metabolic model for glycogen-accumulating organisms in anaerobic/aerobic activated sludge systems

Metabolic Model forGlycogen-Accumulating Organisms inAnaerobic/Aerobic ActivatedSludge Systems

Raymond J. Zeng,1 Mark C. M. van Loosdrecht,2 Zhiguo Yuan,1 Jürg Keller1

1Advanced Wastewater Management Centre (AWMC), The University ofQueensland, St. Lucia, Brisbane 4072, Australia; telephone: 61-7-3365-4727;fax: 61-7-3365-4726; e-mail: [email protected] Laboratory for Biotechnology, Department of BiochemicalEngineering, Delft University of Technology, Julianalaan 67,2628 BC Delft, The Netherlands

Received 8 April 2002; accepted 11 June 2002

DOI: 10.1002/bit.10455

Abstract: Glycogen-accumulating organisms (GAO) havethe potential to directly compete with polyphosphate-accumulating organisms (PAO) in EBPR systems as bothare able to take up VFA anaerobically and grow on theintracellular storage products aerobically. Under anaero-bic conditions GAO hydrolyse glycogen to gain energyand reducing equivalents to take up VFA and to synthe-sise polyhydroxyalkanoate (PHA). In the subsequentaerobic stage, PHA is being oxidised to gain energy forglycogen replenishment (from PHA) and for cell growth.This article describes a complete anaerobic and aerobicmodel for GAO based on the understanding of theirmetabolic pathways. The anaerobic model has been de-veloped and reported previously, while the aerobic meta-bolic model was developed in this study. It is based onthe assumption that acetyl-CoA and propionyl-CoA gothrough the catabolic and anabolic processes indepen-dently. Experimental validation shows that the inte-grated model can predict the anaerobic and aerobic re-sults very well. It was found in this study that at pH 7 themaximum acetate uptake rate of GAO was slower thanthat reported for PAO in the anaerobic stage. On theother hand, the net biomass production per C-mol ac-etate added is about 9% higher for GAO than for PAO.This would indicate that PAO and GAO each have certaincompetitive advantages during different parts of the an-aerobic/aerobic process cycle. © 2002 Wiley Periodicals, Inc.Biotechnol Bioeng 81: 92–105, 2003.Keywords: glycogen accumulating organism (GAO);EBPR; metabolic model; TOGA; stoichiometry; growthyield

INTRODUCTION

Enhanced biological phosphorus removal (EBPR) is widelyaccepted as one of the most economical and sustainableprocesses to remove phosphorus from wastewater. The pro-cess requires alternating anaerobic and aerobic conditions to

enrich the so-called polyphosphate-accumulating organisms(PAO). Under anaerobic conditions, PAO take up volatilefatty acids (VFA) and store them internally in the form ofpolyhydroxyalkanoate (PHA). The reducing power is de-rived from glycolysis of glycogen. The energy for this pro-cess is obtained partly from the glycogen utilisation butmostly from the hydrolysis of the intracellular stored poly-phosphate (polyP). The latter results in the release of ortho-phosphate. In the subsequent aerobic phase, PAO take up anexcessive amount of ortho-phosphate to recover the intra-cellular polyP level by oxidising the stored PHA. Mean-while, they grow and replenish the glycogen pool usingPHA as both carbon and energy sources. Net phosphorusremoval is achieved by wasting sludge after the aerobicperiod, when the biomass contains a high level of polyphos-phate.

Regularly, poor performance or complete failure ofEBPR processes have been reported even under favourableconditions for EBPR (Cech and Hartman, 1990, 1993; Liuet al., 1994; Satoh et al., 1994; Bond et al., 1995). In manyof these cases, a particular group of microorganisms, calledglycogen-accumulating organisms (GAO) (Liu et al., 1996),are often found. GAO are able to take up VFA withoutanaerobic P release and subsequent aerobic P uptake, form-ing a strong potential competitor to PAO in EBPR systems.For this reason, a better understanding of GAO is needed toimprove the performance and reliability of EBPR systems.

The metabolic pathway of GAO has attracted consider-able interest in the past (Cech and Hartman, 1990; Liu et al.,1994; Satoh et al., 1994; Bond et al., 1995). It has beenfound that GAO degrade glycogen anaerobically to supplythe required energy (as ATP) and reducing power for thesynthesis of PHA from volatile fatty acids. Under the sub-sequent aerobic conditions, GAO oxidise PHA for cellCorrespondence to: J. Keller

© 2002 Wiley Periodicals, Inc.

growth and glycogen replenishment. A metabolic model forthe anaerobic processes of GAO has been developed re-cently by Filipe et al. (2001a) and amended by Zeng et al.(2002). A full GAO model has also been proposed by Filipeet al. (2001c), but without providing details of the underly-ing pathways. The aim of this study is to develop and dem-onstrate an aerobic metabolic model for GAO, so that anintegrated anaerobic and aerobic GAO model is obtained.Experiments were performed to validate this metabolicmodel.

ANAEROBIC MODEL

The model for the anaerobic GAO metabolism from Filipeet al. (2001a), with adjusted stoichiometric coefficientsfrom Zeng et al. (2002), is schematically represented inFigure 1. The pH-dependent energy requirement (in mol ofATP) to transport 1 C-mol HAc across the cell membrane isrepresented by alpha (�). In the model, acetate is taken upand activated to acetyl-CoA. Glycogen is hydrolysed andconverted to pyruvate, which provides all the required ATPin the overall process and some of the reducing power(NADH2). Then a part of the pyruvate is decarboxylated toform acetyl-CoA, providing additional NADH2 as well asgenerating CO2. The remaining pyruvate is converted topropionyl-CoA via the succinate-propionate pathway,which consumes NADH2. The succinate-propionate path-way is used to balance the redox potential inside the cell.This step produces no ATP as explained by Zeng et al.(2002) in contrast to the ATP production initially proposedby Filipe et al. (2001a). The acetyl-CoA and propionyl-CoAproduced are assumed to randomly condense to polyhy-droxybutyrate (PHB), polyhydroxyvalerate (PHV), andpolyhydroxy-2-methylvalerate (PH2MV).

At pH 7, which is the operating pH in this study, � is

approximately 0.06 mol ATP/C-mol HAc (Filipe et al.,2001a), so the overall stoichiometry can be described as(C-mol based):

–Acetate –1.12Glycogen +1.358PHB + 0.456PHV +0.0367PH2MV + 0.27CO2 � 0 [I]

AEROBIC MODEL

Under aerobic conditions, GAO oxidise the PHA accumu-lated in the anaerobic phase to gain energy for the replen-ishment of the glycogen pool and for cell growth. The re-actions include PHA catabolism, glycogen production, bio-mass growth, oxidative phosphorylation, as well asmaintenance. PHA comprises of PHB, PHV, and PH2MV,which hydrolyse to acetyl-CoA and propionyl-CoA beforethe above reactions take place:

−PHA + �CH1.5O0.5*�acetyl-CoA*� (A)+ �CH5�3O1�3*�propionyl-CoA*� = 0

Since the ratio between the amounts of acetyl-CoA andpropionyl-CoA formed by the hydrolysis process has to beequal to the acetyl to propionyl ratio in the PHA polymer,the values for � and � can be found from the known an-aerobic stoichiometry (see Appendix A). For pH � 7, asused in this study, the values for � and � are: � � 0.832, �� 0.168 (see Appendix A).

