location of amylase on the surface of the cell has not been reported in any bacterial system. The high starch degrading activity (Table 8) of the soluble cell component was perhaps due to the presence of intracellular amylase or phosphorylase. The presence of intracellular amylase in some systems has been proved beyond doubt (85), In S, bovis cell extract Ushijima and McBee (86) reported the presence of a phosphorylase capable of degrading starch. No reports of extracellular phosphorylase have been made and all phosphorylase systems are. perhaps intracellular. Therefore s the phos- phorylase could also be present inside the cell, and no attempts have been made to characterize the exact nature of the enzymatic system in the soluble ceil component responsible for the degradation of starch. The finding of Nomura et al, (74) (87), that extracellular amylase production in B, subtilis was low in the log growth phase but high in the stationary growth and autolytic phase, was also tested. Figure 36 and 144 Table 5 show that sludge filtrates in the endogenous phase had a much higher starch-COD removal rate compared to sludge filtrates in the log growth phase. But even in the case of endogenous sludge filtrates the starch-COD removal rate was much lower than with whole cells. In the studies with endogenous sludge filtrates no measurements were made to see the amount of autolysis of cells, so the higher activity of enzymes could be due to liberation of intracellular enzymes or surface bound enzymes by autolysis or changed physiological state of the cells. From the preceding discussion it would be fair to infer that the major part of the enzyme system responsible for starch degradation (amy- lases) was situated on the cell surface with minor amounts liberated in the medium. 7.1.5 Synthesis of Sludge In the acclimated systems studied, the total amount of sludge synthesis from the starch substrate fed in cases where the total-COD was completely utilized after six hours of aeration (except the residual stable organic end product COD) varied from an average of 64 to 59 percent of the total-COD fed at 20°C and 30°C respectively for constant substrate level systems. For the system with constant sludge concentration, the percent of substrate concentration converted to sludge mass was 58. The values are referred to as "a" value in Equation 2. Placak and Ruchhoft (9) and Sawyer (88) have reported similar values of sludge synthesis with carbo- hydrate substrate. 145 7.2 Starch Removal in Non-Acclimated Systems In these experiments there were three classes of organic compounds to which activated sludge from the basic seed unit was acclimated. The first was carbohydrates which constitute monomers for the starch molecule ; i.e., glucose and maltose, the second class of compounds was a complex com- pound Metrecal, and the third class was a protein; i.e., bovine serum albu- min. 7.2.1 Starch Removal by Glucose and Maltose Systems As has been mentioned earlier, starch on enzymatic degradation may produce substantial quantities of glucose and maltose molecules besides other oligosaccharides. Further, in order to utilize maltose as a substrate compared to glucose, a cell would have an extra enzyme, the maltase. It has been reported that maltose and maltodextrins have a stimulatory effect on a-amylase formation in B. stearothermophilus (89), whereas glucose had no such effect. The amylase formation of B. subtilis is influenced by the carbon source but in the presence of casein as nitrogen source, most sugars are available as carbon source (82), However, glucose at high concentration is inhibitory to amylase formation in B. subtilis (78), at low concentration it is available for enzyme formation. Fukumoto et al. (78) concluded that different carbon sources in the production of amylases from B. subtilis merely served as energy suppliers, and the observed divergence of the effect of carbon source on amylase formation was due to the competition between the amylase forming systems and other enzyme systems of the cell for the same nitrogen compounds. The degradation rate of starch in maltose system at F/M ratios 146 1.2 and 2.9 are presented in Figures 40 and 41 The degradation rate was slower than a comparable starch acclimated system but there was no lag observed in either case. This clearly indicates that the amylase system of maltose acclimated sludge was present even at the start; i.e., the enzyme was constitutive. However, the lower rate of starch and total-COD reduction indicate that the activity or amount of enzyme present under the conditions was lower than the comparable starch-acclimated system. The total-COD removal characteristic in Figure 40 was slightly different than observed in the starch-acclimated system. After a linear total-COD removal the first two hours, the rate suddenly changed to another linear rate. (This peculiarity was not observed at the higher F/M in Figure 41.) Apparently the starch-COD removal rate was unaffected by this sudden change in the total-COD removal rate. Thus, the elimination rate of starch degra- dation products was altered, not the starch degradation itself. The high rate of build-up of some starch degradation products was inhibitory to the cell at