DEVELOPMENT OF COMBINED ULTIMATE HYBRID RESPIROMETER ... · respirometer-titrate meter for wastewater characterization and kinetic parameters estimation of microorganisms in activated

Suranaree J. Sci. Technol. 16(3):221-233

DEVELOPMENT OF COMBINED ULTIMATE HYBRIDRESPIROMETER-TITRATE METER TO ESTIMATEKINETIC PARAMETERS OF ACTIVATED SLUDGE

Patikorn Saensing1* and Sunthorn Kanchanatawee2

Received: Mar 20, 2009; Revised: Jun 30, 2009; Accepted: Sept 14, 2009

Abstract

The aim of this paper is to present a new development and an application of the ultimate hybridrespirometer-titrate meter for wastewater characterization and kinetic parameters estimation ofmicroorganisms in activated sludge. Currently, respirometry and titrimetry are considered highperformance tools with accuracy and frequency measurement. A new implementation of ultimatehybrid respirometer combined with titrate meter was developed. The new set-up of instrument wasdeveloped based on the previous knowledge and needed only sensor electrodes. The user interfaceprogramming to system control and monitor was developed based on LabVIEW 8.2 (student edition)software packages equipped with NI DAQ USB-6210 (A/D 16 bit). Finally, a laboratory implementationwas tested with readily biodegradable substrate, such as sodium acetate and ammonium chloride, toestimate the essential kinetic parameters of heterotrophic as yield, maximum specific growth rate, decayrate, saturation constant for substrate and initial ammonium concentration in activated sludge samples.The parameter estimation results from using respirometric measurements data were close to the defaultvalues in Activated Sludge Model No. 1 (ASM1) and other reported values. The ammonium concentrationcalculate by using base information were much correlated with the amount of ammonium applied to theactivated sludge sample. This demonstrated that the new instrument set-up was successfully applied toestimate kinetic parameters of activated sludge and measurement of ammonium concentration inactivated sludge samples.

Keywords: Activated sludge, kinetic, oxygen uptake rate, respirometry, titrimetry

Introduction

Respirometry is the measurement andinterpretation of the respiration rate of activatedsludge under a well-defined experimentalcondition, and is defined as oxygen per unit of

volume and time that is consumed by themicroorganisms in activated sludge (Oxygenuptake rate, OUR). It is a frequently and widelyused tool for characterization of wastewater and

1 School of Environmental Engineering, Institute of Engineering, Suranaree University of Technology, 111University Avenue, Muang District, Nakhon Ratchasima,30000, Thailand, E-mail: [email protected]

2 School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, 111University Avenue, Muang District, Nakhon Ratchasima 30000, Thailand, E-mail: [email protected]

* Corresponding author

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222 Combined Ultimate Hybrid Respirometer-Titrate Meter

activated sludge kinetic parameters. The theoryof a hybrid respirometric measurement wasproposed by Vanrolleghem and Spanjers (1998).In the hybrid respirometer, the principles of aflowing gas-static liquid (LFS) and static gas-flowing liquid (LSF) respirometer are combined(L is measurement in liquid phase). Table 1compares the four principles in terms of theseproperties. Several details on types and measure-ment principles of respirometer, can be foundin Spanjers et al. (1998). Further information inthe development and evaluation of the hybridrespirometer can be found in Petersen (2000).

Besides respirometry, titration experimentscan also yield information on the kineticparameters of activated sludge in biologicalnitrogen removal processes. Indeed, the pHvalue of a biological responds to microbialreactions. The nitrification process (NH4

+ + 2O2

➔ NO3- + H2O + 2H+) causes pH to decrease

because of two moles of proton production(Gernaey et al., 1997). Ammonium concentrationthat interprets from proton information produceddue to nitrification reaction was highly correlatedwith amount of ammonium applied to activatedsludge sample (Massone et al., 1996). Thetitrimetric measurement is to keep constantpH in activated sludge which results frombiological reactions to monitor acid or baseconsumption rate.

The reliability, versatility and precision oftitrimetric principle were successfully applied

to estimate kinetic parameters of activated sludge(Ramadori et al., 1980; Massone et al., 1996;Gernaey et al., 1998). The titrimetric principlewas applied to measurement under anoxiccondition for estimation kinetic parameters ofdenitrifying bacteria and evaluation performanceof denitrification process (Onnis et al., 2001;Artiga et al., 2005). This principle was alsoapplied to the measurement under anaerobiccondition for evaluation methanogenic activity(Rozzi et al., 2001).