For the subsequent reactions, it will be assumed that theratios of acetyl-CoA and propionyl-CoA used for catabo-lism, glycogen production, and biomass growth are identi-cal and all equal to �/�. The validity of this assumption willbe discussed below. The reactions involved in the aerobicGAO metabolism are first given in terms of CH1.5O0.5*(activated acetyl-CoA or acetyl-CoA*) and CH5/3O1/3*(activated propionyl-CoA or propionyl-CoA*). The reactions

Figure 1. Schematic description of the anaerobic metabolic model for GAO with modified stoichiometry (adapted from Filipe et al., 2001a).

ZENG ET AL.: METABOLIC MODEL FOR GAO 93

are then integrated with Eq. [A] to obtain the overall PHAcatabolism, glycogen formation, and biomass growth. Detailsfor each individual reaction are given in Appendix B.

r1: PHA Catabolism

Acetyl-CoA* Catabolism

Acetyl-CoA* is converted to CO2 via the tricarboxylic acidcycle (TCA) (Gottschalk, 1986):

−CH1.5O0.5*�acetyl-CoA*� − 1.5 H2O + CO2 + 0.5 ATP+ 2.25 NADH2 = 0 (B)

Propionyl-CoA* Catabolism

Propionyl-CoA* is first converted to pyruvate, which sub-sequently is decarboxylated to acetyl-CoA. Reaction [B]then follows (Gottschalk, 1986). The overall reaction is de-scribed as:

−CH5�3O1�3*�propionyl-CoA*� − 5�3 H2O + CO2

+ 2�3 ATP + 2.5 NADH2 = 0 (C)

(A)+�*(B)+�*(C) gives the overall PHA catabolism:

−PHA − �1.5� + 5��3� H2O + �� + �� CO2

+ �0.5� + 2��3� ATP+ �2.25� + 2.5�� NADH2 = 0 (1)

r2: Glycogen Production from PHA

Glycogen Production from Acetyl-CoA*

Two acetyl-CoA* are condensed to produce one oxalo-acetate through the glyoxylate cycle. Oxaloacetate, which isalso the starting material for biosynthesis, forms glycogen(CH10/6O5/6) via gluconeogenesis (Gottschalk, 1986)

−4/3CH1.5O0.5*(acetyl-CoA*� − 4�6ATP − 5�6H2O+ CH10�6O5�6 + 1�3CO2 + NADH2 = 0 (D)

Glycogen Production from Propionyl-CoA*

Propionyl-CoA forms phosphoenolpyruvate (PEP) eithervia pyruvate or oxaloacetate, which then forms glycogen viagluconeogenesis (Gottschalk, 1986). These two pathwaysend up with the same results (details are given in Appendix B):

−CH5�3O1�3*�propionyl-CoA*� − 0.5H2O − 1�3ATP+ CH10�6O5�6 + 0.5NADH2 = 0 (E)

(A)+3�/4*(D)+�*(E) gives the overall reaction for gly-cogen formation from PHA:

−PHA − ��2 + ��3� ATP − �5��8 + 0.5�� H2O+ �3��4 + �� CH10�6O5�6 + ��4 CO2

+ �3��4 + 0.5�� NADH2 = 0 (2)

r3: GAO Biomass Synthesis from PHA

Biomass Synthesis from Acetyl-CoA*

When acetate (or acetyl-CoA) is used as substrate for bio-mass growth, the synthesis of 1 C-mol biomass produces0.27 mol CO2 and requires 1.7 mol ATP (denoted as K1

hereafter) (Gommers et al., 1988) . Elemental analysis of theexperimentally generated GAO sludge in this study showsthat the biomass composition excluding the intracellularPHA and glycogen can be expressed as CH1.84O0.5N0.19.The biomass synthesis from acetyl-CoA* is thus repre-sented as:

−1.27 CH1.5O0.5*�acetyl-CoA*� − 0.19 NH3 − K1 ATP− 0.405 H2O + CH1.84O0.5N0.19 + 0.723 NADH2

+ 0.27 CO2 = 0 (F)

where the stoichiometry for reducing power (NADH2) wascalculated from a redox balance.

Biomass Synthesis from Propionyl-CoA*

There are no reports in literature that identify the amount ofdecarboxylation for biomass synthesis from propionate.However, in the biosynthesis propionyl-CoA is first con-verted to succinate, which is subsequently used for biomasssynthesis. It is known that 0.409 mol CO2 is produced perC-mol biomass synthesised from succinate (Gommers et al.,1988). Succinate is formed through the reverse succinate-propionate pathway. Therefore, the following reactions areused to determine the CO2 production during biomassgrowth on propionate

1.409 succinate �C-mol� → Biomass �C-mol� + 0.409CO2 (a)

0.75 propionate �C-mol� + 0.25 CO2 → succinate �C-mol� (b)

(a) + 1.409*(b) gives:

1.06 Propionate �C-mol� → Biomass �C-mol� + 0.06 CO2 (c)

Biomass production from propionyl-CoA* can be ex-pressed as:

−1.06 CH5�3O1�3* �propionyl-CoA*� − 0.19 NH3

− K2 ATP − 0.267 H2O + CH1.84O0.5N0.19

+ 0.515 NADH2 + 0.06 CO2 = 0 (G)

The value for K2(the amount of ATP required for bio-mass production from propionate) could not be found in theliterature either, but may be calculated as (C-mol based):

1.27 Acetyl-CoA �C-mol� + 1.7 ATP → 1C-mol biomass (d)

Acetyl-CoA → succinate �the first step of acetyl-CoAfor biosynthesis� (e)

3�4 propionyl-CoA→ succinate + 1�4 ATP (f)

(d) + [(f) - (e)]*1.27 gives:

1.27*3�4 Propionyl-CoA + 1.38 ATP → 1 C−mol biomass (g)

94 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 81, NO. 1, JANUARY 5, 2003

Therefore, K2 is expected to be 1.38 mol ATP per C-molbiomass. The reliability of K2 will be discussed below.

