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Ind. Eng. Chem. Res. 2001, 40, 5110-5119
Sequencing Batch Reactor: Influence of Periodic Operation on Performance of Activated Sludges in Biological Wastewater Treatment Davide Dionisi,† Mauro Majone,*,† Valter Tandoi,‡ and Mario Beccari† Department of Chemistry, University “La Sapienza”, Piazzale Aldo Moro 5, 00185 Rome, Italy, and Water Research Institute, National Research Council, Via Reno 1, 00198 Rome, Italy
This paper presents the main results obtained during several years of studies with periodically fed biological reactors (sequencing batch reactors, SBRs) from the perspective of wastewater treatment. Substrate (acetate) removal mechanisms under transient conditions were studied both in the SBR and in batch tests, as were their dependence on the applied operating conditions in the SBR (organic load rate and/or sludge age, feed length, aerobic or anoxic conditions). The most general evidence was the relevant role of storage (usually representing about 70% of the overall observed yield) and the negligible role of growth (usually less than 10%) during acetate removal for all tested conditions. However, when acetate was present for a long enough time, physiological adaptation could occur, and the growth contribution could become more important. The applied operating conditions affected the biomass behavior. In particular, with an increase in the applied organic load rate, the observed yield in the SBR and the acetate removal rate in the batch tests decreased, whereas with an increase in the feed length (other conditions being the same), the relevance of the storage response decreased. The role of dynamic conditions in selecting a floc-forming or filamentous biomass was also investigated. Even though a floc-forming biomass usually developed, filament growth was also sometimes observed. Thus, the usual assumption that filaments are less able than floc-formers to store the substrate should not be considered as an absolute rule. Finally, an empirical kinetic model, including growth and storage both in parallel and in sequence, was defined and applied to describe and interpret the experimental results. Introduction Periodic processes (e.g., processes carried out in sequencing batch reactors, SBRs) have proven to be highly effective for the biological treatment of wastewaters. As an example, high rates, good bulking control, and nutrient removal have been reported as useful features of periodic processes for water treatment.1,2 The application of periodic processes is widening to several polluted systems, also including air and soil. Periodic processes are highly flexible. Sequencing batch reactors allow for the performance, in a single reactor, of the sequence of steps needed to reach the required effluent standards. Thus, spatial sequences of steps, typical of traditional activated sludge plants, are replaced by temporal sequences. Typical cycles in SBRs include feed-reaction-settling-draw, where the reaction time can be further split into different phases (e.g., anoxic and aerobic for nitrogen removal). This temporal sequence of events can be changed and adapted to influent conditions much more easily than spatial sequences. Moreover, a periodic process has some additional degree of freedom with respect to the corresponding continuous-flow steady-state process: as an example, the biomass behavior in a continuous-flow stirred tank reactor (CFSTR) is fully defined by the sludge residence time (the reciprocal of the growth rate), whereas in a SBR, the length of the cycle and the length * Corresponding author. E-mail:
[email protected]. Tel.: +390649913646. Fax: +3906490631. † University “La Sapienza”. ‡ National Research Council.
of the feed can be varied for the same sludge residence time. These variables are key performance factors of SBRs, which, until now, have been mainly designed and operated on the basis of practical experience. In periodic processes, the biomass grows under transient (unsteady) conditions. As an example, within each cycle, the biomass is exposed to an excess of substrate during the feed period and then to its deficiency during most of the reaction period (the so-called feast-andfamine regime). Under such conditions, growth becomes unbalanced, which means that microorganisms are not able to adapt their growth rates to the changing conditions on the time scale of the change. Different mechanisms (e.g., internal accumulation of the substrate, storage of substrates as internal polymers, excretion of intermediate metabolites) can become important as possible responses to the transient conditions. The response of the biomass to the transient conditions depends on its microbial composition and physiological state, which are, in turn, defined by the operating conditions in the SBR. For that reason, periodic processes exert a strong pressure on microbial populations, thus establishing a selection in favor of more adaptable microorganisms. Usually, the selection of floc-forming microorganisms over filamentous ones is achieved, and the ability of the sludge to settle is often improved in systems with large substrate gradients, such as SBRs. However, the physiological reasons for such different behaviors have not yet been clarified. From the modeling point of view, the mathematical description of the transient behavior of the biomass as a result of its previous physiological adaptation requires
10.1021/ie001008k CCC: $20.00 © 2001 American Chemical Society Published on Web 06/12/2001
