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Water Research 36 (2002) 702–712

Aerobic granulation in a sequencing batch airlift reactor
J.J. Beun, M.C.M. van Loosdrecht*, J.J. Heijnen
Kluyverlaboratory for Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands Received 27 July 2000; received in revised form 15 March 2001; accepted 23 April 2001

Abstract Aerobic granular sludge was cultivated in an intensely mixed sequencing batch airlift reactor (SBAR). A COD loading of 2.5 kg Acetate-COD/(m3 d) was applied. Granules developed in the reactor within one week after inoculation with suspended activated sludge from a conventional wastewater treatment plant. Selection of the dense granules from the biomass mixture occurs because of the differences in settling velocities between granules (fast settling biomass), and filaments and flocs (slow settling biomass). At ‘steady state’ the granules had an average diameter of 2.5 mm, a biomass density of 60 g VSS/l of granules, and a settling rate of >10 m/h. The biomass consisted of both heterotrophic and nitrifying bacteria. The reactor was operated over a long period during which the granular sludge proved to remain stable. The performance of the intermittently fed SBAR was compared to that of the continuously fed biofilm airlift suspension reactor (BASR). The most importance difference was that the density of the granules in the SBAR was much higher than the density of the biofilms in the BASR. It is discussed that this could be due to the fact that the SBAR is intermittently fed, while the BASR is continuously fed. r 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Aerobic granules; Sequencing batch reactor; Airlift reactor; Biofilms

1. Introduction Most wastewater treatment systems have some disadvantages such as a high surplus biomass production, low flexibility with respect to fluctuating loading rates, a large area requirement for reactors and clarifiers and a relatively low volumetric conversion capacity (0.5–2 kg COD/(m3 d) for activated sludge or conventional biofilm systems). For anaerobic processes much more efficient reactors have been developed (40 kg COD/ (m3 d) for an UASB (upflow anaerobic sludge blanket) reactor [1]). In these reactors the biomass grows as well settling granules, which allows the accumulation of high amounts of active biomass in the reactor. Moreover clarifiers are not needed because sludge separation is integrated in the UASB reactor itself.

*Corresponding author. Tel.: +31-15-2781618; fax: + 31-152782355. E-mail address: mark.vanloosdrecht@tnw.tudelft.nl (M.C.M. van Loosdrecht).

Despite the widespread use of UASB processes, the mechanism of biomass granulation is still subject of discussion. Our basic assumption is that, because of substrate diffusion limitations, bacteria prefer growing in suspension over growing in a floc, which has again preference over growth in a biofilm or granule. Growing in suspension is the most favorable form because in a floc, and even more in a biofilm or granule, the bacteria experience diffusion limitations for all kind of components involved. Growing as a floc, a biofilm, or a granule only occurs when the bacteria are otherwise washed out [2]. We consider granular growth as a special case of biofilm growth. Granulation in low turbulent systems by acidifying bacteria, nitrifying bacteria [3] and denitrifying bacteria [4] has been observed. Granulation by aerobic heterotrophs has been observed in high turbulent systems [2,5]. It has been hypothesized that the structure of biofilms is the net result of biomass growth and detachment processes [6]. Growth of the biomass is mainly influenced by the substrate loading rate and the growth yield. Detachment in these highly turbulent

0043-1354/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 3 - 1 3 5 4 ( 0 1 ) 0 0 2 5 0 - 0

