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Evolution of size distribution and transfer of mineral particles between flocs in activated sludges: an insight into floc exchange dynamics
Water Research 36 (2002) 676–684
Evolution of size distribution and transfer of mineral particles between flocs in activated sludges: an insight into floc exchange dynamics V. Chaignona, B.S. Lartigesa,*, A. El Samrania, C. Mustinb a
LEM-ENSG/UMR CNRS 7569, Po#le de l’Eau, 15 Avenue du Charmois, BP 40, 54501 Vandoeuvre Cedex, France b CPB CNRS UPR 6831, 17, Rue Notre Dame des Pauvres, BP 5, F-54501 Vandoeuvre Cedex, France Received 8 November 2000; accepted 30 April 2001
Abstract The aggregation behavior of activated sludge flocs was investigated by monitoring the size distribution of flocs and transfer of mineral particles between flocs, under various conditions of agitation and dilution. The results showed that (i) the shape of the floc size distribution can be fitted with a gamma function, (ii) a steady-state mean floc size is reached for a given stirring rate, (iii) this stable floc size is shifted towards floc growth as sludge concentration is increased, (iv) under cycled-shear conditions, microbial aggregates break up and re-form in an almost reversible manner, (v) blending of raw sludge and sludge spiked with Aquatal mineral particles results in particle exchange between flocs and (vi) the detailed study of exchange kinetics indicates that some flocs do not participate to the aggregation dynamics. These experimental results suggest that the activated sludge floc size is governed by a flocculation/deflocculation balance, implying an exchange of floc constituents between microbial aggregates. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Activated sludge; Bioflocculation; Floc size distribution
1. Introduction The activated sludge floc is an association of microorganisms, microcolonies, and exopolymeric substances secreted by bacteria [1–3]. Expressions commonly encountered in the literature such as ‘‘floc surface’’ [4,5], ‘‘floc core’’ (Characklis, Eriksson), ‘‘central region and outer parts of floc’’ [6,7], suggest a blob-like arrangement of floc constituents. Accordingly, the process of activated sludgeFsorption of colloid organic matter and soluble substrates onto flocs, settling characteristics of microbial aggregatesFis usually analyzed from the properties of individual flocs, while mass
*Corresponding author. Tel.: +33-3-8359-6294; fax: +33-38359-6255. E-mail address: [email protected] (B.S. Lartiges).
transport within aggregates is generally assumed to be purely diffusive in nature [8]. On the basis of recent studies dedicated to exploring the structuring, binding, and biological activity of floc compounds, it appears that such a view of activated sludge aggregates needs to be re-examined. Optical microscopy and Transmission Electron Microscopy (TEM) observations of resin-embedded floc sections do not reveal any morphological differentiation of microbial flocs in contrary with what could be expected in the case of concentration gradients of substrate [1,2,9]. Laser confocal microscopic examination of activated sludge flocs hybridized with oligonucleotide fluorescent probes indicates that bacterial activity does not depend on bacteria location within microbial aggregates [10]. In parallel, numerous papers have evidenced that a reversible floc dissociation can then be triggered through the addition of either monovalent ions or complexing agents [3,7,11–13]. In this context, Mikkelsen and
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 6 6 - 4
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Keiding [14] recently provided a novel picture of activated sludge flocs by suggesting that the effect of shear and solids content on floc size could be interpreted as resulting from a flocculation–deflocculation balance. Using a similar conceptual framework, the present study aims at examining in detail the dynamic equilibrium between floc growth and floc breakup by monitoring the evolution of activated sludge floc size distributions as a function of both dilution and stirring conditions. In addition, further insight into floc dynamics is obtained by following the transfer of mineral particles (used as floc markers) between bacterial aggregates.
2. Materials and methods 2.1. Sample collection Activated sludge samples were collected from the aeration tank of the local municipal wastewater treatment plant (Max!eville, France). This plant has a capacity of 300 000 population equivalent and follows a conventional process with primary physical treatment and secondary activated sludge treatment (sludge age about 8 days). The grab samples, collected in 2 l polyethylene containers, were transported to the laboratory and used for experiments within 2 h of collection. The sludge was not aerated after sampling or during the experiments which were carried out at room temperature. Total suspended solid contents were determined by drying a 50 ml centrifuged sludge sample (3000 g for 5 min) at 1051C. The values averaged 3.5 g/l during the period of the study. The pH measurements showed values around 7.2.
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the reactor. A pumping flow rate of 55 ml/min, and a plastic tubing of 4.6 mm internal diameter and 16 cm long, were selected. Obviously, shearing in the transport tubing somehow affects measured floc size distribution. However, previous experiments (using similar operating conditions) with shear-sensitive aggregates such as Namontmorillonite coagulated with CaCl2 [15] or silica nanoparticles flocculated with Al13-based coagulants [16], revealed that relative variations in floc size are linked to changes in agitation conditions in the reactor rather than to shearing in the transport tubing. Such setup is then appropriate to investigate size evolution upon cyclic changes in stirring. Size measurements were averaged over 1 s and taken every 2 s to allow for a complete renewal of flocs present in the laser beam. Results are volume based and are represented as suspended particle volume versus sphere diameter of equivalent volume. 2.3. Floc structure from floc size measurements In addition to size distribution, the particle sizer provides an estimate of the apparent volume fraction f of particles contained in the sample cell. In the Fraunhoffer range, the aggregates are analyzed as compact particles, and f can be determined from the obscuration of the laser beam using a Beer-Lambert law [17]. The fractal structure of flocs is then characterized ð3Df Þ from the equation fBdm ; where dm is the mean floc size and Df the mass fractal dimension of aggregates [18–20]. In our study, f and dm are obtained during cycled-shear experiments, thus providing a floc structure averaged over the duration of either floc growth or floc breakup.