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a structured approach in which the active biomass is divided into several categories (e.g., precursors, enzymes, RNAs, DNA) in reciprocal relationships with each other to give the overall behavior.3 Nevertheless, because of the complexity and the requirement of too much knowledge about the internal composition of the biomass, structured models have not yet found wide application in wastewater treatment. On the other hand, the presently used models do not take any account of the physiological adaptation of the biomass and give a much more simplified description, as in ASM 3,4 where storage is considered the only phenomenon responsible for substrate removal and growth occurs only for stored products, independent of the operating conditions. To take into account the effect of physiological adaptations of the biomass on the transient response without a detailed knowledge of the biomass internal composition, the use of time-dependent kinetic parameters has been also proposed.5 The present research group has done extensive research on the behavior of biomass that has been cultivated under periodic conditions (by means of SBRs). The aim of the research was to investigate substrate removal mechanisms under transient conditions, with primary attention to the role of storage phenomena in overall solids formation. To this end, a SBR was used to cultivate mixed cultures (coming from activated sludge inoculum) on acetate under a range of different operating conditions (organic load rate, cycle length, feed length, aerobic or anoxic conditions). The transient response of such adapted and selected mixed culture was also studied by means of batch tests performed under conditions that were the same as or different from the conditions of cultivation (in terms of substrate/ biomass ratio and redox condition). The different mechanisms contributing to substrate removal (oxidation, growth, storage, and accumulation) were determined on the basis of the COD balance. The main results obtained by this research group6-9 are summarized and discussed in this paper. Finally, a kinetic model is defined and applied to describe most observed features of biomass behavior under dynamic conditions. Experimental Section SBR Operation. A SBR was utilized to cultivate mixed biomass under different operating conditions. A typical cycle of the SBR consisted of a preselected time (usually a few minutes) of feeding substrate, a substantially longer period (usually several hours) of reaction, and the final withdrawal of the mixed liquor from the mixed vessel. No settling phase was used, so all excess biomass was withdrawn with the mixed liquor. In this way, the solid retention time was equal to the hydraulic retention time, thus making possible very good control of the age and reciprocal specific growth rate of the sludge. This particular arrangement was also chosen to study population dynamics in the sludge resulting only from biological competition for the substrate between floc-formers and filaments, with no additional selection effect due to the settling phase. The temperature was set at 25 °C, the pH was 7.5, the electron acceptor (oxygen or nitrate) was always in excess, and acetate was the only carbon source available in a well-defined mineral medium. The operating conditions that were varied during the experiments were the dilution rate and/or the organic load rate (OLR), the feed length, the number of cycles per day, and the identity
of the electron acceptor (oxygen or nitrate). Runs A were performed with the same OLR and sludge age but with continuous (A1) or intermittent (A2) feed.6 Run B was the same as run A2 but starting from a different inoculum.7 Within runs C and D, the OLR was changed. Within runs C (C1-C4), the OLR was increased by increasing the number of cycles per day (thus also decreasing the sludge age), whereas within runs D, the OLR was increased by increasing the acetate concentration in the feed while maintaining the same sludge age and cycle length (D1-D4).8 To avoid reaching inhibiting acetate concentrations in the medium during the feed, runs D1-D4 with increasing OLRs were carried out with a simultaneous increase in the feed length. In runs E (E1-E3), the biomass was cultivated under anoxic conditions,9 and the OLR was increased by increasing the acetate concentration in the feed (as in run D). In run E4, the operating conditions were the same as in run E2, but the feed length was longer. The SBR cycles were characterized by measurements of the biomass concentration and sludge volume index (SVI) at the end of each cycle. The pseudo-steady state (i.e., a periodic steady-state referred to that particular time of the cycle) was assumed to be reached after at least three sludge ages from the initial inoculum provided that the solids concentration was constant with time (within 15% standard deviation). The pseudosteady state was further maintained for at least five sludge ages and, in any case, for the time necessary to carry out batch tests. The morphotypes of filamentous microorganisms were identified by optical microscopy.10 The observed yield at the end of each cycle was calculated according to the ratio between the biomass concentration and the influent acetate concentration. Less frequently, time profiles of acetate, storage products, and electron acceptors were monitored during the cycle. Further details on the single SBR runs, as well as on the following procedures, are given in the references cited.6-9 Batch Tests. To better investigate the effect of different SBR cultivation conditions on the transient behavior of the biomass (with particular reference to the feast period), batch tests were also performed for each SBR run after pseudo-steady state