One cycle consisted of 2 min influent addition. It has been shown experimentally that a good balance between substrate loading and shear can result in smooth and strong biofilms [7. Reactor set-up and operation The airlift reactor had a working volume of 3.J. 3 min settling.1. aerated sequencing batch reactor (SBR) [9–11]. Activated sludge from a conventional nutrient (N and P) removing wastewater treatment plant was used as inoculum. Effluent was withdrawn at 50 cm from the bottom of the reactor. Aerobic granular sludge can be cultured in a well mixed. In these cases granular sludge with an SVIo50 ml/g were obtained. Materials and methods 2. Fast settling granular sludge greatly benefits the SBR operation. had an internal Fig. and was positioned at a distance of 1.5 kg COD/(m3 d). The HRT was 5. wastewater is treated aerobically in successive cycles of a few hours.J.2 using 1 M NaOH and 1 M HCl. [12] also report the occurence of granular sludge in their system. The riser was 90 cm in height. In a SBR. For many applications a discontinuous operated system is advantageous. pH and off-gas CO2 were monitored continuously. Dangcong et al. 1. Air was introduced by a fine bubble aerator at the bottom of the reactor at a superficial air velocity of 80 m/h (4. The results are compared with biofilm growth in airlift reactors. The reactor was well mixed and highly turbulent with a liquid circulation time of about 6 s. 170 min aeration. The settling time is here chosen such that only particles with a settling velocity larger than 10 m/h are effectively retained in the reactor. settling of the biomass takes place before the effluent is withdrawn. The reactor was operated as a sequencing batch reactor and was therefore called a Sequencing Batch Airlift Reactor (SBAR). to keep the biomass in the reactor. Dissolved oxygen (DO). The air-flow rate was controlled by a mass-flow controller (mfc).070. The temperature of the reactor was maintained at 201C using a water jacket and a thermostat bath.8]. .4 Nl/ min).25 cm from the bottom of the down-comer. The settling time was chosen such that only particles with a settling velocity larger than 10 m/h were effectively retained in the reactor. The aim of this research was to further develop a SBR process with granular sludge growth. Slow growing bacteria with a low growth yield are more easily forced to grow as granules than fast growing aerobic heterotrophic bacteria. Beun et al. diameter of 4 cm. the pH was maintained at 7. The rest of the biomass does not settle fast enough and will be taken out with the effluent. and 5 min effluent withdrawal. / Water Research 36 (2002) 702–712 703 Nomenclature a BASR COD d HRT IA MWX n reactor surface specific area of granules (m2 granule surface area/m3 reactor) biofilm airlift suspension reactor chemical oxygen demand average granule diameter (mm) hydraulic retention time image analysis molecular weight of biomass=25 g/Cmol total number of granules in the reactor (À) SBAR SRT Vr Vx VSS X sequencing batch airlift reactor sludge retention time working volume reactor (l) total volume of granules in the reactor (l) volatile suspended solids biomass Greek symbols d penetration depth (mm) r biomass density (g VSS/l granules) systems is mainly influenced by shear forces [6]. The internal diameter of the down-comer was 6. The reactor was operated in successive cycles of 3 h each.0 l (Fig. Dry weight of the reactor 2. All these results were obtained in continuous fed systems. Schematic representation of the SBAR. Selection of the granules from a biomass mixture in a SBR can be easily based on the difference in settling velocity between the granules (fast settling biomass) and the filaments and flocs (slow settling biomass).25 cm.6 hours and the substrate load was 2. At the end of every cycle. 1). although the SVI indicated in their paper of 100 ml/g seems to point to flocculated sludge.

