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Evolution of bioaggregate strength during aerobic granular sludge formation
Biochemical Engineering Journal 58–59 (2011) 69–78
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Evolution of bioaggregate strength during aerobic granular sludge formation Junfeng Wan a,b,c , Irene Mozo a,b,c , Ahlem Filali a,b,c , Alain Liné a,b,c , Yolaine Bessière a,b,c , Mathieu Spérandio a,b,c,∗ a b c
Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France CNRS, UMR5504, F-31400 Toulouse, France
a r t i c l e
i n f o
Article history: Received 7 January 2011 Received in revised form 23 July 2011 Accepted 25 August 2011 Available online 31 August 2011 Keywords: Aerobic granular sludge Aggregate strength Shear Cohesion
a b s t r a c t This work investigated the modification of aggregate properties during the formation of granular sludge in a sequencing batch airlift reactor (SBAR). The cohesion of biological aggregates was quantified by subjecting sludge samples to two different controlled shear stresses in a stirred reactor. For reference sludge (without granules), flocs broke and reformed easily, indicating that floc size was controlled by the turbulence micro-scale (Kolmogorov scale, here from 17 m to 62 m). In contrast, granules showed high strength which enabled them to resist turbulence and their size was no longer imposed by the Kolmogorov micro-scale. Different steps were observed during the granulation process: a first increase of aggregate cohesion associated with a decrease in sludge volume index (SVI), a growth of aggregates with detachment of fragile particles from the surface and, finally, an increase in the sizes of small and large granules to reach a pseudo-stable size distribution. Results suggest that small particles could have formed the seeds for new granules, as they were maintained in the bioreactor. Here, granular sludge was formed in an SBAR with a conventional settling time (30 min), i.e. without particle washout, and with a low superficial air velocity (SAV = 0.6 cm s−1 ): it is thus demonstrated that high SAV and low settling time are not necessary to produce granules, but probably only accelerate the accumulation of granules. It is shown that the increase of cohesion is the initial phenomenon explaining the granule formation concomitantly with bacterial aggregates densification. It seems important, in the future, to investigate the reasons for this cohesion increase, which is possibly explained either by bacterial bounding interactions or the excretion of extracellular polymeric substances (EPS). © 2011 Elsevier B.V. All rights reserved.
1. Introduction Since the phenomenon of aerobic granular sludge was first reported in the 1990s [1,2], research on the subject has flourished and this new technology has shown its significant perspectives in wastewater treatment [3–5]. Compared with conventional activated sludge, aerobic granular sludge presents a variety of attractive properties such as more compact structure, higher specific activity and better settling properties as required to improve solid–liquid separation. In most of the studies, granular sludge was formed in a sequencing batch reactor [6]. Aerobic granules were obtained by consecutively inoculating with conventional activated sludge and applying operating conditions which favoured the development and selection of granules. Because of its compact structure, this particular aggregate is expected to present similarities with biofilm, which is mainly composed of bacteria
∗ Corresponding author. Tel.: +33 561559785; fax: +33 561559760. E-mail address: [email protected] (M. Spérandio). 1369-703X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2011.08.015
and extracellular polymeric substances [7,8]. However, if mechanisms involved in the initial formation of granules have been the subject of many researches, relationships between microbial aggregation mechanisms, reactor operational conditions and the resulting physical properties of granules still need to be clarified. So far, the literature proposes biological and process conditions leading to granular sludge formation. Concerning the biological selection pressure, stability of granules is found to be improved when selecting slow growing bacteria. This can be done either when increasing the substrate N/COD ratio [9] to favour nitrifying bacteria or by alternating feast/famine and anaerobic/aerobic periods [10] to promote selection of phosphate or glycogen accumulating organisms. Concerning the process conditions, the literature [11–14] underlines that aeration rate is an important factor, influencing granular sludge formation through three main, simultaneous mechanisms: (i) oxygen transfer, which modifies the dissolved oxygen in the bulk and hence within the granule, (ii) mixing, which influences mass transfer of substrates in the liquid boundary layer and (iii) hydrodynamic shear stress, which controls erosion and
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fragmentation. A high superficial gas velocity (SGV) has been seen as a major factor enhancing the formation of aerobic granular sludge but its roles (shear stress, mixing and oxygen transfer) are still controversial due to the interdependency of these processes [12]. Investigations have focused more on biological aspects and aerobic granulation has not been thoroughly studied from the fluid mechanics point of view [15]. So far, little information concerning the influence of mechanical stresses on granule size and shape has been reported. The results of Zima-Kulisiewicz et al. [15] suggest that the shear stress (tangential force) and elongation stress (normal force) together with stresses induced by fluctuating velocities are the main forces influencing granule building. Although the origin and significance of mechanical stresses in the bioreactor have been clarified, only few approaches has been developed to estimate whether bioaggregate strength and size are altered by these stresses, whereas a strong relationship has been reported between the size of physicochemical flocs and the Kolmogorov microscale [16]. Particle strength (or cohesion) has rarely been measured on aerobic granular sludge but it is reasonable to think that this property plays a role in granule formation and stability as underlined on Anammox granules where an increase of input specific power was shown to affect activity and biomass retention [17]. Because it generally controls surface detachment (erosion) and particle breakage, aggregate strength should receive more attention. Detachment of particles is a crucial phenomenon for aerobic granular sludge, as it limits the growth rate and controls the stability of granules (as well as the specific activity of supported bacteria). Detachment rate also controls the microbial competition between fast-growing heterotrophic bacteria and slow-growing autotrophic bacteria as has been shown numerically and experimentally in the case of biofilms [18,19]. This phenomenon is of importance as both kinds of bacteria are necessary for simultaneous carbon and nitrogen removal. The significance of detachment is strongly related to the cohesion of granules. Cohesion is a physical property of the aggregate defining how it can resist shear and elongation force, which induce erosion and/or break-up. It is well known that the strength of aggregates depends on the strength and number of the bonds holding the aggregates together [20]. Aggregate breakage is likely to occur if the stress applied to the surface of the aggregate can counterbalance the bonding strength [21], which means that the more compact the structure of aggregates is, the more difficult they are to break. Thus, cohesion is directly related to structure and is, therefore, highly dependent upon the aggregate formation process. Techniques for measuring granule strengths can be derived from those developed for measuring floc strength. Floc strength has been largely studied [22,23] using various methods. Most of the methods used have been impeller-based techniques [24–29]. Floc strength may be estimated through floc size analysis by examining its breakup consecutively by exposing to known stresses [30]. The method consists of subjecting flocs to single or increasing levels of shear within a vessel and comparing the ratio of the floc size before and after breakage, thus quantifying the energy input necessary for floc breakage [31]. Other techniques, such as ultrasonic methods, have also been adapted to test aggregate strength but are more suitable for application to mineral flocs than to bioaggregates due to the effect of ultrasound on bacterial components [32]. Similar studies have recently focused on characterising granules from anaerobic processes in the upflow anaerobic sludge blanket (UASB) system. For example, the resistance to compression forces has been measured recently [33]. In a previous work [34], anaerobic granules were subjected to strong mechanical stirring (200 rpm) for 5 min. The granular strength of anaerobic granules was deduced from the ratio of solids in the supernatant to the total weight of the granular sludge. This ratio gives an indicative index of the granules’ resistance when subjected to abrasion and shear, but gives no infor-
mation on the modification of the particle sizes. This method has been combined with image analysis to compare two different types of aerobic granules developed in aerobic sequencing batch reactors (SBRs) [35]: fungal granules from one SBR were larger and weaker, with a loosely packed, fluffy structure whereas the bacterial granules from another SBR were smaller and stronger, with a compact structure and higher decantation feasibility. Little information is available to understand how the operating conditions can affect the particle strength in an aerobic granular sludge process. A study carried out by Di Iaconi et al. [36] investigated the influence of hydrodynamic shear forces on the properties of granular biomass developed in a sequencing batch aerobic biofilter reactor. They found that granular biomass density increased quasi-linearly with the increase of shear forces in the reactor and that EPS content and composition were not affected by hydrodynamic shear forces. More recently, an energy-dissipation-based model has been established by Ren et al. [37] to quantify the shear stresses in an aerobic granular sludge SBR and predict the rate of particle detachment. Simulations indicate that shear is induced mainly by gas bubbles or granule collisions and only slightly by the fluid. The possible modification of particle strength has not been integrated in this model. Because the detachment is quantified by absorbance measurement, no information is provided on the size of the detached particles and remaining granules. In conclusion, little is known at present about the evolution of cohesion during granular sludge formation, and yet this should be helpful in understanding the mechanism of granule formation. Consequently, the goal of this study is to characterise the evolution of aggregate cohesion during the formation of aerobic granular sludge, and the behaviour of granules under given hydrodynamic conditions. To determine aggregate strength, biological aggregates were subjected to a calibrated stress in a stirred reactor and the particle size distribution was measured. Flocculated biomass was first characterised and considered as a reference point. Then granular sludge was developed in a statically filled sequencing batch airlift reactor (SBAR) by applying substrate feast and famine periods coupled with alternating anoxic/aerobic conditions. The analysis focused on the progressive conversion from a floc-like structure to granule-like aggregates, and non-significant hydraulic particle selection was applied during this period (using a 30 min settling phase). Finally, at the end of the study, granules were collected after sieving and their specific behaviour was characterised in the stirred reactor.