2.2. Floc size measurements
2.4. Floc spiking with Aquatal particles
Floc size distributions were measured on-line with a Malvern Mastersizer using a particle size detection range of 1.2–600 mm. To avoid multiple scattering in the measurement cell, activated sludge samples were diluted with centrifuged mixed liquor (7120 g for 20 minFSorvall RC50 Plus, Dupont) to yield suspended solid concentrations varying from 3.5 to 140 mg/l. The suspensions thus obtained were stirred in a standard 1 l baffled reactor (90 mm in diameter, 150 mm high), with a 15 mm 54 mm blade positioned at 1/3 the height of the reactor. An adjustable-speed motor (Janke & Kundel RW 20 DZM) was used to provide stirring rates of 100 or 200 rpm, which corresponds to spatially averaged velocity gradients G of 135 and 370 s1, respectively. The agitated suspension was continuously withdrawn from the bottom of the reactor, passed through the analyzer beam with a peristaltic pump located downstream the measurement cell, before being recycled to
Aquatal particles, graciously provided by Luzenac Europe, were used in this work as floc markers. Aquatal, a talc-chlorite interstratified mineral with a negative surface charge at neutral pH, has been shown to be an efficient ballasting agent for sludges with poor settleability [21]. Therefore, it seemed very appropriate for floc spiking although the exact mechanisms of particle adhesion to flocs are yet to be established [22,23]. Activated sludge floc spiking was conducted under the same conditions of sludge dilution as those used in size measurements. Aquatal particles (ground to 11 mm in diameter) were first dispersed in the centrifuged supernatant under strong agitation, and an aliquot of sludge was then added to yield a 35 mg/l suspended solid concentration with a given wt% of ballasting agent. After a chosen period of mixing, flocs were sampled at the lower third of the reactor with a wide mouth pipet, placed on a glass slide, and examined with a Nachet NS
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400 optical microscope equipped with a Leitz Wild MPS 52 camera. Activated sludge flocs were observed between crossed polarizer and analyzer at a magnification of 1250. In that case, the mineral particles incorporated inside the flocs induce a rotation of the polarization plane, and appear as bright spots within the floc organic matrix (Fig. 1). The amount of markers associated with a floc can then be assessed from the exposure time required to take a photograph in spot mode at the center of the floc studied. At least 130 flocs, randomly selected, were examined in each experiment, and the exposure time data were presented as histograms or cumulative frequency curves.
between clusters in suspension, the mathematical solution of von Smoluchowski aggregation equations yields a gamma function [28–31]. Gamma functions are also applicable to steady-state size distributions obtained for various simultaneous binary aggregation and fragmentation processes [32–36]. Therefore, the gamma function fit of activated sludge floc size distribution suggests that floc growth is due to a cluster–cluster aggregation mechanism.
3.2. Influence of agitation on floc size
Fig. 2 presents a typical size distribution of bacterial flocs diluted in centrifuged mixed liquor. Some authors have observed bimodal number-based sized distributions (e.g. [24]). for activated sludge flocs. The volume size distribution obtained in our case is monomodal and shows a slight skewness towards smaller floc sizes which may result in a distinct population when plotted as number. Previous investigators have described activated sludge floc size distributions with either a lognormal equation [2,25] or a power-law model [26,27]. However, the shape of the size distribution is here best fitted by the more general gamma function f ðdÞ ¼ kd a ebd where d is the aggregate size, and k; a; and b are constants. Numerous papers have shown that, in the asymptotic regime of floc growth predicted on binary encounters
The effect of agitation on floc size was explored by submitting the diluted sludge to consecutive cyclic step changes in stirrer speeds from 100 to 200 rpm and vice versa during 40 min periods. When the stirring intensity is increased, flocs obtained using a slow stirring speed, break up first rapidly and then more slowly to reach a steady-state aggregate size of about 70 mm (Fig. 3). When the stirrer speed is set back to its initial value, the flocs re-form to a size slightly greater than the initial floc size. It must be pointed out however, that the floc size observed after 20 min re-growth is close to that obtained just before initiating the first increase in shearing at t ¼ 20 min. Further cycling of agitation conditions between high and low levels of intensity, reveals that the process of activated sludge floc fragmentation and re-aggregation is largely reversible at the macroscopic scale. This type of aggregation behaviorFdefinite and reversible stable average floc size for a given shear rateFis usually reported for inorganic particles destabilized with the addition of either a simple electrolyte or a colloid of opposite charge [18,37]. In that case, the
Fig. 1. Micrograph of an activated sludge floc spiked with Aquatal particles between crossed polarizer and analyzer.