8 mM. 2. Nitrification and denitrification processes were studied during operation of the reactor.3 Cmmol/l. 2.5 kg COD/(m3 d). Medium A: NaAc 97. Analytical procedures The DO concentration in the reactor was measured online with a DO-electrode as a percentage of air saturation. The dry weight was determined by drying a sample for at least 24 h at 1051C. Acetate. From a representative sample of granules the following parameters were measured: * * * d =average diameter measured as surface and volume based average. 1=circle).16] according to the method described in Appendix A. On all the other days the DO of the reactor was not regulated. shape factor=capriciousness of the particle surface (0=line. H. The reactor specific surface area of the granules (a) was calculated as follows: a¼ À3 2 4pð1 2 d Â10 Þ n À Vr Â10 3 ðm2 =m3 Þ: ð3Þ . Changes in morphology of the granules were followed by image analysis (IA) [17]. The bedvolume of the settled biomass granules in the reactor was determined by reading the height of the biomass bed directly from a scale on the reactor column at the end of the settling period.45 mm filters) effluent sample. ash content of the biomass. trace element solution according to Vishniac and Santer 10 ml/l [13]. and r as the biomass density (g VSS/l of granules). 1=circle). The influent NH+ 4 -N concentration was 2. and NO2 concentrations of filtered samples were measured spectrophotometrically at 630 nm with an auto analyzer (Skalar 5010). The oxygen content of the biomass was obtained by subtracting the percentages of C.5N0. aspect ratio=roundness of the particle (0=line. N. 150 ml of medium B and 1300 ml tap water were added to the reactor. total organic carbon (TOC) in the effluent. each measurement. Media The composition of the concentrated media were as follows. The CO2 content of the off-gas was measured online with an infrared CO2 analyzer.8 mmol/l. dw the dry weight of the reactor content (g VSS/l).4. In order to evaluate optimization of the denitrification process in the reactor. These results will be discussed in a future paper. This resulted in an influent acetate concentration of 18. The elemental biomass composition was taken as CH1. and À NO3 were measured occasionally during one cycle to determine the cyclic profiles of these compounds. The morphology of the granules was measured regularly À using Image Analysis (IA).2. The acetate concentration of filtered samples from the reactor was measured by gas À À chromatography. The total number of granules (n) present in the reactor was calculated as follows: Vx n¼4 1 À2 3 p ð d 3 2 Â10 Þ ð2Þ in which d is the surface average diameter of the granules (mm). In each cycle 150 ml of medium A.3. KCl 4. / Water Research 36 (2002) 702–712 content (dw).2. The biomass concentration of the effluent was obtained by measuring the total TOC concentration of the effluent and the TOC concentration of a filtered (0. Calculation procedures The sludge retention time (SRT) of the granular sludge was calculated from the biomass concentration in the reactor (dry weight) and the biomass concentration in the effluent (there was no separate no excess sludge removal). At day 42 a few milligrams of nitrifying sludge was added to the reactor to accelerate the accumulation of nitrifiers in the reactor. The biomass density (r in g biomass 1 of granules) was determined with dextran blue [15. NH+ 4 . from day 66 to day 71 the DO in the reactor was decreased to a constant level of 50% air saturation by mixing the air flow into the reactor with N2 gas. MgSO4 Á 7H2O 3. NH4Cl 30 mM. The total gas flow rate in the reactor was kept constant at 80 m/h.J. For. Photographs of the granules were taken to follow and show the progress of granulation. NO3 .704 J.4 mg NH+ 4 -N/l. 500 granules were analyzed. Beun et al. Particle size distribution curves were made using the measured diameters. or 39. KH2PO4 10 mM. S and ash from 100%.8O0.7 mM. biomass bedvolume and biomass density (r) were measured almost daily. The reactor was operated over 140 days.7 mM. The elemental composition (CHNS) of the biomass was determined by flash combustion in a partial oxygen atmosphere at 10201C using a Carlo Erba EA1108 elemental analyzer. corresponding with a COD loading rate of 2. The total volume of granules present in the reactor Vx was calculated as follows: Vx ¼ Vr dw ðl Þ r ð1Þ with Vr as the working volume of the reactor (l). Medium B: K2HPO4 20 mM. 2. NO2 . and subtracting these two TOC values. and the NH+ 4 . Ash content was determined according to Dutch standard method NEN6621 [14].