(A)+�/1.27*(F)+�/1.06*(G) yields:

−PHA − �0.19��1.27 + 0.19��1.06� NH3

− �K1��1.27 + K2��1.06� ATP− (0.405��1.27 + 0.267��1.06) H2O+ ��1.27 + ��1.06� CH1.84O0.5N0.19

+ �0.723��1.27 + 0.515��1.06� NADH2

+ �0.27��1.27 + 0.06��1.06� CO2 = 0 (3)

r4: Oxidative Phosphorylation

In the oxidative phosphorylation, ATP is produced fromNADH2. The efficiency of this process, which is defined asmoles of ATP produced per mole of NADH2 oxidised, isexpressed by the P/O ratio or �.

−NADH2 − 0.5 O2 + H2O + �ATP = 0 (4)

r5: Maintenance

−ATP = 0 (5)

The rate of this reaction (moATP) is the specific ATP con-

sumption rate due to maintenance.

Determination of Overall Stoichiometry of PHAConversion Rate

rPHA = −r1 − r2 − r3

rGly = �3��4 + �� r2

rNADH2 = �2.25� + 2.5�� r1 + �3��4 + 0.5�� r2

+ �0.723��1.27 + 0.515��1.06� r3 − r4

rATP = �0.5� + 2 ��3� r1 − ��2 + ��3� r2

− �K1��1.27 + K2��1.06� r3 + � r4 − r5

r5 = moATP

rCO2 = �� + �� r1 + ��4 r2 + �0.27��1.27 + 0.06��1.06� r3

rO2 = −0.5 r4

rX = ��1.27 + ��1.06� r3

rH2O = − �1.5� + 5��3� r1 − �5��8 + 0.5�� r2

− �0.405��1.27 + 0.267��1.06� r3 + r4

rNH3 = −�0.19��1.27 + 0.19��1.06� r3 (6)

The model consists of 14 unknowns (r1…r5; rPHA…rNH3)and 10 linear relations as presented in Eq. [6]. Therefore,there are 4 degrees of freedom. For most situations the cellsare in quasi-steady-state and it can be assumed that:

rATP = rNADH2= 0

These restrictions reduce the degrees of freedom to 2.Therefore, the overall equation for the conversion of PHA inthe aerobic stage can be expressed as:

−rPHA =1

Ysglymax rgly +

1

Ysxmax rx + msCx (7)

where Ysglymax and Ysx

max are the maximum yields of glycogenand biomass production on PHA respectively, ms is thespecific PHA demand for maintenance.

Solving the above equations using Maple (WaterlooCanada), the yields are found as:

1

Ysglymax =

24�3�� + 2� + 2� + 4��

�3� + 4��6� + 27�� + 8� + 30��(8)

1

Ysxmax =

201930� + 318000K1� + 678771��+ 813435�� + 269240� + 381000K2

250�106� + 127��6� + 27�� + 8� + 30��(9)

ms =12mo

ATP

6� + 27�� + 8� + 30��(10)

Substituting � � 0.832, � � 0.168 (values for pH � 7) andK1 � 1.7, K2 � 1.38 into the above equations, the yieldsare simplified as:

1

Ysglymax =

2� + 1.26

2.29� + 0.53(11)

1

Ysxmax =

2.13� + 2.29

2.29� + 0.53(12)

ms =mo

ATP

2.29� + 0.53(13)

With a similar approach to that used for PHA conversion,the oxygen consumption rate can be determined as a func-tion of rgly and rx:

−rO2=

1

Yoglymax rgly +

1

Yoxmax rx + mosCx (14)

where Yoglymax and Yox

max are the yields of glycogen and bio-mass production on oxygen, respectively, mos is the specificoxygen demand for maintenance. Their values for pH � 7are obtained as:

1

Yoglymax =

0.92

2.29� + 0.53(15)

1

Yoxmax =

2.057

2.29� + 0.53(16)

mos =1.146mo

ATP

2.29� + 0.53(17)

All the yields are a function of the ATP/NADH2 ratio (�).In PAO systems it was possible to determine this parameterdirectly from experiments (Smolders et al., 1994b). How-ever, in GAO systems it is not possible to determine �independently unless no biomass growth but only glycogenformation could be achieved in the aerobic phase. Addition-ally, � has a strong cross-correlation with the maintenance


coefficient mos and the K values (ATP needed for biosyn-thesis) (van der Heijden et al., 1994). From the literature,the estimated values for � vary from 1.6–1.85 (Smolders etal., 1994b; Beun et al., 2000; Dircks et al., 2001a). Here,lacking any direct means to determine �, an average (1.73)of these literature values is chosen for �. The thus calculatedYsx

max, Ysglymax, Yox

max, and Yoglymas are shown in Table I. The

maintenance coefficient (moATP) will be determined experi-

mentally later by measuring the oxygen consumption rateduring the maintenance stage.

MATERIALS AND METHODS

Reactor Setup and Operation

Biomass was enriched in a lab-scale anaerobic-aerobic se-quencing batch reactor (SBR). The SBR had a workingvolume of 4.2 L. Feed, decant, and sampling ports werepositioned at different levels on the reactor. Seed sludgecame from Caboolture Sewage Treatment Plant, Queen-sland, Australia. The SBR was operated with six 4-h cyclesper day in a temperature-controlled room at 18–22°C. Eachcycle consisted of 1.5 h anaerobic, 2 h aerobic, 25 minsettling, and 5 min decant periods. In the first 10 min of theanaerobic stage 2.1 L synthetic wastewater (compositiongiven below) was pumped into the reactor. Nitrogen gas wasbubbled through the reactor during the whole anaerobicstage at a gas flow rate of 0.5 L/min to strip any oxygen thatmay be present in the liquid phase; 0.5 L/min air was pro-vided during the aerobic stage. After the settling period, 2.1L supernatant was removed, resulting in a hydraulic reten-tion time (HRT) of 8 h; 100 mL mixed liquor was wasted atthe end of the aerobic phase in each cycle. This, togetherwith the known suspended solids in the effluent (∼10 mg/L),allowed the calculation of the solid retention time (SRT),which is approximately 6.6 days. The reactor was constantlymixed with a magnetic stirrer (250 rpm) except during set-tling and decant phases.

The pH in the system was maintained at 6.85–7.05through the use of a one-way pH controller. The controllerstarted to dose 0.5 M HCl when pH exceeded the set-pointof 7. The addition of acid proved sufficient to control thepH, as it tended to increase.