Fig. 2. Example of floc size distribution of activated sludge. The broken line represents the natural distribution; the solid line is the fit based on the gamma curve y ¼ kxa ebx ; with k ¼ 2:05 103, a ¼ 2:3; b ¼ 0:02; y the particle volume, and x the particle diameter. Sludge concentration=35 mg/l and stirring speed=100 rpm.
3. Results and discussion 3.1. Significance of floc size distribution
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Fig. 3. Variation of average floc size as a function of time during consecutive cyclic step changes in agitation intensity. Sludge concentration=35 mg/l.
steady-state aggregate size is interpreted as the result of a dynamic equilibrium between floc growth and floc breakup. On the other hand, when aggregation occurs through a bridging of particles and polymeric flocculant species, the flocs have been shown to re-form only partially after shearing [20,38,39]. Particle-flocculant bond breakages during floc fragmentation [38,40] and/ or re-structuring of flocs leading to more compact structures [20,39] are invoked for explaining such irreversible floc dynamics. Microbial flocculation is generally attributed to exopolymeric bridging of bacterial cells [41,42]. Bridging of floc components involves ionic interactions between charged functional groups of biopolymers and divalent cations [11,12,43,44], and may also be mediated through hydrophobic moieties of exopolymers [45,46], specific protein–polysaccharide interactions [47], or simple physical enmeshing [48]. In addition, electrical double layer effects have been proposed to play an important role in floc stability [7]. The reversibility in aggregate size observed during cycled-shear experiments substantiates a charge neutralization mechanismFeither double-layer compression and/or ionic bridgingFfor activated sludge floc binding. Furthermore, as it is unlikely that activated sludge flocs once fragmented re-form with the same material, an exchange of floc constituents must occur within the reactor. Typical values of mean velocity gradients reported in the literature for aeration tanks (20– 150 s1) [49], are in the same range as the G-values used in this study. As a consequence, the dynamics of activated sludge floc evidenced here should also take place in wastewater treatment process. Fractal dimensions determined from the log–log plot of suspended floc volume versus floc mean diameter, are indicated on Fig. 3 for each agitation step while the corresponding data are presented in Fig. 4. A 2.4570.05 Df value, consistent with fractal dimensions previously
Fig. 4. Determination of the floc mass fractal dimension Df from the slope of the volume fraction versus mean floc size log–log plot. Sludge concentration=35 mg/l.
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reported for activated sludge aggregates [50–52], can be found during both floc fragmentation and floc regrowth. Although the range of floc size investigated (less than an order of magnitude) precludes a self-similar fractal statement [53], it is clear from the constant slope value that no major floc re-structuring occurred during increased shear conditions.
3.3. Influence of activated sludge concentration on median floc size Fig. 5a shows the temporal evolution of dm at constant stirrer speed (100 rpm/135 s1) for various sludge concentrations. In all cases, the average floc size increases slightly with time to reach a steady-state size after about 30 min. Such an increase is particularly marked for high sludge concentrations. This change in floc size with time is likely due to a slow response of aggregates to continuing agitation. Interestingly, both the initial and steady-state mean floc size depend strongly on sludge concentration. As illustrated in Fig. 5b, the aggregate size measured
immediately after dilution can be linearly correlated with the suspended solid content in the 3–140 mg/l concentration range. The formation of smaller microbial flocs upon dilution may arise from changes in ionic strength. Indeed, it was noted that centrifugation of the mixed liquor yields a supernatant of slightly reduced conductivity. As the structural properties of activated sludge flocs are known to be highly sensitive to small changes in ionic composition [3,7,12,13], it could be assumed that the dilution of sludge with a supernatant of lower ionic strength induces some deflocculation of the suspension. Still, the most straightforward explanation is to relate the decrease in stable floc size to a lower amount of suspended matter within the reactor. Indeed, at a given intensity of agitation, for various coagulated inorganic suspensions, the degree of aggregation has been shown to increase markedly with increasing particle concentration [18,37,54]. This concentration dependence results from the dynamic equilibrium between floc formation and floc destruction which governs aggregate mean size, and is shifted in the direction of floc growth as particle concentration increases [37,55]. Therefore, the response of mean floc size to a variation in sludge concentration found in this study, supports a dynamic description of activated sludge aggregates in agreement with the tendencies observed by Mikkelsen and Keiding [14] on the basis of turbidity measurements. However, a linear correlation between mixed liquor suspended solids and floc size of opposite trend was derived by Li and Ganczarczyk [27] during field studies. This suggests that sludge concentration is one of the many factors controlling floc size during the wastewater process. 3.4. Aquatal particles as an exchange tracer
Fig. 5. (a) Time evolution of mean floc size for a constant shear rate of 135 s1 at different sludge concentrations: (J) 3.5 mg/l, (K) 35 mg/l, (+) 70 mg/l, and (}) 140 mg/l. (b) Effect of activated sludge concentration on initial mean floc size.
