A Metabolic Model for Biological

Phosphorus Removal by
Denitrifying Organisms
T. Kuba, E. Murnleitner, M . C. M. van Loosdrecht,* and J. J. Heijnen
Department of Biochemical Engineering, Delft University of Technology,
Julianalaan 67, 2628 BC Delft, The Netherlands

Received December 13, 1995/Accepted April 29, 1996

A metabolic model for biological phosphorus removal
under denitrifying conditions has been established. The
model is based on previous work with aerobic phospho-
rus removal. The form of the kinetic equations used is the
same as for the aerobic model. The main difference is the
value of PJNADH, ratio i n the electron transport phos-
phorylation with nitrate (S,,,). This value was determined
independently from batch tests with an enriched culture
of denitrifying phosphorus-removing bacteria. The mea-
sured S,,, was approximately 1.0 m o l ATP/mol NADH,.
This indicates that the energy production efficiency with
nitrate compared t o oxygen is approximately 40% lower.
These batch tests were also used t o identify a proper set
of kinetic parameters. The obtained model was subse-
quently applied for the simulation of cyclic behavior i n an
anaerobic-anoxic sequencing batch reactor at different
biomass retention times. The simulation results showed
that the metabolic model can be used successfully for the
denitrifying dephosphatation process. The obtained ki-
netic parameters for denitrifying enrichment cultures,
however, deviated from those obtained for the aerobic
formation are derived from the conversion of glycogen
(Arun et al., 1989; Mino et al., 1987; Smolders et al.,
1994a). In the aerobic zone oxygen is utilized as an electron
acceptor for the oxidation of stored PHA, which leads to
growth, phosphate uptake (polyp regeneration), and synthe-
sis of glycogen by the phosphorus-removing bacteria.
In conventional processes, oxygen is utilized as an elec-
tron acceptor for phosphorus uptake. Nitrate is considered to
disturb the phosphorus removal process. However, from a
microbiological point of view there is no restriction against
utilization of nitrate instead of oxygen. Recently denitrify-
ing phosphorus-removing bacteria (DPB) have been found
in activated sludge systems and laboratory cultures (Kerrn-
Jespersen and Henze, 1993; Kuba et al., 1993; Vlekke et al.,
1988; Wanner et al., 1992). In these bacteria nitrate is uti-
lized for the oxidation of stored PHA and is removed as
dinitrogen gas from wastewater. Thus besides phosphorus,

-
enrichment cultures. 0 1996 John Wiley & Sons, Inc.
Key words: phosphorus removal denitrifying dephos-
phatation stoichiometry metabolic model sequenc-
nitrogen, which also induces the eutrophication of surface
waters, is removed simultaneously by DPB. Previous re-
search has shown that DPB have similar potential for phos-
ing batch reactor
phorus removal (Kuba et al., 1993) and a similar biological
metabolism based on intracellular PHA and glycogen (Kuba
INTRODUCTION et al., 1994) as the phosphorus-removing bacteria in the
conventional anaerobic-aerobic ( N O ) processes.
In order to prevent surface waters from eutrophication, bio-
Several mathematical models of the phosphorus removal
logical phosphorus removal has been introduced in waste-
process are proposed, but only few studies have so far been
water treatment plants with activated sludge for several de-
made at modeling or denitrifying dephosphatation. For in-
cades. The biological phosphorus removal is caused by bac-
stance, phosphorus-removing bacteria in the Activated
teria which can accumulate phosphate as polyphosphate
Sludge Model No. 2 proposed by the IAWQ Task Group
(polyp) granules in the cells. To achieve phosphorus re-
does not incorporate denitrifying dephosphatation (Gujer et
moval, activated sludge is circulated through anaerobic and
al., 1995). Moreover, the model does not take the intracel-
aerobic zones. In the anaerobic zone where no electron ac-
lular glycogen metabolism into account.
ceptors (like oxygen and nitrate) are present, the phospho-
A structured metabolic model of the phosphorus removal
rus-removing bacteria take up lower fatty acids such as
process under conventional N O conditions has been pro-
acetic acid and store them as polyhydroxy-alkanoates
posed by Smolders et al. (1995a,b). The model is based on
(PHA; for instance, poly-P-hydroxy-butyrate or -valerate).
three anaerobic and five aerobic metabolic reactions, in-
The energy (ATP) for this process is derived from glycogen
cluding the glycogen metabolism. Smolders et al. showed
conversion and the stored polyp by hydrolysis and excretion
that their model is very well capable of describing the com-
of phosphate (van Groenestijn, 1987, 1989; van Veen et al.,
plex conversions of the biological phosphorus removal pro-
1993). The reduction equivalents (NADH,) for the PHA
cess. Their model, developed for the N O process, can be
modified for the denitrifying dephosphatation process, be-
* To whom all correspondence should be addressed. Telephone: 31-15- cause the anaerobic metabolism is completely identical in
2781618; fax: 3 1-15-2782355; e-mail: marK.vl@stm.tudelft.nl both processes, and the difference between the aerobic and