Synthetic Feed

A 2.1 L synthetic wastewater feed included 50 mL solutionA and 2.05 L solution B. Solution A contained per litre(adapted from Smolders et al., 1994a): 35.05 g NaAc � 3H2O,

2 g MgSO4 � 7H2O, 0.9 g CaCl2 � H2O, 3 g NH4Cl, 1 gpeptone, 12 mL nutrient solution, and 0.025 g allyl-N thio-urea (ATU) to inhibit nitrification. It was adjusted to pH 5.5with 2M HCl and autoclaved. Solution B contained per litre:3.8 mg KH2PO4 and 4.3 mg K2HPO4, which was adjustedto pH 10 with 2M NaOH. One-litre nutrient solution con-tained (based on Smolders et al., 1994a): 1.5 gFeCl3 � 6H2O; 0.15 g H3BO3; 0.03 g CuSO4 � 5H2O; 0.18 gKI; 0.12 g MnCl2 � 4H2O; 0.06 g Na2MoO4 � 2H2O; 0.12 gZnSO4 � 7H2O; 0.15 g CoCl2 � 6H2O; 10 g ethylene-diamine tetra-acetic acid (EDTA). The mixed influent (fromsolutions A and B) therefore contained 400 mg COD/L and2 mg PO4-P/L.

Batch Tests on TOGA Sensor

The titration and off-gas analysis (TOGA) sensor, schemati-cally shown in Figure 2, is a powerful tool to study thestoichiometry of biological processes via the integration ofthe titrimetric and off-gas analysis (Pratt et al., submitted).It consists of a bioreactor, a pH control system, and anoff-gas measurement arrangement. The bioreactor had aworking volume of 1.3 L. Liquid temperature and dissolvedoxygen are both measured using a dissolved oxygen elec-trode (YSI model 5739; Yellow Springs, Youngstown, OH,USA). The pH system consists of a pH electrode (IonodeIJ44; TPS, Brisbane, Australia) and high accuracy dosingpumps (Prominent Beta4, Heidelberg, Germany). Thepumps are used to maintain system pH at 7 ± 0.01 byaddition of NaOH or HCl (0.03 M) (Pratt et al., submitted).The rate of base/acid dosage is thus equivalent to the hy-drogen ion/hydroxide production rate (HPR) due to the bio-logical as well as physical and chemical processes occurringin the reactor. The off-gas measurement technique is basedon a quadrupole mass spectrometer (OmniStar, Balzers AG,Liechtenstein) in conjunction with a number of in-line massflow controllers (Bronkhorst Hi-tech, El-Flow, Netherlands)to measure the oxygen and carbon dioxide concentration inboth inlet and outlet gases, allowing the calculation of oxy-gen uptake rate (OUR) and CO2 transfer rate (CTR) (Gapesand Keller, 2001). The reactor is completely sealed duringmeasurement.

For the cyclic study in TOGA, 600 mL mixed liquor wasremoved from the parent SBR at the end of aerobic stage tothe 1.3 L TOGA bioreactor. This sludge was mixed with690 mL standard growth media. The mixed liquor was thenbubbled with nitrogen gas. When the experiment started, 10mL concentrated acetate was added into the reactor toachieve 200 mg/L COD in the reactor at the start of the

Table I. Maximum yields for PHA and oxygen conversions (pH � 7, � � 1.73).

PHA conversion Oxygen conversion

Growth Ysxmax 0.75 (C-mol X/C-mol PHA) Yox

max 2.18 (C-mol X/mol O2)Glycogen Ysgly

max 0.95 (C-mol Gly/C-mol PHA) Yoglymax 4.89 (C-mol Gly/mol O2)


experiment; 1.5 hours later, oxygen replaced nitrogen bub-bling to start the aerobic stage for 2 h.

Analyses

Ammonia nitrogen (NH4-N), nitrate nitrogen (NO3-N), ni-trite nitrogen (NO2-N), and orthophosphate (PO4-P) wereanalysed using Lachat QuikChem8000 Flow InjectionAnalyser. MLSS and MLVSS were analysed according toAPHA Standard Methods (1992). Acetate was measured bygas chromatography with DB-FFAP column at 140°C andFID detector at 250°C.

Glycogen was determined by the modified method ofBond et al. (1995). Five mL of 0.6 M HCl was added toweighed freeze-dried biomass in screw-topped glass tubes,then heated at 100°C for 5 h. After cooling and centrifuga-tion, 1 mL of the supernatant was transferred to HPLC vialfor glucose analysis. Fifty �L was injected into Hewlett-Packard X-87H 300 × 7.8 mm, BioRad Aminex ion exclu-sion HPLC column at 70°C.

For PHB/PHV determination, weighed freeze-dried bio-mass was put into screw topped glass tubes. The biomasswas suspended in 2 mL acidic methanol solution (3%H2SO4) and 2 mL chloroform. The tube was screwed tightlyand heated to 100°C for 6 h. Within 1 h, the glass tubeneeded to be retightened to avoid any leakage. After cool-ing, 1 mL of Milli-Q water was added and shaken vigor-ously for 10 min. When the phases were separated, around1 mL of the bottom organic layer was removed to the GCvials. Three �L of organic phase was injected on GC withcolumn DB-5 (30 m × 0.25 mm × 0.25 �m) at 100°C withan FID detector at 240°C.

For elemental composition analysis, around 0.5 mgfreeze-dried biomass was used to determine the carbon, hy-drogen, oxygen, and nitrogen contents using a Perkin-Elmer(Norwalk, CT) 240 Elemental Analyser. The elemental

composition of active biomass was determined by subtrac-tion of PHA and glycogen and correction of the composition.

RESULTS

General Results of SBR Experiments

The SBR reached a quasi-steady-state within 100 days afterits conversion from a PAO to a GAO reactor. This wasachieved through reducing the influent P gradually, suchthat the culture had just sufficient phosphate for cell growth.MLSS and MLVSS were about 3.6 g/L and 3.49 g/L, re-spectively; the P-content was 2.2%, indicating that, indeed,P was only available for cell synthesis and not for poly-phosphate accumulation. Cyclic studies were carried out onthe SBR, during which both solid and liquid phases weremeasured. Figure 3 shows the results of a typical cyclicstudy. During the anaerobic stage, acetate was taken upwithin 35 min, which was accompanied by the consumptionof glycogen and production of PHA. No phosphorus release

Figure 2. Schematic of the TOGA sensor (Pratt et al., submitted).

Figure 3. A cyclic study on the parent SBR, showing the phenotype ofGAO. The conditions were changed at 1.5 h from anaerobic to aerobic.


was observed. In the subsequent aerobic stage, PHA wasoxidised and glycogen replenished.

The ammonia profile in the aerobic phase, in combinationwith the experimentally determined biomass composition(CH1.84O0.5N0.19), can be used to calculate the biomassgrowth rate as nitrification was inhibited by ATU. Indeed,no NOx-N was detected in the reactor at any time (data notshown). Figure 3 shows 0.485 mmol/L NH4

+-N was con-sumed in the cycle, which, based on the chemical formula ofactive biomass, implied that 62.6 mg/L active biomasswould have been produced per cycle. The effective SRTwas 6.6 days, which means that 374 mg VSS was wastedper cycle. Glycogen and PHA stored in the biomass con-tributed 29% w/w of VSS (from direct analysis). Therefore,the active biomass that was wasted per cycle was about 262mg or equivalent to 62.4 mg/L reactor volume, whichmatches very well with the estimation based on the ammo-nia uptake.