was reached. The biomass was withdrawn at the end of a cycle, transferred to a smaller reactor, diluted to the chosen concentration (usually 200 mg of COD/L), and then spiked with the substrate (usually 100 mg of COD/L in short-term batch tests and at least 1000 mg of COD/L in long-term tests). The sludge in the batch reactor was sampled at regular intervals for analytical determinations of acetate, storage compounds (specifically, polyhydroxybutyrate, PHB), and ammonia. Ammonia consumption was related to active biomass formation, on the basis of an average biomass composition (see Material Balances); the possible interference of ammonia consumption for dissimilative purposes (nitrification) was avoided by inhibihiting nitrification with thiourea. Before (at least 1 h) and during the test, the batch reactor was maintained under bubbling of air (aerobic tests) or of nitrogen (anoxic tests). The oxygen or nitrate/ nitrite consumption associated with substrate removal for energy production was also measured and used to calculate the overall observed yield (see Material Balances). To measure the oxygen uptake rate (OUR), aeration was interrupted at intervals, and the dissolved
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oxygen decrease was measured as a function of the time. The OUR was calculated by also taking into account oxygen transfer through the air-medium interface. The oxidized COD was directly calculated by integrating the OUR versus time. For each set of cultivation conditions, at least two replicated batch tests were performed. In run D2, four replicated batch tests were performed, and the obtained standard deviation of the mean was used to calculate the standard deviation associated with each set of batch tests (taking into account the different number of replicates). Analytical Methods. The anions (acetate, nitrate, and nitrite) were measured on filtered samples (0.45 µm porosity) by ion chromatography (Dionex, AG14 and AS14 columns, eluent 4.8 mM Na2CO3/0.6 mM NaHCO3, flow rate 1.5 mL/min, regenerant 50 mN H2SO4, flow rate 1.0 mL/min). The ammonium was measured on filtered samples by the direct Nessler method (APHA, 1995). For PHB determination, the sludge was immediately treated with a NaClO solution (7% active Cl2). Then, PHB was extracted, hydrolyzed, and esterified to 3-hydroxybutyric methyl ester to be determined by gas chromatography.11 Material Balances. The obtained data were used to calculate the different substrate removal mechanisms by means of the COD balance. It was assumed that the substrate removed from the liquid phase (-∆S, acetate) could be recovered in solids as PHB (∆XPHB, storage), other internal intermediates or precursors (∆Xacc, accumulation), and active biomass (∆Xgro, growth) or lost (-∆O2, oxidized for energy needs resulting from accumulation, storage, growth, and maintenance). Each term of the balance was expressed as COD. On the basis of preliminary experiments, the possibility of release into the liquid medium of metabolic intermediates was not considered. The COD balance was then defined by the equation
(-∆S) ) ∆XPHB + ∆Xacc + ∆Xgro + (-∆O2) The acetate and PHB concentrations were converted to mg of COD/L according to oxidation stoichiometry. The substrate transformed into active biomass was calculated from the ammonia uptake using an assumed average biomass nitrogen content (10% of the dry weight). The accumulation term was calculated as the difference to redress the balance. In early experiments where the ammonia concentration was not measured, the contribution of accumulation and growth were lumped together. By referring each contribution to the overall substrate removal, “observed yields” were calculated for storage, accumulation and growth via
YSTO )
∆XPHB ∆Xacc ∆Xgro , YACC ) , YGRO ) (-∆S) (-∆S) (-∆S)
The overall observed yield, YOBS (formation of solid COD, independently from its composition), lumping all contributions together was calculated according to
YOBS ) 1 -
(oxidized COD) (removed COD)
Results and Discussion General Evidence. In Figures 1 and 2, respectively, typical cycles of aerobic and anoxic SBR are given. (In
Figure 1. Typical SBR cycle under aerobic conditions (biomass from run C3): (a) acetate and PHB, (b) dissolved oxygen, (c) contributions to the overall COD removed. Solid lines refer to simulated profiles according to the kinetic model (Figure 8, Tables 2 and 3).
all figures, the solid lines show simulation results according to the model presented in Figure 8 and Tables 2 and 3, while the dashed lines simply link experimental points.) It can be seen that, in both cases, the acetate is present in the medium only for the first few minutes of the cycle; indeed, it is quickly removed while PHB is stored. PHB is the only source of carbon and energy for most of the cycle (with the exception of material coming from cellular decay). Correspondingly, in the first part of the aerobic cycle where acetate is present, dissolved oxygen undergoes a sharp drop, whereas in the second part where acetate has been depleted, dissolved oxygen rises again to its initial value. In the anoxic cycle, nitrate in the feed is quickly removed, and nitrite is formed during acetate depletion, whereas both nitrate and nitrite are slowly removed after acetate depletion. Both observations clearly confirm that acetate consumption is significantly faster than PHB consumption. The typical conditions of feast and famine are therefore applied to the biomass.
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Figure 2. Typical SBR cycle under anoxic conditions (biomass from run E2): (a) acetate and PHB, (b) nitrate and nitrite.