some stage involving their assimilation. Figures 37 and 38 represent the starch removal characteristics of glucose acclimated sludge. In Figure 37 (F/M 1.24) the starch-COD and total-COD removal rates did not have any lag and the removal characteris- tics were very similar to those of the starch acclimated systems, although the removal rates were lower. However, in Figure 38 (F/M 2.54), the total- COD removal rate was retarded initially, but the starch-COD removal pro- ceeded at a slow rate without any lag. The assimilation of the starch degradation product in this system was retarded. This is attributed to the low concentration of the enzymes responsible for assimilation of the starch degradation product (consequently the removal of total-COD); the time re- quired for the induction of more enzymes caused the lag in the total-COD removal. 147 The total-COD and starch-COD removal rates of glucose acclimated sludges were lower than the comparable maltose or starch acclimated systems (Figure 39, Table 5 and Table 7), but there was no lag involved in the starch utilization,, This would indicate that the enzymes responsible for removal were constitutive in the glucose systems, although the total amount of the enzymes produced initially was less than that in the maltose or starch acclimated systems. The starch removal rate by the glucose sludges was lower than that of maltose sludges. This may be expected, since the glucose sludge may have to evolve or reactivate in quantities at least two enzymes, amy- lases and maltases, before the starch can be utilized. In maltose systems it may be possible to get by with the evolution or reactivation of only one enzyme, amylases, for the utilization of starch. Thus, maltose sludges can be expected to degrade starch at a faster rate initially. An attempt was also made to see the change in starch removal rate when a glucose acclimated sludge was gradually becoming acclimated to starch. Figures 44, 45 and 46 show that within 4- days the rate of total- COD and starch-COD was of the same order of magnitude as starch-acclimated systems (Table 5). This change in starch removal rates was brought about either by increased induced enzyme production by the starch substrate or by change in predominant microorganisms most suitable for utilizing starch as substrate. It will be difficult to pinpoint what was actually happening from the data obtained. However, from the point of engineering considera- tions, it is evident that glucose sludges can be completely acclimated to starch substrate in a relatively short time. Thus, a qualitative shock load of this kind in an activated sludge system can be taken care of in a matter of days. 148 7.2,2 Starch Removal by Metrecal System The Metrecal substrate is a mixture of various natural ingre- dients, each quite complex in chemical composition. Table 1 lists a few of the properties. It will be evident that Metrecal has about 11,6 percent carbohydrate and the manufacturer even mentions the presence of starch on the packaged product but information about the amount of starch is not made available. Thus, the feeding of starch substrate to Metrecal- acclimated sludge was a partial shock load since microorganisms in the sludge were familiar with such compounds. The starch removal characteristic of Metrecal-acclimated sludge has been presented in Figure 42, There was a long lag period in the total -COD removal, whereas starch-COD removal rate was very slow but did not indicate an appreciable lag. This would indicate that perhaps the cell assimilative system for the starch degradation products was not devel- oped until the end of the lag period. In the Metrecal system, too, the amylolytic enzymes seemed to be constitutive since there was no appreciable lag in the starch-COD removal rates. The starch removal rates when com- pared with maltose and glucose acclimated sludge were generally lower for the Metrecal system (Table 7), 7.2,3 Starch Removal by Bovine Serum Albumin System This substrate is chemically very different from the starch molecule, so it would be expected that there would be a long lag before any degradation of starch by this system occurs. In Figure 43 it will be seen that there was quite a marked lag in both starch-COD and total-COD removal. The total-COD removal rate was 149 lower than the acclimated system even in the linear removal phase following the lago The enzyme system responsible for starch removal with bovine serum albumin acclimated sludge was adaptive. The presence of starch induced the amy lo lytic enzymes in the sludge after a period of lag. This shows that the enzyme systems responsible for starch degradation are quite easily inducible even with sludges acclimated to quite different structural compounds. 7.3. Adsorption of Starch Substrate by Sludges In all the systems using starch substrate, especially with starch acclimated systems, it was reported that there was immediate removal of substrate on contact with sludge. This initial removal was referred to as adsorption. The removal of substrate from the system by sludge was invari- ably indicated by a comparable increase in the weight of sludge. The possibility cf the substrate being removed on the filter paper and giving a false initial adsorption phenomenon was eliminated by use of substrate previously filtered through a similar membrane filter. Thus, any immediate removal of the starch substrate by the sludge was attributed to adsorption. 