The results obtained from titrimetricexperiments show reliability of this methodin estimating kinetic parameters of activatedsludge samples. Also, the advantages of eachmeasurement principle are combined to improveperformance of instrument with higher accuracyand frequency measurement (Gernaey et al.,2001; Gernaey et al., 2002a, 2002b). Theinstrument was applied to estimate kineticparameters for Activated Sludge Model No. 1(ASM1) calibration, monitoring and control ofbiological nutrient removal processes (Guisasolaet al., 2007; Sin and Vanrolleghem, 2007).

The aim of this paper is to develop andrealize the combined ultimate hybrid respirometer-titrate meter to support the advancement inwastewater research with demonstrating the datainterpretation from respirometric-titrimetricexperiments. In addition, the main goal of thisimplementation is to construct the instrumentwhich needs only the sensor electrodes to

Table 1. Advantages and disadvantages of different respirometric principles (KLa isoxygen mass transfer coefficient)

Respirometer type Advantages Disadvantages

Static gas-static Easy to operate Danger for oxygen limit Lowliquid (LSS) OUR measurement frequency

Flowing gas-static High OUR measurement KLa estimation neededliquid (LFS) frequency

Static gas-flowing No KLa needed Low OUR measurementliquid (LSF) frequency

Hybrid respirometer No KLa needed Two dissolved oxygen electrodeHigh OUR measurementfrequency

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223Suranaree J. Sci. Technol. Vol. 16 No. 3; July - September 2009

connect to the system.

Materials and Methods

A new implementation set-up is combinedultimate hybrid respirometer-titrate meter(Vanrolleghem and Spanjers, 1998; Petersen,2000). An overview of the instrument is shownin Figure 1. The set-up consists of an aerationvessel (V = 1.86 L) and a respiration chamber(V = 0.84 L). The latter is made strictly airtight,while the aeration vessel was open. For theaeration, fine-bubble aeration was provided inthe aeration vessel by using a simple air stonediffuser commercially available for fish tanks.Atmospheric air, dried and compressed to 4 barpressure, was used as air supply. Stirrers withadjustable speed mixed the content of bothvessels. A peristaltic pump (six-roller) withadjustable speed was used to continuous pumpthe activated sludge to both vessels. The averagedosage of acid and base pump (diaphragm) was0.031 ± 0.001 and 0.032 ± 0.001 mL/pulse,respectively. Aeration vessel, respiration cham-ber, transferring tube, measuring chamber anddissolved oxygen (DO) electrodes were submergedin the water bath to reduce the difference intemperature between aeration vessel andrespiration chamber.

A pH electrode (HI-1230B, HannaInstruments) was placed at aeration vessel andtwo polarographic DO electrodes (YSI-200, YSIIncorporated) were placed in measuring chambersbefore and after respiration chamber. Signalsfrom two DO, pH and temperature electrodeswere amplified by sensor signal conditionersto the range of amplitude (± 10 VDC). Allelectrode signals were noise filtered with 10Hertz low passed analog filter circuits beforebeing converted to digital signals by usingNI-6210 (USB-DAQ, A/D 16 bit, I/O, NationalInstruments). The user interface for dataprocessing and control was developed, basedon LabVIEW 8.2 software package (Studentedition, National Instruments). The dataacquisition frequency of the sensors was set to3 seconds. In the implementation set-up,calibration of DO electrodes was done in twosteps while aerated distilled water was pumpedthrough the set-up. First, the two DO electrodesin the measuring chamber were calibrated withDO saturation in distilled water, and thencalibrated with zero DO. Calibration of pHelectrode was done by using standard pH buffersolutions (4.0, 7.0, and 10.0 pH).

For the experimental work, activatedsludge was sampling from the MLE pilot scalewastewater treatment plant (Environmental

Figure 1. Overview of the combined ultimate hybrid respirometric-titrimetric set-up

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Engineering Lab, Suranaree University ofTechnology, Nakhon Ratchasima, Thailand),which performs nitrification, denitrification andchemical oxygen demand (COD) removal. It wasfed with municipal synthetic wastewater withaverage COD, TKN (Total kjedahl nitrogen)and TP (Total phosphorus) 238.99 ± 13.15,32.47 ± 2.98, and 4.40 ± 0.59 mg/L, respectively.The hydraulic retention time (HRT), solidretention time (SRT), temperature and pHwere maintained at 6 h 5 days, 28 ± 0.10oC and7.5 ± 0.03 respectively.