In Figure 3, a typical profile of an aerobic batch test (performed during run E on biomass cultivated under anoxic conditions) is shown for acetate and PHB (part a) as well as for ammonia and OUR (part b). Table 1 presents a summary of the operating parameters and pseudo-steady-state performance of the SBR, as well as average results of the batch tests. The obtained data in the batch tests show a clear picture of transient substrate metabolism for biomass previously grown under periodic conditions. PHB storage always gives a relevant contribution to solids formation, under both aerobic and anoxic conditions. Storage yields are in the range 0.45-0.69 COD/COD under aerobic conditions (60-100% of the overall observed yield), whereas they are in the range 0.27-0.55 COD/COD (40-90% of the overall observed yield) under anoxic conditions (with the exception of run E4). In run A1 (continuous feeding), the storage yield is the lowest (0.40 COD/COD) among the aerobic tests, and in run E4 (slow and long feed), it is the lowest (0.19 COD/COD) among the anoxic tests. The growth contribution (formation of active biomass) to overall solids formation is usually very low, as can be argued on the basis of the values of the observed yields, which are always much higher than usually assumed for balanced growth on acetate (0.50 COD/ COD).12 Aside from this indirect evidence, in the latest batch experiments (batch tests done either under aerobic or anoxic conditions on biomass cultivated under anoxic conditions), active biomass formation was independently estimated through experimental determination of ammonia consumption (see Experimental Section). Figure 3 (aerobic batch test) shows that there is no relevant change in the ammonia profile at the spike of acetate, whereas the OUR suddenly increases by 1 order of magnitude, i.e., the energy production rate is changing, whereas the growth rate is not. Correspond-
Figure 3. Typical aerobic batch test at low S/X (biomass from run E2): (a) acetate and PHB, (b) OUR and ammonia, (c) contributions to the overall COD removed. Solid lines refer to simulated profiles according to the kinetic model (Figure 8, Tables 2 and 3).
ingly, from the COD balance, the growth contribution to acetate removal is usually very low, under both aerobic and anoxic conditions (in the range 0.0-0.1 COD/COD). Independent estimation of the growth contribution through ammonia consumption also made it possible to show that, under some conditions, growth and storage do not match the overall solids formation. Hence, the hypothesis of internal accumulation of compound(s) was formulated, according to other literature evidence.13,14 Accumulation means that the substrate is transported into the cell and maintained there as such or as a lowmolecular-weight intermediate. According to that hypothesis, accumulation requires less energy than storage, where a polymer is synthesized, but it is less likely because of unfavorable gradients and osmotic pressure.13 Although indirect, strong evidence of accumulation was also found in the time profile of the observed yields. In several experiments, the overall observed yield
0.77 (3.2%) 0.60 (6.4%) 0.17
0.33 (11.5%)
0.46 (8.5%)
0.60 (3.2%) 0.40 (6.4%) 0.20
135 350
>1000 488
750 (8.5%)
3 4 2 oxygen
3 cont cont oxygen
150 (8.5%)
355
355
A2
0.33 1067
0.33 1067
A1
0.68 (3.2%) 0.69 (6.4%) 0
828 (8.5%)
0.52 (8.5%)
>1000 556
3 4 2 oxygen
355
0.33 1067
B
effect of initial inoculum (vs run A2)
effect of continuous vs intermittent feeding C1
C3
0.75 16 3 oxygen
1420
1.32 1067
C4
0.37 32 3 oxygen
2840
2.64 1067
0.68 (3.2%) 0.58 (6.4%) 0.10
670 (8.5%)
0.50 (7.6%)
159 538
0.74 (3.2%) 0.54 (6.4%) 0.20
1197 (8.5%)
0.59 (5.3%)
238 630 0.64 (16.8%)
2028 683
0.76 (3.2%) 0.55 (6.4%) 0.21
1400 (8.5%)
0.74 (3.2%) 0.54 (6.4%) 0.20
993 (8.5%)
Batch Tests
0.60 (7.2%)
231 641
D1
1 12 3 oxygen
1067
1 1067
0.74 (3.2%) 0.50 (6.4%) 0.24
1096 (8.5%)
0.55 (15%)
136 588
SBR Pseudo-Steady State (End of Cycle)
1.5 8 3 oxygen
710
0.66 1067
C2
0.75 (2.2%) 0.47 (4.5%) 0.28
964 (6%)
0.47 (1.7%)
102 1247
1 12 10 oxygen
2668
1 2668
D2
0.76 (3.2%) 0.49 (6.4%) 0.27
603 (8.5%)
0.42 (5.9%)
152 1795
1 12 10 oxygen
4268
1 4268
D3
0.73 (2.6%) 0.60 (5.3%) 0.13
688 (6.9%)
0.36 (5.2%)
294 3047
1 12 10 oxygen
8536
1 8536
D4
effect of increasing OLR by increasing feed concentration
effect of increasing OLR by decreasing sludge age
3 4 3 oxygen
355
0.33 1067
runs D
runs C
0.65 (2.6%) 0.27 (5.3%) 0.08 0.30
756 (6.9%)
0.28 (11.6%)
250 296
6 4 12 nitrate
178
0.17 1067
E1
0.62 (3.2%) 0.55 (6.4%) 0.01 0.06
583 (8.5%)
0.20 (11%)
200 432
6 4 12 nitrate
355
0.17 2133
E2
0.68 (3.2%) 0.40 (6.4%) 0.06 0.22
494 (8.5%)
0.16 (11.1%)
200 692
6 4 12 nitrate
710
0.17 4267
E3
E4
0.55 (3.2%) 0.19 (6.4%) 0.14 0.22
454 (8.5%)
0.21 (7.7%)
200 444
6 4 24 nitrate
355
0.17 2133
effect of increasing OLR by increasing feed concentration under anoxic conditions
runs E
a Batch tests were performed with the same electron acceptor as the SBR. Values in parentheses show the standard deviations of the mean (taking into account the different number of replicates).
