Enzyme and Microbial Technology 33 (2003) 292–298

Towards a rheological parameter for activated sludgebulking characterisation

N. Tixier, G. Guibaud∗, M. BauduLaboratoire des Sciences de l’Eau et de l’Environnement, Faculté des Sciences et Techniques, University of Limoges,

123, Avenue A. Thomas, 87060 Limoges Cedex, France

Received 22 February 2003; received in revised form 3 May 2003; accepted 3 May 2003

Abstract

An examination of the rheological behaviour of activated sludge presenting filamentous bulking was carried out. It was shown thatrheograms from filamentous sludge exhibit a particular behaviour due to its thixotropic characteristic, contrary to non-filamentous ones. Arheological protocol of measurement of thixotropy consisting of submitting sludge to increasing–decreasing shear rates was proposed andoptimised to obtain the greatest sensitivity as possible on the determination of the proliferation of filamentous bacteria in activated sludge.A relevant parameter, consisting in the determination of the reduced hysteresis area developed (rHa) from rheograms, for indicating thepresence and the amount of filamentous bulking was deduced. An application to the study of several bulking developments was carried out.The thixotropic characteristic of filamentous sludge was shown to originate from an irreversible breakdown of the filamentous matrix. Thetotal suspended solid (TSS) content was shown to have a great influence on the value rHa. The morphological characteristics of filamentswere supposed to have an influence on the value rHa too. The carbohydrate content of activated sludge was shown to be linearly linked tothe rheological parameter rHa/TSS during the filamentous growth phase and an increase in rHa/TSS was followed by an increase in SVI.© 2003 Elsevier Inc. All rights reserved.

Keywords:Activated sludge; Rheology; Filamentous bulking; Reduced hysteresis area; Thixotropy

1. Introduction

The activated sludge process is limited by its ability toseparate biomass from the effluent[1,2]. Filamentous bulk-ing is one of the main factors leading to biomass loss inthe effluent. Filamentous micro-organisms form a part of thebacterial population that plays a predominant role in the bac-terial community of the activated sludge, they can serve as“backbones” for flocs and then favour its growth[3,4]. Nev-ertheless, over-proliferation of filamentous micro-organismsis recognised as being the main factor inducing poor abilityof filamentous flocs to settle[5–9]. It is of major interestfor wastewater treatment plant (WWTP) operators to rapidlydetect the appearance of filamentous bulking due to its con-sequences on the quality of the effluent[10].

Sludge rheological properties are dependent on their na-ture, thus rheology should be able to give clues to theirphysical state[11–14]. Some rheological parameters suchas viscosity were shown to depend on factors such as sludge

∗ Corresponding author. Tel.:+33-5-55-45-74-28;fax: +33-5-55-45-72-03.

E-mail address:gguibaud@unilim.fr (G. Guibaud).

surfaces[15,16]and to be sensitive to changes in pH or ionicenvironment [17,18]. Rheological measurements shouldsupply interesting information concerning sludge character-isation and more particularly concerning floc structure.

Wastewater sludges are non-Newtonian fluids[19], i.e.shear stress (τ) exerted on the sludge is not proportional tothe induced shear rate (γ). Various rheological models forinterpretation of sludge characteristics have been proposed[20,21]. Rheology of sludge often presents thixotropicproperties[22], which means that the viscosity of sucha fluid does not remain constant for a given shear rate,it also becomes a function of time. The consistency of athixotropic fluid depends both on shear rate and on shearduration. This property is often encountered with floccu-lated activated sludge[23] and parameters resulting fromthis property can be used as a field control index for charac-terization and control of sludge conditioning with polyelec-tolytes[12].

Thixotropic materials generate shear rate–shear stresscurves that are different whether they are determined for in-creasing or decreasing shear rates. These upward and down-ward curves describe a hysteresis loop and the enclosed areacan be used to evaluate the magnitude of thixotropy[23,24].