Activated sludge was aerated overnightuntil it reached the endogenous respiration phaseand then washed 2 - 3 times with the distilledwater prior to transferring to the set-up (Artigaet al., 2005). At the start of the experiment, theset-up was filled with 2.74 L activated sludge.The activated sludge was aerated until theendogenous respiration phase was reached.During the experiments, small substrate pulses(e.g. 10 mL) of sodium acetate (13 g COD/L)and ammonium chloride (1 g NH4

+-N/L) stocksolutions were dosed to the activated sludge.All experiments, instrument calibration andactivated sludge sample preparation wereperformed at 28 ± 0.10oC. The pH wascontrolled within a narrow set-band 7.5 ± 0.03,by dosing with 0.05N HCl and 0.05N NaOH, asdescribed in detail by Gernaey et al. (2001).

Data Interpretation

Data derived from each experiment wereinterpreted using both a spreadsheet program(Excel) and a model-based data interpretationmethod based on ASM1.

Experimental Results and Discussion

Structure of Combined Ultimate HybridRespirometer-titrate Meter

The schematic diagram of the ultimatehybrid respirometer-titrate meter is presented inFigure 2(a). In this set-up, the DO electrode inthe aeration vessel was moved to the otherconnecting line between the respiration chamberand the aeration vessel. In this way, the DOconcentration of liquid flowing into and out ofthe respiration chamber, SO,1 and SO,2, weremeasured by two DO electrodes, DO1 and DO2,respectively. At the same time, OUR can becalculated by derived through a mass balancedof oxygen over the respiration chamber with asimilar procedure as the static gas-flowing liquidrespirometer (Equation (1)) (Vanrolleghem andSpanjers, 1998). Measuring chamber can beoperated in four different patterns (Lu et al.,2006). So the up-flow with negative pressure(more stabled measurement than each otherpatterns) (Figure 2(b)) was selected as the

Figure 2. Schematic diagram of ultimate (full line) and simple (dotted line) hybridrespirometer (a) and DO measuring chamber (b)

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regular operation pattern for DO measuringchamber.

(1)

where OUR is the oxygen uptake rate (mg/L.min), Q is the flow rate (L/min) and V2 is therespiration chamber volume (L).

In the new set-up, DO electrode did notplace in aeration vessel and respiration chamber.The advantages are reduced hydrodynamicnoise and oscillated readings of DO electrodesin both vessels. A possible reason is becauseof pressure acting on the surface membrane ofDO electrode (Petersen, 2000). A workingvolume of measuring chamber was about 4 mL,0.5% of respiration chamber volume. Hydraulicretention time of measuring chamber atoperating condition, Q = 0.25 L/min was notmore than 1.0 sec. So the oxygen uptake rate inmeasuring chamber could be ignored (Lu et al.,2006). In the new implementation, an accurateand constant temperature water bath was designedto accommodate the whole system (aerationvessel, measuring chamber with DO electrodes,respiration chamber and almost all transfertube) to achieve accurate and reliable DOmeasurement.

Batch experiment where the rapid changeof oxygen uptake rate, the time constant of theelectrode (τelectrode) is then no longer negligible.To deal with this, the DO electrode dynamicsshould be taken into account before Equation(1) is used for calculation of oxygen uptake rate.Using the general accepted first order modeldescribing the dynamics of DO electrode(Equation (2) – (3)) (Vanrolleghem and Spanjers,1998). The DO electrode time constant 8seconds was used in this experiment (YSIIncorporated).

(2)

(3)

The software implementation withcombined ultimate hybrid respirometer wasdeveloped based on the LabVIEW 8.2 package(Student edition, National Instruments).The main content was noise filtering, sensorssignal processing, devices controlling and userinterface. All parameters needed to be set up bythe user such as temperature and pH set-point,respiration chamber volume, internal flowrate, acid and base dosing volume, electrodetime constant, signal filter and measuringfrequency can input into this panel (Figure 3(a)).Meanwhile, the value of DO, oxygen uptakerate, pH, acid or base accumulations andtemperature can be real-time display on panelas shown in Figure 3(b), which provides systemmonitoring. By this way, the user can findpossible fault detection and take effectiveaction in time.