7.3.1 Adsorption by Acclimated Systems The amount of adsorption of the starch by the acclimated sludge at different temperatures has been presented in Tables 4, 5 and 6. The concentration of initial substrate, after adsorption had occurred, is also presented. There was generally higher amount of starch adsorption at 5°C than at 20°C under comparable situations. But the adsorptions at 30°C were generally higher than both 20°C and 5°C. Thus, the generalization that low 150 temperature favors higher adsorption was not wholly applicable here. At- tempts to determine the amount of starch adsorbed at different times after initial contact were not successful, hence it was not possible to determine the equilibrium concentration. However s it must be remembered that adsorp- tion of starch to bacterial cells which degrade it would produce a complex dynamic equilibrium. It would be very difficult to theoretically evaluate such a system where adsorption, degradation and growth are going on simul- taneously o Attempts were made to plot log x/m versus initial substrate concentration for the adsorption data at the three temperatures, according to Equation 13a „ The great scatter of points in Figure 58 indicates that simple assumptions made in Freundlich's derivation are not valid when the starch substrate is adsorbed on bacterial cells. The data plotted in Figure 58 were not obtained under equilibrium conditions, and it was doubtful if such equilibrium conditions did exist in the experimental sys- tem at any time. The adsorption data of 5°C, where the metabolic activity of sludge would be low, were plotted in Figure 59 to see if adsorption in- creased with increased sludge concentration. Meanwhile the starch concen- tration was kept constant. There was a linear relationship in Figure 59, indicating a mass effect rather than an equilibrium situation based on adsorption and desorption. The adsorption of starch on sludge under anaerobic conditions s Figures 33 and 34, was of the same order of magnitude as with the sludge under aerobic conditions. This would mean that even in conditions of no growth the adsorption was present. Adsorption would thus seem a physical process of removal of starch by sludges. 0) o D 30° C A 20° C Constant Substr • 20° C Constant MLSE o 5° C 1 1 1 4 1 1 □ 3 O c • < < 0=3 □ □ < < < < 1 • D b CM O r- o m o CM O o 8 151 o 8 8o> 6 guj oO cvj 2 O o X o cc s< - o £ CVJ o >- o c/> o C\J UJ «K e> \ Q o o> => o E «*» o o 10 C/5 O 8^ _l 5s O CO lo Q ,-J p UJ p < o o O ^ cc CM z < K UJ 0- c/) 3 «§ (f) 5S or q: o o o o < CD ZD K UJ O en o _J Q O _j UJ X Z 10 o o 2 O 1- <3" o (/) Q < 0> if) //6w '(HGUOSGV HOdVlS or Z) u_ 153 7.3,2 Adsorption by Non-Acclimated Systems The amount of starch adsorbed in almost all non-acclimated systems was very much lower than the acclimated systems under similar F/M conditions. This is evident by comparing initial starch adsorption data in Table 5 and Table 7„ Thus it would appear that adsorption of starch on activated sludge was also dependent on the acclimation of the sludge. This was further demonstrated in the Figures 45 and 46 „ The change in starch substrate removal characteristics was determined with glucose acclimated sludge, over a period where the sludge became finally acclimated to starch substrate. The change in initial adsorption with succeeding days was plotted in Figure 60. There was an immediate increase in adsorption of starch substrate by the glucose sludge as it was getting acclimated to starch substrate. However, after getting completely acclimated, there was a drop of initial starch adsorbed on the 6th day. This was very unexpected and unexplainable. But it would seem that adsorption of starch en the sludge was dependent on the acclimation characteristics of the sludge to starch. So, on the whole, adsorption of starch on sludge constitutes a valid means of its removal but the exact amount would depend on many factors like temperature, amount of sludge, acclimation and physiological condition of the sludge. The influence of acclimation on the amount of starch ad- sorbed would indicate a more complex situation than a mere physical phe- nomenon. It would be tempting to hypothesize that increased adsorption of substrate on cells after acclimation was facilitated by cellular enzyme systems on the surface of the cell, since it has been found that in our case the amylolytic enzyme systems of starch acclimated sludges were located on the cell surface. However, no proof of such a hypothesis is available at 154 E o" UJ CD rr o in Q < X o rr n) 600 400 200 Sludge Acclimated to Starch Sludge Acclimated to Glucose J I I L J L 12345678 TIME , days FIGURE 60: CHANGE IN STARCH ADSORPTION BY GLUCOSE SLUDGE WITH ACCLIMATION TO STARCH 155 the present moment , The removal of starch from the medium was due to two processes, adsorption of starch on the cells followed by its enzymatic degradation on the cell wallo The high positive values of Q 1n > the tem- perature coefficient for the starch-COD removal, indicated