2. Materials and methods 2.1. Sequencing batch airlift reactor (SBAR) The reactor employed was an airlift column with a working volume of 17 L (internal diameter = 15 cm, total height = 105 cm, H/D ratio = 7). A plate baffle (length: width = 83:15 cm) placed vertically in the middle of the reactor divided the column into two zones: riser and downcomer. Air was introduced through a fine bubble aerator at the bottom of the reactor, and induced the circulation of air, liquid and solid. The system is schematically presented in Fig. 1a. The column was inoculated with 10 L of conventional activated sludge taken from the aeration tank of a local municipal wastewater treatment plant at a mixed liquor suspended solid (MLSS) concentration of 4.5 g L−1 and a sludge volume index after 30 min (SVI30 ) of 172 mL g−1 . The reactor was fed with a synthetic wastewater having a mixed organic carbon source: glucose, ethanol, sodium propionate and sodium acetate, each contributing 25% of the total COD (total = 750–1000 mgCOD L−1 ). The other components of the wastewater (mg L−1 ) were as follows: NH4 Cl
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Fig. 1. Schematic diagram of the SBAR (a) and cohesion test vessel (b).
(143–191), NaNO3 (610), NaHCO3 (100), KH2 PO4 (100), CaCl2 (30), MgSO4 ·7H2 O (4) and other necessary elements were similar to the trace solution described elsewhere [38]. The organic and ammonium loading rates were simultaneously increased during the study with a constant COD/N ratio of 20 (Table 1) by increasing the influent concentration or decreasing the cycle duration. The system was first operated by cycles of 6 h including influent filling (30 min), preanoxic phase (30 min), aeration (240 min), settling (30 min) and effluent withdrawal (30 min). Reduction of the cycle to 4 h was performed by a proportional reduction of the filling, anoxic and aerobic phases. A relatively low particle selection pressure was imposed compared to those used in previous studies on granular sludge [3,11,14]. The process was initially operated with a high aeration rate (superficial air velocity SAV = 2.23 cm s−1 ) and a purely aerobic reaction phase. From day 80, the SAV was reduced to 0.63 cm s−1 , and the pre-anoxic phase was introduced from day-120 (by means of nitrogen gas sparging at the same gas velocity).
In this study, the optical index of the dispersant was set to 1.330, which corresponded to the value for the water. As biological aggregates are mainly composed of organic matter, the optical index was set to 1.596 as indicated in the work of Lambert et al. [40]. Qualitative observations were performed by using either a microscope or a stereoscopic microscope depending on the size of the aggregates. 2.3. Cohesion tests Cohesion tests were performed in a stirred vessel, equipped with an impeller (5 cm in diameter), consisting of a 145 mm × 245 mm (inner diameter × height) cylinder providing a testing volume of 2.5 L (Fig. 1b). The shear level corresponding to the operating conditions induced during this experiment can be approximated by the average velocity gradient value (G) for isotropic and homogeneous turbulence as follows:
P V
2.2. Analytical methods
G=
Sludge samples taken from the column were characterised in terms of suspended solids (SS) and volatile suspended solids (VSS) using standard methods [39]. Sludge volume index (SVI) was measured in a 1000 mL graduated cylinder with the sludge sample taken from the mixed liquor at the end of the aerobic phase. SVI5 and SVI30 were measured after 5 and 30 min, respectively, so as to compare aggregates properties with the one proposed for granular sludge in literature. The particle size distribution (PSD) of aggregates was analysed using a laser diffraction technique (Mastersizer, Malvern 2000, UK) with a particle size ranging from 0.02 m to 2000 m.
where G is the velocity gradient (s−1 ), P is the power input (W), is the dynamic viscosity (Pa s) and V is the suspension volume (m3 ). The power input (P) was calculated as the energy dissipated by the impeller:
Table 1 Strategy for organic and ammonium loading rates. Time
Duration of cycle (h)
OLR (kg COD d−1 m−3 )
ALR (kg N-NH4 d−1 m−3 )
d70–120 d120–175 d175–256
6 6 4
1.412 1.882 2.822
0.071 0.094 0.141
P = Np N 3 d5
(1)
(2)
where Np is the power number given by the manufacturer (4 in our case: turbine, 4 flat blades), is the mass density of the fluid (kg m−3 ), N is the rotation speed (s−1 ) and d is the impeller diameter (m). The mechanical stirring system provided an average velocity gradient of up to 3400 s−1 . By considering the energy dissipation (ε in m2 s−3 ) in terms of a gradient (G) dependent expression: ε = · G2
(3)
where is the kinematic viscosity (m2 s−1 ), the Kolomogorov microscale ( in m), corresponding to the size of the smallest
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Fig. 2. Hydrodynamic test conditions in the vessel and the measurement time: (a) theoretical hydrodynamic characterisation of the system (vessel + impeller); (b) hydrodynamic test conditions during the three phases: low mixing intensity (phases I and II) and high mixing intensity (phase II) and symbols used for different times.
eddies during mixing (linked to the shear level in the vessel), could be determined:
=
3 ε
1/4
(4)
Based on the previous definitions, a theoretical characterisation of the cohesion test system was performed in terms of average velocity gradient and Kolmogorov microscale resulting from a rotation speed as presented in Fig. 2a. As shown in Fig. 2b, the cohesion test consisted of a three-phase procedure: the vessel was first operated at a low mixing intensity (300 rpm/250 s−1 ) for 45 min (phase I); then the shear intensity was increased (1700 rpm/3400 s−1 ) for 45 min (phase II) and, finally, decreased to the original condition for 45 min (phase III). Those operating conditions lead to turbulent flow regime with impeller’s Reynolds numbers of 12 500 and 70 000, respectively. During the entire period, PSD was measured 5 min before the end of each phase (Fig. 2b). The Kolmogorov microscale corresponding to the applied G values was 62 m for a G value of 250 s−1 (300 rpm) during phases I and III, and 17 m for 3400 s−1 (1700 rpm) during phase II. For each test, the sludge was sampled at the end of the aerobic phase of the operational cycle of the SBAR and systematically diluted to 1 g L−1 by using the ultra centrifuged supernatant (10 000 × g, 15 min) in order to avoid any modification of the environment, such as the ionic strength, which is known to have a significant effect on flocculation processes. 3. Results 3.1. Characterisation of floc behaviour (reference) during the cohesion test As a starting point, the cohesion test was performed on a flocculated sludge (Fig. 3a) developed in a purely aerobic SBAR fed by the same synthetic influent as for the anoxic/aerobic SBAR. Sludge was sampled from the reactor and placed in the stirred reactor before various stirring rates were imposed. As can be observed in Fig. 3b, the particle size distribution of this flocculated biomass was clearly influenced by the hydrodynamics: an increase in impeller rotation speed resulted in a significant reduction in median diameter from 80 to 18 m. After reduction of the velocity gradient, an increase in the median diameter was
observed (Fig. 3c) indicating a reflocculation of the particles and resulting in full recovery of the initial particle size. Furthermore, it can be seen that the suspension seemed to be calibrated by the turbulence scale as the average diameter (70 and 16 m) was close to the Kolmogorov microscale (62 and 17 m, respectively). Similarly, it has been demonstrated that chemically induced flocculation is controlled by hydrodynamic turbulence in stirred coagulation reactors [41]. The present results suggest that the approach proposed for physicochemical flocs is still valid for biological flocs developed in the laboratory with soluble synthetic substrates.
3.2. Evolution of aggregate properties during granular sludge formation in SBAR 3.2.1. Suspended solid concentration, SVI and particle size As shown in Fig. 4, the profiles of MLSS and SVI versus time varied inversely during the study. Before day 120, MLSS first increased from 2.5 to 4.5 g L−1 whereas SVI30 decreased from 150 to 100 mL g−1 . Then the pre-anoxic mixing phase was introduced in the cycle after the static filling phase, from day-120 (by supplying nitrogen gas instead of air at the same gas flow rate). Later again, a significant decrease of SVI30 from 100 to 45 mL g−1 and an increase of MLSS up to around 9.5 g L−1 were observed. These data are thoroughly described in Wan et al. [42]. From day-175, the organic, ammonium and nitrate loading rates were increased, as the cycle time was reduced from 6 to 4 h. After a temporary drop for a short period, MLSS recovered the same level as before, and larger and larger particles (>200 m) were observed in the airlift column. MLSS continued to increase during the following months and stabilised around 15 g L−1 after 350 days. Simultaneously, SVI decreased to 30 mL g−1 indicating typical properties of granular sludge. Aggregates’ PSD in the column was measured on different days (Fig. 5). The median floc size (d0.5 ) was around 100 m on day-71 (similar to the reference sludge) and decreased slightly during the first phase. On day-212, bimodal peaks were observed, indicating that two populations of aggregates were present in the reactor. On day-260, the size of both these types of aggregates had increased significantly and the median size of the biggest population (granules) was around 900 m.