Ygro (COD/COD) Yacc (COD/COD)
Ysto (COD/COD)
specific acetate uptake rate (mg of COD/ g of CODh) Yobs (COD/COD)
SVI (mL/g) biomass (mg of COD/L) observed yield (COD/COD)
dilution rate (day-1) influent acetate concentration (mg of COD/L) organic load rate (mg of COD/Ld) sludge age (day) cycles/day feed length (min) electron acceptor
SBR operating conditions
run B
runs A
Table 1. Summary of Experimental Evidencea
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Figure 5. Effect of organic load rate applied to the SBR on observed yield at the end of the cycle: (a) runs with costant sludge age (runs D, 1 day; runs E, 6 days), (b) runs with varying sludge age (runs C, 0.37-3 days). Error bars at (1 standard deviation (when not visible, they are within the experimental point).
Figure 4. Typical aerobic test at high S/X (biomass from run B): (a) removed acetate and PHB, (b) OUR, (c) contributions to the overall COD removed. Solid lines refer to simulated profiles according to the kinetic model (Figure 8, Tables 2 and 3).
continuously decreased during acetate removal, whereas the storage observed yield continuously increased, so in the first part of the experiment where the overall observed yield was very high and the storage yield accounted for only a minor part of it, the missing part of the COD balance was higher.6 Other indirect evidence of accumulation lies in the PHB maximum values, which are often reached some time after acetate depletion.14 Such behavior is consistent with a picture in which accumulation comes first and storage then follows. In other words, accumulation is the first mechanism to remove the substrate and maintain it inside the cells with no lag phase and low energy demands. The accumulated substrate is then stored with some (though short) delay and with some extra requirement of energy. Nevertheless, the accumulated compound(s) has (have) not yet been individuated, so other possible explanations might arise. The described substrate metabolism was typical of batch tests performed with a low initial substrate/
biomass ratio (0.5 COD/COD, short-term tests). At these ratios, the time necessary for acetate depletion was short (usually less than 2 h), and the acetate consumption and PHB storage rates were fairly constant. Batch tests at higher initial substrate/biomass ratios (longterm tests) have also been performed7 (Figure 4). In these cases, the main initial removal mechanism was still storage, but the storage rate decreased with time. This decrease was attributed to saturation of the storage capacity when PHB was about 50% of the overall dry weight of the biomass. In contrast, the acetate and oxygen uptake rates increased with time, and the COD balance showed that growth was becoming more important. This indicates that the transient behavior of the biomass is highly dependent on the time scale of the experiment, which, in turn, is determined by the initial substrate/biomass ratio. Effect of Operating Conditions on Biomass Behavior. Along with this general evidence, a primary aim of the presented research was to investigate how the applied periodic conditions influence the biomass behavior. With reference to pseudo-steady-state performance, Figure 5 compares the observed yields as functions of the OLR at constant (part a) or varying (part b) sludge age. It is evident that the observed yield decreases as the OLR increases when the sludge age is maintained constant (runs D and E, where the increase in the OLR was obtained by increasing the substrate concentration). This behavior is not predicted by the standard chemostat theory: according to this theory, the observed yield is fully defined by the sludge age (or by its reciprocal, the specific growth rate), whereas it is independent of the substrate concentration (i.e., the organic load rate). In contrast, in runs C where the OLR
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Figure 6. Effect of organic load rate applied to the SBR on acetate uptake rates in low-S/X batch tests: (a) runs with costant sludge age (runs D, 1 day; runs E, 6 days), (b) runs with varying sludge age (runs C, 0.37-3 days). Error bars at (1 standard deviation.
Figure 7. Effect of the length of SBR feed on the acetate uptake rate and yield in low-S/X batch tests.