that the rate controlling reaction was enzymatic reaction rather than physical process „ The effect of temperature on physical processes like adsorption is usually inverse, i.e., increase of temperature decreases the adsorption,, However, there still is a possibility that the adsorption of starch on the cell surface was occurring at sites where the exoenzymes were situated „ In this case the high positive value of Q would indicate the total effect of two enzymatic processes , There is also a possibility of formation of "active sites" for starch adsorption after acclimation of a particular sludge. However, here also no proof of existence of such sites is available. The average size of a starch molecule in solution was not avail- able at the present time. However, the molecular dimensions of glycogen, a polysaccharide of similar structure as amylopectin fraction of starch, has been reported as 15 my in diameter (90), The shape of the glycogen molecule is globular as opposed to the linear shape of starch molecules. If the size of starch molecules is assumed to be the same as that of gly- cogen , then approximate calculation can be made to see what amount of starch can be adsorbed per gram of sludge if monomolecular coverage of the cell surface is assumed. Projected area of starch molecule = 175 my 2 5 o Surface area of 1 y diameter cocci cells = 3,14 x 10 my^ Number of molecules of starch in a monomolecular coverage of the cell = = 18000 156 From Table 2 the average molecular weight of potato starch, assuming 23 percent amy lose and 77 percent amylopectin, was 185,000. Therefore 1 molecule of starch would weigh 3.1 x 10 mg and the amount of monomolecular starch adsorbed per cell = 18000 x 3.1 x 10~ mg = 5.6 -12 -12 x 10 mg. If the weight of a cell was assumed to be 1 x 10 g (91), then the amount of starch adsorbed on cells (monolayer adsorption) would be 5.6 mg/gram of cell. However, the experimental value of the starch adsorbed per gram of sludge at 20°C was on an average 150 mg. This dis- crepancy can be explained, either by the fact that multilayer starch ad- sorption was occurring on the cells or the linear starch molecule oriented in such a way had much smaller surface area of contact as compared to that assumed for globular glycogen. From the calculations above it is evident that adsorption of starch on the cells covers at least a monolayer and the possibility of starch adsorption only on the specific sites where amylolytic enzymes were situated on the cell surface would be much less. Therefore, it appears that adsorption of starch on sludge is a physical phenomenon. The acclimation of sludge to starch causing high starch adsorption is perhaps either due to a change in the predominant microbial population of the system, or due to a change in the surface characteristics of the cells. In the field of microbiology, various references have been made in which macromolecular substrates are adsorbed and degraded by enzymes located on the surface, in confirmation to our findings with starch. The breakdown of cellulose by Cytophaga has been attributed to surface located enzymes (92) and adsorption of the cells on cellulose micelles was the pre- dominant mechanism of degradation of cellulose. 157 The hydrolysis of ribonucleic acid by Pasteurella pestis has also been reported to be due to surface adsorption of the substrate and its subsequent degradation by the surface located enzyme (92). In passing, mention may be made about the adsorption of some antiseptics, like phenols and esters of acids, to bacterial cells. It has been reported that the mechanism of toxicity in such cases is due to the adsorption (93). The random adsorption of bacteriophage to cells has already been mentioned earlier. 7.4 Mechanism of Starch Removal In the systems investigated the mechanism of removal of starch has been postulated as follows: (i) a portion of starch was adsorbed on the sludge, depending on the conditions (temperature, acclimation, amount of sludge, physiological condition of sludge). (ii) the adsorbed starch was degraded by cell-wall associated enzyme systems into smaller degradation products. (iii) the starch in the medium was either adsorbed on the cell sites vacated by degraded starch molecules, or was degraded by small amounts of extracellular enzyme system in the medium. The former was much more effective compared to the latter in starch removal. (iv) the starch degradation products were then utilized by the cell for assimilation. In non-acclimated systems induction or reactivation of enzyme system was required before any starch degradation occurred by the mechanism proposed in the above paragraph. 