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Fig. 4. Evolutions of MLSS, MLVSS and SVI30 .
tion curves and the associated microscopic observations are shown in Fig. 6. Additionally, Kolmogorov microscales (17 and 62 m) corresponding to the two rotation speeds used are presented together with PSD (Fig. 6). In this experiment, the particle size distribution should be interpreted considering that two types of bio-particles co-exist in the reactor. On day-162 (Fig. 6a), microscopic observation indicated the appearance of homogeneous ovoid aggregates with co-existence of smaller flocs and grazing protozoa. During the cohesion test, the PSD clearly showed that the size of the aggregates remained almost constant whatever the shear rate applied. The curves were centred on a diameter of 80 m with a marked tail on the left which showing the existence of smaller particles (diameter around 10 m). Thus there was no apparent aggregate break-up under the different hydrodynamic conditions imposed in the stirred reactor. Hence, compared to the reference sludge, the resistance of aggregates to mechanical stress was definitely much higher. This means that strong aggregates were formed in the SBAR due to the new growth conditions imposed. The size of the aggregates was no
day-71
day-161
day-212
day-260
10 9 8
% Vol
7
Fig. 3. Example of the cohesion test on a flocculated biomass from a purely aerobic SBAR: (a) microscopic observation before cohesion test; (b) evolution of size distribution during the shear test (cf. Fig. 1b for the colour legend). Kolmogorov microscale for high ( ) and low ( ) rotation speed; (c) evolution of median diameter (d0.5 ) in volume during the cohesion test.
3.2.2. Bioaggregate cohesion During aerobic granular sludge formation, aggregate cohesion was assessed on different days in the stirred vessel. The characterisations were performed on samples collected on day-162 (i), day-212 (ii) and day-260 (iii) (arrows in Fig. 4). The size distribu-
6 5 4 3 2 1 0 1
100
10
d ( m) Fig. 5. Evolution of PSD in the SBAR.
1000
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Fig. 6. Variation of cohesion of aggregates during the formation of aerobic granules (cf. Fig. 1b for the colour legend of size distribution) and corresponding microscopic observation (bar: 200 m). Kolmogorov microscale for high ( ) and low ( ) rotation speed.
longer calibrated by the turbulence and stabilised well above the Kolmogorov microscale (17 and 62 m). This result demonstrates that the initial step of granulation corresponds to an increase in aggregate cohesion and it should be pointed out that the greatest decrease of SVI was observed during this initial phase (from 150 to 50 mL g−1 ). At the same time, observations indicated that aggregates became progressively more compact than the initial flocs (and would probably show higher density). On day-212 (Fig. 6b) the results suggested that two types of particles co-existed and presented different behaviours with respect to hydrodynamic strain. After sampling, the initial particle size distribution was clearly bimodal with two populations, one around 50 m (dmax1 ) and one around 210 m (dmax2 ). As shown by microscopic observation at this stage, large, dense aggregates
(100–600 m) clearly co-existed with small flocculated particles (10–100 m). Fig. 7a shows the evolution of dmax1 (small aggregates) and dmax2 (large aggregates) during the test. On the one hand, the size dmax1 is controlled by the turbulence change, i.e. the Kolmogorov microscale. Like the fragile flocs from the reference sludge (cf. Section 3.1), small aggregates seem to be broken at high velocity gradient and re-flocculated at low velocity gradient, reaching the same size as at the beginning. On the other hand, the value dmax2 is not significantly affected by modification of the velocity gradient. These results indicate a second phase in the granulation process: during this phase, granules grow rapidly but small fragile particles are still observed. This may be due either to the dispersed growth of flocs or to the detachment of fragile particles due to surface erosion of granules. This phenomenon was specifically
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Fig. 7. Evolution of dmax1 and dmax2 for the two respective peaks of the bimodal distribution during the cohesion test: (a) day-212; (b) day-260.
observed after an increase of organic loading rate (OLR) from 1.9 to 2.8 kg COD m−3 d−1 . In the sample collected on day-260 (Fig. 6c), matured granules were observed (showing a dense internal core). The PSD again shows a bimodal distribution. The new values of dmax1 and dmax2 indicate that both the small and large particles grew in size in the SBR, respectively, to 150 m and 900 m. At this stage, during the cohesion test performed in the stirred vessel, both dmax1 and dmax2 remained constant, independently of the velocity gradient, as shown in Fig. 7b. This means that neither the smaller nor the larger aggregate population seems to be influenced by the hydrodynamic stress induced during the test. Moreover, the size of both types of particles is much higher than the Kolmogorov microscale imposed in the vessel (17–62 m). Therefore, the two types of bioaggregates were very cohesive. Actually, two hypotheses can be proposed: small particles (still fragile on day-212) were either lost or progressively became bigger from day-212 to day-260. The latter phenomenon was probably encouraged in the SBAR because little particle selection (no washout) was imposed. In our study, as the settling time was 30 min, it seems reasonable to think that it was sufficiently long to retain small particles in the bioreactor and to make their growth possible with respect to their cohesion properties. Due to this phenomenon, two types of aggregates still coexisted in the reactor at this stage. Consequently, these results illustrate a third phase of granule formation: when the cohesion of all the particles became sufficient to make them resistant to the hydrodynamic and mechanical strain, particles could all grow above the turbulence micro-scale. Granules progressively reached a size ranging from 500 to 1000 m, which is a common value for aerobic granules [5,43].
ticle volume and thus were not significantly observed in the volume distribution. When focusing on the evolution of size distribution by number (Fig. 9b), we note the effect of the increase of velocity gradient: the distribution, originally monomodal (centred on 300 m), becomes bimodal when granules are subjected to a high rotation speed (G = 3400 s−1 ). A new population of small particles centred on a diameter (based on number) of about 50 m appeared and, even when the low rotation speed (G = 250 s−1 ) was applied again, no reflocculation was observed. Here, it appears clearly that small particles are detached from the surface of the granules and form a new population of particles. It should be pointed out that these new particles are strong enough to resist the micro turbulence as they are not calibrated at the Kolmogorov scale. This behaviour is clearly different from that observed for flocs (cf. Section 3.1). It leads us to think that those detached particles probably contain dense microcolonies from the surface of the granules and can constitute interesting seeds for new granule development. However, more work will be necessary to confirm this hypothesis and to determine the microbial diversity in detached particles. 4. Discussion 4.1. Initial formation of granular sludge Until now, few works have focused on the initial period of granule formation. Usually, granules have been selected as rapidly
3.3. Final granular sludge To characterise the granular sludge at steady state accurately, after 500 days of operation, granules were collected and small particles were removed by sieving at 400 m. Granules had a distinct ovoid shape (Fig. 8) and a size from 400 m to 1000 m. The SVI5 and SVI30 of these granules were similarly measured at 20 mL g−1 , which is comparable to other studies with pure granule suspensions [5]. As presented in Fig. 9, a cohesion test was performed on granule suspension. The PSD by volume (Fig. 9a) brings out the high cohesion of granules, the size of which remained almost constant during the steps of high and low rotation speed. However, a population of small particles (less than 90 m) appeared at the end of phase II (). These small particles contributed only slightly to the total par-
Fig. 8. Final appearance of isolated granules after 500 days (bar = 400 m).
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a
Phase I
Phase II
Phase III
b
Phase I
Phase II
Phase III
20
14
18
12
16 14
Number (%)
Vol (%)
10 8 6
12 10 8 6
4
4 2
2 0
0 1
10
100
1000
d (µm)
1
10
100
1000
d (µm)
Fig. 9. Behaviour of isolated granules when subjected to the cohesion test: (a) PSD by volume; (b) PSD by number.