Biotechnology and Bioengineering, Vol. 52, Pp. 685-695 (1996)

0 1996 John Wiley &Sons, Inc. CCC 0006-3592/96/060685-11
anoxic metabolic reactions is only the electron transport aerobic value of 6 = 1.85 mol ATP/mol NADH, (Smolders
phosphorylation: et al., 1994b).
Aerobic: NADH, + 0.50, + GATP + H,O (1)
Anoxic PHB and Nitrate Conversion Rates
Anoxic: NADH, + 0.4HN0, + 6,ATP + 0.2N,
+ 1.2H,O (2) The linear relation for PHB or nitrate conversion rates as a
Smolders et al. (1994b) determined a value of P/NADH, function of growth, polyp formation, and glycogen synthe-
(P/O) ratio (amounts of produced ATP per oxidized sis is derived similarly as described by Smolders et al.
NADH,), 6, from the difference of measured oxygen con- (1994b) for aerobic phosphorus removal.
sumption rates in the absence and presence of phosphorus. From the assumption that no net accumulation of NADH,
In the same way, a value of the P/NADH, ratio under anoxic and ATP takes place, the overall stoichiometric equation of
conditions, 6 , can be determined. By using the obtained 6 , the PHB conversion rate under anoxic conditions, rphb,can
the metabolic model for the conventional aerobic process be expressed as follows, by using three PHB yield coeffi-
can be modified for denitrifying dephosphatation. cients ( l/Yphb.x, l/Yphb-pp, l/Yphb-gl) and one maintenance
In this research, the 6, and a proper set of kinetic param- coefficient (mphb):
eters are determined in batch tests with different phosphorus -)lphb = (l/yphb-x) . r x + (‘/yphb-pp) ’ ‘pp + (l’yphb-gl)
concentrations using enriched DPB sludge in an anaerobic- . rgl + mphb * cx (3)
anoxic (A,) sequencing batch reactor (SBR). The obtained
anoxic model based on the 6, is applied to denitrifying with
dephosphatation by DPB. Cycle behavior of phosphorus in
l/Yphb, = (0.635 + 2.24256,+ ~)/(2.256,+ 0.5) (4)
the A, SBR is simulated under two hfferent biomass reten-
tion time (SRT) conditions to verify the reliability and va- 1/YPh&,, = (6,/EN + 01,)/(2.256, + 0.5) (5)
lidity of the model. Also a comparison with the aerobic (26, + 1.5)/(2.256, + 0.5)
l/Yphb-gl = (6)
model and the limits of the present metabolic model will be
discussed. mphb = mapN/(2.256, + 0.5) (7)
Equation (3) describes the amount of required PHB for bio-
METABOLIC MODEL mass production (rJ, polyp (rpp), and glycogen synthesis
(rgl) and maintenance (mpbb). Yield and maintenance coef-