Batch Tests

Batch tests with the TOGA sensor were conducted to:

● determine a number of stoichiometric and kinetic param-eters. These included the anaerobic maintenance coeffi-cient (mAn

ATP), the maximum anaerobic acetate uptakerate (qGAO

max ), and the aerobic maintenance coefficient(mo

ATP);● validate the model using both on-line and off-line data.

Only wasted sludge was used to perform the batch tests inorder to avoid disturbing the normal operation of the parentSBR.

Determination of Anaerobic MaintenanceCoefficient (mAn

ATP)

For PAO, it is believed that polyP hydrolysis provides theenergy for maintenance under anaerobic conditions. ForGAO, Filipe et al. (2001a) proposed that glycogen would beused to provide energy for maintenance. In a batch test, 600mL mixed liquor was removed at the end of the aerobicphase from the parent SBR and transferred to the TOGAbioreactor. This sludge was mixed with the standard growthmedia without, however, acetate. The mixed liquor was thenbubbled with nitrogen gas for 10 h to maintain anaerobicconditions. Figure 4 shows the measured glycogen fraction(glycogen concentration divided by biomass concentration)during the experiment. The glycogen hydrolysis rate is thusdetermined as 4.7 × 10−3 C-mol glycogen/C-mol biomass/h(the slope of the regression line in Fig. 4), which is similarto the value (4.13 × 10−3) determined by Filipe et al.(2001a). According to Zeng et al. (2002), 0.5 mol ATPshould be produced per C-mol of glycogen hydrolysed.Therefore, the anaerobic maintenance coefficient(mAn

ATP)is determined as 2.35 × 10−3 mol ATP/C-mol bio-mass/h. This value is in the range of those reported for PAO(1.47 to 2.5 × 10−3) (Smolders et al., 1995; Brdjanovic et al.,

1997). Analysis of the PHA formed during this test indi-cated that glycogen was indeed directly converted to PHAwithout uptake of external carbon substrates (results notshown).

Determination of the Aerobic MaintenanceCoefficient (mo

ATP)

The aerobic maintenance coefficient was determined fromthe oxygen uptake rate (OUR) measured during an extendedaerobic experiment in the TOGA system. The maintenancecondition was achieved by continuing aeration for 8 h aftera normal aerobic batch test. The measured OUR and CTRare shown in Figure 5. The OUR reached a maximum within30 min after the start of aeration and then started to dropwithout reaching a constant rate. This behaviour seems to beconsistent with the literature since PHA oxidation has beenfound to be a multiple order reaction instead of a zero-orderone (Dircks et al., 2001). After 6 h aeration, the OUR be-came quite constant at 0.21 mmol O2/h and overlappedperfectly with the CTR, which is expected under mainte-nance conditions where all oxygen consumed is converted

Figure 4. Experiment to determine the anaerobic ATP maintenancebased on the glycogen degradation in an anaerobic batch test withoutacetate addition.

Figure 5. Oxygen uptake rate (OUR) and CO2 transfer rate (CTR) pro-files in an extended aerobic batch test.


to CO2. From this, the aerobic oxygen demand for mainte-nance could be determined as mos � 3.51 × 10−3 mol O2/C-mol biomass/h. From Eq. [17] mo

ATP can be calculated as0.014 mol ATP/C-mol biomass/h, which is somewhat lowerthan the value reported for PAO (0.019 mol ATP/C-molbiomass/h) (Smolders et al., 1994b).

Maximum Acetate Uptake Rate (qGAOmax )

During normal operation of the parent SBR, acetate wascompletely taken up within 35 min, which did not allowsufficient time for an accurate determination of the maxi-mum acetate uptake rate (qGAO

max ). This rate was thereforedetermined using separate batch tests. In Figure 6, the ac-etate profiles during three batch tests are shown. Three dif-ferent amounts of acetate were added to the TOGA reactor,resulting in initial acetate concentrations of 100, 200, 400mg/L COD, respectively. A consistent qGAO

max was obtainedfrom these profiles, which ranges between 0.16–0.18 C-mol/C-mol biomass/h.

CO2/HAc in Anaerobic Stage

CO2 was produced under both anaerobic and aerobic con-ditions. In the TOGA sensor, the CO2 transfer rate (CTR),not CO2 production rate (CPR), was actually measured on-line due to the high solubility of CO2. In order to determineCPR, the amount of dissolved CO2 needs to be determined.When CO2 dissolves, it immediately equilibrates with bi-carbonate, which affects pH (Eq. [18]). Meanwhile, the ac-etate uptake would also affect the pH during the anaerobicperiod because 1 mol acetate uptake consumes 1 mol hy-drogen ion (H+). The combined pH effect of the two pro-cesses was measured by TOGA as HPR. The relation be-tween HPR, CTR, and CPR can be described as in Eq. [19](Pratt et al., submitted):

CO2�g�

↕KLaCO2

CO2�d�↔H2CO3↔1

H+ + HCO3−↔

2H+ + CO3

2−

pKa(cl� 6.35; pKa�c2� 10.35 (18)

HPR =−rAcetate

2+� CPR − CTR

1 + 10pKa(c1)−pHop� (C-mol based� (19)

where rAcetate is the acetate uptake rate, pHop is the oper-ating pH, which is 7 in this study. CPR is solved as:

CPR = �1 + 10pKa�c1�−pHop��HPR +rAcetate

2 � + CTR (20)

The total amount of CO2 produced can be calculated byintegrating Eq. [20] on both sides.

A typical batch experiment during the anaerobic stage ispresented in Figure 7. The conversions are further sum-marised in Table II. The CO2 production in the anaerobicstage is calculated as 2.35 mmol by integrating Eq. [20],which results in a ratio of CO2 produced to HAc consumedof 0.285. This ratio fits the proposed stoichiometry (0.27) inEq. [I] well.

Furthermore, the maximum CPR is calculated as 2.73C-mmol/h using Eq. [20], resulting in a ratio of maximumCPR to qGAO

max (10.17 C mmol/h) of 0.276, which also fits theproposed stoichiometry (0.27) in Eq. [I] very well.

Figure 6. Acetate uptake in three batch tests with different COD loading.Figure 7. Carbon flow (a), CO2 transfer rate and hydrogen ion produc-tion rate (b) during anaerobic phase in a batch experiment.


As shown in Table II, the carbon balance closes to 99.8%.It can be calculated that the redox balance closes to 99.4%.Both indicate that the measurements are very accurate.

