Ecological and kinetic aspects of amylolysis and proteolysis in activated sludge

J.M.A. Janssen

Research output: Thesisinternal PhD, WU


<p/>An investigation has been made of the enzymic degradation of biopolymers by activated sludge. Starch was chosen as the model substrate; it was administered continuously at different sludge loading values which covered the entire range of loadings applied in sewage purification plants. The acclimatization of activated sludge to loading with starch as the sole source of carbon and energy was followed with regard to substrate removal, growth, amylase activity and numbers and types of amylolytic bacteria. Attention was paid to the nature and the location of the amylolytic enzymes and to the mechanism and kinetics of the amylolysis. The production and degradation of biomass and of amylases, the latter including their kinetics, were studied in relation to the sludge loading with starch and the proteolytic activity of the sludge. The influence of biomass retention on these phenomena was studied by comparing laboratory-grown activated sludge (i.e. sludge I, with retention of flocculated biomass) with sludge II (without retention of biomass).<p/>Sludges I and II, acclimatized to starch, were able to degrade this compound almost completely when it was administered as the sole carbon nutrient over the sludge loading range 0.075 to 2.4 g COD/g (Table 4.3). Immediate and nearly complete removal of starch and other carbohydrates by sludges I grown on glucose or maltose was observed when the initial, preformed, amylolytic activity of the sludge was higher than a distinct critical value; when it was lower, a lag phase was observed in the increase of biomass and of amylolytic activity (Fig. 4.3). Under such conditions incomplete starch removal was observed to occur during a period of maximally one week (Fig. 4.3E).<p/>The concerted action of α-amylases and debranching enzymes was probably the most important mechanism of extracellular starch degradation by sludge bacteria. The starch hydrolases catalyse also the breakdown of dextrins. Enzymes specifically hydrolysing lower oligosaccharides such as maltotriose were not clearly detected in whole starch sludge or in its ultrasonicate (3.2).<p/>The average amylolytic activity calculated per (viable) amylolytic bacterial cell present in starch sludge I, cultivated at pH 7.0, did not vary clearly over the SL range 0.3-2.4 and was about equal to the amylolytic activity of amylolytic bacteria in maltose and glucose sludge I (SL = 0.3). Only at SL = 0.075 with starch it was higher, probably due to active amylases associated with dead cells and cell debris which make up the bulk of the biomass of low-loaded sludge (Tables 4.5 and 4.6; Fig. 4.9).<p/>Enzymes hydrolysing macromolecular substrates like protein and starch were completely associated with the sludge biomass. Only in high-loaded laboratory sludges 0, 5-3 % of the activity was detected in the sludge-free liquid (Table 7. 1). This result is in agreement with the observation that the predominant amylolytic bacteria isolated from continuously loaded starch sludge I (SL = 0.3) did not excrete amylases into the media of batch cultures (Table 8.5). The enzyme-sludge association prevents the continuous wash-out of these enzymes from the sludge tanks. The proteinases are located at the outside of the cell wall of the sludge bacteria and are freely accessible to macromolecular substrates, as it is true of about 50 % of the amylases. Mass transfer resistance of polymers to cell surfacebound amylases and proteinases in sludge flocs, and steric hindrance due to binding of these enzymes to the bacteria were not to a large extent limiting the standard reaction rates with whole activated sludge (7.2).<p/>The remaining 50 % of the amylases is probably located within the periplasmic space i.e. outside the cytoplasmic membrane but inside the molecular sieve constituted by the outer membrane of the cell wall of the Gram-negative bacteria occurring in activated sludge (Chapter 8). The periplasmic: amylases are accessible to oligosaccharides which are able to pass through the outer membrane and are protected against proteolytic attack (7.2).<p/>The relationship between dextrinogenic reaction rate of amylases from activated sludge and starch concentration strictly obeyed Michaelis-Menten kinetics only seldom (cf. the experiment of Fig. 6.4). In almost all of the other experiments (SL = 0.075-2.4) a curvilinear relationship was observed in the Lineweaver-Burk plot (Figs. 6.5 and 6.6), indicating rather large differences between the half-saturating substrate concentrations of the amylases present (6.1). Half-maximum reaction rate constants of the amylases with the greatest substrate affinity were estimated to approximate unrivalled low values, viz. 15 to 20 ing of starch/l (cf. data of Table 6.1 and Fig. 6.7). The <em>K</em><sub><font size="-1">1/2</font></sub><em></em> values were not appreciably affected by SL (0.075-2.4), by the period of starch loading, and by the association of the amylases with the sludge (6.2, 6.3). Starch adsorption by starch sludge was observed to occur (Figs. 6.1 and 6.2) but did not initiate amylolysis at the surface of bacterial cell walls; it is unlikely to play a significant role in continuously operated starch activated sludge (6.2, 6.3).