For the basic derivation of the metabolic model and the
ficients are defined by the metabolic model parameters ( 6 ,
exact reading of most of the reaction stoichiometry under
K, E,, a3,matpN)(Smolders et al., 1994b). The formulas of
denitrifying conditions we refer to Smolders et al. (1994a,b,
these yield coefficients are completely identical for aerobic
1995a,b), who developed a metabolic model for the con-
(Smolders et al., 1994b) and anoxic conditions, when 6 , E,,
ventional A/O process (called A/O model). This model was
and matp, are replaced by 6, E, and inatp.
modified for the denitrifying dephosphatation process
In the same way, the stoichiometric nitrate conversion
(called A, model).
rate under anoxic conditions, rno3,can be expressed by using
three nitrate yield coefficients ( 1/Yno3-x,1/Yno3-pp,l/Yno3-gl)
Anaerobic Metabolism and one maintenance coefficient (mno,):
The anaerobic metabolism is identical in both processes. -rnoj = (1/Yno3-x) . rx + (1/Yno,-pp> rpp + (1/Yno3-gJ
Three basic reactions are involved in the anaerobic metabo- . rgl+ mno, . Cx (8)
lism (Smolders et al., 1994a): (i) the transport and storage of
substrate (acetic acid, HAc) as poly-P-hydroxy-butyrate with
(PHB), (ii) polyp degradation, and (iii) glycogen degrada-
l/Yno,-x = (0.123 + 0.9~)/(2.256,+ 0.5) (9)
tion. These three processes are stoichiometrically coupled,
and only one rate equation is needed. l/Yno3-pp = (0.96,/~, + 0.901,)/(2.256,+ 0.5) (10)
l/Yno3-gl = 0.95/(2.256, + 0.5) (11)
Electron Transport Phosphorylation with Nitrate
m,,, - 0.9ma,/(2.256, + 0.5) (12)
Five basic reactions are involved in the aerobic metabolism
(Smolders et al., 1994b): (i) PHB catabolism, (ii) electron The formulas of these yield and maintenance coefficients
transport phosphorylation, (iii) production of biomass, (iv) are similar between aerobic (Smolders et al., 1994b) and
phosphate transport and polyp synthesis, and (v) glycogen anoxic conditions, but the aerobic coefficients are 5/4 times
synthesis. The difference between the aerobic and anoxic higher than the anoxic coefficients corresponding to the
metabolism is only electron transport phosphorylation with difference of available electrons per mole of electron ac-
oxygen or nitrate [Eqs. (1) and (2)]. The P/NADH, ratio ceptor for oxygen (four electrons) and nitrate (five elec-
under anoxic conditions, however, will be different from the trons).