158 7 5 Engineering Significance It has been shown here that the removal of a colloidal waste, like starch, follows linear kinetics very similar to those of many indus- trial wastes , like pharmaceutical, brewery and refinery wastes, which have high colloidal content Even some simple molecules, like butyrate, glu- cose and aspartic acid 9 have linear removal kinetics under aerobic condi- tions (37), as was pointed out earlier „ Thus, colloidal wastes may follow kinetics quite similar to those of simple compounds, and the design criteria governing the removal of simple compounds in activated sludge would be applicable quite appropriately to the removal of colloidal waste. However, the removal mechanism of a colloidal waste like starch was found to be quite different from that of small molecules, like glucose, lactose, etc In the case of removal of starch, initial adsorption of substrate on sludge was evident in almost all the cases studied „ This phenomenon of adsorption has not been observed (10) (24) with simple small molecules, and high initial removal in these systems has been attributed to high rate of assimilation,, Therefore, the removal of colloidal wastes like starch would be very effective in the contact stabilization process „ A contact time of a few minutes would, in some instances, be able to remove a very high amount of total-COD input if the MLSS is maintained at a fairly high level „ The starch degradation would be proportional to F/M ratio but initial starch adsorption would be proportional to the initial MLSS, Further, the total- COD removal rate, indicative of assimilation of starch degradation product, would be proportional to initial MLSS too Further, in the case of shock loads involving such colloidal wastes to an activated sludge plant $ the resulting upset situation may be overcome 159 by increasing the amount of return sludge, or in other words increasing the amount of MLSS in the system. The increased sludge concentration would have to be such that suitable F/M is maintained for starch degradation. The starch degradation and adsorption was not affected by anaerobiosis. This would indicate that in a conventional activated sludge plant treating starch waste s the aeration of sludge-waste mixture at the head end of the tank would not be necessary, A small mechanical mixer would be sufficient to permit starch degradation, followed by aeration to assimilate the degradation products. In the case of contact stabilization process a short mixing chamber may be sufficient to allow high starch ad- sorption without the need of aeration. The amount of sludge carried in the contact tank under anaerobic conditions will not be limited by the oxygination capacity of the aeration device in aerobic systems. Very high starch removal may be obtained with high MLSS (above 10,000 mg/1) in the contact tank and short contact period. Longer contact time would be harm- ful since the effluent would carry the starch degradation products. It would be apparent from the Table 5 that in the acclimated activated sludge systems with starch as substrate, generally 150 mg/1 of total-COD was adsorbed initially at a MLSS concentration of about 1000 mg/l„ On extrapolation to a contact stabilization process treating starch waste equivalent to 1000 mg/1 COD, a 5-10 minute contact time with 5000 mg/1 mixed liquor suspended solids concentration would give a removal of 750- 800 mg/1 COD by adsorption. Longer contact periods may not be necessary since the starch removal by adsorption is practically instantaneous. The optimum contact periods and MLSS concentration in the contact tank would have to be determined by further experimentation. It would be worthwhile to see if the data presented allows to determine by interpolation the starch removal at other operating conditions. 160 For example s what would be the starch removal characteristics at 12°C S initial mixed liquor suspended solids concentration of 1400 mg/1 and starch-COD concentration of 1800 mg/1? From Figure 57 the "b" and "c" values for 12°C (285°K) was 95 mg/hour/g sludge and 0„330 mg/mg/hr re- spectively „ Using Equation 15, the value of 135 mg/l/hr was obtained for k t , the total-COD removal rate constant for initial MLSS of 1400 mg/1,, Similarly the k , the starch-COD removal rate constant, was found to be 0o256 (hour)" for F/M ratio of 1„285 from Equation 18„ It would take about = 13 „ 4 hours to completely remove the total-COD of 1800 mg/l„ i. J J However, some 40-80 mg/1 COD would still remain in the medium owing to stable metabolic end products „ But this still would give a removal effi- ciency of above 95 percent , For 95 percent starch degradation it would take 5ol hours if the Equation 17 is used, i.e., logarithmic degradation of starch-COD„ Table 11 gives the details of the above calculations „ 161 TABLE 11 CALCULATION DETAILS FOR DETERMINING BY INTERPOLATION THE STARCH DEGRADATION CHARACTERISTICS AT 12°C, 1400 mg/1 MLSS AND STARCH-COD CONCENTRATION OF 1800 mg/1 From Figure 57: AT 12°C (285°K) "b" = 95 mg/hr/g sludge and "c" = 0,330 rng/mg/hr From Equation 15: Total-COD removal rate constant k = b(S ), S = 1400 mg/1 o o ° /. k = 1,^0x95 = 135 mg/l/hr t From Equation 18: Starch-COD removal rate constant k = c (F/M)" 1 , (F/M)" 1 = 0o778 o°o k = 0,256 (hr)" 1 s The time required for 95 percent removal of starch-COD can be obtained from Equation 17 „ log -£.. = k s (t - t Q ) log i2£ = 0,256 (t - t ) o o 1.301 c . . ' • t " X o = 07256 = 5o1 h ° UrS 162 8 CONCLUSIONS lo The removal of starch in an acclimated activated sludge system follows simple logarithmic kinetics for initial periods „ However, in some cases after the initial period there is a divergence from the logarithmic relationship , The starch removal rate constant is dependent on food-to-microorganism ratio (F/M) and