3.20]. and on day 12 to its final length of 3 min for the next 140 days. 2. The period when acetate was present is referred to as feast period. General observations The reactor was operated in successive cycles of 3 h each. during the remainder of the operation period of 140 days. Typical concentration profiles during a steady state cycle of the SBAR (day 64). Biomass granules. 2. 7). In the second cycle after inoculation of the reactor a clear feast period was already observed. 3). On day 1 the feast period was about 100 min. . qmax is the maximal conversion rate of oxygen or acetate film ((C)mol/(Cmol h)). The settling time was chosen such that only particles with a settling velocity larger than 10 m/h were effectively retained in the reactor.J. These small granules quickly increased in diameter. When all acetate was consumed. 7). The walls of the reactor needed to be cleaned every two months only. Observation of granulation during the start-up period (day 1–7) The observations during the start-up period (7 days) of the SBAR are given in Table 1. On day 6 the biomass grown on the wall of the reactor was removed from the reactor. (E) ammonium. A settling period of 5 min was applied during the first 6 days after inoculation to prevent wash out of all the biomass. In general. endogenous respiration occurred.1. Typical concentration profiles during a cycle when the reactor was operating under stable conditions are shown in Fig. During the feast period the DO in the reactor was low (75% air saturation) due to oxygen consumption for acetate uptake and conversion. Within 7 days after inoculation granules were clearly observed in the reactor. On day 7 the settling period was decreased to 4 min. Fig. in which d is the penetration depth (m). and CX is the biomass concentration in the biofilm (=r=MwX (Cmol/m3)) [2]. The remainder of the cycle is named famine period. This will be discussed in a future paper [18]. and this decreased to about 60 min on day 3. / Water Research 36 (2002) 702–712 705 The penetration depths of oxygen and of acetate were calculated according to sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 6CL D . filaments and flocs were present as a mixture in the reactor. D is the diffusion coefficient (=7.2. In the liquid some very small granules were already visible (see also Fig. (normal line) CO2 production rate. As soon as this granulation process started. CL is the concentration of oxygen or acetate in the liquid (mol/ m3).2 Â 10À6 m2/h). Selection of the biomass granules from the mixture occurred during the settling period (Fig. The transition from feast to famine period was directly observed from a sharp increase in the dissolved oxygen (DO) concentration in the reactor.J. the DO immediately increased to almost 100% air saturation. In general the conversions observed in this granular sludge reactor compare well to previous observations in a conventional activated sludge laboratory SBR [19. Nitrification and denitrification occurred in the SBAR. Beun et al. The biomass concentration in the reactor started to increase and consequently the length of the feast period decreased again. d¼ film qmax Cx ð4Þ 3. When acetate was depleted. biomass growth on the wall diminished. On day 1 the reactor was inoculated with 1 l of activated sludge (totally 9 gSS) from the return sludge flow of a conventional nutrient removal wastewater treatment plant (see Fig. all acetate was consumed within a few minutes. (b): (bold line) DO. The wall of the reactor became completely covered with attached biomass in these first 4 days. Results 3. The rapid transition from flocculated to granular sludge shows that the chosen strategy was successful. (m) NOx. From day 3 to day 4 the feast period increased again due to wash out of a large amount of the biomass. (a): (’) acetate.

7 mm at day 30 and had a steady state value of 2. The inoculum consisted of suspended sludge and sludge flocs only. feast period decreases (70 min) Fig. 4). This indicates that the granules were more or less spherical particles with a smooth surface. Hereafter the effluent biomass concentration decreased to about 75 mg VSS/l during the rest of the time (Fig. Photographs show the inoculum and the sludge on different days (Fig. The smaller biomass particles subsequently washed out.706 J. This decrease was likely caused by the fact that biomass granules partly broke up due to the decreased DO (50%) which was applied between day 66 and 71. On day 37 the granules had a more or less ‘steady state’ average diameter of about 2. After increasing the DO the density increased again to 60 g VSS/l at day 142. In general . Development of the granules from day 6 onwards The development of the granules in the reactor was followed by sampling and image analysis for about 140 days. Thereafter no significant change of the granules occurred. Selection of well settling. 6). The biomass concentration in the effluent fluctuated around 250 mg VSS/l until day 40. 5). 7).5 mm during the remainder of the experiment (Fig. The photograph of day 63 only shows some more filaments on the surface of the granules.J. The biomass concentration in the reactor increased to about 10 g VSS/l at day 65 (Fig. This was associated to the deterioration of biomass granules due to the decreased DO applied from day 66 to day 71. feast period is visible in the DO pattern (100 min) 2 3 4 5 6 Feast period decreases (60 min) Biomass is being washed out.8 (Fig. The density of the granules increased to 70 g VSS/l at day 80 (Fig. and were both stable for the rest of the time at a value of 0. feast period increases (100 min) Significant biomass growth on the wall 7 Settling period 4 min In liquid very small granules present Feast period increases due to very low biomass concentration after biomass removal from wall Granules become bigger. The surface average diameter of the granules increased to about 2. 3. 3. / Water Research 36 (2002) 702–712 Table 1 Observations during start-up of the SBAR Day number 1 Action Inoculation with fresh activated sludge Settling period 5 min Settling period 5 min Settling period 5 min Settling period 5 min Settling period 5 min Removal of wall growth from the reactor Settling period 5 min Observation After 1 cycle operation. 4). Small granules were already present after 6 days of operation. After day 80 the density decreased to 40 g VSS/l within 7 days. Inoculation of the reactor with nitrifying biomass at day 42 did not notably influence the density of the granules. Thereafter the biomass concentration decreased to about 7 g VSS/l around day 80. 6). Beun et al. These disappeared again after a few days (Fig. 7). dense granules in the SBAR. This low biomass production was associated to a low sludge loading and consequently high sludge age of 50 days (Fig. The aspect ratio and shape factor of the granules increased in the first 10 days.3. 6). The inner structure of the granules was observed in more detail under the light microscope by cutting the granules in two with a razor blade.7–0.5 mm (see also Fig. 6).