The new implementation workedconsistent, except peristaltic pump, activatedsludge transfer pump between aeration vesseland respiration chamber. A two-roller peristalticpump is the main cause of high pressureoscillating in the activated sludge transferringtube. This problem can be solved by changingtransferring pump to a six-roller peristaltic pumpand increasing the speed of the pump. The testwith distilled water (Figure 4(a)) showed theoscillating of pressure in the activated sludgetransferring tube less affect DO measurementand oxygen uptake rate calculation. Aftertemperature reached the set-point value(28 ± 0.10oC), DO was stable throughout theexperiment period. Standard deviation (0.003mg/L.min) was much narrowband. The test withactivated sludge sample (Figure 4(b)) shows DOprofile and the calculation of oxygen uptake ratewith the same concentration of two pulses ofsubstrate were applied.

Activated Sludge Kinetic ParametersEstimation

Modeling efforts of activated sludge

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processes took off in the early 1970s with thepioneering work of the Marais group from theUniversity of Cape Town. The industry standardASM1 was developed by the IAWPRC taskgroup on modeling activated sludge processes(Henze et al., 2000). Since then, understandingof activated sludge processes increased whichresulted in the development of ASM2 andASM3. Activated sludge models can be appliedfor the following purposes; design of new plants,analysis of existing plants, real-time control ofplant, and serving as a guidance for researcherto achieve more efficient experimental designs(Henze et al., 2000).

Respirometry, the measurement ofoxygen uptake rate of biomass has been awell-established methodology for the calibration

of activated sludge models (Kappeler and Gujer,1992; Spanjers and Vanrolleghemm, 1995;Vanrolleghem et al., 1999). The stoichiometricparameter (Yield, Y), kinetic parameters(Maximum specific growth rate (μ max), decayrate (b) and saturation constant for substrate (K))are the major group parameters to be calibrated.In the mathematical model, the process ratesand the stoichiometric relationship between theprocesses and compounds are formulatedmathematically by the matrix method (Table 2)for dynamic model presentation.

An accurate assessment of the heterotrophicbacteria yield coefficient, YH, is the mostimportant, not only because this parameterinfluences sludge production and oxygen usedbut also because it has an impact on the value of

Figure 4. The experiment with distilled water (a) and activated sludge sample (b)

Figure 3. Software user interface (a) and real-time display panel (b)

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other parameters whose determination requiresa value of YH (Vanrolleghem et al., 1999). Alinear relationship between the substrate (SS)added and oxygen consumed (OC) allows tocalculate biomass yield via OC = (1 - YH)SS byusing the slope of the curve. The respirogramobtained in this experiment is shown in Figure5(a).

The heterotrophic bacteria yield coefficientwas evaluated through a respirometric batchexperiment with 5 different pulses of a completelybiodegradable organic substrate added toendogenous activated sludge sample. Sodiumacetate range from 9.6 – 229.3 mg COD/L wasused in this experiment with an average ofmixed liquor volatile suspended solid (MLVSS)of 1,000 mg/L. A linear increase of OC was

observed when increasing amount of sodiumacetate was added to the respirometer (Figure5(b)). The maximum average yield for this dataseries is 0.69 ± 0.01 mg COD/mg COD whichis much close to the default value in ASM1(0.67) (Henze et al., 2000). Muller et al. (2004)reported the yield on acetate 0.69 (in the rangeof 0.66 - 0.71) mg COD/mg COD. Similarresults were reported by Dricks et al. (1999), avalue of yield on acetate was 0.71 mg COD/mgCOD. Furthermore, the difference observed alsoimplies that in the determination of the readilybiodegradable COD (rbCOD) fraction byrespirometric methods, the yield coefficientshould not be based on a standard valuefrom literature but should be experimentallydetermined from the activated sludge used inthe experiment.