686 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 52, NO. 6,DECEMBER 20, 1996
Kinetics
The conversion rates of HAc, phosphate, nitrate, PHB, and
glycogen under A, conditions were simulated using the
same kinetic equations as proposed by Smolders et al.
(1995a,b). The kinetic equations are summarized in Table I.
Under anaerobic conditions, the HAc uptake rate can be
described by a Monod-type kinetic equation. From the stoi-
chiometry (Smolders et al., 1994a) (Table V), the PHB,
glycogen, and phosphate conversion rate under the anaero-
bic condition can be calculated from the HAc uptake rate. It
was assumed that maintenance energy under anaerobic con-
dition is obtained from polyp degradation. The anaerobic
maintenance coefficient (man) was obtained from the sec-
ondary phosphorus release rate (Kuba et al., 1993) when
both HAc and nitrate are absent.
Under anoxic conditions, three kinetic equations for bio-
mass synthesis, phosphate uptake, and glycogen synthesis
and a maintenance coefficient were defined. From the stoi-
chiometry [Eqs. (3) and (S)], the PHB and nitrate conver-
sion rate under anoxic conditions can be calculated by using
these three kinetic equations.
In the simulation these kinetic equations were switched
on and off with switching functions, depending on anaero-
bic or anoxic conditions. The switching functions were de-
fined to be 1 when the concentration of a component is not
0; otherwise these switches are 0. For instance, under aero-
bic conditions, the anoxic kinetic equations are inactive due
to a switching function of nitrate which is equal to 0.
DETERMINATION OF THE STOlCHlOMETRlC
COEFFICIENTS OF THE A2 METABOLIC MODEL
The yield coefficients for biomass synthesis (1/Y,hb-,
1/Yno3J, polyp formation (l/Y,,hb.pp, l/Yno,-pp),and glyco-
gen synthesis (l/Yphb.gl, l/Yno3-gl) are now expressed as a
function of the PNADH, ratio (S,), the coefficient for the
NADH, requirement of phosphate transport (E,), the re-
quired ATP for polyp synthesis (a,),the polymerization
constant in biomass synthesis (K), and the maintenance en-
ergy (mat,,,). In this anoxic metabolic model, the parameters
ag and K can be assumed equal to the aerobic parameters.
Smolders et al. (1994b) reported a3 = 1 mol ATP/mol P
and K = 1.6 mol ATP/mol C(biomass). The other param-
eters (E, matp,.S,) are determined by the following proce-
dure.
Determination of the Phosphate Transport
Coefficient
Under aerobic or anoxic conditions, phosphate is taken up
inside the cell and polyp is formed. The phosphate transport
into the cell is an energy-consuming process. The energy
required for the phosphate transport is generated by the
import of protons which are subsequently exported over the
cell membrane in the oxidation of NADH, [Eq. (1) or (2)]
(Mitchell, 1986). The ratio of energy consumption of the
phosphate transport under anoxic conditions (E,) to aerobic
conditions will be identical to the ratio of the PNADH,
value under both conditions:
E, = E SJS - (134
Smolders et al. (1994b) obtained S = 1.85 mol ATP/mol
NADH, and E = 7 mol P/mol NADH,, leading to
E, = 7.SJ1.85 ( 13b)
Determination of the Maintenance Coefficient
under the Anoxic Condition (matpJ
Smolders et al. (1994b) determined the ATP maintenance
coefficient (mat,) from the endogenous oxygen consumption
rate. They found mat,, = 0.019 mol ATP/mol C * h. In this
-
research, a value of 0.01 mol ATP/mol C h was chosen for
the A, system, because the anaerobic phosphate release for
maintenance (m,) for the A, system was found to be 50%
of the value in the A/O system too.
Determination of the P/NADH2 Ratio under the
Anoxic Condition (6,)
Determination of the P/NADH, ratio under anoxic condi-
tions (6,) can be achieved by combining measured conver-