as possible by a short settling phase and authors have mainly investigated the mature granules, looking for the best possible performance. Beun et al. [14] proposed a schematic diagram illustrating different phases: pellet formation, growth with development of filaments, detachment due to shear, growth of bacteria embedded in polymers, lysis and growth of dense colonies. Our study provides complementary information and demonstrates that, whereas initial floc size is clearly calibrated by the Kolmogorov microscale, aggregates become stronger and progressively resist the hydrodynamic stress. From the results, successive steps in the granulation process can be highlighted: (1) an initial increase of aggregate cohesion associated with a rapid decrease of SVI, (2) a growth of aggregates with temporary detachment of small fragile particles, and (3) a growth of all the particles to much larger than the turbulence micro-scale. Granular sludge was previously defined as “a particle which can settle rapidly and which does not coagulate under reduced shear stress” [43]. In this definition high settling velocity is pointed out (due to high density and size) but also the fact that granules are stable particles which cannot agglomerate (unlike flocs). Here we can introduce a new, complementary definition: granules are particles whose cohesion is sufficiently high to resist turbulence and shear stress fluctuations. This high cohesion allows granule size to increase to much higher than the turbulence microscale (whereas floc size is controlled by the Kolmogorov scale). Here, granular sludge was formed in an SBAR with a conventional settling time, i.e. without particle washout. We suggest that the modification of particle properties, i.e. higher strength and higher density, which are linked to modifications of microbial growth, initially explains the apparition of granules. In contrast, a reduction in settling time, if applied, would probably only accelerate the accumulation of granules. These data suggest that cohesion is a property which should be considered when characterising granules and investigating aerobic granulation mechanisms. More work is definitely necessary to understand the origin of this high cohesion of granules. In this study, granules were obtained by imposing an anoxic feast period and an aerobic famine period while the aeration rate was relatively moderate. Therefore, it should be pointed out that the initial increase of cohesion and decrease of SVI did not follow an increase of shear stress in the reactor as often reported in the literature but was linked to metabolic selection as proposed by few authors [9,10,42]. However, the results obtained during the cohesion test tend to confirm that an increase in hydrodynamic
constraints in the SBAR could play a role in the erosion of aggregate during granule growth. It is still difficult to give the fundamental reason why cohesion improved. One simple hypothesis is that this phenomena results from densification, dense colonies having more bonding interactions and thus being much difficult to break: Adav et al. [44] demonstrated in pure culture that a high autoaggregation index was a prerequisite for granule formation. It was assumed in our work that nitrate diffusion inside aggregates (where oxygen becomes limiting) could encourage aggregate densification but also that anoxic carbon storage could improve the conversion of readily biodegradable substrate into slowly biodegradable internal compounds (see Wan et al. [42]) favouring aggregate stability by the way of slow growth as already suggested [10]. Furthermore Liu et al. [45] underlined that formation of granules is associated with a sharp increase of hydrophobicity. Zhang et al. [46] also observed an increase of surface charge and hydrophobicity during the formation of aerobic granular sludge together with an increase of protein/polysaccharide ratio: the increase in protein content was found to decrease the surface negative charge of bacteria cells, thus reducing the electrostatic repulsions and favouring bridging between two neighbouring cells. As it is suggested in literature that EPS production can explain the difference between the properties of flocs and granules [45,47,48], more works are currently planned to evaluate the relation between cohesion of granules and EPS properties. At present, it is still difficult to give a physicochemical explanation of the high cohesion of granules even though experiments based on specific enzyme attacks tend to show that either -polysaccharides [49] or ␣-polysaccharides [48] could be responsible for this characteristic. 4.2. Stabilisation of granular sludge In a granular sludge system, aggregates can present heterogeneous retention times as smaller particles are preferentially lost after settling, especially if short settling times are imposed. The steady state is assumed to be reached when external data are stabilised (size, MLSS concentration and activity). This was obtained in our work after 360 days. It seems reasonable to think that the diameter of aerobic granules stabilises when the growth rate is counterbalanced by the detachment rate. Ren et al. [37] found gas bubbles and particle collisions to be the main reason for the detachment. Here, the cohesion test demonstrated that a high shear rate
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generated the release of particles of about 50 m. the dissipated energy in the SBAR is expected to be lower (around 100 s−1 ) than that obtained in the stirred vessel. However, Fig. 2a underlines that similar result could have been obtained with a lower rotation speed (kolmogorov microscale is around 23 m for 1200 rpm). Furthermore, the solid concentration in the airlift reactor is twelve times higher than the one used in the cohesion test. Thus the particle collisions due to the high MLSS concentration could locally induce such detachment, whose resulting small but cohesive particles possibly constitute the seeds for the formation of new granules. In long-term experiments, it could be expected that old granules would show lower cohesion because of deterioration of the internal structure. Adav et al. [50] indeed observed that the centres of granules became porous, showing an empty internal heart after long-term starvation. This was attributed to the fact that the internal part of the aggregate (exopolymers) could be partly biodegraded, i.e. used as a secondary substrate in such famine conditions. Recently, Lemaire et al. [51] also observed large channels and holes, which probably negatively affect granule cohesion, but these channels were partly filled by EPS. In our work, no loss of cohesion was observed after 500 days of operation. Basically, no significant rupture of granules occurred during the test for a range of G between 250 s−1 and 3400 s−1 . The occurrence of these mechanisms is probably linked to the sludge retention time in the sequencing batch reactor and the duration of the experiments. In our case, biomass was wasted with effluent withdrawal (the upper fraction of sludge volume exceeding 50% of the reactor). Calculations showed that the mean total SRT reached 40 days (it is possible that bigger granules were maintained in the system longer because of their settling capacity). With these conditions, granules size may be small enough to avoid too much transfer resistance. Growth is then sufficiently possible in the core of the aggregate to avoid (or slow down) a loss of integrity and a decrease of aggregate strength. This is crucial information for possible industrial applications of aerobic granular sludge as the stability of the granule structure should be guaranteed in order to maintain high performance.
5. Conclusion The aggregate strength of flocculated sludge and granular sludge were assessed in a stirred reactor, by varying the rotation speed. Special attention was paid to the initial period of granular sludge formation in a sequencing batch airlift reactor and some important insights can be underlined: • It is confirmed that high SAV and low settling time are not prerequisites to produce granules if proper metabolic selection (here by alternating anoxic feast/aerobic famine), is applied. • At the initial phase of granular sludge formation, an increase of aggregate cohesion is first observed, whereas SVI decreases rapidly; then aggregates resist high shear imposed in the stirred cell. • Then growth of aggregates is progressively observed. Small particles are temporarily detached, indicating the role of hydrodynamics in particle erosion. • As no aggregate selection was performed, bimodal particle size distribution was observed, the small population of detached particles probably constituting the seeds for new granules. Granules showed a high cohesion even after 500 days of operation. • Tests performed on mature granules (diameter greater than 400 m) indicate that high energy dissipation (ε ≈ 10 m2 s−3 ) leads to surface detachment of particles of about 50 m, these small particles also showing high cohesion.
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These results show that granules can be defined as microbial aggregates with a higher cohesion than flocs, which makes it possible for them to grow to a size much greater than the turbulence micro-scale. This indicates that a key point in granulation is the initial increase in aggregate cohesion, which was observed in the present work after anoxic–aerobic alternance was imposed. Further work will be necessary to understand the reasons for this cohesion increase, in relation with the biological (formation of colonies) and chemical (EPS quantity and properties) modifications occurring inside aggregates. Acknowledgements This work was supported by the UT-INSA programme. It is part of the Ph.D. research of Junfeng WAN. The authors also thank Aldo Moro and Evrard Mengelle for their technical contributions to this work. References [1] H.S. Shin, K.H. Lim, H.S. Park, Effect of shear-stress on granulation in oxygen aerobic upflow sludge bed reactors, Water Sci. Technol. 26 (1992) 601–605. [2] K. Mishima, M. Nakamura, Self-immobilization of aerobic activated-sludge—a pilot-study of the aerobic upflow sludge blanket process in municipal sewagetreatment, Water Sci. Technol. 23 (1991) 981–990. [3] Y. Liu, J.-H. Tay, State of the art of biogranulation technology for wastewater treatment, Biotechnol. Adv. 22 (2004) 533–563. [4] S.S. Adav, D.J. Lee, K.Y. Show, J.H. Tay, Aerobic granular sludge: recent advances, Biotechnol. Adv. 26 (2008) 411–423. [5] M.K. de Kreuk, N. Kishida, M.C.M. van Loosdrecht, Aerobic granular sludge—state of the art, Water Sci. Technol. 55 (2007) 75–81. [6] L.M.M. de Bruin, M.K. de Kreuk, H.F.R. van der Roest, C. Uijterlinde, M.C.M. van Loosdrecht, Aerobic granular sludge technology: an alternative to activated sludge? Water Sci. Technol. 49 (2004) 1–7. [7] B.S. McSwain, R.L. Irvine, M. Hausner, P.A. Wilderer, Composition and distribution of extracellular polymeric substances in aerobic flocs and granular sludge, Appl. Environ. Microbiol. 71 (2005) 1051–1057. [8] M.C.M. van Loosdrecht, M.K.D. Kreuk, J.J. Heijnen, The unity of biofilm structures, in: S. Bathe, M.K.D. Kreuk, B.S. McSwain, N. Schwarzenbeck (Eds.), Aerobic Granular Sludge, IWA Publishing, 2005, pp. 1–6. [9] Y. Liu, S.-F. Yang, J.-H. Tay, Improved stability of aerobic granules by selecting slow-growing nitrifying bacteria, J. Biotechnol. 108 (2004) 161. [10] M.K. de Kreuk, M.C.M. van Loosdrecht, Selection of slow growing organisms as a means for improving aerobic granular sludge stability, Water Sci. Technol. 49 (2004) 9–17. [11] J.H. Tay, Q.S. Liu, Y. Liu, The effects of shear force on the formation, structure and metabolism of aerobic granules, Appl. Microbiol. Biotechnol. 57 (2001) 227–233. [12] B.S. McSwain, R.L. Irvine, Dissolved oxygen as a key parameter to aerobic granule formation, Water Sci. Technol. 58 (2008) 781–787. [13] Y. Liu, J.H. Tay, The essential role of hydrodynamic shear force in the formation of biofilm and granular sludge, Water Res. 36 (2002) 1653–1665. [14] J.J. Beun, A. Hendriks, M.C.M. Van Loosdrecht, E. Morgenroth, P.A. Wilderer, J.J. Heijnen, Aerobic granulation in a sequencing batch reactor, Water Res. 33 (1999) 2283–2290. [15] B.E. Zima-Kulisiewicz, L. Díez, W. Kowalczyk, C. Hartmann, A. Delgado, Biofluid mechanical investigations in sequencing batch reactor (SBR), Chem. Eng. Sci. 63 (2008) 599–608. [16] C. Coufort, C. Dumas, D. Bouyer, A. Line, Analysis of floc size distributions in a mixing tank, Chem. Eng. Process. 47 (2008) 287–294. [17] B. Arrojo, A. Mosquera-Corral, J.L. Campos, R. Méndez, Effects of mechanical stress on Anammox granules in a sequencing batch reactor (SBR), J. Biotechnol. 123 (2006) 453. [18] D. Elenter, K. Milferstedt, W. Zhang, M. Hausner, E. Morgenroth, Influence of detachment on substrate removal and microbial ecology in a heterotrophic/autotrophic biofilm, Water Res. 41 (2007) 4657–4671. [19] E. Morgenroth, P.A. Wilderer, Influence of detachment mechanisms on competition in biofilms, Water Res. 34 (2000) 417–426. [20] D.H. Bache, C. Johnson, J.F. McGilligan, E. Rasool, A conceptual view of floc structure in the sweep floc domain, Water Sci. Technol. 36 (1997) 49–56. [21] M. Boller, S. Blaser, Particles under stress, Water Sci. Technol. 37 (1998) 9–29. [22] L.A. Glasgow, J.P. Hsu, An experimental-study of floc strength, AIChE J. 28 (1982) 779–785. [23] P. Jarvis, B. Jefferson, J. Gregory, S.A. Parsons, A review of floc strength and breakage, Water Res. 39 (2005) 3121–3137. [24] B.M. Wilen, K. Keiding, P.H. Nielsen, Flocculation of activated sludge flocs by stimulation of the aerobic biological activity, Water Res. 38 (2004) 3909–3919. [25] C. Selomulya, G. Bushell, R. Amal, T.D. Waite, Aggregate properties in relation to aggregation conditions under various applied shear environments, Int. J. Miner. Process. 73 (2004) 295–307.