A continuous reversible aggregation of microbial flocs can also be verified by monitoring the transfer of floc markers during the mixing of spiked and unspiked sludges. Preliminary cycled shear experiments with spiked flocs indicate that the incorporation of Aquatal particles does not significantly affect the dynamics and structure of activated sludge aggregates. As illustrated in Fig. 6, both the floc size profile obtained during cyclic step changes in agitation intensity and the aggregate structure, are equivalent to those exhibited for an unspiked sludge. Nevertheless, it should be noted that the steady-state floc size is attained more rapidly when floc markers are added, suggesting that those particles may act as nuclei during floc growth [21]. The amount of Aquatal particles associated with a floc can be assessed from the exposure time required to take a photograph in spot mode at the center of the floc studied. The exposure time frequency histograms obtained for sludges spiked with 0, 15, and 30 wt% of Aquatal particles are shown in Figs. 7a–c. All histo-
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grams are monomodal and, as expected, the distributions are shifted towards lower values of exposure time as the concentration in floc markers increase. A small proportion of long exposure times can be observed even at high spiking concentration, indicating the existence of flocs incorporating no or little Aquatal particles. Fig. 7d presents the histogram obtained after mixing a raw sludge with a 30 wt% spiked sludge. Both suspen-
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sions were first conditioned separately for 20 min, and then mixed and further agitated for 20 min. The histogram is very similar to the one observed for flocs spiked directly with 15 wt% of Aquatal particles, evidencing that floc markers, initially entrapped in the spiked sludge have been redistributed over all the unspiked activated sludge aggregates. The same data, plotted as cumulative frequency curves, illustrate more clearly the transfer of Aquatal particles between flocs (Fig. 8). Indeed, the curve associated with a blend of a 30 wt% spiked sludge with a raw sludge is an intermediate between the curves of the two unblended sludges, and is nearly superimposed with that determined for a 15 wt% spiked sludge.
3.5. Kinetics of Aquatal transfer between microbial aggregates
Fig. 6. Effect of cycled-shear on the evolution of average diameter of activated sludge flocs spiked with 20 wt% of Aquatal particles.
Floc spiking was also examined as a function of mixing time to gain information about the kinetics of the exchange process. 20 wt% of Aquatal particles were added to the diluted sludge, and samples were then collected at 20 min intervals for microscopic examination. As shown in Fig. 9, the cumulative frequency curves vary with time, the narrower distribution
Fig. 7. Frequency distribution of exposure times for sludges spiked with 0 wt% (a), 15 wt% (b), 30 wt% (c), and for the blend of the raw sludge with the 30 wt% spiked sludge (d). Sludge concentration=35 mg/l.
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Fig. 8. Cumulative frequency curves of exposure times demonstrating the transfer of Aquatal particles between flocs.
‘‘young’’ sludges, whereas more compact and spherical flocs are characteristic of ‘‘older’’ sludges [6,11,56]. Variations in floc morphology have been invoked to explain the differences in sludge volume obtained between agitated and non-agitated settling tests [6,56]. These observations can be readily re-interpreted on the basis of a subjacent aggregation dynamics. Irregular ‘‘young’’ flocs with weakly bound biopolymers could be ascribed to a cluster–cluster reversible aggregation, while round and compact ‘‘old’’ flocs would reflect a low or no aggregation dynamics. Experimental results such as increased extraction of polymeric material under high speed stirring [3,56], variations in sludge filterability [6], or improvement of sludge settling properties with an increase in extractable polysaccharide concentration [57], could be similarly re-evaluated and are compatible with an exchange process occurring between activated sludge flocs.
4. Conclusion
Fig. 9. Time evolution of the cumulative frequency curve of exposure times for a sludge spiked with 20 wt% of Aquatal particles. Stirring speed=100 rpm and sludge concentration=35 mg/l.
obtained at 60 min stirring indicating a more homogeneous repartition of floc markers between microbial aggregates. In addition, the different curves intersect for an exposure time of about 0.5 s. This suggests that, at short mixing times, a small proportion of flocs take up a large amount of Aquatal particles, and that these floc markers are then gradually redistributed among the other aggregates with continuing agitation. In other terms, all the activated sludge flocs do not participate to the same extent to the exchange dynamics. While some flocs undergo rapid formation and de-aggregation facilitating the incorporation of Aquatal particles, other aggregates exhibit much smaller rates of aggregation and fragmentation, and are then less active towards the transfer of floc markers. Interestingly, activated sludge flocs associated with high exposure times and a presumably low aggregation dynamics, were found to present a more rounded shape. Floc morphology is usually related to sludge age, strongly irregular aggregates being observed for
The results reported in this study were repeated in separate experiments on sludges from the same wastewater treatment plant. Although some variations in the magnitude of variables occurred, the trends observed were similar to those presented in the paper. In all cases, the response of aggregate size to variations in agitation and sludge concentration, and the exchange of floc markers between spiked and unspiked sludges, supported the concept of reversible aggregation between activated sludge flocs. To complete the physico-chemical description of bioflocculation, the nature of units exchanged between flocs should be better defined. As exopolymeric substances are known to be the main floc-forming constituents [1,2,9], it is reasonable to speculate that the exchange units correspond essentially to biopolymers or bundles of exopolymeric material. Such a view would be consistent with earlier experimental work showing that soluble exopolymers are chemically similar to polymeric material from the floc [58,59]. Hence, bacteria and microcolonies would be essentially enmeshed in the network of exopolymeric material, in agreement with the earlier model proposed by Friedman et al. [48]. Clearly, further work is needed to clarify the nature of exchange units and to quantify aggregation dynamics. The role of aggregation dynamics should also be related to sludge properties.
Acknowledgements The authors gratefully acknowledge the authorization of sampling granted by the Grand Nancy urban community. We also thank the staff of Max!eville wastewater treatment plant for experimental assistance.