Fig. 5.4. 8 the distributions on day 6 and day 63 are shown. and between 115 mm (completely aerobic conversion) and 520 mm (completely anoxic conversion) for acetate. (4) to be between 17 mm (in the feast period) and 20 mm (in the famine period) for oxygen. The outer layer had a rather dense structure. two layers could be observed in the granule: (1) the center of the granule. The penetration depths were calculated according to Eq. 4.J. Sludge retention time (SRT) of the SBAR (’) and reactor specific surface area of the granules (m) from day 6 until day 142. Due to the penetration depth of acetate biomass growth mainly occurred in this outer layer. Particle size distribution Particle size distribution in the SBAR was followed using image analysis. The center of the granule had a more fluffy and gelly structure and was more transparent compared to the outer layer of the granule. 3. Beun et al.4 mm. which was about 1.7 min in diameter. This corresponds with the thickness of 400 mm of the denser outer layer of the granules. biomass concentration in the SBAR (E) and in the effluent of the SBAR (m) from day 6 until day 142. In Fig. / Water Research 36 (2002) 702–712 707 Fig. No big empty holes resulting from complete lysis of the interior biomass were observed in the center of the granule. Day 6 is the first day after . and (2) the outer layer with a thickness of about 0. Total number of granules in the SBAR (K).J.

the aspect ratio (E).5 mm. the shape factor (*). Beun et al. and kept in stable operation in the SBAR for months. Most of the granules were smaller than 0. 7. . and the density r of the granules (’) from day 6 until day 142. 8). On day 15.5 mm present. 7). The particle size distribution curve of day 63 is representative for the steady state situation of the SBAR. The median value of the granule diameter was around 3 mm. momentary increase of the pH to 9 caused disruption of particles. It is a normal distribution curve with some more small granules of about 0.1. 6. inoculation of the reactor that granules were visible (Table 1 and Fig. Photographs of the sludge in the SBAR on different days after inoculation (bar=1 mm). On day 16 the distribution curve was already similar to the one on day 14. Development of the average diameter of the granules in the SBAR (K).5 mm. The distribution curve of this day showed that there were mainly small granules of around 0.708 J. The SBAR It was demonstrated that aerobic granular sludge could be cultivated on defined medium. an unexpected. the average granule diameter was around 2. The particle size distribution curves on many different days showed that the system is able to recover very fast after disturbances. Fig. 4.J. / Water Research 36 (2002) 702–712 Fig.5 mm in diameter on this day (Fig. Discussion 4.