Figure 5. Yield determination respirogram (a) and linear relationship between oxygenconsumed with substrate added (b)

Table 2. The matrix notation for heterotrophic bacteria (XBH) growth and decay in ASM1

Component i 1 2 3 4Process rate, ρj

j Process SS XBH SO SNH

1 Aerobic growth of 1

heterotrophs

2 Decay of -1 1

heterotrophs

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Saturation constant for substrate, KS, isdetermined following the method describedby Cech et al. (1984). This method is basedon Monod equation (Equation (4) - (5)) andanalysis of the oxygen uptake rate at differentsubstrate concentration. Exogenous oxygenuptake rate (OURex) was measured as thedifference between endogenous oxygen uptakerate (OURend) and the total oxygen uptake rateregistered in every experiment.

(4)

(5)

The exogenous oxygen uptake rate wasdivided by the maximum value of exogenousoxygen uptake rate registered in the wholeexperiment to calculate the relative activity(μ /μ maxH), depending on substrate concentration.The resulted of respirometric experimentalconcurrent with data fitted by simulation isshown in Figure 6. The saturation constant forsubstrate (KS) obtained is 19.1 ± 2.7 mg/L, whichis close to the default value in ASM1 (20 mg/L)(Henze et al., 2000). The observed Ks value wasclosed to 20.4 mg/L (at 20oC) that was reported

by Penya-roja et al. (2002). The different invalue of KS is 15 mg/L (at 30oC) that wasreported by Dosta (2007).

The decay coefficient for heterotrophicbacteria with lineal death, bH, was determinedfollowing the method proposed by Spanjers andVanrolleghem (1995). For this experiment,activated sludge was aerated without substrateapplied. The method was based on measuringthe oxygen uptake rate obtained after additionof an optimal mixture of sodium acetate (Figure7(a)). This was repeated 3 times over a periodof 9 days. If yield and maximum growth rate areconstant, the value of the parameter combinationswill only depend on the active biomass. Thedecay coefficient is calculated from the slope ofa plot of LnOURmax versus time (Figure 7(b)).

As shown in Figure 7(b), LnOURmax

versus time shows a straight line with the decay

Figure 6. Assessment of saturationconstant for substrate

Figure 7. The respirogram obtained froma decay experiment (a) and a plotof LnOURmax versus time (b)

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coefficient for heterotrophic bacteria(lineal-death), bH

Lineal-death, as slope. The valuedetermined was 0.1 day-1, where obtainedcoefficient differs from the one in the ASM1.The death-regeneration concept, adopted in theASM1, implies that the classical methods fordetermination of the decay of biomass cannot be applied directly. The parameter oflineal-death concept has to be translated tothe parameter of death-regeneration conceptby applying Equation (6) (Orhon and Artan,1994), where fp is the particulate inert fractionof biomass (0.08). The decay rate was,bH

Death-regeneration, obtained 0.31 ± 0.02 day-1. Thisdecay rate value very close to 0.28 day-1

(at 20oC) was reported by Manser et al. (2006).This decay rate is lower than the defaultvalue in ASM1 (0.62 day-1), but still comparableto the range of decay rate values (0.05 – 1.6day-1) in the literature (Jeppsson, 1996), andbH

Lineal-death is about 5% of maximum specificgrowth rate value (Kappeler and Gujer, 1992).

(6)

The maximum specific growth rate ofheterotrophic bacteria, μ maxH, under aerobicconditions, is concomitant with the saturation

constant for substrate, KS, an essential kineticparameter to characterize the COD removalcapacity and the biomass production of theactivated sludge under study. This parameter wasassessed with the procedure proposed byKappeler and Gujer (1992) based on addinghigh readily biodegradable substrate to lowconcentration of activated sludge with nonelimiting of DO concentration. Sodium acetate1,402 mg COD/L was used in this experimentwith an average of MLVSS 720 mg/L. Therespirogram obtained in this experiment is shownin Figure 8(a). A linear increase of oxygenuptake rate was observed when sodium acetatewas added to the respirometer. The possibilityof assessing (μmaxH - bH) is the slope of the plotof LnOUR versus time (Figure 8(b)). The μmaxH

for this experimental data is 2.2 ± 0.3 day-1. ThisμmaxH value close to 1.68 day-1 (at 25oC) wasreported by Guisasola (2005). This μmaxH ismuch lower than the default value in ASM1 (6day-1), but still comparable to the range of μmaxH

values (0.6 – 13.2 day-1) in the literature(Jeppsson, 1996). The results of all experimentsare shown in Table 3.