Table I. Kinetic equations for anaerobic and anoxic reactions of biological phosphorus-removing organisms.

Reaction Equation Parameter Value Unit A/O value"

Anaerobic
HAc uptake 0.2 mol C h o l C.h 0.4
1.o mmol C/L 1.0
0.05 mmOVL -
Maintenance 2.5x 10-3 mol P/mol C.h 4 x lo-3
Anoxic
Biomass synthesis 0.05 mol C/mol C.h 0.14

Phosphate uptake 0.1 mol P h o l C.h 0.2

0.1 mmol P L 0.1
0.3 mol P h o l C 0.3
Glycogen formation 0.8 rnol C h o l C.h 0.8
0.3 rnol C h o l C 0.21
Maintenance 3.6 x 10-3 mol C/mol C.h 4x

"Reported values for organisms enriched in an anaerobic-aerobic SBR (Smolders et al., 1994b, 1995b)

KUBA ET AL.: METABOLIC MODEL OF DPB 687

sion rates of nitrate (rno,), biomass (rx),PHB (Ip+ and
polyp (rpp) in batch tests with the enriched denitrifying
dephosphatation sludge from the following equation, which
is obtained from Eqs. (3) and (8) by elimination of the
glycogen conversion rate (rgl):
= rnoj + (1/Yno3-J . r x + (1/Yno3-pp) . rpp
0 ZnSO, * 7H,O, and 0.15 g CoC1, 6H,O.
- {rphb + ( yph&x) ' rx + ( Yphb-pp)
'rpp -tmphb * cx} . (Yphb-gl/Yno,-gl) + %o, ' cx
Because all conversion yield (Y)values and E, are linked to
6, [Eqs. (4)-(7) and (9)-(12) and Eq. (13b)l and because K,
a, and matp, are known, Eq. (14) only contains 6, and
measured conversion rates (r). This equation then allows us
to evaluate the best estimate for .6,
MATERIALS AND METHODS
Continuous Operation of the Anaerobic-Anoxic
Sequencing Batch Reactor
The study has been carried out in a laboratory-scale SBR
with a working volume of 3.5-3.6 L at room temperature
(20-25°C). The SBR was operated in a cycle of 6 h. The
cycle consisted of three phases: anaerobic (2.0 h), anoxic
(3.5 h), and a settling period (0.5 h). Inoculum for the an-
aerobic-anoxic (A2) SBR was obtained from a phosphorus
and nitrogen removal process operating on municipal waste-
water. Of the synthetic wastewater containing HAc and
phosphate, 1.75 L was pumped into the reactor during the
first 10 min of the anaerobic phase. In the anoxic phase, 100
mL NaNO, solution (117 mmol/L) was pumped into the
SBR with a constant flow rate during the first 110 min. At
the end of the anoxic phase, 64 or 112 mL of excess sludge
was removed, resulting in a biomass retention time (SRT) of
14 or 8 days, respectively. After settlement (15 min), 1.85 L
supernatant as treated effluent was pumped out from the
SBR. The overall hydraulic retention time was 0.5 days. The
SBR was continuously mixed by impellers at 450 rpm, ex-
cept for the settling period. Dinitrogen gas continuously
flowed through the SBR head space with a flow rate of 60
L h , to prevent introduction of oxygen in the system. The
pH was strictly controlled at 7.0 f 0.5 by addition of 1 M
HCl or NaOH to avoid chemical phosphate precipitation.
For the batch experiments, enriched sludge from the SBR
was used.
The A, SBR was operated under 14 days SRT for 10
months; thereafter the SRT was decreased to 8 days. After
steady-state conditions were achieved at each SRT, HAc,
phosphate, nitrate, NH,+, PHB, and glycogen concentra-
tions were regularly measured in a cycle.
Media
Synthetic medium was used containing, per liter, 0.375 g
HAc [400 mg chemical oxygen demand (COD)L] as car-
bon source, 0.049 g K,HP04 and 0.028 g KH2P04 as phos-
688 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 52, NO. 6, DECEMBER 20, 1996
phorus source (15 mg PL), 0.227 g NH4Cl (60 mg NL),
0.6 g MgSO, * 7H,O, 0.07 g CaCl, * 2H20, 0.01 g ethylene
diamine tetraacetic acid (EDTA), and 2 mL nutrient solu-
tion. The nutrient solution contained, per liter, 1.5 g
FeCl, * 6H,O, 0.15 g H,BO,, 0.03 g CuSO, * 5H,O, 0.03 g
-
KI, 0.12 g MnCl, 4H,O, 0.06 g Na,MoO, * 2H20, 0.12 g