not on the initial solids „ 2, The removal of total-COD, which includes starch degradation products s has a linear relationship with time as opposed to the logarithmic relationship of starch removal,, In some cases there is a brief lag in the starch removal rate which is attributed to quantitative shock loading of the systems. The rate constant of total-COD removal is dependent on the initial mixed liquor suspended solids (MLSS) and not on F/M, 3, The effect of temperature on the removal rate of starch- and total-COD is quite significant „ The energy of activation, AE, is of the order of 16,500 and 20,400 cal per mole for starch-COD and total-COD removal respectively, 4„ In the aerobic systems there is no accumulation of starch breakdown products other than carbohydrates in the medium indicating that the further breakdown of these small carbohydrates is inside the cello 5 Anaerobiosis of the acclimated activated sludge has no effect on starch degradation and initial adsorption of starch on the sludge, as long as the contents are well mixed. The rate of removal of starch for anaerobic conditions is similar to that for aerobic conditions. The total- COD and total-carbohydrate removal is very much retarded, and there is hardly any growth in the anaerobic systems, 6, Acclimation of the sludge is extremely important for high starch removal rates. The enzyme system responsible for starch degradation 163 (amylase) is constitutive in the case of glucose, maltose and Metrecal systems, and is adaptive in the case of bovine serum albumin system,, 7, The large proportion of enzymes responsible for starch degradation in the systems tested are located on the cell walls with an insignificant amount liberated in the medium, especially during the log- growth phase o There is no perceptible inhibition of starch degrading enzyme systems by high concentration of substrate „ Use of phosphate buf- fer in the systems tested allows less liberation of the starch degrading enzyme systems in the medium from the cell wall when compared to the systems with acetate buffer of the same pH„ There is an indication of an intracellular starch degrading enzyme in the acclimated sludge which was not characterized . 8 The amount of starch substrate utilized for the synthesis of sludge varied from 58 to 64 percent „ 9, Adsorption of starch by sludge is evident in all the systems tested but there is no good correlation between the amounts adsorbed per unit weight of sludge and the concentration of starch added or with the temperature. The amount of starch adsorbed on sludges varies from a high of 24 percent for acclimated systems to a low of 1,5 percent for non- acclimated systems. The amount of starch adsorbed is dependent upon the initial sludge concentration, temperature and acclimation characteristic of the sludge, but independent of the presence of oxygen, 10, Mechanism of starch removal in activated sludge system is the initial adsorption of starch on cells, followed by its degradation by the cell-wall-situated enzyme systems. The degradation products are liberated in the medium and subsequently assimilated by the cell. Thus, the mechanism of removal of colloidal substrates in activated sludge unit differs signifi- cantly when compared to simple small molecule substrates. 164 9, SUGGESTIONS FOR FUTURE WORK The complexity of the removal of colloidal waste in an activated sludge system has already been emphasized „ Only a small segment concerning the basic aspects of this problem has been investigated in this study„ The investigation can be pursued further in two ways. One line of approach would be to probe further into some of the aspects of the problem dealt with here,, These may involve studying the removal of starch in the activated sludge system under different operating conditions o The operating variables that could be studied are effect of different pH, effect of nutrient deficiency and effect of the presence of other substrates in conjunction with starch , The location of the starch degrading enzymes on the cell walls can be further tested by degradation of the cell walls by lysozyme under conditions where the protoplasts are protected from disintegration and the cytoplasmic membranes are intact, Extraction of the enzyme from cell wall by lysozyme would undoubtedly pinpoint its location. The effect of starch adsorption on acclimation of sludge can also be studied in more detail , and reasons for the decreased adsorption after some days of acclimation may be investigated. The use of contact stabilization process for the treatment of starch wastes may also be investigated in light of the findings of the present and future research. Alternatively s the kinetics and mechanism of removal of colloidal substrates other than starch could be investigated. The substrates which could be studied are carbohydrates (glycogen, cellulose and pectin), pro- teins (albumins and casein) and lipids (lecithin and cephalin). The study of these different colloidal substrates in an activated sludge system would give a wider perspective to the results. 165 Further, it would be worthwhile to investigate the removal characteristics of industrial wastes having a large undefined colloidal content. Would the results of such an experimentation have any correlation with those obtained with pure colloidal substrates? 