and the cells deeper in the biofilm will be deprived of acetate. Acetate can therefore not penetrate completely into the biofilm (o20 mm). and subsequently recovered very fast. It is not fully clear how these granules in the very beginning were formed.J. Comparing the granules and biofilms formed in the SBAR and the BASR. Another possibility is some fast settling flocs/material from the inoculum. Evaluating the difference in granule formation in both reactors (Table 2) makes it possible to discuss the mechanisms involved.1 Cmmol/l). The density of the granules and/or the number of granules in the reactor increased with increasing biomass content. All biomass above this point. the SBAR was operated mainly as a COD removal reactor.2. continuously for the BASR and intermittently for the SBAR. The granules were able to deal with disturbances (e. and reached a high density of about 60 g VSS/l of granules. 8. and a combination of heterotrophic and autotrophic biofilms.5 mm for the remainder of the operation period.e. the granules usually partly broke up. One possibility is that they originate from the biofilm grown on the wall of the reactor. Optimization for N-removal and further enrichment of autotrophic biomass was not carried out yet. After disturbances. Due to the high liquid circulation velocity small pieces could be broken from the biofilm. the most striking difference is the density of the biomass in the biofilm that is much higher in the SBAR than in the BASR. Slow settling biomass was taken out with the effluent. The granules in the reactor had a smooth surface. In principle granules are biofilms without a carrier.J. the granules get penetrated with acetate over a depth of 500 mm because the acetate concentration in the liquid is high due to pulse feeding.17]. Until now. the feast period).e. The granules had a stable diameter of 2. In that case there was no inoculum used at all. Subsequent development of new granules could occur out of broken pieces from these initial granules [5]. Particle size distribution in the SBAR at day 6 (start of granulation) and day 63 (steady state). In the SBAR the cells in the . The settling time was chosen such that only particles with a settling velocity larger than 10 m/h were effectively retained in the reactor. When there is substrate present in the SBAR (i. the effluent port was just above the settled sludge layer. the acetate concentration in the liquid is always very low (o0. 4. The SBAR was easily started-up with suspended activated sludge from a normal wastewater treatment plant. We did not observe the typical fungal intermediate phase as observed previously [11]. momentary increase of pH) and fluctuations (decreased DO). After 1 month of operation a pseudo steady state granule diameter was reached. The SBAR compared with the biofilm airlift suspension reactor The formation of aerobic biomass granules has been reported previously in a continuously fed turbulent system.g. after 3 min settling. / Water Research 36 (2002) 702–712 709 Fig. Almost all other operational factors (such as volume loading rate and DO) were similar. In the continuously fed BASR. In the BASR data were gathered for heterotrophic biofilms. The biomass concentration in the reactor still increased after 1 month of operation. Selection of the granules from the biomass mixture in the reactor occurred by utilizing the difference in settling velocity between the granules (fast settling biomass) and the filaments and flocs (slow settling biomass). These pieces could be starting material for formation of granules. Granules developed in the reactor within 1 week after inoculation. The reason for this large difference is probably the way the reactors are fed i. the biofilm airlift suspension reactor (BASR) [5. was removed from the system. although during cleaning that biofilm was removed completely from the reactor. autotrophic biofilms. In the BASR granules developed from biofilm fragments that originated from broken up biofilm particles. Beun et al. In later stages of the experiment both heterotrophic and autotrophic biomass were present in the SBAR.