-
(14)
Batch Tests for Determination of the P/NADH,
Ratio under Anoxic Conditions
In order to determine the P/NADH, ratio in electron trans-
port phosphorylation with nitrate @,) batch tests with vari-
able initial phosphate concentrations were conducted using
the enriched denitrifying dephosphatation sludge (SRT 8
days). The conversion rates of phosphate, nitrate, PHB,
glycogen, and NH,+ and the active biomass concentration
were obtained from five batch tests. The formula
CH,,,Oo,~,No,,$,,o,, was assumed as the active biomass
formula (Smolders et al., 1995b). It was assumed that the
measured MLVSS (mixed liquor volatile suspended solids
concentration) is the sum of PHB, glycogen, and the active
biomass (C,) (Smolders et al., 1995b). The active biomass
conversion rate (rx)was calculated from the measured NH,+
conversion rate (rnh,):
rx = -r nh4 /0.2 (15)
For each batch test, 400 mL sludge was taken from the A,
SBR at the end of the anaerobic phase. Thus, much PHB
was stored inside denitrifying phosphorus-removing bacte-
ria (DPB), and little polyphosphate was stored due to an-
aerobic phosphorus release with HAc consumption. After
centrifugation (3500 rpm) of the sludge for 15 min, the
supernatant was replaced by a medium without HAc and
phosphate, and the resuspended sludge was transferred into
a 1-L laboratory fermentor. After addition of proper
amounts of phosphate and nitrate to the pretreated sludge,
the batch test was started. The initial phosphate and nitrate
concentrations in the batch tests are summarized in Table 11.
All batch tests were conducted in the 1-L laboratory fer-
mentor at 25°C using the pretreated DPB sludge. The pH
was controlled at 7.0-7.1 by addition of 0.5 M HC1 or
NaOH. Dinitrogen gas was flushed through the head space
of the fermentor with a flow rate of 30 L/h to prevent
oxygen contamination.
Table XI. Initial phosphate and nitrate concentrations in the batch tests.
Initial P concentration Initial NO,- concentration
Run No. (=OW (rnrnollL)
0.0 6.0
1.1 6.3
2.1 6.4
1.1 6.2
2.1 3.0
Analyses
Orthophosphate was determined by the ascorbic acid
method. HAc was determined on a glass chromatograph
(GC) with a Hayesep Q 80-100 mesh column (185°C) and
a flame ionization detector (FID) (185°C). Nitrate was ana-
lyzed on a liquid chromatography (LC) column (Skalar In-
struments) by Skalar methods. The NH,+ concentrations
were measured with an ammonia-selective gas electrode
(METROHM, 605060 10).
For dry weight determination, a 20-mL sample of the
sludge was filtered on a Whatman glass microfiber (GF/C)
filter. The filter was dried for 24 h at 105°C and weighed on
a microbalance. The organic content (MLVSS) was deter-
mined by incinerating the dry filters in an oven at 550°C
for 1 h.
The extraction and analyses of intracellular PHB and gly-
cogen were carried out as described by Smolders et al.
(1994a,b).
RESULTS
fying conditions in comparison with aerobic conditions is in
the expected range (John and Whatley, 1970).
The obtained value of 6, ( = 1.0) was used in the meta-
bolic model. The conversion of each component was simu-
lated to verify the reliability and validity of the anaerobic-
anoxic model (A2 model).
Stoichiometry under Anaerobic and Anoxic
Conditions for the A2 Model
From the obtained 6, ( = 1.O), all yield coefficients, Y [Eqs.
(4)-(7) and (9)-(12)], can be calculated. The overall rela-
tions for PHB and nitrate conversion in the anoxic phase are
+
-rphb = 1.628rx + 0.460rpp 1 . 2 7 3 + ~ ~ x 10-3Cx
~ 3.64
(16)
-r,,, +
= 0.568rX 0.414rpp + 0.346rg, + 3.27 x 10-3Cx
(17)
The yield coefficients for the metabolic reactions under an-
oxic conditions are given in Table V.