<p/>A comparison of the respiration rates of starch-grown sludge supplied with glucose or starch revealed that dextrins rather than glucose are the end products of extracellular starch hydrolysis (Fig. 6.10). The same conclusion was drawn on the basis of the kinetics of starch hydrolysis in sludge, viz. of the ratio of the critical (potential) to the actual amylase activity being only 1.2 (Fig. 4.3E; 6.5).<p/>Assuming that dextrins passing through the outer membrane of Gramnegative (amylolytic) bacteria consisted on the average of 5 glucose residues, that the kinetic parameters of sludge amylases were the same with starch and with dextrins with a DP of 6 or higher as the substrate, and that <em>K</em><sub><font size="-1">1/2</font></sub> approached <em>K</em><sub><font size="-1">diss</font></sub><em>,</em> the following statements relating to continuous starch loading of sludge I could be made (Table 6.4, section 6.5). a) Inadequate starch degradation occurred only if more than about 1/6 of the amylases was occupied with substrate. All amylolytic activities observed at SL = 0.075 and 0.24 far exceeded the critical values, but those at SL = 1.2 and 2.4 incidentally were lower than the critical activities (Figs. 4.6A and 4.7). b) On the average about 2 % (SL = 0.075) to 5.5 % (SI, = 2.4) of the surface- bound amylases was occupied with substrate. c) Average concentrations of higher dextrins (with a DP of about 6 or higher) ranged from only 0.4-0.5 (SIL = 0.075) to 1.2- 1.5 ing (SL = 2.4) glucose equivalents/litre of sludge-free liquid (cf. Table 4.3). d) The average residence times of these dextrins ranged from about 4 min (SL = 0.075) to 0.4 min (SL = 2.4). e) Average concentrations of total residual substrate (lower and higher dextrins) likely amounted to 5 - 10 % or less of the COD present in the sludgefree liquid, over the whole range of loadings applied; the remainder, corresponding with several per cents of the COD of the influent, probably consisted of refractory bacterial products which limited the process of purification (cf. Table 4.3).<p/>The acclimatization of glucose- or maltose-grown sludge I to starch loading was achieved by a population shift rather than by induction of enzyme synthesis of an existing population. When the activity of the preformed amylases was high enough to ensure immediate and complete starch removal, i.e. was higher than the critical value, the amylolytic bacteria increased from about 5 % of the total (viable) cell count to almost 100 % within 4 days (Figs. 4.3B and 4.4). DA <sub><font size="-1">spec</font></sub> of this sludge attained the value of starch-grown sludge after 3 - 5 days (Figs. 4.3 and 4.6A). When the preformed amylase activity was lower than the critical value, the increase of the amylolytic bacteria to almost 100 % was attained after 7 - 10 days. This retardation was probably due to concomitant growth of non-amylolytic bacteria at the expense of accumulated dextrins (Figs. 4.3E and 4.5). At continuous starch loading (SL = 0.3-2.4) the proportion of the amylolytic bacteria remained at 80-90 % of the total (viable) bacteria. At SL = 0.075 only about 60 % was amylolytic and about 1/3 of the viable bacteria apparently utilized only substrates derived from decaying cell material (entirely cryptic growth). Spill over of starch degradation products by amylolytic bacteria was concluded hardly to occur under conditions of complete starch removal (Tables 4.5 and 8.4).<p/>Starch sludge I did not attain a steady state with respect to its population composition (8.3 and 8.4), predominant regulation mechanism of amylase synthesis (8.5) and, dependent on SL, amylolytic enzyme activity (Fig. 4.6A). Several morphologically distinct flavobacteria usually succeeded each other as the predominant bacteria (8.4). The control of amylase synthesis in pure cultures of these bacteria was not very strict (8.4). Apparently, the amylase synthesis in the majority of the bacteria of starch sludge I was partly constitutive. However, experiments with whole starch sludge I indicated that the predominant regulatory mechanism of amylase synthesis can vary during the time of starch loading (8.5). Hence it was concluded that the regulation mechanisms of amylase synthesis had no predominant effect on the competition between amylolytic bacteria in activated sludge.<p/>Sludge I, deprived of added protein for a long period and supplied with starch and ammonium sulphate as only carbon and nitrogen nutrients (C/N = 5), displayed a high proteolytic activity which on the average decreased by 50 % with increasing SL with starch (Fig. 5.2), whereas the average amylolytic activity increased more than tenfold (Table 5.2). Therefore PA <sub><font size="-1">spec</font></sub> , at least in non-protein-fed activated-sludge systems, and DA <sub><font size="-1">spec</font></sub> can be regarded as intrinsic and extrinsic parameters, respectively (5.2. 