This difference is probably due to the fact that . i. leading to a denser biofilm. The difference in shear forces between the SBAR and BASR is not clear. The superficial gas flow rate in both reactors was similar. center of the granules can therefore take up acetate and grow (using either oxygen or nitrate as electron acceptor). The average specific growth rate of the biomass in the SBAR was much lower than in the BASR (higher penetration depth of substrate. and maintained under stable operation for months.5 60 5 Â 104 375 dc ¼ diameter carrier. This was observed when applying a DO of 50% in the reactor between day 66 and day 71. smaller then the acetate penetration depth. The cells below this penetration depth can use NOÀ 3 as electron acceptor. The balance between biomass growth and shear determines the final structure of the granules [6].67 0.710 J. The density of the granules in the SBAR was much higher than the density of comparable biofilms in the BASR.4 Â 106 470 BASR (Autotrophic biomass) [5] F 80 1 0. since the density of the biomass in the SBAR was much higher than in the BASR. The balance between these two factors is obviously more favorable in the SBAR than in the BASR.02–0.3 1.41 (dc ¼ 0:26)a 20 1. i. leading to a less dense biofilm.e.e. Considering the total number of particles in the reactor (Table 2) the detachment should in principle be highest in the BASR. an electron acceptor (O2 or NOÀ 3 ) is needed. The O2 penetration depth in the SBAR is in the feast period around 20 mm.6 80 0. For the BASR the O2 penetration depth is around 80 mm.75 (dc ¼ 0:26)a 60 4 Â 106 2300 2. Beun et al. lower effective sludge loading).67 0.0008 8 2. The BASR contains basalt as a carrier material.8 Â 105 390 BASR (Heterotrophic biomass) [16] 4. since a higher concentration of particles leads to a higher collision frequency and thus higher detachment forces. Conclusions In a sequencing batch airlift reactor. O2 penetration will be even less. The density of the biofilms can also be influenced by the specific growth rate of the biomass.0015 25 0. aerobic granular sludge could easily be cultivated. For conversion of acetate.054 7. For the biofilm structure the influence of shear forces has to be taken into account as well. larger then the acetate penetration depth.2 0. in continuously fed systems like the BASR) develop a surface with filamentous structures with a high porosity and a low density.16 1. These observations can easily be understood.22] showed that in growing systems the detachment rate is higher when more bare carriers are present. In both the SBAR as the BASR O2 will not penetrate completely into the biofilm. i. A lower specific growth rate leads to more dense biofilms [22]. Modeling studies [21] also showed that more substrate limited biofilms (i. It has been demonstrated that for non-growing biofilms in an airlift reactor the detachment forces increased with increasing bare carrier concentration [23.5 80 5. At decreased DO as applied for 1 week in the SBAR.e. The biofilm will get a less dense structure and finally break up into pieces. 5.6 0. Summarizing it can be said that the specific biomass growth rate in the SBAR was lower than in the BASR. This might also have contributed in a higher density in the SBAR.e. The detachment forces in the SBAR were probably lower than in the BASR.J. / Water Research 36 (2002) 702–712 Table 2 Comparison of the SBAR with the BASR SBAR (Heterotrophic and autotrophic biomass) Acetate load (kg COD/(m3 d)) Superficial gas flow rate (m/h) HRT (h) Average specific biomass Formation rate (dÀ1) Dry weight (g VSS/l) d (mm) r (g VSS/l granules) n a (m2/m3) a BASR (Heterotrophic and autotrophic biomass) [16] 1 80 0. When this is not available.56 (dc ¼ 0:26)a 20 1. lysis of the cells in the center of the biofilm will occur. Growth of the cells in the center of the granules leads to an increasing biomass density of the granules. since the total number of particles was a factor 10 higher than in the SBAR. Granules developed in the reactor within 1 week after inoculation with suspended activated sludge. The difference in settling velocity between the granules (fast settling biomass) and the filaments and flocs (slow settling biomass) is seen as the dominant factor to obtain granular sludge.