0141-0229/$ – see front matter © 2003 Elsevier Inc. All rights reserved.doi:10.1016/S0141-0229(03)00124-8

N. Tixier et al. / Enzyme and Microbial Technology 33 (2003) 292–298 293

This paper examines the thixotropic behaviour of filamen-tous activated sludges in relation to their filament content. Asuitable rheological measurement protocol is optimised anda potential rheological indicator of the presence of filamen-tous bulking is proposed.

2. Materials and methods

2.1. Activated sludge samples

The activated sludges were collected in the aeration tankof two domestic WWTPs working at low organic loading[F/M]. Sludge samples are noted A and B according to thesampling location and a number is indexed according to thesampling date. WWTP A treats 285,000 inhabitant equiva-lent (in.eq.) and receives mainly domestic wastewaters anda low part of industry wastewaters. WWTP B treats 22,500in.eq., it receives domestic wastewaters and it also treats agreat amount of effluents originating from a dairy industryand a cardboard factory.

The total suspended solid (TSS) content was measured ac-cording to the standard method NFT 90-105-2[25]. Sludgevolume index (SVI) was measured in a 1 l glass tube after30 min of settling. Activated sludges that were sampled didnot show filamentous bulking, therefore they were placed inlab-scale plant, immediately after sampling (without dilutionor thickening), in such a way that favoured filamentous bact-erial development. The activated sludge lab-scale plant cons-isted of an 11 l cylinder aeration tank equipped with a bubblediffuser and a 10 l settling tank. The lab-scale plant operat-ing conditions were as follow: the feeding mode consistedof a continuous supply of a solution with a chemical oxygendemand (COD) of 2.1 g l−1 using a peristaltic pump thatdelivered a 0.25 l h−1 nutrient flow. The composition of thissolution was the following: tap water containing 5.4 ml l−1

of meat extract, 0.29 g l−1 of glucose, 0.23 g l−1 of ammo-nium chloride and 0.16 g l−1 of di-sodium hydrogenophos-phate. Sludge collected in the settling tank was re-injectedinto the aeration tank in a discontinuous way (once per day).

2.2. Analytical methods

The rheological tests were carried out using a rotationalrheometer (PAAR Physica system MC100) with a coaxialcylindrical measurement device with a double gap measur-ing system. The sample volume required was 17 ml and thetemperature was maintained constant at 20±2 ◦C during allexperiments. The software US200 (PAAR Physica) recordedthe rheograms shear stress (τ) as a function of shear rate (γ)and allows subsequent data analysis.

The rheological measurements were carried out in steadyflow in such a way that laminar shear flow conditions couldbe respected. For that device, the valueγmax (maximumvalue reached forγ) must not exceed 1100 s−1. The protocolenvisaged is composed of three steps:

(1) Increasingγ linearly from 0 toγmax in 3 min.(2) Maintainingγ constant atγmax for 1 min.(3) Decreasing linearlyγ from γmax to 0 in 3 min.

The parameterγmax has to be determined in order to obtainthe greatest sensitivity for the rheological index. The hys-teresis loop area (Ha) developed from rheograms is givenby Eq. (1):

Ha =∫ t

0τ dγ(t) (1)

whereτ (Pa) is the shear stress andγ (s−1) is the shear rate.It can be reduced to the volume unit by dividing Ha with

sample volume, it is then noted as rHa and expressed inPa s−1 ml−1. This value can be considered as a measure ofthe degree of thixotropy[26]. Five replicate measurementswere conducted for each rHa value presented, the averagevalue was determined and the accuracy is considered asbeing the average standard deviation between these fivemeasurements.

The microscopic examination and image capture ofsludge were performed during the evolution of the filamen-tous growth phase with an optical microscope (Zeiss Axi-olab) equipped with a video camera (SONY SSC DC 18P)connected to a computer. This device was used at a 400×magnification and allowed to assess the relative amountof filamentous bacteria in the different samples using theEikelboom scale[27].

The determination of the carbohydrate content in sludgesamples was carried out with the phenol/acid sulphuricmethod according to Dubois et al.[28]. Prior to the de-termination of the carbohydrate content, sludge samplescollected were submitted to centrifugation (4000×g, 5 min)using a centrifuge JOUAN MR23i and their supernatantwas replaced by an identical volume of ultra-pure water.Finally, sludge samples were ground using an Ultra-TurraxT25 (IKA-Kalabortechnik) to allow homogeneous samplesto be obtained.