1). Proteolytic bacteria on the average amounted to about 50 % of the total (viable count at SL = 0.075 and 0. 3, and to about 20 % at SL = 2.4 (Table 5.5A; Fig. 5.3). At SL = 0. 3, about half of the amylolytic bacteria was also proteolytic and the majority of the latter was also amylolytic (partly cryptic growth). The average proteolytic activity, calculated per (viable) proteolytic cell, sharply increased with decreasing SL (Fig. 5.4), probably due to the great stability of proteinases (as compared to that of amylases) in combination with large numbers of dead cells at low loadings. It is concluded that degradation of cells and reutilization of released cell material (turnover) play an important role in the metabolism of sludge I. PA <sub><font size="-1">spec</font></sub> can be regarded as a measure of the intensity of extracellular turnover of cell protein. An important part of the proteolytic enzymes in activated sludge of outdoor plants apparently served also turnover as was concluded from the fact that lowloaded Bennekom sludge showed the same PA <sub><font size="-1">spec</font></sub> as high-loaded Zeist sludge, whereas DA <sub><font size="-1">spec</font></sub> of the former was half of that of the latter (Table 5.3). The adverse effect of winter conditions on PA <sub><font size="-1">spec</font></sub> of these sludges did not or only to some extent result in the production of greater amounts of proteolytic enzymes, as contrasted to the compensation for the low-temperature effect on the amylolytic activity by the production of greater amounts of amylases (Fig. 4.12).<p/>Starch sludge II, consisting mainly of bacteria growing in suspension, showed a low PA <sub><font size="-1">spec</font></sub> and contained about 15 % proteolytic bacteria. Both parameters were hardly affected by SL with starch and mean cell residence time (Figs. 5.2 and 5.3; Table 5.5B). This indicates that PA <sub><font size="-1">spec</font></sub> cannot be increased by rising sludge age only, but that retention of flocculated biomass, as it occurs in sludge 1, is an important condition for attaining high PA <sub><font size="-1">spec</font></sub> values. This conclusion was confirmed by the observation that PA <sub><font size="-1">spec</font></sub> of the flocculated biomass fraction of high-loaded starch sludge I was severalfold higher than that of the suspended fraction of the same sludge, in contrast to DA <sub><font size="-1">spec</font></sub> which, on the average, was about equal in both fractions (Table 7.5). The high PA <sub><font size="-1">spec</font></sub> of sludge flocs apparently results from relatively high rates of death and/or autolysis of cells in flocs due to relatively unfavourable growth conditions, rather than from long mean cell residence times (7.3).<p/>Numbers of viable bacterial cells in starch sludge 1 amounted to about 130 x 10 <sup><font size="-1">7</font></SUP>bacteria/mg dry biomass at SL = 2.4 (t <sub><font size="-1">s</font></sub> = 1 day) as contrasted to about 6 x 107 bacteria/mg at SL = 0.075 (t <sub><font size="-1">s</font></sub> = c. 60 days); these viable counts were assessed to correspond with 90 and 4.5 %, respectively, of the bacterial cell mass (Table 5.6). The graph of the relationship between the viable fraction and the net specific growth rate observed at the different SL values tended to go through the origin (Fig. 5.6). The latter relation allowed the calculation of t* <sub><font size="-1">d</font></sub> (mean doubling time of viable cells) of 0.65 day at t <sub><font size="-1">s</font></sub> = 1 day and of an average maximum t* <sub><font size="-1">d</font></sub> of roughly 3 days. The latter is the actual one in sludges with a t <sub><font size="-1">s</font></sub> of more than about 10 days. The results suggest that very slow growing or dormant bacteria do not emerge in very low-loaded starch sludges I. The bacteria apparently are obliged either to multiply or to die and cannot maintain themselves for more than 3 days without growth and division. The viable fraction of low-loaded starch sludge II seemed to be higher than that of comparable sludge I, indicating higher maximum t* <sub><font size="-1">d</font></sub> values. As t <sub><font size="-1">s</font></sub> values of less than about 3 days do not occur in sludges of most of the sewage purification plants, specific growth rates of viable cells in these sludges may be assessed to vary maximally with a factor of about 2 above the minimum growth rate over the whole range of loadings applied. The parameter which reacts sharply to variation of SL, is the viability.<p/>From the relation between the growth yield of starch sludge I and t <sub><font size="-1">s</font></sub> , a maximum growth yield of 0.49 g dry biomass per g glucose equivalent and a biomass turnover coefficient of 0.05 day <sup><font size="-1">-1</font></SUP>were calculated (Table 5.7; Figs. 5.7 and 5.8). In very low-loaded sludge ( <em>Y</em> = 0.20 g biomass per g GE) the value of the turnover coefficient was smaller, probably mainly due to the great refractoriness of dead cell material. Autolytic degradation of the cell wall of dead cells was apparently the rate- limiting step in the extracellular turnover of the protein of these dead cells in sludge I. Heterobacteriolysis was not demonstrated in sludge.