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Evolution of size distribution and transfer of mineral particles between flocs in activated sludges: an insight into floc exchange dynamics V. Chaignona, B.S. Lartigesa,*, A. El Samrania, C. Mustinb a
LEM-ENSG/UMR CNRS 7569, Po#le de l’Eau, 15 Avenue du Charmois, BP 40, 54501 Vandoeuvre Cedex, France b CPB CNRS UPR 6831, 17, Rue Notre Dame des Pauvres, BP 5, F-54501 Vandoeuvre Cedex, France Received 8 November 2000; accepted 30 April 2001
Abstract The aggregation behavior of activated sludge flocs was investigated by monitoring the size distribution of flocs and transfer of mineral particles between flocs, under various conditions of agitation and dilution. The results showed that (i) the shape of the floc size distribution can be fitted with a gamma function, (ii) a steady-state mean floc size is reached for a given stirring rate, (iii) this stable floc size is shifted towards floc growth as sludge concentration is increased, (iv) under cycled-shear conditions, microbial aggregates break up and re-form in an almost reversible manner, (v) blending of raw sludge and sludge spiked with Aquatal mineral particles results in particle exchange between flocs and (vi) the detailed study of exchange kinetics indicates that some flocs do not participate to the aggregation dynamics. These experimental results suggest that the activated sludge floc size is governed by a flocculation/deflocculation balance, implying an exchange of floc constituents between microbial aggregates. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Activated sludge; Bioflocculation; Floc size distribution
1. Introduction The activated sludge floc is an association of microorganisms, microcolonies, and exopolymeric substances secreted by bacteria [1–3]. Expressions commonly encountered in the literature such as ‘‘floc surface’’ [4,5], ‘‘floc core’’ (Characklis, Eriksson), ‘‘central region and outer parts of floc’’ [6,7], suggest a blob-like arrangement of floc constituents. Accordingly, the process of activated sludgeFsorption of colloid organic matter and soluble substrates onto flocs, settling characteristics of microbial aggregatesFis usually analyzed from the properties of individual flocs, while mass
*Corresponding author. Tel.: +33-3-8359-6294; fax: +33-38359-6255. E-mail address: [email protected] (B.S. Lartiges).
transport within aggregates is generally assumed to be purely diffusive in nature [8]. On the basis of recent studies dedicated to exploring the structuring, binding, and biological activity of floc compounds, it appears that such a view of activated sludge aggregates needs to be re-examined. Optical microscopy and Transmission Electron Microscopy (TEM) observations of resin-embedded floc sections do not reveal any morphological differentiation of microbial flocs in contrary with what could be expected in the case of concentration gradients of substrate [1,2,9]. Laser confocal microscopic examination of activated sludge flocs hybridized with oligonucleotide fluorescent probes indicates that bacterial activity does not depend on bacteria location within microbial aggregates [10]. In parallel, numerous papers have evidenced that a reversible floc dissociation can then be triggered through the addition of either monovalent ions or complexing agents [3,7,11–13]. In this context, Mikkelsen and
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 6 6 - 4
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Keiding [14] recently provided a novel picture of activated sludge flocs by suggesting that the effect of shear and solids content on floc size could be interpreted as resulting from a flocculation–deflocculation balance. Using a similar conceptual framework, the present study aims at examining in detail the dynamic equilibrium between floc growth and floc breakup by monitoring the evolution of activated sludge floc size distributions as a function of both dilution and stirring conditions. In addition, further insight into floc dynamics is obtained by following the transfer of mineral particles (used as floc markers) between bacterial aggregates.
2. Materials and methods 2.1. Sample collection Activated sludge samples were collected from the aeration tank of the local municipal wastewater treatment plant (Max!eville, France). This plant has a capacity of 300 000 population equivalent and follows a conventional process with primary physical treatment and secondary activated sludge treatment (sludge age about 8 days). The grab samples, collected in 2 l polyethylene containers, were transported to the laboratory and used for experiments within 2 h of collection. The sludge was not aerated after sampling or during the experiments which were carried out at room temperature. Total suspended solid contents were determined by drying a 50 ml centrifuged sludge sample (3000 g for 5 min) at 1051C. The values averaged 3.5 g/l during the period of the study. The pH measurements showed values around 7.2.
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the reactor. A pumping flow rate of 55 ml/min, and a plastic tubing of 4.6 mm internal diameter and 16 cm long, were selected. Obviously, shearing in the transport tubing somehow affects measured floc size distribution. However, previous experiments (using similar operating conditions) with shear-sensitive aggregates such as Namontmorillonite coagulated with CaCl2 [15] or silica nanoparticles flocculated with Al13-based coagulants [16], revealed that relative variations in floc size are linked to changes in agitation conditions in the reactor rather than to shearing in the transport tubing. Such setup is then appropriate to investigate size evolution upon cyclic changes in stirring. Size measurements were averaged over 1 s and taken every 2 s to allow for a complete renewal of flocs present in the laser beam. Results are volume based and are represented as suspended particle volume versus sphere diameter of equivalent volume. 2.3. Floc structure from floc size measurements In addition to size distribution, the particle sizer provides an estimate of the apparent volume fraction f of particles contained in the sample cell. In the Fraunhoffer range, the aggregates are analyzed as compact particles, and f can be determined from the obscuration of the laser beam using a Beer-Lambert law [17]. The fractal structure of flocs is then characterized ð3Df Þ from the equation fBdm ; where dm is the mean floc size and Df the mass fractal dimension of aggregates [18–20]. In our study, f and dm are obtained during cycled-shear experiments, thus providing a floc structure averaged over the duration of either floc growth or floc breakup.