3. Results and discussion

3.1. Presentation of typical rheograms of filamentoussludge and non-filamentous one’s

Fig. 1 presents an example of rheograms experimentallyobtained for two sludge samples (originated from sam-ple A1) having a very close TSS content but a stronglydifferent filamentous bacteria content. In comparison tonon-filamentous one, the rheogram of filamentous sampledevelops a great hysteresis area (Ha), due to its strongthixotropic characteristic. Non-filamentous activated sludgeis weakly thixotropic in the TSS working range of thestudy (inferior to 7 g l−1). Classical rheological models usedfor non-Newtonian fluids such as Bingham’s or Herschel-Bulkley’s [12] can be used to describe its behaviour. On

294 N. Tixier et al. / Enzyme and Microbial Technology 33 (2003) 292–298

Fig. 1. Rheograms of a filamentous sludge (- - -, TSS: 5.5 g l−1) and of anon-filamentous one (—, TSS: 5.8 g l−1).

the contrary, no classical model can be used to describe therheological behaviour presented by filamentous sludge be-cause of the specificity of the flow curve during increasingshear rate, a great resistance to flow appears even from lowshear rates. The major difference noted in the value of rHaaccording to the amount of filamentous bacteria presentin activated sludge was investigated as a potential tool forevaluating sludge quality.

3.2. Optimisation of the protocol

The aim of this part was to determine the parameterγmaxdescribed previously allowing the greatest efficiency for theparameter rHa for detecting filamentous bulking. By effi-ciency, we mean that the difference between the values ofrHa for a filamentous sludge and a non-filamentous one(value close to zero) has to be the greatest as possible. Fur-thermore, optimum accuracy in the measurement of rHa isexpected. With this aim in view, it was decided to study theevolution of rHa and its standard deviation with the varia-tion of γmax in the range 400–1000 s−1 for two sludge sam-ples conditioned in lab-scale plant (A1 and B1) presentingfilamentous bulking (4 on the Eikelboom scale). Results ofthat experiment are presented inFig. 2.

It appeared that as theγmax rose, the rHa increased andthe standard deviation decreased. rHa values obtained forγmax = 400, 500 and 600 s−1 were too low and the stan-dard deviation was too high to allow sensitive enough mea-surements. Forγmax = 900 and 1000 s−1, the value rHaremained nearly constant for sample A1 and increased forsample B1. In the meantime, for these values ofγmax, thestandard deviation increased for sample A1 and for sampleB1 (γmax= 900 s−1) and decreased forγmax= 1000 s−1. Thevalue γmax= 800 s−1 was then chosen because it generateda low standard deviation and good sensitivity allowing to

Fig. 2. Evolution of rHa and its standard deviation vs.γmax for sludgeA1 (TSS= 6.1 g l−1) and sludge B1 (TSS= 3.1 g l−1).

determine the parameter rHa with a good accuracy. Further-more, this value is distant enough from the limit determinedpreviously to prevent problems linked to a change in flowregime and possible appearance of vortexes.

3.3. Influence of TSS content on rHa and elementsconcerning the cause of thixotropy

Fig. 3presents the evolution of rHa with the TSS contentof two filamentous sludge (samples A2 and B2 conditionedin lab-scale plant). An exponential law well describes thevariation of rHa with TSS. It indicates the way how filamen-tous flocs interactions influence rHa. The power and expo-nential laws are the most often encountered in the literatureto describe the evolution of rheological parameters such asyield stress or apparent viscosity with TSS[13,16,18]. TheTSS content is recognised as being the main factor affectingsludge rheology, therefore it is important to compare sludgerheological properties at identical or closest as possible TSScontent.