Titrimetry, the indirect measurementof the pH effects resulting from the biomassmetabolic activities, has recently reached a levelof precision which makes it useful for quantifyingthe kinetic parameters of activated sludge

Figure 8. Maximum specific growth rate determination respirogram (a) and plot of LnOURversus time (b)

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processes (Sin and Vanrolleghem, 2007),Titrimetric measurement principle is based onadding small amount of a titration solution tokeep constant pH at a fixed set-point valueduring the reaction.

In this study, the simultaneouslyrespirometric and titrimetric measurementswere tested with activated sludge samples todetermine oxygen uptake rate (data not shown)and amount of NaOH (0.05N) to compensatefor the proton production by autotrophic bacteriain nitrification process. An initial ammoniumconcentration can be calculated based on thedifference between B2 and B1, according toEquation (7)

(7)

where SNH is an initial NH4+-N concentration in

the vessel of titration unit (mg N/L), B1 is thevolume of base needed to increase the pH ofsludge sample to the pH set-point (mL), B2 issum of B1 and volume of base necessary tocompensate for the protons produced duringnitrification (mL), N is base normality (mequiv/mL), 7 is the conversion factor (mg N/mequiv),and Vreactor is the volume of sludge in the vesselof the titrate meter (L) (Gernaey et al., 1997).

The experiment was carried out with anaverage of MLVSS 2,180 mg/L at pH set-point7.8 ± 0.03. Ammonium chloride of 1.9 and 3.7mg NH4

+-N/L were used in this experiment. As

shown in Figure 9(a) and 9(b), the profiles ofbase dosage were almost a straight line duringthe period of nitrification reaction. The resultsof titration experiments should theoretically beequal to the amount of NH4

+-N added at thebeginning of the titration experiment. Thecalculation of NH4

+-N concentration of 2.0 and3.6 mg NH4

+-N/L were obtained. The resultsshow that the set-up allows for determination ofNH4

+-N concentration in the activated samplesusing a simple and robust titration method.

Conclusions

The combined ultimate hybrid respirometer-titrate meter was developed and designed to bean advanced tool for wastewater engineeringresearch based on the previous knowledge andonly required sensors electrode to connect to thesystem. The user interface was developedbased on LabVIEW 8.2 (Student edition) forcontrolling and experimental monitoring withreal-time display. The major sources of noisefor oxygen sensing and oxygen uptake ratecalculation were minimized by increasednumber of peristaltic pump roller. The differencein temperature in each vessel was solved bymeans of submersed aeration vessel, dissolvedoxygen measuring chamber with DO electrodes,respiration chamber and almost all transfer tubesinto water bath with high accuracy and reliabletemperature control.

An essential kinetic parameters ofheterotrophic bacteria (XBH) are yield (YH),

Table 3. The kinetic parameters obtained from respirometric experiments

Kinetic parameters of ASM1/ ASM3 Literature heterotrophic bacteria Value Unit (20oC) (Jeppsson,

(Henze et al., 2000) 1996)

Yield coefficient 0.69 ± 0.01 mg/mg 0.67/0.85 0.38 - 0.75

Max. spec. growth rate 2.2 ± 0.3 day-1 6/2 0.6 - 13.2

Saturation constant for COD 19.1 ± 2.7 mg/L 20/2 5 - 30

Decay rate 0.31 ± 0.02 day-1 0.62/0.2 0.05 - 1.6

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maximum specific growth rate (μmaxH), decayrate (bH) and saturation constant for substrate(KS) which were estimated via respirometricmeasurements. The results of parameterestimation using respirometric measurementsdata were close to the default values in ASM1and other reported values. Even though someparameter was lower than the default value,they were still comparable within the range ofvalues in the literature. The difference of eachparameter from default value may be due to thedifference of temperature, sludge sources,location, operating conditions, experimentaldesign, and experimental time. The results ofall the experiments show that the developedcombined ultimate hybrid respirometer-titratemeter was successfully applied to estimatekinetic parameters of activated sludge andthe measurement of ammonium concentrationin activated sludge samples.

Acknowledgments

Financial supports for this work are fromSuranaree University of Technology ResearchFund, Rianchai Cement Block Part. Ltd andSHELL Centennial Education Fund.

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