2.2. Floc size measurements
2.4. Floc spiking with Aquatal particles
Floc size distributions were measured on-line with a Malvern Mastersizer using a particle size detection range of 1.2–600 mm. To avoid multiple scattering in the measurement cell, activated sludge samples were diluted with centrifuged mixed liquor (7120 g for 20 minFSorvall RC50 Plus, Dupont) to yield suspended solid concentrations varying from 3.5 to 140 mg/l. The suspensions thus obtained were stirred in a standard 1 l baffled reactor (90 mm in diameter, 150 mm high), with a 15 mm 54 mm blade positioned at 1/3 the height of the reactor. An adjustable-speed motor (Janke & Kundel RW 20 DZM) was used to provide stirring rates of 100 or 200 rpm, which corresponds to spatially averaged velocity gradients G of 135 and 370 s1, respectively. The agitated suspension was continuously withdrawn from the bottom of the reactor, passed through the analyzer beam with a peristaltic pump located downstream the measurement cell, before being recycled to
Aquatal particles, graciously provided by Luzenac Europe, were used in this work as floc markers. Aquatal, a talc-chlorite interstratified mineral with a negative surface charge at neutral pH, has been shown to be an efficient ballasting agent for sludges with poor settleability [21]. Therefore, it seemed very appropriate for floc spiking although the exact mechanisms of particle adhesion to flocs are yet to be established [22,23]. Activated sludge floc spiking was conducted under the same conditions of sludge dilution as those used in size measurements. Aquatal particles (ground to 11 mm in diameter) were first dispersed in the centrifuged supernatant under strong agitation, and an aliquot of sludge was then added to yield a 35 mg/l suspended solid concentration with a given wt% of ballasting agent. After a chosen period of mixing, flocs were sampled at the lower third of the reactor with a wide mouth pipet, placed on a glass slide, and examined with a Nachet NS
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400 optical microscope equipped with a Leitz Wild MPS 52 camera. Activated sludge flocs were observed between crossed polarizer and analyzer at a magnification of 1250. In that case, the mineral particles incorporated inside the flocs induce a rotation of the polarization plane, and appear as bright spots within the floc organic matrix (Fig. 1). The amount of markers associated with a floc can then be assessed from the exposure time required to take a photograph in spot mode at the center of the floc studied. At least 130 flocs, randomly selected, were examined in each experiment, and the exposure time data were presented as histograms or cumulative frequency curves.
between clusters in suspension, the mathematical solution of von Smoluchowski aggregation equations yields a gamma function [28–31]. Gamma functions are also applicable to steady-state size distributions obtained for various simultaneous binary aggregation and fragmentation processes [32–36]. Therefore, the gamma function fit of activated sludge floc size distribution suggests that floc growth is due to a cluster–cluster aggregation mechanism.
3.2. Influence of agitation on floc size
Fig. 2 presents a typical size distribution of bacterial flocs diluted in centrifuged mixed liquor. Some authors have observed bimodal number-based sized distributions (e.g. [24]). for activated sludge flocs. The volume size distribution obtained in our case is monomodal and shows a slight skewness towards smaller floc sizes which may result in a distinct population when plotted as number. Previous investigators have described activated sludge floc size distributions with either a lognormal equation [2,25] or a power-law model [26,27]. However, the shape of the size distribution is here best fitted by the more general gamma function f ðdÞ ¼ kd a ebd where d is the aggregate size, and k; a; and b are constants. Numerous papers have shown that, in the asymptotic regime of floc growth predicted on binary encounters
The effect of agitation on floc size was explored by submitting the diluted sludge to consecutive cyclic step changes in stirrer speeds from 100 to 200 rpm and vice versa during 40 min periods. When the stirring intensity is increased, flocs obtained using a slow stirring speed, break up first rapidly and then more slowly to reach a steady-state aggregate size of about 70 mm (Fig. 3). When the stirrer speed is set back to its initial value, the flocs re-form to a size slightly greater than the initial floc size. It must be pointed out however, that the floc size observed after 20 min re-growth is close to that obtained just before initiating the first increase in shearing at t ¼ 20 min. Further cycling of agitation conditions between high and low levels of intensity, reveals that the process of activated sludge floc fragmentation and re-aggregation is largely reversible at the macroscopic scale. This type of aggregation behaviorFdefinite and reversible stable average floc size for a given shear rateFis usually reported for inorganic particles destabilized with the addition of either a simple electrolyte or a colloid of opposite charge [18,37]. In that case, the
Fig. 1. Micrograph of an activated sludge floc spiked with Aquatal particles between crossed polarizer and analyzer.
Fig. 2. Example of floc size distribution of activated sludge. The broken line represents the natural distribution; the solid line is the fit based on the gamma curve y ¼ kxa ebx ; with k ¼ 2:05 103, a ¼ 2:3; b ¼ 0:02; y the particle volume, and x the particle diameter. Sludge concentration=35 mg/l and stirring speed=100 rpm.