As evidence, the presence of filamentous flocs is consid-ered as the most important parameter for the thixotropic be-haviour noted for these sludges. More precisely, disruptionof filaments seems to be at the origin of the hysteresis loopsdeveloped by filamentous sludges, thixotropy represents theenergy spent in solid–solid structure disruption[26]. A con-firmation of this assertion is given inFig. 3, the dotted linecurve represents the evolution of rHa for sludge sample A2subjected twice consecutively to the measurement protocol.A very strong decrease in rHa happened in comparison to thefirst application of the protocol, indicating the irreversiblebreakage of the filamentous network structure when a suffi-cient shear rate is reached.Fig 4a and b, resulting from pho-tographs of filamentous flocs, respectively, before and afterhaving been submitted to shear rate, show the breakage of the

N. Tixier et al. / Enzyme and Microbial Technology 33 (2003) 292–298 295

Fig. 3. Evolution of rHa with the TSS content for two filamentous sludge samples A2 (�) and B2 (�) and after two consecutive applications of theprotocol for sample A2 (�).

filamentous network structure. Many fragments of filamentsare observed after application of shear rate (Fig. 4b). If werefer to a typical rheogram of filamentous sludge (Fig. 1),we can suppose that for the lowest shear rate, filaments forma network structure able to oppose flow resulting in a largeincrease in shear stress. When a higher shear rate is reached,the shear stress starts to decrease indicating certainly thatthe energy furnished was sufficient to disrupt most of thefilament network opposing flow.

As suggested by the strong difference in the two equa-tions describing the evolution of rHa for samples A2 andB2 with the TSS content, the filaments morphology (size,length) and their density in sludge sample play an impor-tant role in determining the rheological behaviour of sludgesamples because they determine the strength of the networkstructure that oppose to flow. The type of filaments and theirstructure are supposed as responsible for the differencesnoted in rHa for samples A2 and B2 rather than the flocstructure.

Fig. 4. Flocs and filaments before (a) and after (b) application of shear rate (sample A2, magnification 400×).

3.4. Application to the study of the development ofseveral filamentous bulkings

The comparison between the evolution of the parameterrHa/TSS and SVI during two filamentous bulking devel-opments (samples A3 and B3, originating from WWTPs Aand B and conditioned in lab-scale plant) was carried out.The results are, respectively, shown inFigs. 5 and 6forsamples A3 and B3. SVI is an index of sludge settleability,its increase indicates a degradation in sludge settleability.As filamentous bacteria proliferation is recognised as be-ing a factor leading to the decrease in sludge settleability(i.e. an increase in SVI), the comparison of the evolutionof rHa/TSS and SVI should furnish interesting informationconcerning the characterisation of bulkings.

For sample A3, the parameter rHa/TSS starts to increasefrom 6th day, indicating the proliferation of filamentousbacteria. Then, it increases regularly until the 18th day.The parameter SVI remains quite constant (between 50 and

296 N. Tixier et al. / Enzyme and Microbial Technology 33 (2003) 292–298

Fig. 5. Evolution of rHa/TSS (�) and SVI (�) during the evolution ofbulking for sludge sample A3.

Fig. 6. Evolution of rHa/TSS (�) and SVI (�) during the evolution ofbulking for sludge sample B3.

Fig. 7. Evolution of rHa/TSS with time during the development of two filamentous bulking A4 (�) and B4 (�).

100 ml g−1) for the nine first days and strongly increasesafter, indicating a degradation of sludge settleability. Forsample B3, rHa/TSS starts to increase from fourth day. SVIvaries from 70 to 115 ml g−1 the first 6 days and increasesstrongly after this delay, indicating the weak settleability ofsludge.

The increase in rHa/TSS indicates a filamentous bacteriaproliferation, this one is accompanied by a decrease in sludgesettleability as shown by the increase in SVI for the twobulkings investigated. Nevertheless, the increase in rHa/TSSwas shown to be faster in indicating filaments proliferationthan the increase in SVI due to its greater variation range atthe moment of the appearance of filamentous bulking.

Activated sludge samples A4 and B4 with no initial fila-mentous bacteria proliferation were collected from WWTPsA and B and conditioned in the continuous lab-scale plantdescribed previously. Regular rheological measurementswere carried out on these sludge samples to investigate thechanges in sludge quality with time.Fig. 7presents the evo-lution of the parameter rHa/TSS with time, this parameterwas used to investigate the filaments content because of theexponential relationship between rHa and TSS.