increase is obtained by decreasing the sludge age (by the shortening of the cycle length), the observed yield slightly increases, as expected. Also the transient behavior seems to be influenced by the OLR applied to the parent SBR. Figure 6 shows acetate uptake rates in batch tests. It is evident that there is a tendency toward lower uptake rates with increasing OLR at constant sludge age (runs D and E). In runs C where the OLR was increased by decreasing the sludge age, the acetate uptake rate shows a maximum. A maximum value of acetate uptake rate vs sludge age has already been reported.12,16 The length of the feed period also affects the transient response of the biomass. Figure 7 compares the batch test results for biomass coming from runs E2 and E4, which differ only in the length of the feed period. Both a lower acetate uptake rate and a lower storage yield are obtained for the biomass adapted to the slower feed rate in the parent reactor. This effect of feed length can
be easily explained: the longer the feed length, the less dynamic the conditions for biomass growth, and therefore, the less relevant the storage response. In the limit of very slow feed, the system will evolve to a continuousflow steady state, as in run A1 where the storage role is minimized. Hence, it is clear that the previous growth conditions affect the dynamic response of the biomass. In a mixed culture, this effect can be due to a change in both the microbial composition of the mixed culture and the physiological state. In fact, in mixed cultures, a different microbial composition corresponds to each set of applied cultivation conditions (see also the next paragraph), and different species dominate the biocenosis. To distinguish between the two effects, pure culture studies are needed. Indeed, in a pure culture, only physiological adaptation can occur with a change in the applied growth conditions. As an example, the dynamic responses of A. globiformis and S. natans cultivated under continuous and intermittent feed of glucose in the range of culture age 0.13-2.1 days have been compared.16 For both microorganisms, the previous culture age affected the maximum rate of substrate removal in batch tests. This shows that physiological adaptation for a single microorganism also certainly occurs. The presence of aerobic vs anoxic conditions does not seem to exert a strong effect on the transient response of the biomass. In particular, parallel batch tests performed under aerobic and anoxic conditions on biomass cultivated under only anoxic conditions (runs E) showed very similar behaviors in terms of both rate and relative importance of storage. Effects on Microbial Composition of the Biomass. Another effect that is usually reported for biomass grown in the presence of a substrate gradient is kinetic pressure in favor of floc-forming microorganisms over filamentous ones, with a consequent improvement in the settling properties of the sludge. The positive effect of the substrate gradient is usually explained by higher storage rates under transient conditions for flocformers than filaments.17 SVI data in Table 1 and microscopic morphological examinations confirmed that a floc-forming biomass usually developed under the transient conditions that were applied. However, the kinetic advantage of floc-forming organisms over filamentous ones cannot be taken as a general rule, as runs B and C4, showed that a filamentous biomass can sometimes proliferate. In both runs, the characterization of the filamentous biomass that developed showed that the prevailing filaments were able to store PHB at a high rate,7,8 as is typical of floc-forming organisms. The fast storage response was therefore the reason for the occurrence of filaments in the periodic system. However, in runs A2 and B, identical operating conditions were applied, but only in the latter case did filaments prevail; hence, it is evident that the occurrence of filaments can strongly depend on the initial reactor inoculum. Modeling. To describe the experimental behavior observed under such a large range of operating conditions, a kinetic model has been developed. A conceptual sketch of the model is presented in Figure 8, and the processes, material balances, and rates are listed in Table 2 in the form of a stoichiometric matrix (the kinetics of formation or consumption of each component in each metabolic process is obtained by multiplying the corresponding stoichiometric coefficient for the process rate expression).5
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Figure 8. Scheme of the kinetic model of substrate removal under dynamic conditions.
The proposed model assumes two or three steps in series for overall substrate metabolism. The first step is always internal accumulation (i.e., the substrate is transported into the cell and maintained inside as such or in slightly metabolized form). Then, the accumulated compound can be used for growth either directly (dashed line) or through previous storage and subsequent use of the stored product (solid line). From the kinetic point of view, growth (of either accumulated or stored compounds) requires physiological adaptation, so the growth rate is time-dependent. In the model, the initial maximum growth rate depends on the previous famine period (in that is expressed as a function of residual PHB) and increases according to a shifting-order dependence on time. In contrast, the transient response in terms of accumulation and storage is immediate (i.e., there is neither a lag phase nor adaptation of the biomass physiology), so time-invariant parameters apply for this part of the model. On the other hand, a “saturation” term is included for both accumulation and storage, which describes the expected or observed decrease of the formation rate when the internal concentration is increasing (of course, there is no saturation term for growth that is an autocatalytic phenomenon). Thus, the two- and three-step pathways are simultaneously available, their relative importance being dependent on the time scale of the transient condition (where the external substrate is present), which is, in turn, dependent on the initial S/X ratio. As usual, reactions on external soluble substrates are represented as shifing-order reactions with respect to the substrate concentration, whereas reactions on intermediate substrates are represented as shifting-order with respect to the internal substrate/active biomass ratio.3,12 For the sake of simplicity, the decay and maintenance metabolisms are represented only in terms of consumption of the stored polymer. To describe the observed decrease in the overall yield with the increase in the organic load rate, an empirical term was added that links the maintenance coefficient to the intracellular fraction of the accumulated compound. It is assumed that maintaining higher concentrations of accumulated compounds (as happens at higher organic load rates) requires more maintenance energy to overcome unfavorable gradients and osmotic pressure. The proposed model was used to describe the kinetics of substrate metabolism and related variables under a range of operating conditions, namely, time profiles of acetate, PHB, and oxygen in a typical SBR cycle (Figure 1); time profiles of acetate, ammonia, PHB, and OUR in typical low-S/X batch tests (Figure 3); and time profiles of removed acetate, PHB, and OUR in typical high-S/X batch tests (Figure 4).