3. Results and discussion 3.1. Significance of floc size distribution
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Fig. 3. Variation of average floc size as a function of time during consecutive cyclic step changes in agitation intensity. Sludge concentration=35 mg/l.
steady-state aggregate size is interpreted as the result of a dynamic equilibrium between floc growth and floc breakup. On the other hand, when aggregation occurs through a bridging of particles and polymeric flocculant species, the flocs have been shown to re-form only partially after shearing [20,38,39]. Particle-flocculant bond breakages during floc fragmentation [38,40] and/ or re-structuring of flocs leading to more compact structures [20,39] are invoked for explaining such irreversible floc dynamics. Microbial flocculation is generally attributed to exopolymeric bridging of bacterial cells [41,42]. Bridging of floc components involves ionic interactions between charged functional groups of biopolymers and divalent cations [11,12,43,44], and may also be mediated through hydrophobic moieties of exopolymers [45,46], specific protein–polysaccharide interactions [47], or simple physical enmeshing [48]. In addition, electrical double layer effects have been proposed to play an important role in floc stability [7]. The reversibility in aggregate size observed during cycled-shear experiments substantiates a charge neutralization mechanismFeither double-layer compression and/or ionic bridgingFfor activated sludge floc binding. Furthermore, as it is unlikely that activated sludge flocs once fragmented re-form with the same material, an exchange of floc constituents must occur within the reactor. Typical values of mean velocity gradients reported in the literature for aeration tanks (20– 150 s1) [49], are in the same range as the G-values used in this study. As a consequence, the dynamics of activated sludge floc evidenced here should also take place in wastewater treatment process. Fractal dimensions determined from the log–log plot of suspended floc volume versus floc mean diameter, are indicated on Fig. 3 for each agitation step while the corresponding data are presented in Fig. 4. A 2.4570.05 Df value, consistent with fractal dimensions previously
Fig. 4. Determination of the floc mass fractal dimension Df from the slope of the volume fraction versus mean floc size log–log plot. Sludge concentration=35 mg/l.
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reported for activated sludge aggregates [50–52], can be found during both floc fragmentation and floc regrowth. Although the range of floc size investigated (less than an order of magnitude) precludes a self-similar fractal statement [53], it is clear from the constant slope value that no major floc re-structuring occurred during increased shear conditions.
3.3. Influence of activated sludge concentration on median floc size Fig. 5a shows the temporal evolution of dm at constant stirrer speed (100 rpm/135 s1) for various sludge concentrations. In all cases, the average floc size increases slightly with time to reach a steady-state size after about 30 min. Such an increase is particularly marked for high sludge concentrations. This change in floc size with time is likely due to a slow response of aggregates to continuing agitation. Interestingly, both the initial and steady-state mean floc size depend strongly on sludge concentration. As illustrated in Fig. 5b, the aggregate size measured
immediately after dilution can be linearly correlated with the suspended solid content in the 3–140 mg/l concentration range. The formation of smaller microbial flocs upon dilution may arise from changes in ionic strength. Indeed, it was noted that centrifugation of the mixed liquor yields a supernatant of slightly reduced conductivity. As the structural properties of activated sludge flocs are known to be highly sensitive to small changes in ionic composition [3,7,12,13], it could be assumed that the dilution of sludge with a supernatant of lower ionic strength induces some deflocculation of the suspension. Still, the most straightforward explanation is to relate the decrease in stable floc size to a lower amount of suspended matter within the reactor. Indeed, at a given intensity of agitation, for various coagulated inorganic suspensions, the degree of aggregation has been shown to increase markedly with increasing particle concentration [18,37,54]. This concentration dependence results from the dynamic equilibrium between floc formation and floc destruction which governs aggregate mean size, and is shifted in the direction of floc growth as particle concentration increases [37,55]. Therefore, the response of mean floc size to a variation in sludge concentration found in this study, supports a dynamic description of activated sludge aggregates in agreement with the tendencies observed by Mikkelsen and Keiding [14] on the basis of turbidity measurements. However, a linear correlation between mixed liquor suspended solids and floc size of opposite trend was derived by Li and Ganczarczyk [27] during field studies. This suggests that sludge concentration is one of the many factors controlling floc size during the wastewater process. 3.4. Aquatal particles as an exchange tracer
Fig. 5. (a) Time evolution of mean floc size for a constant shear rate of 135 s1 at different sludge concentrations: (J) 3.5 mg/l, (K) 35 mg/l, (+) 70 mg/l, and (}) 140 mg/l. (b) Effect of activated sludge concentration on initial mean floc size.