It proved necessary to consider the contribution of an-aerobic maintenance to the overall anaerobic stoichiometry.The glycogen consumption due to this process representsabout 5% of the total glycogen consumption (Fig. 4). Sincethe internal redox balance has to be closed the production ofPHB, PHV, and PH2MV from the maintenance process canbe described as follows (Filipe et al., 2001a):

1C mol Glycogen → 1�6 PHB + 5�12 PHV (21)+ 1�4 PH2MV + 1�6 CO2

The comparison between the measured and predictedchanges of various compounds in the anaerobic phase is

also given in Table II. Clearly, the proposed metabolismpredicts very well the anaerobic conversions of GAOs.

Aerobic Batch Experiment

A typical aerobic experiment is shown in Figure 8. Theconversions of various compounds are summarised in TableIII. The hydrogen ion production is due to the consumptionof ammonia for bacteria growth and CO2 production andtransfer. The relation between HPR and CPR can be ex-pressed as:

HPR = rNH4+ � CPR − CTR

1 + 10pKa�c1�−pHop� (22)

where rNH4 is the ammonium consumption rate.CPR is calculated as:

CPR = �1 + 10pKa�c1�−pHop](HPR − rNH4� + CTR (23)

Integrating Eq. [23] on both sides allows the calculation ofthe amount of CO2 produced. The thus calculated CO2 pro-duction during the whole aerobic period is 1.30 mmol.

Based on the data in Table III, the carbon balance iscalculated to close to 98% while the redox balance closes to104%. Again, both indicate that the measurements have ahigh accuracy.

Evaluation of the Yields in the Aerobic Model

According to the metabolic model developed in this study,the conversion rates of PHA and oxygen depend on theglycogen formation and biomass growth rates in the follow-ing way (pH � 7):

−rPHA = 1.05 rgly + 1.33 rx + 3.06 × 10−3 Cx (24)

−rO2 = 0.20 rgly + 0.46 rx + 3.51 × 10−3 Cx (25)

where the last terms of two equations represent the PHA andoxygen consumption rates due to the maintenance process.rx can be calculated from the ammonium consumption rate(rx � rNH4/0.19).

Theoretically, the above two equations could be directlyused to evaluate the yield coefficients. However, the dis-crete PHA, glycogen, and ammonia nitrogen data did not

Table III. Measured compounds during aerobic stage in a typical batchexperiment.

Measured compound Measured change Unit

Glycogen production 10.97 C-mmolPHB consumption −10.54 C-mmolPHV consumption −4.12 C-mmolAmmonium consumption* −0.41 N-mmolBiomass production 2.13 C-mmolO2 consumption −3.75 mmolCO2 production 1.30 mmol

*Includes 0.06 mmol taken up during the anaerobic stage.Figure 8. Carbon flow (a), Oxygen uptake, CO2 transfer and hydrogenion production rates (b) during the aerobic phase in a batch experiment.

Table II. Measured compounds during the anaerobic phase in a typicalbatch experiment.

Measured compoundMeasured

changeModel predict

(incl. maintenance) Unit

HAc consumed −8.24 −8.24(base) C-mmolPHB formation 11.47 11.27 C-mmolPHV formation 4.22 3.96 C-mmolGlycogen hydrolysis −9.83 −9.72 C-mmolCO2 production 2.35 2.30 mmol


allow an accurate determination of the time varying rates ofrPHA, rgly, and rx. To make the evaluation possible, bothsides of Eqs. [24] and [25] were integrated with respect totime from 0 (beginning of the aerobic phase) to a varyingupper limit t (up to the end of the aerobic phase):

�0

trPHA��d� = 1.05 �

0

trgly��d�

+ 1.33 �0

trx��d� + 3.06 × 10−3 CXt (26)

�0

trO2��d� = 0.20 �

0

trgly��d� + 0.46 �

0

trx��d�

+ 3.51 × 10−3 CXt (27)

The left-hand sides of the above two equations are the mea-sured accumulated PHA and O2 consumption to time t,respectively, while the right-hand sides are the same accu-mulated consumption calculated based on the measured gly-cogen and nitrogen data. In Figure 9, the calculated PHAand oxygen consumptions using the right-hand side of theformulas for different times t are plotted against those ob-tained from the left-hand sides for the same times. In thecalculations, the PHA, glycogen, and nitrogen data mea-sured during both cyclic studies on the parent SBR andbatch tests were used, while the oxygen uptake data wereonly available during batch tests. Figure 9 shows that themeasured PHA (Fig. 9a) and oxygen consumption (Fig. 9b)match the calculated PHA and oxygen consumption verywell over a relatively wide range, indicating that the yieldsderived from metabolic pathways are reasonable.

DISCUSSION

Model Evaluation

Both the anaerobic and aerobic metabolic models have beendemonstrated to reproduce the stoichiometric relationshipsmeasured during cyclic studies in the SBR and in batchexperiments. The anaerobic model is further compared to

literature data in Table IV. The model-predicted glycogen toacetate ratio compares well with the values reported in Liuet al. (1994). However, it fails to predict the values reportedby Filipe et al. (2001a) for the same ratio. It has to be noted,however, that this reported ratio is considerably lower than1, which is difficult to understand from a stoichiometricpoint of view. A glycogen hydrolysis ratio of less than oneC-mol per C-mol acetate uptake is inconsistent with themetabolic pathway for anaerobic processes proposed by Fil-ipe et al. (2001a) and also used in this model. In particular,it violates the energy balance whereby it is assumed thatadditional energy (�� ATP) is required for acetate uptake,which would have to be negative in order to get a glycogenutilisation to acetate uptake ratio of less than 1. The PHA toacetate ratios reported in both Filipe et al. (2001a) and Liuet al. (1994) match reasonably well with the model predic-tion. However, there is a relatively large deviation of themodel predicted PHV: PHB ratio (0.34) from that reportedin Liu et al. (1994) (0.58), which cannot be explained on thebasis of the data provided.

The maximal yields of PAO and GAO are compared inTable V. This shows that during the aerobic stage, bothmaximum yields of biomass and glycogen are higher forGAO than for PAO. This is caused by the fact that propio-nyl-CoA, which is more reduced (i.e., more energetic) com-pared to acetyl-CoA, is involved in the metabolism of GAO.Furthermore, as also shown in Table V, the net biomassgrowth per HAc consumed is about 9% higher for GAOthan for PAO. This is consistent with the fact that GAOmaintain fewer metabolic processes than PAO during theaerobic period. GAO only need to convert PHA to glycogenapart from growth, while PAO have to also take up PO4 andsynthesise poly-P, requiring additional energy consumption.However, it is worthwhile mentioning that the net biomassgrowth yield for GAO is 17% lower when compared to thatof ordinary heterotrophs, which grow directly on acetateaerobically (reported as 0.45 C-mol biomass/C-mol HAc byvan Aalast-van Leeuwen et al., 1997).