A microscopic observation has shown that fresh acti-vated sludge samples (A4 and B4) contained a very fewfilamentous bacteria, no more than 1 on the Eikelboomscale[27]. The values of the parameter rHa/TSS for freshsamples A4 and B4 are very close to zero, no exceeding2 × 103 Pa s−1 g−1. For sample A3, rHa/TSS starts to in-crease significantly from 4th day (4.3 × 103 Pa s−1 g−1) tothe 18th day (18.6×103 Pa s−1 g−1). For sludge sample B3,a significant increase in rHa/TSS is detected only from 9thday and reach 14.0×103 Pa s−1 g−1 the 12th day. Then it de-creased the 14th day until the value of 7.3×103 Pa s−1 g−1,

N. Tixier et al. / Enzyme and Microbial Technology 33 (2003) 292–298 297

Table 1Evolution of the rheological parameter and of the filament content asa function of time during the study of the development of filamentousbulking

Sludgesample

Time(days)

rHa/TSS(×103 Pa s−1 g−1)

Filament contentexpressed asEikelboom scale

A4 0–4 2–4 0–14–15 4–10.5 2–3

>15 >11 4

B4 0–9 2–3 0–19–11 3–4 2

>11 >5 3–4

corresponding to a filament content of 3 on the Eikelboomscale, this evolution indicates a decrease in the content offilamentous bacteria. The sample A4 (6th day) presents anirregular shaped curve too, a sudden increase in rHa/TSSis noticed followed by a decrease (from 6th to 8th day)before increasing again. The change in the balance betweenfilamentous bacteria and floc forming ones may have lots ofcauses and it is difficult to determine them in this particularcase, even small variations in lab-scale pilot plant drivingshould induce strong variations in filament content. Themicroscopic examination of sludge (Table 1) shows thatthe Eikelboom’s index and the parameter rHa/TSS have thesame evolution trend, they increase with the filamentousbacteria content in activated sludge. The filaments appear-ance in activated sludge is clearly indicated by a valuerHa/TSS increasing from a value very close to zero to amuch greatest one.

For these two sludge samples, the evolution of the carbo-hydrate content in flocs was measured from the appearanceof filamentous bulking and compared to the rheologicalparameter rHa/TSS. The determination of the carbohydratecontent aims to quantify indirectly the amount of filaments

Fig. 8. Evolution of the carbohydrate content in sludge as a function ofrHa/TSS during the filamentous growth phase, samples A4 (�) and B4

(�).

in activated sludge flocs because filamentous bacteria andmore particularly the sheaths of filaments are known ascontaining a high amount of carbohydrate[29]. The resultsobtained for the two sludge samples are presented inFig. 8.It appears that the carbohydrate content increases withrHa/TSS, a linear correlation between these two parametersgives satisfactory determination coefficients (R2 = 0.77). Itindicates that these two parameters may be linked and thatthe proliferation of filaments may be mainly responsiblefor the increase in carbohydrate content in sludge. Never-theless, variations in internal and external reserve productsof non-filamentous micro-organisms or in the filamentousmicro-organism species involved in bulking may play a rolein increasing the carbohydrate content too.

4. Conclusion

The particular rheological behaviour of filamentous ac-tivated sludge was investigated. The rheological parame-ter rHa was considered as an evaluation of the degree ofthixotropy of sludge. Its determination after the optimisationof a suitable rheological measurement protocol was shownto offer interesting perspectives for the monitoring of sludgequality as far as its filament content is concerned.

Among the factors influencing rHa, the TSS contentwas shown to play a major role, an exponential relation-ship between these parameters was found. Furthermore,the thixotropy of such filamentous samples was shown tooriginate in filaments breakage during increasing shear ratephase, the filaments morphology was supposed as importantin determining the degree of thixotropy too.

A rheological application to the study of several filamen-tous bulking evolution has shown that the use of rheologicalmeasurement could constitute an operational tool in detect-ing the appearance of bulking in activated sludge aerationtanks.

Acknowledgments

The authors thank the Regional Council of Limousin forits financial support.

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