Biofilm structures. Noyola A. The four fractions and the original dextran blue solution are analyzed by a spectrophotometer at 620 nm. Selected dyes for residence time distribution evaluation in bioreactors. A known amount of the liquid above the settled granules is removed and a sample is taken from it (fraction 1).J. Zeeman G.31(12):3191–4. 1988. Influence of biomass production and detachment forces on biofilm structures in a biofilm airlift suspension reactor. in a SBR process. 1993. Water Research 1999. Water Science Technology 1995. Aerobic granular sludge-a case report. Van Loosdrecht MCM. Van Loosdrecht MCM. Biotechnology Progress 1996. Wageningen. Garrido-Fernandez JM. Heijnen JJ. Dextran blue colorant as a reliable tracer in submerged filters. Beun JJ. Van Loosdrecht MCM. / Water Research 36 (2002) 702–712 711 the SBAR is intermittently operated. Beun JJ. Van Loosdrecht MCM. The mixture is mixed gently. Heuvel JC. In the BASR acetate penetration is less than 20 mm. Jimenez B.21:195–213.22(10):1253–7. A known amount of demineralized water is added to the granules. Biotechnology Techniques 1988. Delgenes J-P. The Netherlands. Feeding substrate as a pulse results in penetration of acetate over a depth of 500 mm into the granules.58(4):400–7. and subsequently the granules are allowed to settle. Faup G.12(6):764–72.44:595–608. Delft. Microelectrode measurements of the activity distribution in [18] [19] [20] nitrifying bacterial aggregates. Heijnen JJ. Eikelboom D. Mulder A. Aerobic granular sludge in a sequencing batch reactor. Hendriks A. Van Loosdrecht MCM. Heijnen JJ. Granules density determination by dextran blue A known amount of a dextran blue solution (1 g/l) is added to a representative sample (and known amount) of the reactor. Grotenhuis JTC. Formation and detachment of biofilms and granules in a nitrifying biofilm airlift suspension reactor. Van Benthum WAJ. The mixture is gently mixed. the SBAR should have a high H/D ratio (column height/column diameter) to improve selection of granules by the difference in settling velocity.2(2):77–82. submitted for publication. Van Loosdrecht MCM. Biotechnology & Bioengineering. in a volume ratio of about 1 : 1. Biological wastewater treatment anaerobic treatment (in Dutch). In: Lettinga G. Measuring also the dry weight of the reactor sample allows calculation of the density of the granules (g biomass/1 of granules).45:7–17. Van Loosdrecht MCM. activated sludge cultures. Bepaling van de asrest. Heijnen JJ. (submitted) N-removal in a granular sludge sequencing batch airlift reactor. Heijnen JJ. Water Research 1999. slow growing. Biotechnology & Bioengineering 1994. The Netherlands. Van Loosdrecht MCM. Ng WJ. The granules are allowed to settle. Van den Ottengraf SPP. Tijhuis L. since dextran blue only binds to the water and not to the biomass. Pudoc: Wageningen. Van Loosdrecht MCM. a short standstill time for settling. Nederlands Normalisatie Instituut. Picioreanu C. Biotechnology & Bioengineering 2000. Tijhuis L. The additional advantage is that a high H/D ratio and the absence of an external settler result in a reactor with a small footprint. This last step is repeated until four fractions are obtained. Morgenroth E. Beun JJ. allowing a more efficient use of reactor volume. substrate loading and reactor scale on the formation of biofilms in airlift reactors. 1982. Van Benthum WAJ. The good settling characteristics of the biomass allow.1. Sherden T. Moletta R. Hijman B. Santer M.32:35–43. [4] [5] [6] [7] Appendix A A. Heijnen JJ. Biotechnology & Bioengineering 1994. Wilderer PA. Applied and Environmental Microbiology 1993. Hulshoff Pol LW. [2] Tijhuis L. Subsequently. Ong SL.67(4): 379–89.J. Heijnen JJ. Biotechnology & Bioengineering 1998. Influence of detachment. Hulshoff Pol LW. Verhoef EV. Applied Microbiology and Biotechnology 1995. A known amount of the liquid above the settled granules is removed and a sample is taken from it (fraction 2).59(2):573–9. p. Noyola A. Aerobic granulation in a sequencing batch reactor. Water Research 1997. Stoichiometry and kinetics of poly-b-hydroxybutyrate metabolism in aerobic. Department of Environmental Technology. Bacteriological Reviews 1975. NNI NEN 6621. while the BASR is continuously fed with substrate. Tijhuis L. Water Research 1988. Kwok WK.33(10): 2283–90. The Thiobacilli. 203–10. Bernet N.44(8):867–79. Wageningen Agricultural University. Formation and growth of heterotrophic aerobic biofilms on small suspended particles in airlift reactors. [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] References [1] Lettinga G. Heijnen JJ. Van Loosdrecht MCM. Beun et al. the volume of the biomass in the reactor sample could be calculated. Roustan M. Van Loosdrecht MCM. Capdeville B. Beun JJ. Morgenroth E. Gjaltema A. European patent 1998. editors. Jimenez B. Vishniac W. Zehnder AJB. Werkwijze voor het verkrijgen van korrelvormige groei van een micro-organisme in een reactor. Heijnen JJ. [3] De Beer D. Tijhuis L. Acetate will not reach the cells deeper and no growth will occur there. Van Loosdrecht MCM. Granular Aerobic Sludge. Dangcong P. Granulation of denitrifying sludge. Paletta F. For practical application. Solids retention time in spherical biofilms in a biofilm airlift suspension reactor. Heijnen JJ. Heijnen JJ. Heijnen JJ. Stoichiometry and kinetics of poly-b-hydroxybutyrate . Van der Hoek JP.33:890–3. Capdeville B.

1999. Van Loosdrecht MCM.712 J. Van Loosdrecht MCM. Biotechnology & Bioengineering 2000.45:258–69. Influence of different substrates on the . IAWQ/IWA Conference on Biofilm systems. Effect of diffusive and convective substrate transport on biofilm structure formation: a two-dimensional modeling study. 69. Heijnen JJ. Biotechnology & Bioengineering 1995. Heijnen JJ. Van Loosdrecht MCM. Picioreanu C. [23] Gjaltema A. metabolism under denitrifying conditions in activated sludge cultures. Detachment of biomass from suspended non-growing spherical biofilms in airlift reactors. Biotechnology & Bioengineering 2000. [22] Villasenor JC. Beun et al. New York. [21] Picioreanu C. Tijhuis L. Heijnen JJ. / Water Research 36 (2002) 702–712 formation of biofilms in a biofilm airlift suspension reactor.J.68(5):496–507.