A continuous reversible aggregation of microbial flocs can also be verified by monitoring the transfer of floc markers during the mixing of spiked and unspiked sludges. Preliminary cycled shear experiments with spiked flocs indicate that the incorporation of Aquatal particles does not significantly affect the dynamics and structure of activated sludge aggregates. As illustrated in Fig. 6, both the floc size profile obtained during cyclic step changes in agitation intensity and the aggregate structure, are equivalent to those exhibited for an unspiked sludge. Nevertheless, it should be noted that the steady-state floc size is attained more rapidly when floc markers are added, suggesting that those particles may act as nuclei during floc growth [21]. The amount of Aquatal particles associated with a floc can be assessed from the exposure time required to take a photograph in spot mode at the center of the floc studied. The exposure time frequency histograms obtained for sludges spiked with 0, 15, and 30 wt% of Aquatal particles are shown in Figs. 7a–c. All histo-
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grams are monomodal and, as expected, the distributions are shifted towards lower values of exposure time as the concentration in floc markers increase. A small proportion of long exposure times can be observed even at high spiking concentration, indicating the existence of flocs incorporating no or little Aquatal particles. Fig. 7d presents the histogram obtained after mixing a raw sludge with a 30 wt% spiked sludge. Both suspen-
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sions were first conditioned separately for 20 min, and then mixed and further agitated for 20 min. The histogram is very similar to the one observed for flocs spiked directly with 15 wt% of Aquatal particles, evidencing that floc markers, initially entrapped in the spiked sludge have been redistributed over all the unspiked activated sludge aggregates. The same data, plotted as cumulative frequency curves, illustrate more clearly the transfer of Aquatal particles between flocs (Fig. 8). Indeed, the curve associated with a blend of a 30 wt% spiked sludge with a raw sludge is an intermediate between the curves of the two unblended sludges, and is nearly superimposed with that determined for a 15 wt% spiked sludge.
3.5. Kinetics of Aquatal transfer between microbial aggregates
Fig. 6. Effect of cycled-shear on the evolution of average diameter of activated sludge flocs spiked with 20 wt% of Aquatal particles.
Floc spiking was also examined as a function of mixing time to gain information about the kinetics of the exchange process. 20 wt% of Aquatal particles were added to the diluted sludge, and samples were then collected at 20 min intervals for microscopic examination. As shown in Fig. 9, the cumulative frequency curves vary with time, the narrower distribution
Fig. 7. Frequency distribution of exposure times for sludges spiked with 0 wt% (a), 15 wt% (b), 30 wt% (c), and for the blend of the raw sludge with the 30 wt% spiked sludge (d). Sludge concentration=35 mg/l.
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Fig. 8. Cumulative frequency curves of exposure times demonstrating the transfer of Aquatal particles between flocs.
‘‘young’’ sludges, whereas more compact and spherical flocs are characteristic of ‘‘older’’ sludges [6,11,56]. Variations in floc morphology have been invoked to explain the differences in sludge volume obtained between agitated and non-agitated settling tests [6,56]. These observations can be readily re-interpreted on the basis of a subjacent aggregation dynamics. Irregular ‘‘young’’ flocs with weakly bound biopolymers could be ascribed to a cluster–cluster reversible aggregation, while round and compact ‘‘old’’ flocs would reflect a low or no aggregation dynamics. Experimental results such as increased extraction of polymeric material under high speed stirring [3,56], variations in sludge filterability [6], or improvement of sludge settling properties with an increase in extractable polysaccharide concentration [57], could be similarly re-evaluated and are compatible with an exchange process occurring between activated sludge flocs.
4. Conclusion
Fig. 9. Time evolution of the cumulative frequency curve of exposure times for a sludge spiked with 20 wt% of Aquatal particles. Stirring speed=100 rpm and sludge concentration=35 mg/l.
obtained at 60 min stirring indicating a more homogeneous repartition of floc markers between microbial aggregates. In addition, the different curves intersect for an exposure time of about 0.5 s. This suggests that, at short mixing times, a small proportion of flocs take up a large amount of Aquatal particles, and that these floc markers are then gradually redistributed among the other aggregates with continuing agitation. In other terms, all the activated sludge flocs do not participate to the same extent to the exchange dynamics. While some flocs undergo rapid formation and de-aggregation facilitating the incorporation of Aquatal particles, other aggregates exhibit much smaller rates of aggregation and fragmentation, and are then less active towards the transfer of floc markers. Interestingly, activated sludge flocs associated with high exposure times and a presumably low aggregation dynamics, were found to present a more rounded shape. Floc morphology is usually related to sludge age, strongly irregular aggregates being observed for
The results reported in this study were repeated in separate experiments on sludges from the same wastewater treatment plant. Although some variations in the magnitude of variables occurred, the trends observed were similar to those presented in the paper. In all cases, the response of aggregate size to variations in agitation and sludge concentration, and the exchange of floc markers between spiked and unspiked sludges, supported the concept of reversible aggregation between activated sludge flocs. To complete the physico-chemical description of bioflocculation, the nature of units exchanged between flocs should be better defined. As exopolymeric substances are known to be the main floc-forming constituents [1,2,9], it is reasonable to speculate that the exchange units correspond essentially to biopolymers or bundles of exopolymeric material. Such a view would be consistent with earlier experimental work showing that soluble exopolymers are chemically similar to polymeric material from the floc [58,59]. Hence, bacteria and microcolonies would be essentially enmeshed in the network of exopolymeric material, in agreement with the earlier model proposed by Friedman et al. [48]. Clearly, further work is needed to clarify the nature of exchange units and to quantify aggregation dynamics. The role of aggregation dynamics should also be related to sludge properties.
Acknowledgements The authors gratefully acknowledge the authorization of sampling granted by the Grand Nancy urban community. We also thank the staff of Max!eville wastewater treatment plant for experimental assistance.
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