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[40] S. Lambert, A. Thill, P. Ginestet, J.M. Audic, J.Y. Bottero, Structural interpretations of static light scattering patterns of fractal aggregates. I. Introduction of a mean optical index: numerical simulations, J. Colloid Interface Sci. 228 (2000) 379–385. [41] D.N. Thomas, S.J. Judd, N. Fawcett, Flocculation modelling: a review, Water Res. 33 (1999) 1579–1592. [42] J. Wan, Y. Bessière, M. Spérandio, Alternating anoxic feast/aerobic famine condition for improving granular sludge formation in sequencing batch airlift reactor at reduced aeration rate, Water Res. 43 (2009) 5097–5108. [43] M. de Kreuk, B. McSwain, S. Bathe, S. Tay, N. Schwarzenbeck, P. Wilderer, Discussion Outcomes, in: S. Bathe, M.K. de Kreuk, B.S. McSwain, N. Schwarzenbeck (Eds.), Aerobic Granular Sludge, IWA Publishing, 2005, pp. 155–159. [44] S.S. Adav, D.-J. Lee, J.-Y. Lai, Intergeneric coaggregation of strains isolated from phenol-degrading aerobic granules, Appl. Microbiol. Biotechnol. 79 (2008) 657–661. [45] Y. Liu, S.-F. Yang, Q.-S. Liu, J.-H. Tay, The role of cell hydrophobicity in the formation of aerobic granules, Curr. Microbiol. 46 (2003) 270–274. [46] L. Zhang, X. Feng, N. Zhu, J. Chen, Role of extracellular protein in the formation and stability of aerobic granules, Enzyme Microb. Technol. 41 (2007) 551–557. [47] Y.Q. Liu, Y. Liu, J.H. Tay, The effects of extracellular polymeric substances on the formation and stability of biogranules, Appl. Microbiol. Biotechnol. 65 (2004) 143–148. [48] T. Seviour, M. Pijuan, T. Nicholson, J. Keller, Z.G. Yuan, Understanding the properties of aerobic sludge granules as hydrogels, Biotechnol. Bioeng. 102 (2009) 1483–1493. [49] S.S. Adav, D.J. Lee, J.H. Tay, Extracellular polymeric substances and structural stability of aerobic granule, Water Res. 42 (2008) 1644–1650. [50] S.S. Adav, D.J. Lee, J.H. Tay, Activity and structure of stored aerobic granules, Environ. Technol. 28 (2007) 1227–1235. [51] R. Lemaire, R.I. Webb, Z.G. Yuan, Micro-scale observations of the structure of aerobic microbial granules used for the treatment of nutrient-rich industrial wastewater, ISME J. 2 (2008) 528–541.
Contents lists available at SciVerse ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Evolution of bioaggregate strength during aerobic granular sludge formation Junfeng Wan a,b,c , Irene Mozo a,b,c , Ahlem Filali a,b,c , Alain Liné a,b,c , Yolaine Bessière a,b,c , Mathieu Spérandio a,b,c,∗ a b c
Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France CNRS, UMR5504, F-31400 Toulouse, France
a r t i c l e
i n f o
Article history: Received 7 January 2011 Received in revised form 23 July 2011 Accepted 25 August 2011 Available online 31 August 2011 Keywords: Aerobic granular sludge Aggregate strength Shear Cohesion
a b s t r a c t This work investigated the modification of aggregate properties during the formation of granular sludge in a sequencing batch airlift reactor (SBAR). The cohesion of biological aggregates was quantified by subjecting sludge samples to two different controlled shear stresses in a stirred reactor. For reference sludge (without granules), flocs broke and reformed easily, indicating that floc size was controlled by the turbulence micro-scale (Kolmogorov scale, here from 17 m to 62 m). In contrast, granules showed high strength which enabled them to resist turbulence and their size was no longer imposed by the Kolmogorov micro-scale. Different steps were observed during the granulation process: a first increase of aggregate cohesion associated with a decrease in sludge volume index (SVI), a growth of aggregates with detachment of fragile particles from the surface and, finally, an increase in the sizes of small and large granules to reach a pseudo-stable size distribution. Results suggest that small particles could have formed the seeds for new granules, as they were maintained in the bioreactor. Here, granular sludge was formed in an SBAR with a conventional settling time (30 min), i.e. without particle washout, and with a low superficial air velocity (SAV = 0.6 cm s−1 ): it is thus demonstrated that high SAV and low settling time are not necessary to produce granules, but probably only accelerate the accumulation of granules. It is shown that the increase of cohesion is the initial phenomenon explaining the granule formation concomitantly with bacterial aggregates densification. It seems important, in the future, to investigate the reasons for this cohesion increase, which is possibly explained either by bacterial bounding interactions or the excretion of extracellular polymeric substances (EPS). © 2011 Elsevier B.V. All rights reserved.
1. Introduction Since the phenomenon of aerobic granular sludge was first reported in the 1990s [1,2], research on the subject has flourished and this new technology has shown its significant perspectives in wastewater treatment [3–5]. Compared with conventional activated sludge, aerobic granular sludge presents a variety of attractive properties such as more compact structure, higher specific activity and better settling properties as required to improve solid–liquid separation. In most of the studies, granular sludge was formed in a sequencing batch reactor [6]. Aerobic granules were obtained by consecutively inoculating with conventional activated sludge and applying operating conditions which favoured the development and selection of granules. Because of its compact structure, this particular aggregate is expected to present similarities with biofilm, which is mainly composed of bacteria
∗ Corresponding author. Tel.: +33 561559785; fax: +33 561559760. E-mail address: [email protected] (M. Spérandio). 1369-703X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2011.08.015
and extracellular polymeric substances [7,8]. However, if mechanisms involved in the initial formation of granules have been the subject of many researches, relationships between microbial aggregation mechanisms, reactor operational conditions and the resulting physical properties of granules still need to be clarified. So far, the literature proposes biological and process conditions leading to granular sludge formation. Concerning the biological selection pressure, stability of granules is found to be improved when selecting slow growing bacteria. This can be done either when increasing the substrate N/COD ratio [9] to favour nitrifying bacteria or by alternating feast/famine and anaerobic/aerobic periods [10] to promote selection of phosphate or glycogen accumulating organisms. Concerning the process conditions, the literature [11–14] underlines that aeration rate is an important factor, influencing granular sludge formation through three main, simultaneous mechanisms: (i) oxygen transfer, which modifies the dissolved oxygen in the bulk and hence within the granule, (ii) mixing, which influences mass transfer of substrates in the liquid boundary layer and (iii) hydrodynamic shear stress, which controls erosion and
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fragmentation. A high superficial gas velocity (SGV) has been seen as a major factor enhancing the formation of aerobic granular sludge but its roles (shear stress, mixing and oxygen transfer) are still controversial due to the interdependency of these processes [12]. Investigations have focused more on biological aspects and aerobic granulation has not been thoroughly studied from the fluid mechanics point of view [15]. So far, little information concerning the influence of mechanical stresses on granule size and shape has been reported. The results of Zima-Kulisiewicz et al. [15] suggest that the shear stress (tangential force) and elongation stress (normal force) together with stresses induced by fluctuating velocities are the main forces influencing granule building. Although the origin and significance of mechanical stresses in the bioreactor have been clarified, only few approaches has been developed to estimate whether bioaggregate strength and size are altered by these stresses, whereas a strong relationship has been reported between the size of physicochemical flocs and the Kolmogorov microscale [16]. Particle strength (or cohesion) has rarely been measured on aerobic granular sludge but it is reasonable to think that this property plays a role in granule formation and stability as underlined on Anammox granules where an increase of input specific power was shown to affect activity and biomass retention [17]. Because it generally controls surface detachment (erosion) and particle breakage, aggregate strength should receive more attention. Detachment of particles is a crucial phenomenon for aerobic granular sludge, as it limits the growth rate and controls the stability of granules (as well as the specific activity of supported bacteria). Detachment rate also controls the microbial competition between fast-growing heterotrophic bacteria and slow-growing autotrophic bacteria as has been shown numerically and experimentally in the case of biofilms [18,19]. This phenomenon is of importance as both kinds of bacteria are necessary for simultaneous carbon and nitrogen removal. The significance of detachment is strongly related to the cohesion of granules. Cohesion is a physical property of the aggregate defining how it can resist shear and elongation force, which induce erosion and/or break-up. It is well known that the strength of aggregates depends on the strength and number of the bonds holding the aggregates together [20]. Aggregate breakage is likely to occur if the stress applied to the surface of the aggregate can counterbalance