<p/>The standard amylase activity of starch sludges I decreased rapidly during starvation of these sludges (Figs. 5.9 and 5.10). Proteolytic enzymes only were responsible for the (initial) inactivation of the (cell-surface-bound) amylases (Figs. 5.10 and 5.12). This inactivation was prevented by saturating the amylases with starch (Fig. 5.10C). However, the very rapid and apparently irreversible inactivation of enzymes like Fungamyl 1600 (an α-amylase) and lysozyme, when added to activated sludge, was not attributed to proteolysis but to adsorption by the sludge (Figs. 7.3, 7.4 and 7.5; Table 7.6). During the enzymic degradation of amylases in starch sludge I, both amylases and proteinases probably remain cellwall-bound (5.5.2). The (initial) rate of inactivation of amylases in starving starch sludges I with different amylase activities obeyed first order kinetics according to the equation - d(DA)/dt = <em>k</em> i (DA) (Figs. 5.12, 5.13 and 5.14A and B). This is in agreement with the high <em>K</em><sub><font size="-1">1/2</font></sub><em></em> value of proteolytic enzymes of activated sludge (Fig. 6.11) and the very small amounts of cell surface-bound amylases in starch sludge, viz. roughly 0.006% of the biomass of the viable amylolytic bacteria which produced the enzymes (4.6.3). In fact, actual degradation rates of surface-bound amylases in situ might approximate 1/10,000 of the potential rates (6.6). The specific amylase inactivation rate ( <em>k</em> i) was proportional to PA <sub><font size="-1">spec</font></sub> (Fig. 5.14A). Under reactor conditions, at 20°C, the average relation was defined as <em>k</em> i = 1.25 PA<font size="-1"><sup>30°C</SUP><sub>spec</sub></font>over the whole range of PA <sub><font size="-1">spec</font></sub> values (0.038 to 0.87) found in the SL range of 0.075 to 2.4. The values of <em>k</em> i were much higher than those of <em>a</em> *, i.e. the decay rates of amylase were much higher than those of the entire biomass.<p/>As the initial amylase inactivation rates as observed in starch sludge I under starvation conditions will also be valid under conditions of continuous operation (5.5.1), the following model was proposed: gross rate of amylase synthesis rate of amylase wastage + rate of proteolytic inactivation of amylase = (μ <sub><font size="-1">net</font></sub> + <em>k</em> i) DA <sub><font size="-1">spec</font></sub> (Table 5.8). The relatively high turnover rate of amylases in low-loaded starch sludges is illustrated by the following calculation. The daily net production of amylases at SL = 0.075 was only roughly 11550 of that at SL = 2.4, whereas the daily gross production at the former loading was almost 1/30 of that at the latter, i.e. roughly proportional to SL. Consequently, imaginary DA <sub><font size="-1">spec</font></sub> values ( <em>k</em> i = 0 and <em>a</em> * = 0) were roughly equal for all SL values applied (Table 5.8; Fig. 4.8A). The decrease of DA <sub><font size="-1">spec</font></sub> actually observed with decreasing SL (Fig. 4.8A) is explained only by the much higher decay rate of amylases of dead cells compared to that of biomass. Starch sludge II is less proteolytic than comparable sludge I and obviously is considerably less dynamic, at least with respect to amylase degradation (Fig. 5.9B).<p/>Starch sludges I and II of comparable SL or t <sub><font size="-1">s</font></sub> generally differed in many respects. Sludges I grew mainly in flocs and contained many bacterial species, usually predominated by a few morphologically distinct flavobacteria. These sludges were highly proteolytic, contained a large proportion of proteolytic bacteria, especially at low SL, and showed a high amylase degradation rate; they had a large fraction of dead cells and showed a relatively low growth yield at low SL. Sludges II consisted mainly of dispersed or loosely aggregated bacterial cells, were predominated by one or a few bacterial species, once even for 500 days by a <em>Nocardia</em> -like bacterium (Plates 4.9, 4.10 and 4.11), showed a low PA <sub><font size="-1">spec</font></sub> , contained a relatively small fraction of proteolytic bacteria, showed a low amylase inactivation rate, probably a large viable fraction, and a high growth yield.<p/>It is concluded that the retention (feedback) of flocculated biomass is a condition necessary for the predominance of the flocculated bacteria over dispersed cells. The relatively adverse growth conditions within the flocs, creating different ecological niches, are the cause of most of the differences observed between sludges I and II.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Mulder, E.G., Promotor, External person
Award date24 Oct 1979
Place of PublicationWageningen
Publication statusPublished - 1979


  • sewage sludge
  • properties
  • microbiology
  • waste water treatment
  • water treatment
  • activated sludge
  • aeration
  • carbohydrates

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