Figure 9. The calculated PHA (a) and oxygen (b) consumptions from glycogen and ammonium data match well with the measured consumptions. Thesolid lines represent ideal matches


Kinetic Comparison

The maximum specific acetate uptake rate for GAO (qGAOmax )

was found to be 0.16–0.18 C-mol/C-mol biomass/h in thisstudy. This value is about 29% lower than the value (0.24C-mol/C-mol biomass/h) observed by Filipe et al. (2001b).They reported that, in their enriched PAO and GAO sys-tems, qPAO

max (0.19 C-mol/C-mol biomass/h) was smaller thanqGAO

max at pH 7, and therefore concluded that GAO were ableto compete effectively with PAO during anaerobic substrateuptake. On the other hand, as shown in Table V, the netgrowth yield of GAO is 21% higher than that of PAO. Thesedifferences in substrate uptake rate and biomass yield wouldmean that PAO have a competitive disadvantage on bothmeasures and should be out-competed by GAO easily.Therefore, GAO would likely dominate in such mixed sys-tems, which is the not case in reality. However, it has to benoted that the values for PAO reported in the literature(Table VI) vary widely and the two values measured forGAO so far are within the range of these PAO values.Further investigations into these significant differences be-tween the various experimental studies are required sincethis is likely an important factor in the PAO/GAO compe-tition.

Impact of Propionyl-CoA Fraction on the Yields

An assumption made in the derivation of the aerobic stoi-chiometry is that acetyl-CoA and propionyl-CoA gothrough the catabolic and anabolic processes independently.This assumption has yet to be verified experimentally.

However, it can be demonstrated that the assumption shouldnot result in significant errors in the yield coefficients. Forthe two extreme cases, where PHA comprises either PHB orPHV only, the calculated yield coefficients are shown inTable VII. The yields for both glycogen and biomass differby only about 10%, despite the fact that the amount ofpropionyl-CoA used in the two cases are 0 and 60%, re-spectively. Based on the anaerobic stoichiometry and sup-ported by the experimental results, the content of propionyl-CoA in PHA is typically only 10–20% (depending on pH)when acetate is used as the substrate. Therefore, the aboveassumption should not introduce significant errors in theyield coefficients. Indeed, the yield coefficients obtained inthis study fall well within the extreme values.

Impact of K2 on the Biomass Growth Yield

A theoretical value of 1.38 was obtained for parameter K2,the amount of ATP required for biomass growth on propio-nyl-CoA. Although this value is yet to be confirmed experi-mentally, sensitivity analysis suggests that this value hasonly a marginal impact on the overall stoichiometry.

According to the stoichiometry developed, the biomassyield (Ysx

max) is the only parameter that is affected by K2:

1

Ysxmax =

2.13� + 2.02 + 0.195K2

2.29� + 0.53(28)

The dependency of Ysxmax on K2 for K2 values ranging from

0.5–1.7 is shown in Figure 10. As propionyl-CoA is morereduced than acetyl-CoA, it can be reasonably assumed thatthe K2 value would be no larger than that of K1 (1.7). Ifplotting K2 deviation vs. Ysx

max deviation based on K2 �1.38 (Fig. 10), the slope of the line is about 0.025, whichmeans that the change in Ysx

max for the entire range of K2

values shown is well less than 3%. Therefore, the choice ofthis theoretical value would be adequate for most situations.

Overall Conversions During Anaerobic andAerobic Phases

The overall stoichiometry of anaerobic and aerobic GAOprocesses with SRT � 7 days and pH � 7.0 is shown inFigure 11. With 1 C-mol HAc added anaerobically, 1.12C-mol glycogen is hydrolysed and 1.85 C-mol PHA is pro-duced, accompanied by 0.27 mol CO2 release. In the aerobic

Table V. Comparison of maximum growth yields of PAO and GAO.

PAOSmolders et al.

(1994b)GAO

This study Unit

� 1.85 1.73 mol ATP/mol OYsx

max 0.74 0.75 C-mol/C-molYsgly

max 0.90 0.95 C-mol/C-molms 4.0 × 10−3 3.06 × 10−3 C-mol/C-mol/hNet biomass* growth

per HAc added 0.34 0.37 C-mol/C-mol

*Biomass excluding polyphosphate and carbon reserves.

Table VI. Comparison of the maximum acetate uptake rate of GAO andPAO at T � 20°C.

Reference qHAcmax Unit

PAO Smolders et al. (1995) 0.43 C-mol/C-mol/hMurnleitner et al. (1997) 0.30 C-mol/C-mol/hBrdjanovic et al. (1997) 0.17 C-mol/C-mol/hFilipe et al. (2001b) 0.19 C-mol/C-mol/h

GAO Filipe et al. (2001a) 0.24 C-mol/C-mol/hThis study 0.17 C-mol/C-mol/h

Table IV. Comparison of anaerobic stoichiometry with literature.

C-molAnaerobic

modelFilipe’s data

(2001a) This studyLiu’s data

(1994)

pH � 7 7 7 7 Not listedAcetate −1 −1 −1 −1Glycogen −1.12 −0.83 −1.20 −1.11 ∼ −1.25PHA 1.85 1.65 1.91 1.76 ∼ 1.91PHB 1.36 1.26 1.39 1.17PHV 0.46 0.38 0.52 0.68PH2MV 0.037 Undetected Not measured 0.063PHV:PHB 0.34 0.31 0.38 0.58CO2 0.27 Not listed 0.29 Not listed


phase, 1.26, 0.50, and 0.09 C-mol PHA are used for glyco-gen formation, biomass growth, and maintenance, respec-tively. In each cycle, 0.37 C-mol active biomass, 0.01 C-mol PHA, and 0.08 C-mol glycogen are removed as excessorgan matter. The oxygen required for glycogen formation,biomass growth, and maintenance process is 0.25, 0.17, and0.10 mol, respectively. The amount of CO2 produced bythese same processes is 0.06, 0.13, 0.09 mol, respectively.

CONCLUSIONS

This article reports on the complete model for glycogen-accumulating organisms (GAO) based on known metabolicpathways and supported by experimental studies and litera-ture values. The main outcomes from this study are:

● The anaerobic model proposed by Filipe et al. (2001a)and amended by Zeng et al. (2002) predict the experi-mental results very well, including the maintenance con-siderations.

● A model for the aerobic processes is proposed and vali-dated in several ways. This model links in directly withthe anaerobic one to provide a complete description ofthe proposed metabolic behaviour of GAO in an anaero-bic/aerobic reaction cycle.

● The yields derived in the aerobic model match the ex-perimental results well and also are in agreement withmost data from the literature.

● An assumption is made in the aerobic model that acetyl-CoA and propionyl-CoA go through the catabolic andanabolic processes independently. This needs to be ex-

perimentally verified, but theoretical considerations pro-vide good justification for this assumption.