the bonding strength [21], which means that the more compact the structure of aggregates is, the more difficult they are to break. Thus, cohesion is directly related to structure and is, therefore, highly dependent upon the aggregate formation process. Techniques for measuring granule strengths can be derived from those developed for measuring floc strength. Floc strength has been largely studied [22,23] using various methods. Most of the methods used have been impeller-based techniques [24–29]. Floc strength may be estimated through floc size analysis by examining its breakup consecutively by exposing to known stresses [30]. The method consists of subjecting flocs to single or increasing levels of shear within a vessel and comparing the ratio of the floc size before and after breakage, thus quantifying the energy input necessary for floc breakage [31]. Other techniques, such as ultrasonic methods, have also been adapted to test aggregate strength but are more suitable for application to mineral flocs than to bioaggregates due to the effect of ultrasound on bacterial components [32]. Similar studies have recently focused on characterising granules from anaerobic processes in the upflow anaerobic sludge blanket (UASB) system. For example, the resistance to compression forces has been measured recently [33]. In a previous work [34], anaerobic granules were subjected to strong mechanical stirring (200 rpm) for 5 min. The granular strength of anaerobic granules was deduced from the ratio of solids in the supernatant to the total weight of the granular sludge. This ratio gives an indicative index of the granules’ resistance when subjected to abrasion and shear, but gives no infor-
mation on the modification of the particle sizes. This method has been combined with image analysis to compare two different types of aerobic granules developed in aerobic sequencing batch reactors (SBRs) [35]: fungal granules from one SBR were larger and weaker, with a loosely packed, fluffy structure whereas the bacterial granules from another SBR were smaller and stronger, with a compact structure and higher decantation feasibility. Little information is available to understand how the operating conditions can affect the particle strength in an aerobic granular sludge process. A study carried out by Di Iaconi et al. [36] investigated the influence of hydrodynamic shear forces on the properties of granular biomass developed in a sequencing batch aerobic biofilter reactor. They found that granular biomass density increased quasi-linearly with the increase of shear forces in the reactor and that EPS content and composition were not affected by hydrodynamic shear forces. More recently, an energy-dissipation-based model has been established by Ren et al. [37] to quantify the shear stresses in an aerobic granular sludge SBR and predict the rate of particle detachment. Simulations indicate that shear is induced mainly by gas bubbles or granule collisions and only slightly by the fluid. The possible modification of particle strength has not been integrated in this model. Because the detachment is quantified by absorbance measurement, no information is provided on the size of the detached particles and remaining granules. In conclusion, little is known at present about the evolution of cohesion during granular sludge formation, and yet this should be helpful in understanding the mechanism of granule formation. Consequently, the goal of this study is to characterise the evolution of aggregate cohesion during the formation of aerobic granular sludge, and the behaviour of granules under given hydrodynamic conditions. To determine aggregate strength, biological aggregates were subjected to a calibrated stress in a stirred reactor and the particle size distribution was measured. Flocculated biomass was first characterised and considered as a reference point. Then granular sludge was developed in a statically filled sequencing batch airlift reactor (SBAR) by applying substrate feast and famine periods coupled with alternating anoxic/aerobic conditions. The analysis focused on the progressive conversion from a floc-like structure to granule-like aggregates, and non-significant hydraulic particle selection was applied during this period (using a 30 min settling phase). Finally, at the end of the study, granules were collected after sieving and their specific behaviour was characterised in the stirred reactor.
2. Materials and methods 2.1. Sequencing batch airlift reactor (SBAR) The reactor employed was an airlift column with a working volume of 17 L (internal diameter = 15 cm, total height = 105 cm, H/D ratio = 7). A plate baffle (length: width = 83:15 cm) placed vertically in the middle of the reactor divided the column into two zones: riser and downcomer. Air was introduced through a fine bubble aerator at the bottom of the reactor, and induced the circulation of air, liquid and solid. The system is schematically presented in Fig. 1a. The column was inoculated with 10 L of conventional activated sludge taken from the aeration tank of a local municipal wastewater treatment plant at a mixed liquor suspended solid (MLSS) concentration of 4.5 g L−1 and a sludge volume index after 30 min (SVI30 ) of 172 mL g−1 . The reactor was fed with a synthetic wastewater having a mixed organic carbon source: glucose, ethanol, sodium propionate and sodium acetate, each contributing 25% of the total COD (total = 750–1000 mgCOD L−1 ). The other components of the wastewater (mg L−1 ) were as follows: NH4 Cl
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Fig. 1. Schematic diagram of the SBAR (a) and cohesion test vessel (b).
(143–191), NaNO3 (610), NaHCO3 (100), KH2 PO4 (100), CaCl2 (30), MgSO4 ·7H2 O (4) and other necessary elements were similar to the trace solution described elsewhere [38]. The organic and ammonium loading rates were simultaneously increased during the study with a constant COD/N ratio of 20 (Table 1) by increasing the influent concentration or decreasing the cycle duration. The system was first operated by cycles of 6 h including influent filling (30 min), preanoxic phase (30 min), aeration (240 min), settling (30 min) and effluent withdrawal (30 min). Reduction of the cycle to 4 h was performed by a proportional reduction of the filling, anoxic and aerobic phases. A relatively low particle selection pressure was imposed compared to those used in previous studies on granular sludge [3,11,14]. The process was initially operated with a high aeration rate (superficial air velocity SAV = 2.23 cm s−1 ) and a purely aerobic reaction phase. From day 80, the SAV was reduced to 0.63 cm s−1 , and the pre-anoxic phase was introduced from day-120 (by means of nitrogen gas sparging at the same gas velocity).
In this study, the optical index of the dispersant was set to 1.330, which corresponded to the value for the water. As biological aggregates are mainly composed of organic matter, the optical index was set to 1.596 as indicated in the work of Lambert et al. [40]. Qualitative observations were performed by using either a microscope or a stereoscopic microscope depending on the size of the aggregates. 2.3. Cohesion tests Cohesion tests were performed in a stirred vessel, equipped with an impeller (5 cm in diameter), consisting of a 145 mm × 245 mm (inner diameter × height) cylinder providing a testing volume of 2.5 L (Fig. 1b). The shear level corresponding to the operating conditions induced during this experiment can be approximated by the average velocity gradient value (G) for isotropic and homogeneous turbulence as follows:
P V
2.2. Analytical methods
G=
Sludge samples taken from the column were characterised in terms of suspended solids (SS) and volatile suspended solids (VSS) using standard methods [39]. Sludge volume index (SVI) was measured in a 1000 mL graduated cylinder with the sludge sample taken from the mixed liquor at the end of the aerobic phase. SVI5 and SVI30 were measured after 5 and 30 min, respectively, so as to compare aggregates properties with the one proposed for granular sludge in literature. The particle size distribution (PSD) of aggregates was analysed using a laser diffraction technique (Mastersizer, Malvern 2000, UK) with a particle size ranging from 0.02 m to 2000 m.
where G is the velocity gradient (s−1 ), P is the power input (W), is the dynamic viscosity (Pa s) and V is the suspension volume (m3 ). The power input (P) was calculated as the energy dissipated by the impeller:
Table 1 Strategy for organic and ammonium loading rates. Time
Duration of cycle (h)
OLR (kg COD d−1 m−3 )
ALR (kg N-NH4 d−1 m−3 )
d70–120 d120–175 d175–256
6 6 4
1.412 1.882 2.822
0.071 0.094 0.141
P = Np N 3 d5
(1)
(2)
where Np is the power number given by the manufacturer (4 in our case: turbine, 4 flat blades), is the mass density of the fluid (kg m−3 ), N is the rotation speed (s−1 ) and d is the impeller diameter (m). The mechanical stirring system provided an average velocity gradient of up to 3400 s−1 . By considering the energy dissipation (ε in m2 s−3 ) in terms of a gradient (G) dependent expression: ε = · G2
(3)
where is the kinematic viscosity (m2 s−1 ), the Kolomogorov microscale ( in m), corresponding to the size of the smallest
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Fig. 2. Hydrodynamic test conditions in the vessel and the measurement time: (a) theoretical hydrodynamic characterisation of the system (vessel + impeller); (b) hydrodynamic test conditions during the three phases: low mixing intensity (phases I and II) and high mixing intensity (phase II) and symbols used for different times.