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Nomura, M„, Hosoda, J„ s Maruo, B M and Akabori, S„, "Studies on Amylase Formation by Bacillus subtilis . II, Effect of Amino Acid Analogues on Amylase Formation," Journal of Biochemistry ( Tokyo ) $ 43 , 841 (1956), 171 75, McKinney. R E„, "Biological Oxidation of Organic Matter s " Advances in Biologacal W aste Treatment , edited by Eckenfelder s W W, , Jr,, and McCabe, J,, (Proceed ings of the Third Conference on Biologic al Waste Treatment^ Manhattan College, New York, 1960) , Pergamon Press, London, 1 (1963), 76, Fischer s E, H, , and Stein. 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F. , Jr., "Colorimetric Determination of Proteins and Carbo- hydrates," Industrial Water and Waste , 1^ 1, 17 (1962). 95. Seifter, S., Dayton, S„, Novic, B s and Muntwyler, E , "Estimation of Glycogen with the Anthrone Reagent," Archives of Biochemistry , 25 , 191 (1950). 173 11, APPENDIX A 174 DETAILS OF ANALYTICAL PROCEDURE Chemical Oxygen Demand Test (62) Reagents i Standard potassium dichromate solution, 0,25 N ii„ Standard ferrous ammonium sulphate solution, 0,05 N iii. Sulfuric acid reagent This was prepared by adding 22 grams of silver sulfate crystals to a nine-pound bottle of concentrated sul- furic acid iv„ Ferroin indicator solution v Mercuric sulfate crystals Procedure A 10-ml sample or an aliquot diluted to 10 ml with distilled water was mixed with 5 ml of standard potassium dichromate and 15 ml of sulfuric acid reagent. One gram of mercuric sulfate was then added. The mixture was refluxed for two hours with boiling chips in an Erlenmeyer flask o The flask was cooled, and 40 ml of distilled water was added. The excess dichromate in the mixture was titrated with standard ferrous ammo- nium sulfate using the ferroin indicator, A blank consisting of 10 ml of distilled water instead of the sample, together with the reagents, was refluxed in the same manner. The calculations were done in the same manner as mentioned in the reference (61), 175 Starch-Iodine Test (66) Reagents i. Potassium iodide-iodine reagent This was prepared by dissolving 2 grams of solid iodine in 20 ml of a solution containing 20 grams of potassium iodide, and then diluting to 1 liter with distilled water, Procedure Samples containing up to 2 mg/ml of starch were added to a 100-ml volumetric flask containing about 80 ml of distilled water „ One ml of potassium iodide-iodine reagent was added „ The intensity of the blue color formed was determined in the Lumetron colorimeter (Photovolt Corpora- tion, Model 401) at 650 millimicrons after 15 minutes of color development, against a distilled water reagent blank „ A set of standards (containing 0,5, 1„0 9 1„5 and 2„0 mg of starch) were also treated in the same manner as the samples, in order to obtain a standard curve for estimating the amount of starch present in the samples „ Figure 61 represents a standard curve for the potato starch substrate used in this study. 176 0.1 0.2 OPTICAL DENSITY FIGURE 61. STANDARD CURVE FOR POTATO STARCH- IODINE TEST 177 Total Carbohydrates-Anthrone Test Reagents io Anthrone reagent This reagent was prepared by dissolving 0,2 grams of anthrone in 100 ml of concentrated sulfuric acid. It was freshly prepared every time the test was performed and refrigerated for an hour prior to use, as the reagent is claimed to darken on aging. Procedure The test involved the use of a sample containing 10-100 micrograms of carbohydrate per ml. The sample was placed in a chilled test tube and made up to 5 ml with distilled water „ Ten ml of freshly prepared anthrone reagent were quickly added and the reactants mixed by blowing air through the mixture. The tubes were covered with aluminum caps and placed in a boiling water bath for 10 minutes. After cooling the tubes, the optical density of the colored solution was determined at 620 my wave length in the Bausch and Lamb Spectronic 20 spectrophotometer against a reagent blank prepared with distilled water, A set of glucose standards was run each time the test was performed. Chilling of the reagents and the samples, prior to boiling, was practiced as recommended by several investigators (94) (95), 178 Folin Ciocalteu Test (69) Reagents i« Folin A reagent This solution contained 20 g of sodium carbonate and 4 g of sodium hydroxide in 1 liter of distilled water. The reagent was stable for 2 to 3 months s if it was not contaminated,, ii, Folin B reagent The reagent was prepared by dissolving 100 mg of sodium tartrate and 50 mg of copper sulfate in 10 ml of dis- tilled water. In this form s the reagent was stable for a week, but if the 2 constituents were not mixed, the individual solutions were stable for longer periods, iii. Folin C reagent One ml of Folin-phenol reagent (supplied by Fischer Scientific Co,) was added to 1,2 ml of distilled water. This reagent was freshly prepared each time the test was performed, as it was stable only for an hour, iv, Folin mixture This reagent contained 1 ml of Folin A solution added to 49 ml of Folin B solution. Procedure One ml of a sample, or an aliquot diluted to 1 ml, containing 50 to 150 micrograms of protein was mixed with 5 ml of Folin 's mixture in a test tube. The contents of the tube were allowed to stand for 15 minutes at room temperature, 0,5 ml of Folin C reagent was added to the tube and the contents mixed quickly in a vortex mixer. The mixture was allowed to 179 stand at room temperature for 30 minutes „ If any organic debris was present, it was removed at this stage by centrifugation. The intensity of the color developed was measured in the Bausch and Lamb Spectronic 20 spectrophotometer at 700 my wave length against a reagent blank prepared with distilled water. A set of standards prepared with bovine serum albumin fraction V was assayed every time in order to obtain a standard curve. 