● The energy needed for maintenance under both anaerobicand aerobic conditions for GAO is similar as for PAO.

● The maximal acetate uptake rate for GAO (qGAOmax ) was

found to be lower in this study than most reported valuesfor qPAO

max , which indicates that PAO might take up acetatefaster than GAO in the anaerobic stage.

● The growth yield of GAO is slightly higher than for PAO.The net biomass production per C-mol acetate for GAOis 9% higher than for PAO but 17% lower than for theordinary heterotrophs directly growing on acetate (vanAalast-van Leeuwen et al., 1997).

NOMENCLATURE

Cx biomass concentration, excluding storage products [C-mmol/L]

Ysxmax maximum yield of biomass on PHA [C-mol/C-mol]

Ysglymax maximum yield of glycogen on PHA [C-mol/C-mol]

Yoxmax maximum yield of biomass on oxygen [C-mol/mol O2]

Yoglymas maximum yield of glycogen on oxygen [C-mol/mol O2]

ms maintenance for growth on PHA [C-mol/C-mol/h]mos maintenance for growth on oxygen [mol O2/C-mol/h]mAn

ATP anaerobic maintenance coefficient based on ATP [mol/C-mol]

moATP aerobic maintenance coefficient based on ATP [mol/C-mol]

PHA polyhydroxyalkanoatePHB polyhydroxybutyratePHV polyhydroxyvaleratePH2MV polyhydroxy-2-methylvalerate

Greek Symbols

� ATP required transporting acetate during anaerobic stage[mol ATP/C-mol]

� percent of Acetyl-CoA* in PHA [C-mol/C-mol]� percent of Propionyl-CoA* in PHA [C-mol/C-mol]� P/O ratio [mol ATP/mol O]

REFERENCES

APHA (American Public Health Association). 1992. Standbook methodsfor the examination of water and waste water, 18th ed. Washington,DC: APHA.

Table VII. Impact of propionyl-CoA (C-mol) percentage on Ysxmax Ysgly

max

and ms.

Case study Propionyl-CoA Ysxmax Ysgly

max ms

PHB only 0% 0.729 0.916 3.19 × 10−3

This study 16.8% 0.752 0.952 3.06 × 10−3

PHV only 60% 0.810 1.040 2.95 × 10−3

Figure 10. Impact of variance of K2 on Ysxmax and its relative deviation

Figure 11. Conversions, expressed in mol, during the anaerobic andaerobic phases of GAO system (SRT � 7 days and pH � 7) after additionof 1 C-mol HAc.


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APPENDIX A

P H A f o r m u l a c a n b e e x p r e s s e d a s : P H A �PHBaPHVbPH2MVc (all are based on C-mol)

aPHB � a CH1.5O0.5* (Acetyl-CoA*)

bPHV � 2b/5 CH1.5O0.5* (Acetyl-CoA*) + 3b/5CH5/3O1/3*(Propionyl-CoA*)

cPH2MV � c CH5/3O1/3*(Propionyl-CoA*)

PHA � (a+2b/5) CH1.5O0.5* + (3b/5 + c)/ CH5/3O1/3*

a+2b/5 � �; (3b/5 + c) � �

a =243 + 216� + 48�2

315 + 552� + 240�2 b =67.5 + 300� + 120�2

315 + 552� + 240�2

c =4.5 + 36� + 72�2

315 + 552� + 240�2

� =270 + 336� + 96�2

315 + 552� + 240�2 � =45 + 216� + 144�2

315 + 552� + 240�2

When pH � 7, � � 0.06, therefore:

a � 0.734, b � 0.246, c � 0.02; � � 0.832, � �0.168.

APPENDIX B

Model Development With DetailMetabolic Pathways

Acetyl-CoA* Catabolism

− Acetyl-CoA − 3 H2O + 2CO2 + 4 NADH2 + ATP = 0 (1�)

− CH1.5O0.5*(acetyl-CoA*) + Acetyl-CoA (C-mol)+ 0.25 NADH2 = 0 (2�)

(2�) + (1�)/2

− CH1.5O0.5*(acetyl-CoA*) − 1.5 H2O) + CO2

+ 0.5 ATP + 2.25 NADH2 = 0 (1)

Propionyl-CoA* Catabolism

Propionyl-CoA → Pyruvate + ATP + 2 NADH2 (3�)

Pyruvate → Acetyl-CoA + CO2 + NADH2 (4�)

(3�) + (4�) + (1�)

Propionyl-CoA → 3 CO2 + 2 ATP + 7 NADH2 (5�)


Propionyl-CoA* → Propionyl-CoA + 0.5 NADH2 = 0 (6�)

(5�) + (6�)

Propionyl-CoA* → 3 CO2 + 2 ATP + 7.5 NADH2 (7�)

− CH5�3*(propionyl-CoA*) − 5�3 H2O + CO2 + 2�3 ATP+ 2.5 NADH2 = 0 (2)

Glycogen Production from Acetyl-CoA*

Acetyl-CoA → 0.5 Oxaloacetate + 1.5 NADH2 (8�)

Oxaloacetate + ATP → PEP + CO2 (9�)

2 PEP + 2 ATP + 2 NADH2 → C6H10O5 (Glycogen) (10�)

(8�) + (9�)/2 + (10�)/5

Acetyl-CoA + 1 ATP → 0.25 C6H10O5

+ 0.5 CO2 + NADH2 (11�)

((2�)–(11�)/2)*4/6

−4/3 CH1.5O0.5*(acetyl-CoA*) − 4/6 ATP − 5/6 H2O+ CH10/6O5/6 + 1/3 CO2 + NADH2 � 0 (3)

Glycogen Production from Propionyl-CoA*

� Propionyl-CoA via Pyruvate, then to PEP, finally to Gly-cogen

Propionyl-CoA → Pyruvate + ATP + 2NADH2 (12�)

Pyruvate + ATP → PEP (13�)

(12�) + (13�) + (10�)/2

Propionyl-CoA + 1 ATP → 0.5 C6H10O5 + NADH2 (14�)

� Propionyl-CoA carboxylates to Oxaloacetate, then toPEP, finally to glycogen

Propionyl-CoA + CO2 → Oxaloacetate+ 2 NADH2 + ATP (15�)

(15�) + (19�) + (10�)/2

Propionyl-CoA + 1 ATP → 0.5 C6H10O5

+ NADH2 (14�)

Two pathways result in the same Eq. (14�).

((6�)–(14�))/3

−CH5/3O1/3*(propionyl-CoA*) − 0.5 H2O − 1/3 ATP+ CH10/6O5/6 + 0.5 NADH2 � 0 (4)


metabolic model for glycogen-accumulating organisms in anaerobic/aerobic activated sludge systems

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