eddies during mixing (linked to the shear level in the vessel), could be determined:
=
3 ε
1/4
(4)
Based on the previous definitions, a theoretical characterisation of the cohesion test system was performed in terms of average velocity gradient and Kolmogorov microscale resulting from a rotation speed as presented in Fig. 2a. As shown in Fig. 2b, the cohesion test consisted of a three-phase procedure: the vessel was first operated at a low mixing intensity (300 rpm/250 s−1 ) for 45 min (phase I); then the shear intensity was increased (1700 rpm/3400 s−1 ) for 45 min (phase II) and, finally, decreased to the original condition for 45 min (phase III). Those operating conditions lead to turbulent flow regime with impeller’s Reynolds numbers of 12 500 and 70 000, respectively. During the entire period, PSD was measured 5 min before the end of each phase (Fig. 2b). The Kolmogorov microscale corresponding to the applied G values was 62 m for a G value of 250 s−1 (300 rpm) during phases I and III, and 17 m for 3400 s−1 (1700 rpm) during phase II. For each test, the sludge was sampled at the end of the aerobic phase of the operational cycle of the SBAR and systematically diluted to 1 g L−1 by using the ultra centrifuged supernatant (10 000 × g, 15 min) in order to avoid any modification of the environment, such as the ionic strength, which is known to have a significant effect on flocculation processes. 3. Results 3.1. Characterisation of floc behaviour (reference) during the cohesion test As a starting point, the cohesion test was performed on a flocculated sludge (Fig. 3a) developed in a purely aerobic SBAR fed by the same synthetic influent as for the anoxic/aerobic SBAR. Sludge was sampled from the reactor and placed in the stirred reactor before various stirring rates were imposed. As can be observed in Fig. 3b, the particle size distribution of this flocculated biomass was clearly influenced by the hydrodynamics: an increase in impeller rotation speed resulted in a significant reduction in median diameter from 80 to 18 m. After reduction of the velocity gradient, an increase in the median diameter was
observed (Fig. 3c) indicating a reflocculation of the particles and resulting in full recovery of the initial particle size. Furthermore, it can be seen that the suspension seemed to be calibrated by the turbulence scale as the average diameter (70 and 16 m) was close to the Kolmogorov microscale (62 and 17 m, respectively). Similarly, it has been demonstrated that chemically induced flocculation is controlled by hydrodynamic turbulence in stirred coagulation reactors [41]. The present results suggest that the approach proposed for physicochemical flocs is still valid for biological flocs developed in the laboratory with soluble synthetic substrates.
3.2. Evolution of aggregate properties during granular sludge formation in SBAR 3.2.1. Suspended solid concentration, SVI and particle size As shown in Fig. 4, the profiles of MLSS and SVI versus time varied inversely during the study. Before day 120, MLSS first increased from 2.5 to 4.5 g L−1 whereas SVI30 decreased from 150 to 100 mL g−1 . Then the pre-anoxic mixing phase was introduced in the cycle after the static filling phase, from day-120 (by supplying nitrogen gas instead of air at the same gas flow rate). Later again, a significant decrease of SVI30 from 100 to 45 mL g−1 and an increase of MLSS up to around 9.5 g L−1 were observed. These data are thoroughly described in Wan et al. [42]. From day-175, the organic, ammonium and nitrate loading rates were increased, as the cycle time was reduced from 6 to 4 h. After a temporary drop for a short period, MLSS recovered the same level as before, and larger and larger particles (>200 m) were observed in the airlift column. MLSS continued to increase during the following months and stabilised around 15 g L−1 after 350 days. Simultaneously, SVI decreased to 30 mL g−1 indicating typical properties of granular sludge. Aggregates’ PSD in the column was measured on different days (Fig. 5). The median floc size (d0.5 ) was around 100 m on day-71 (similar to the reference sludge) and decreased slightly during the first phase. On day-212, bimodal peaks were observed, indicating that two populations of aggregates were present in the reactor. On day-260, the size of both these types of aggregates had increased significantly and the median size of the biggest population (granules) was around 900 m.
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Fig. 4. Evolutions of MLSS, MLVSS and SVI30 .
tion curves and the associated microscopic observations are shown in Fig. 6. Additionally, Kolmogorov microscales (17 and 62 m) corresponding to the two rotation speeds used are presented together with PSD (Fig. 6). In this experiment, the particle size distribution should be interpreted considering that two types of bio-particles co-exist in the reactor. On day-162 (Fig. 6a), microscopic observation indicated the appearance of homogeneous ovoid aggregates with co-existence of smaller flocs and grazing protozoa. During the cohesion test, the PSD clearly showed that the size of the aggregates remained almost constant whatever the shear rate applied. The curves were centred on a diameter of 80 m with a marked tail on the left which showing the existence of smaller particles (diameter around 10 m). Thus there was no apparent aggregate break-up under the different hydrodynamic conditions imposed in the stirred reactor. Hence, compared to the reference sludge, the resistance of aggregates to mechanical stress was definitely much higher. This means that strong aggregates were formed in the SBAR due to the new growth conditions imposed. The size of the aggregates was no
day-71
day-161
day-212
day-260
10 9 8
% Vol
7
Fig. 3. Example of the cohesion test on a flocculated biomass from a purely aerobic SBAR: (a) microscopic observation before cohesion test; (b) evolution of size distribution during the shear test (cf. Fig. 1b for the colour legend). Kolmogorov microscale for high ( ) and low ( ) rotation speed; (c) evolution of median diameter (d0.5 ) in volume during the cohesion test.
3.2.2. Bioaggregate cohesion During aerobic granular sludge formation, aggregate cohesion was assessed on different days in the stirred vessel. The characterisations were performed on samples collected on day-162 (i), day-212 (ii) and day-260 (iii) (arrows in Fig. 4). The size distribu-
6 5 4 3 2 1 0 1
100
10
d ( m) Fig. 5. Evolution of PSD in the SBAR.
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Fig. 6. Variation of cohesion of aggregates during the formation of aerobic granules (cf. Fig. 1b for the colour legend of size distribution) and corresponding microscopic observation (bar: 200 m). Kolmogorov microscale for high ( ) and low ( ) rotation speed.
longer calibrated by the turbulence and stabilised well above the Kolmogorov microscale (17 and 62 m). This result demonstrates that the initial step of granulation corresponds to an increase in aggregate cohesion and it should be pointed out that the greatest decrease of SVI was observed during this initial phase (from 150 to 50 mL g−1 ). At the same time, observations indicated that aggregates became progressively more compact than the initial flocs (and would probably show higher density). On day-212 (Fig. 6b) the results suggested that two types of particles co-existed and presented different behaviours with respect to hydrodynamic strain. After sampling, the initial particle size distribution was clearly bimodal with two populations, one around 50 m (dmax1 ) and one around 210 m (dmax2 ). As shown by microscopic observation at this stage, large, dense aggregates
(100–600 m) clearly co-existed with small flocculated particles (10–100 m). Fig. 7a shows the evolution of dmax1 (small aggregates) and dmax2 (large aggregates) during the test. On the one hand, the size dmax1 is controlled by the turbulence change, i.e. the Kolmogorov microscale. Like the fragile flocs from the reference sludge (cf. Section 3.1), small aggregates seem to be broken at high velocity gradient and re-flocculated at low velocity gradient, reaching the same size as at the beginning. On the other hand, the value dmax2 is not significantly affected by modification of the velocity gradient. These results indicate a second phase in the granulation process: during this phase, granules grow rapidly but small fragile particles are still observed. This may be due either to the dispersed growth of flocs or to the detachment of fragile particles due to surface erosion of granules. This phenomenon was specifically
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Fig. 7. Evolution of dmax1 and dmax2 for the two respective peaks of the bimodal distribution during the cohesion test: (a) day-212; (b) day-260.
observed after an increase of organic loading rate (OLR) from 1.9 to 2.8 kg COD m−3 d−1 . In the sample collected on day-260 (Fig. 6c), matured granules were observed (showing a dense internal core). The PSD again shows a bimodal distribution. The new values of dmax1 and dmax2 indicate that both the small and large particles grew in size in the SBR, respectively, to 150 m and 900 m. At this stage, during the cohesion test performed in the stirred vessel, both dmax1 and dmax2 remained constant, independently of the velocity gradient, as shown in Fig. 7b. This means that neither the smaller nor the larger aggregate population seems to be influenced by the hydrodynamic stress induced during the test. Moreover, the size of both types of particles is much higher than the Kolmogorov microscale imposed in the vessel (17–62 m). Therefore, the two types of bioaggregates were very cohesive. Actually, two hypotheses can be proposed: small particles (still fragile on day-212) were either lost or progressively became bigger from day-212 to day-260. The latter phenomenon was probably encouraged in the SBAR because little particle selection (no washout) was imposed. In our study, as the settling time was 30 min, it seems reasonable to think that it was sufficiently long to retain small particles in the bioreactor and to make their growth possible with respect to their cohesion properties. Due to this phenomenon, two types of aggregates still coexisted in the reactor at this stage. Consequently, these results illustrate a third phase of granule formation: when the cohesion of all the particles became sufficient to make them resistant to the hydrodynamic and mechanical strain, particles could all grow above the turbulence micro-scale. Granules progressively reached a size ranging from 500 to 1000 m, which is a common value for aerobic granules [5,43].