The parameter values (Table 3) were adjusted according to the following criteria: (1) The four stoichiometric yields, which give accumulated compound per unit of substrate removed (YXacc/S, YXPHB/Xacc, YXh/XPHB,YXh/Xacc) were assumed to be invariant for all tested conditions and were preliminarly adjusted on the basis of experimental profiles of all tests. (2) The parameters Kacc, ∞ were found not to be very influential bPHB, and µmax and were therefore considered invariant for all tested conditions and fixed according to literature results. (3) The parameter facc was fixed at the maximum value experimentally determined from the COD lack in batch tests and was considered invariant for all tested conditions. (4) The parameters fPHB and KPHB, relevant only in high-S/X batch test, were adjusted according to the experimental profile of that test and then held fixed in the other tests. (5) The remaining parameters (qacc, qPHB, KgPHB, Kgacc, Kt) were adjusted independently in each test. Parameter estimation was performed by multivariate nonlinear regression according to experimental profiles of all available experimental variables (Scientist, Micromath, 1994). From Figures 1, 3, and 4, it can be seen that the proposed model describes the experimental profiles quite well for all tested conditions. Accordingly, parts c of Figures 1, 3, and 4 show time profiles of the COD balance. In SBR cultivation or in low-S/X batch tests, the time scale of the feast period is very short, so both physiological adaptation and internal saturation are very small and the storage response is the main mechanism of substrate removal. On the other hand, when the time scale is longer (e.g., in high-S/X batch tests), both physiological adaptation and internal saturation occur, and direct growth with no previous storage becomes more relevant. In that case, the model describes the time sequence of the two phenomena well. First, both the acetate and oxygen uptake rates decrease as a result of saturation of the storage capacity (as also shown from the PHB profile), and then, they both increase again because of the increase in the growth rate. The presence of intermediate accumulation is evident in the early part of Figure 1c: the accumulated compound(s) acts (act) as an internal pool of substrate that increases when external substrate is present and quickly decreases after the external substrate’s depletion. This possible presence of a low-capacity accumulation “buffer”, even if it still has to be experimentally demonstrated, can conceptually justify a variety of experimental evidence, such as the formation of PHB after acetate depletion,14 the high yield and quick decrease in the substrate removal rate when storage is slow,6 and the lack of recovery in the COD balance.9 Such experimental evidence seems to be linked to stronger stress conditions such as low organic load or long starvation. It is noteworthy that most parameters can be held constant among the different tests. The different physiological states for sludges cultivated under different operating conditions is thus represented through adjustment of a relatively low number of parameters. However, those parameters have to be adjusted on the basis of each experimental profile, thus reflecting the empirical nature of the kinetic expressions that have been used. As yet, we have not been able to link the parameters to the previous operating conditions (i.e., to the “history of the biomass"). A further step in the
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Ind. Eng. Chem. Res., Vol. 40, No. 23, 2001
Table 2. Stoichiometric Matrix Relative to the Model Described in Figure 8 components process
So
1
accumulation of acetate
1-
storage of accumulated compound
1-
1 YXPHB/Xacc
growth on stored producta
1-
1 YXh/XPHB
growth on accumulated compounda
1-
1 YXh/Xacc
maintenance a
YXacc/S
S
-
XPHB
Xacc
1
Xh
1
-
-
1 YXh/XPHB -
-1
(
)
Xacc/Xh S qacc X 1Kacc + S h facc
1
YXacc/S
process rate
(
)
XPHB/Xh Xacc/Xh qPHB X 1KPHB + Xacc/Xh h fPHB
1 YXPHB/Xacc -1
1
XPHB/Xh µmax(t) X KgPHB + XPHB/Xh h
1 YXh/Xacc
1
Xacc/Xh µmax(t) X Kgacc + Xacc/Xh h
(
bPHBXh 1 +
-1
)
Xacc/Xh facc
t Kt + t
Time dependence of growth rate is expressed as µmax(t) ) µmax(to) + [µ∞ max - µmax(to)]
Table 3. Parameter Values of the Model in Figure 8 parameter qacc Kacc facc qPHB KPHB fPHB ∞ µmax KgPHB Kgacc Kt bPHB YXacc/S YXPHB/Xacc YXh/XPHB YXh/Xacc
SBR cycle
low-S/X batch test
high-S/X batch test
units
1.81 4 0.1 1.50 0.038 1 0.25 0.08 0.024 0.025 0.008 0.97 0.77 0.66 0.54
0.43 4 0.1 1.92 0.038 1 0.25 0.1 8 46 0.008 0.97 0.77 0.66 0.54
0.58 4 0.1 6.45 0.038 1 0.25 330 0.008 7.5 0.008 0.97 0.77 0.66 0.54
mg of CODS/(mg of CODXh h) mg of COD/L mg of CODXacc/mg of CODXh mg of CODXPHB/(mgXh h) mg of CODXPHB/mg of CODXh mg of CODXPHB/mg of CODXh 1/h mg of CODXPHB/mg of CODXh mg of CODXacc/mg of CODXh h 1/h mg of CODXacc/mg of CODS mg of CODXPHB/mg of CODXacc mg of CODXh/mg of CODXPHB mg of CODXh/mg of CODXacc