180 12. APPENDIX B 181 ACCLIMATION OF UNITS Determination of Solids Balance The seed sludge (500 mg) from the continuous Metrecal fed activated sludge unit was used to start the acclimation units for each of the substrates used. The concentration of substrate fed every day was about 1500 mg/1 in the case of starch, maltose and glucose units (1000 mg/1 in the case of bovine serum albumin unit). A third of the mixed liquor was wasted every day. The mixed liquor suspended solids, representing the biologically active mass, was determined from time to time The steady state conditions were attained when the amount of synthesis of sludge by utilization of substrate was balanced by the amount of sludge wasted every day. Figure 62 indicates the attainment of acclimation of Metrecal sludge to starch substrate over a period of time. The steady state MLSS concen- tration for starch substrate was 1330 mg/1 under the conditions of opera- tion. Figures 64, 65 and 66 represent the attainment of steady state condition MLSS in the glucose, maltose and bovine serum albumin substrate systems respectively starting with Metrecal sludge. In the case of starch, acclimation was also determined by the attainment of optimum substrate removal kinetics as shown in Figure 63. It was evident it took only about 5 days for the Metrecal sludge to get acclimated to starch substrate. 182 r X O o o o o CD e o o to 1 >» ii ■o < o or cd CD LU h- z CD n _1 2 - 1 1 \ - V - o V • 1 ^a* \ o 1 _ OJ _ O - 00 - CD >> o LU - _J CD o h- LU CD O => 1 (0 LU O _l z < < o _J LU < ir m h- iij co ^ CO o 2 i o LU < 1- < a: _J co o GO o Z> < CO ^ CD LU m Z) CD U_ i/6 uj l sanos Q3QN3dsns yonon Q3xiiai 185 O o> 0) E o O O CO ro >% ii ■o CO o 0) CO 1 CO S O O \ \ O" No CM 00 CD o ■o UJ CO o o IT) O O o o o m i/6w'sanos Q3QN3dsns yonon q3xiim UJ if) O 1- _l < ^ o 1- UJ o Q => _J (.n LU _i o < 2 o < LU _l a: < h- 00 Ul S C/) U_ _J o S 2 1 o UJ h- 1- < < ^ ir »— _J (0 o 00 o Z> < (/) in C£> UJ q: Z> O Lu 186 1 1 1 1 1 o 1 1 1 ~ 1 0) u» 1 o E 55 g 1 CD =* .1 1 o 1 tead LSS 1 V) 5 1 1 1 I ™ o \ \ - \ o / / / / / rS 1 « o o o <\J - o - 00 - to - sj- - 0J o o m o ■o UJ LU . LU > o o z 00 < o _l < CD UJ o Q ID _l CO CO CO 1 LU _l h- < o GO < _J < ^ S _J Z> o or o LU < CO C£> CD LU or Z) o Ll I/6uj 'SQIIOS 03QN3dSnS blOnOll Q3XIIM 187 13. VITA The author was born at Lucknow, India, on January 25, 1936 , After finishing his pre-college education at Colvin Collegiate School in Lucknow, he qualified to study at Bengal Engineering College, Sibpur, Howrah (affiliated to the University of Calcutta, India) in 1953. During the entire period of study at Bengal Engineering College, he was awarded a merit Scholarship for high scholastic grades. In 1957, he received the Bachelor of Engineering degree in Civil Engineering from the University of Calcutta, graduating with a First Class, From 1957 to I960, he was engaged in civil engineering practice, primarily design and construction of water supply and sewerage facilities for the Durgapur Steel Project Township, In 1960, he undertook graduate work at the University of Illinois He was awarded the degree of Master of Science in Sanitary Engineering, The title of his Master's thesis was "Effect of Biological Slime on the Re- tention of Alkyl Benzene Sulfonates on Granular Media," Since 1960, he has held a Research Assistantship, Initially, he was associated with a research project, "Fate of Synthetic Detergents in Soils and Ground Waters 9 " which was supported by the United States Public Health Service, and later he was associated with the research project, "Initial Rapid Removal of Organic Matter by Microorganisms in Treatment of Waste Waters," also supported by the United States Public Health Service, He was elected a member of Sigma Xi, The author has co-published the following papers: 1, "Retention of ABS on Soils and Biological Slimes," Ewing, B, B , Lefke $ L, W„ , and Banerji, S. K, , Proceedings of the 1961 Symposium, Ground Water Contamination, Technical Report W61-5, Robert A, Taft Sanitary Engineering Center, U,S,P,H,S,, Cincinnati, Ohio s 166 (1961), 188 "Effect of Biological Slimes on the Retention of Alkyl Benzene Sulfonates on Granular Media," Ewing, B„ B„, and Banerji, S, K„ , Proceedings 17th Industrial Waste Conference, Purdue University, Extension Series 112, 351 (1962), The author is a member of the following professional societies; American Society of Civil Engineering (Associate Member) American Water Works Association Water Pollution Control Federation \