ticle volume and thus were not significantly observed in the volume distribution. When focusing on the evolution of size distribution by number (Fig. 9b), we note the effect of the increase of velocity gradient: the distribution, originally monomodal (centred on 300 m), becomes bimodal when granules are subjected to a high rotation speed (G = 3400 s−1 ). A new population of small particles centred on a diameter (based on number) of about 50 m appeared and, even when the low rotation speed (G = 250 s−1 ) was applied again, no reflocculation was observed. Here, it appears clearly that small particles are detached from the surface of the granules and form a new population of particles. It should be pointed out that these new particles are strong enough to resist the micro turbulence as they are not calibrated at the Kolmogorov scale. This behaviour is clearly different from that observed for flocs (cf. Section 3.1). It leads us to think that those detached particles probably contain dense microcolonies from the surface of the granules and can constitute interesting seeds for new granule development. However, more work will be necessary to confirm this hypothesis and to determine the microbial diversity in detached particles. 4. Discussion 4.1. Initial formation of granular sludge Until now, few works have focused on the initial period of granule formation. Usually, granules have been selected as rapidly
3.3. Final granular sludge To characterise the granular sludge at steady state accurately, after 500 days of operation, granules were collected and small particles were removed by sieving at 400 m. Granules had a distinct ovoid shape (Fig. 8) and a size from 400 m to 1000 m. The SVI5 and SVI30 of these granules were similarly measured at 20 mL g−1 , which is comparable to other studies with pure granule suspensions [5]. As presented in Fig. 9, a cohesion test was performed on granule suspension. The PSD by volume (Fig. 9a) brings out the high cohesion of granules, the size of which remained almost constant during the steps of high and low rotation speed. However, a population of small particles (less than 90 m) appeared at the end of phase II (). These small particles contributed only slightly to the total par-
Fig. 8. Final appearance of isolated granules after 500 days (bar = 400 m).
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a
Phase I
Phase II
Phase III
b
Phase I
Phase II
Phase III
20
14
18
12
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Vol (%)
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4 2
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0 1
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Fig. 9. Behaviour of isolated granules when subjected to the cohesion test: (a) PSD by volume; (b) PSD by number.
as possible by a short settling phase and authors have mainly investigated the mature granules, looking for the best possible performance. Beun et al. [14] proposed a schematic diagram illustrating different phases: pellet formation, growth with development of filaments, detachment due to shear, growth of bacteria embedded in polymers, lysis and growth of dense colonies. Our study provides complementary information and demonstrates that, whereas initial floc size is clearly calibrated by the Kolmogorov microscale, aggregates become stronger and progressively resist the hydrodynamic stress. From the results, successive steps in the granulation process can be highlighted: (1) an initial increase of aggregate cohesion associated with a rapid decrease of SVI, (2) a growth of aggregates with temporary detachment of small fragile particles, and (3) a growth of all the particles to much larger than the turbulence micro-scale. Granular sludge was previously defined as “a particle which can settle rapidly and which does not coagulate under reduced shear stress” [43]. In this definition high settling velocity is pointed out (due to high density and size) but also the fact that granules are stable particles which cannot agglomerate (unlike flocs). Here we can introduce a new, complementary definition: granules are particles whose cohesion is sufficiently high to resist turbulence and shear stress fluctuations. This high cohesion allows granule size to increase to much higher than the turbulence microscale (whereas floc size is controlled by the Kolmogorov scale). Here, granular sludge was formed in an SBAR with a conventional settling time, i.e. without particle washout. We suggest that the modification of particle properties, i.e. higher strength and higher density, which are linked to modifications of microbial growth, initially explains the apparition of granules. In contrast, a reduction in settling time, if applied, would probably only accelerate the accumulation of granules. These data suggest that cohesion is a property which should be considered when characterising granules and investigating aerobic granulation mechanisms. More work is definitely necessary to understand the origin of this high cohesion of granules. In this study, granules were obtained by imposing an anoxic feast period and an aerobic famine period while the aeration rate was relatively moderate. Therefore, it should be pointed out that the initial increase of cohesion and decrease of SVI did not follow an increase of shear stress in the reactor as often reported in the literature but was linked to metabolic selection as proposed by few authors [9,10,42]. However, the results obtained during the cohesion test tend to confirm that an increase in hydrodynamic
constraints in the SBAR could play a role in the erosion of aggregate during granule growth. It is still difficult to give the fundamental reason why cohesion improved. One simple hypothesis is that this phenomena results from densification, dense colonies having more bonding interactions and thus being much difficult to break: Adav et al. [44] demonstrated in pure culture that a high autoaggregation index was a prerequisite for granule formation. It was assumed in our work that nitrate diffusion inside aggregates (where oxygen becomes limiting) could encourage aggregate densification but also that anoxic carbon storage could improve the conversion of readily biodegradable substrate into slowly biodegradable internal compounds (see Wan et al. [42]) favouring aggregate stability by the way of slow growth as already suggested [10]. Furthermore Liu et al. [45] underlined that formation of granules is associated with a sharp increase of hydrophobicity. Zhang et al. [46] also observed an increase of surface charge and hydrophobicity during the formation of aerobic granular sludge together with an increase of protein/polysaccharide ratio: the increase in protein content was found to decrease the surface negative charge of bacteria cells, thus reducing the electrostatic repulsions and favouring bridging between two neighbouring cells. As it is suggested in literature that EPS production can explain the difference between the properties of flocs and granules [45,47,48], more works are currently planned to evaluate the relation between cohesion of granules and EPS properties. At present, it is still difficult to give a physicochemical explanation of the high cohesion of granules even though experiments based on specific enzyme attacks tend to show that either -polysaccharides [49] or ␣-polysaccharides [48] could be responsible for this characteristic. 4.2. Stabilisation of granular sludge In a granular sludge system, aggregates can present heterogeneous retention times as smaller particles are preferentially lost after settling, especially if short settling times are imposed. The steady state is assumed to be reached when external data are stabilised (size, MLSS concentration and activity). This was obtained in our work after 360 days. It seems reasonable to think that the diameter of aerobic granules stabilises when the growth rate is counterbalanced by the detachment rate. Ren et al. [37] found gas bubbles and particle collisions to be the main reason for the detachment. Here, the cohesion test demonstrated that a high shear rate
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generated the release of particles of about 50 m. the dissipated energy in the SBAR is expected to be lower (around 100 s−1 ) than that obtained in the stirred vessel. However, Fig. 2a underlines that similar result could have been obtained with a lower rotation speed (kolmogorov microscale is around 23 m for 1200 rpm). Furthermore, the solid concentration in the airlift reactor is twelve times higher than the one used in the cohesion test. Thus the particle collisions due to the high MLSS concentration could locally induce such detachment, whose resulting small but cohesive particles possibly constitute the seeds for the formation of new granules. In long-term experiments, it could be expected that old granules would show lower cohesion because of deterioration of the internal structure. Adav et al. [50] indeed observed that the centres of granules became porous, showing an empty internal heart after long-term starvation. This was attributed to the fact that the internal part of the aggregate (exopolymers) could be partly biodegraded, i.e. used as a secondary substrate in such famine conditions. Recently, Lemaire et al. [51] also observed large channels and holes, which probably negatively affect granule cohesion, but these channels were partly filled by EPS. In our work, no loss of cohesion was observed after 500 days of operation. Basically, no significant rupture of granules occurred during the test for a range of G between 250 s−1 and 3400 s−1 . The occurrence of these mechanisms is probably linked to the sludge retention time in the sequencing batch reactor and the duration of the experiments. In our case, biomass was wasted with effluent withdrawal (the upper fraction of sludge volume exceeding 50% of the reactor). Calculations showed that the mean total SRT reached 40 days (it is possible that bigger granules were maintained in the system longer because of their settling capacity). With these conditions, granules size may be small enough to avoid too much transfer resistance. Growth is then sufficiently possible in the core of the aggregate to avoid (or slow down) a loss of integrity and a decrease of aggregate strength. This is crucial information for possible industrial applications of aerobic granular sludge as the stability of the granule structure should be guaranteed in order to maintain high performance.
5. Conclusion The aggregate strength of flocculated sludge and granular sludge were assessed in a stirred reactor, by varying the rotation speed. Special attention was paid to the initial period of granular sludge formation in a sequencing batch airlift reactor and some important insights can be underlined: • It is confirmed that high SAV and low settling time are not prerequisites to produce granules if proper metabolic selection (here by alternating anoxic feast/aerobic famine), is applied. • At the initial phase of granular sludge formation, an increase of aggregate cohesion is first observed, whereas SVI decreases rapidly; then aggregates resist high shear imposed in the stirred cell. • Then growth of aggregates is progressively observed. Small particles are temporarily detached, indicating the role of hydrodynamics in particle erosion. • As no aggregate selection was performed, bimodal particle size distribution was observed, the small population of detached particles probably constituting the seeds for new granules. Granules showed a high cohesion even after 500 days of operation. • Tests performed on mature granules (diameter greater than 400 m) indicate that high energy dissipation (ε ≈ 10 m2 s−3 ) leads to surface detachment of particles of about 50 m, these small particles also showing high cohesion.
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