development of the model would be to link the rate of the different processes to the intracellular composition of the biomass, in terms of key constituents (e.g., RNA, specific enzymes). Conclusions When biomass grows under dynamic conditions, as happens in the periodic processes applied in advanced water treatment, substrate is removed with mechanisms others than simple growth and oxidation. Under these conditions, storage in the form of PHB is the main mechanism of acetate removal, thus also becoming a key factor in the competition between microorganisms in the activated sludge. Biomass behavior is affected by the applied periodic conditions of cultivation. Related effects can be of practical importance in SBR operation and design, such as effects on the overall observed yield, the maximum acetate uptake rate, and the settling properties. All effects present some unexpected trends. In particular, an increase in the applied OLR at constant sludge age causes a decrease in the average solids yield in the SBR. This leads to a decrease in sludge production per unit of removed substrate under dynamic conditions, whereas the opposite trend would be expected under steady-state conditions. Analogously, the maximum acetate uptake rate under transient conditions decreases when the OLR increases (sludge age being constant). Moreover, for the
same amount of substrate fed to the reactor (i.e., the same OLR), the maximum acetate uptake rate and storage yield are reduced when the feed length is increased. With reference to microbial population dynamics, our experimental work confirms that a flocforming biomass is usually selected in SBRs. However, there are important exceptions, and in two runs, a filamentous biomass also developed. The reason for the occurrence of filaments was found to be their high storage rate. Thus, the usual assumption that filaments are less able than floc-formers to store the substrate should not be considered as an absolute rule, and mechanisms of kinetic selection in activated sludge systems should be more fully investigated. On the basis of the reported results, an empirical kinetic model was deleoped and applied to typical tests in order to have a quantitative mathematical description of most observed features of biomass behavior under dynamic conditions. The model considers the possible competition of two pathways for substrate removal, with or without intermediate storage before growth. The relative relevance of the two mechanisms is established from the time scale of the experiments and the speed of physiological adaptation. The latter is simply described through a time-dependent parameter for growth (the maximum growth rate). Even though the model provides a good description of the results obtained under different conditions, some parameters (actually depend-
Ind. Eng. Chem. Res., Vol. 40, No. 23, 2001 5119
ing on the unknown biomass intracellular composition) had to be adjusted to fit the experimental data, thus reflecting the empirical nature of the kinetic expressions that have been used. In that sense, the model aims to be an acceptable compromise toward a more rigorous structured approach linking the rate of different processes to the intracellular composition of the biomass (e.g., RNA, precursors, and specific enzymes)3. Of course, the transfer of reported results to SBR design and operation must be done with caution, because these results were obtained using a synthetic medium with a single carbon source. Thus, confirmation is still necessary on more complex and/or real media. In that sense, the mathematical description supplied by the model can be used as a tool for exploring possible effects in the design and operation of SBRs. Nomenclature of the Kinetic Model bPHB ) maintenance coefficient, 1/h facc ) maximum intracellular content of accumulated compounds, mg of CODXacc/mg of CODXh fPHB ) maximum intracellular PHB content, mg of CODXacc/ mg of CODXh Kacc) semisaturation constant for accumulation, mg of COD/L Kgacc ) semisaturation constant for growth of accumulated intermediate, mg of CODXacc/mg of CODXh KgPHB ) semisaturation constant for growth of stored product, mg of CODXPHB/mg of CODXh KPHB ) semisaturation constant for PHB storage, mg of CODXacc/mg of CODXh Kt ) semisaturation constant for time dependence of maximum growth rate, h qacc ) maximum specific accumulation rate, mg of CODS/ (mg of CODXh h) qPHB ) maximum specific PHB storage rate, mg of CODXPHB/ (mg of CODXh h) S ) substrate (acetate) concentration, mg of COD/L So ) oxygen concentration, mg/L Xacc ) accumulated compound concentration, mg of COD/L Xh ) active biomass concentration, mg of COD/L XPHB ) PHB concentration, mg of COD/L YXacc/S ) accumulated compound per unit of removed substrate, mg of CODXacc/mg of CODS YXh/Xacc ) active biomass formed per unit of accumulated substrate, mg of CODXh/mg of CODXacc YXh/XPHB ) active biomass formed per unit of stored PHB, mg of CODXh/mg of CODXPHB YXPHB/Xacc )stored PHB per unit of accumulated substrate, mg of CODXPHB/mg of CODXacc µmax(t) ) time dependent maximum specific growth rate, 1/h µ∞max ) maximum specific growth rate, 1/h All other symbols are defined in the text.
Acknowledgment The authors acknowledge the National Agency for the Protection of the Environment (ANPA) for partial financial support of this research.
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Received for review November 30, 2000 Revised manuscript received April 17, 2001 Accepted April 26, 2001 IE001008K