Simulation of the Conversion in the Batch Tests

P/NADH, Ratio under Anoxic Conditions
In order to determine the P/NADH, ratio (aN),the conver-
sion of each component and the active biomass concentra-
tions were obtained from five batch tests with the enriched
DPB sludge under anoxic conditions. Two data sets for the
determination of 6, were prepared from the results in each
batch test. One data set was the converted amounts of each
component which were calculated from the difference be-
tween the initial and the final concentration. Another data
set was the initial conversion rates of each component. The
data sets are shown in Tables I11 and IV.
According to Eq. (14), 6, was calculated in each batch
test. As shown in Tables 111 and IV, the average of obtained
6, values was approximately 1.0 (mol ATP/mol NADH,)
with a standard deviation of 0.19. Smolders et al. (1994b)
reported 1.85 (mol ATP/mol NADH,) under aerobic con-
ditions. Therefore, the energy (ATP) production in electron
transport phosphorylation with nitrate is approximately 40%
lower than with oxygen. The lower efficiency for denitri-
Simulations were performed with the software package
AQUASIM (version 1.Ob) (Reichert, 1994). AQUASIM al-
lows the stoichiometry and kinetic equations to be defined
in relation to biological conversions, hydraulic effects, and
reactor configurations. Also parameter estimation and sen-
sitivity analyses can be made.
Kinetic parameters under anoxic conditions were esti-
mated with AQUASIM (weighted least-squares estimation)
by using the experimental results of the batch tests. The best
estimated kinetic parameters under anoxic conditions are
shown in Table I.
By using the kinetic (Table I) and stoichiometric equa-
tions [Eqs. (16) and (17)] and the obtained optimal kinetic
parameters under anoxic conditions, it is possible to calcu-
late the conversion rate of each component in the batch
tests. Figure 1 shows the measured and calculated phos-
phate, nitrate, NH,+, PHB, and glycogen concentrations in
the batch tests. The simulation results indicated that the
model could properly simulate the measured conversion of

Table 111. The converted amounts of each component in the batch tests.

Run polyp NO,- PHB Biomass” Cxb 6, (mmol ATFV

No. (mmol/L) (mmol/L) (mmol C L ) (mmol CIL) (mmol C L ) mol NADH,)

0 0.00 -4.40 -1 1.89 2.15 35.77 0.87

1 0.95 -1.63 -4.92 2.15 32.25 1.41
2 2.51 -4.69 -12.34 2.10 30.20 1.00
3 3.42 -4.85 -1 1.65 1.80 24.10 1.03
4 1.19 -2.20 -5.47 1.50 3 1.40 0.97

Average 1.06

aThe active biomass conversion was calculated from the NH; conversion (rx = rnb/0.20).
bBiomass excluding polyp and carbon reserves.

KUBA ET AL.: METABOLIC MODEL OF DPB 689

 Table IV. The initial conversion rates of each component in the batch tests.

Run ‘PP ‘w rphb r, C, 6,(molATFV

No. (mm0VL.h) (mmol/L.h) (mmol C/L.h) (mmol C L h ) (mmol C/L) mol NADH,)

0 0.00 -1.79 -4.60 0.90 35.77 0.74

1 0.98 -1.74 -5.27 1.15 32.25 1.26
2 1.12 -2.17 -5.62 0.83 30.20 0.94
3 1.01 -1.45 -4.21 0.45 24.10 1.16
4 1.23 -2.27 -5.70 1.03 31.40 0.93

Average 1.01

each component at various initial concentrations of phos- DISCUSSION

phate.
Application of the A, Model to Cycle Behavior in
the A2 SBR
Phosphate, nitrate, NH,+,HAc, PHB, and glycogen concen-
trations were measured in a steady-state cycle at 8 and
14 days SRT. The cycle behavior of each component was
simulated by the above-mentioned model, and the simula-
tion results were compared with the measured data sets of
each component conversion under 8 and 14 days SRT. Un-
der anoxic conditions, the calculation was done by using
the same kinetic equations, stoichiometric relations, and
kinetic parameters as derived from the batch tests. The ki-
netic parameters for the anaerobic conditions were obtained
from the cycle behavior by parameter estimation with
AQUASIM. These are included in Table I.
Now, all kinetic equations, stoichiometric relations, and
kinetic parameters under anaerobic and anoxic conditions
are specified. Identical kinetic and stoichiometric param-
eters were used for the simulation under 8 and 14 days SRT.
The calculation was done until steady states were obtained
(25 A, cycles = 150 h). The measured and simulated cycle
behaviors at each SRT are shown in Figures 2 and 3.
The A, model could predict the concentration of all dis-
solved components (HAc, phosphate, nitrate, NH,+). How-
ever, a large difference between the measured and simulated
organic intracellular polymer (PHB and glycogen) concen-
tration was observed.
P/NADH2 Ratio under Denitrifying Conditions
The P/NADH, ratio under anoxic conditions (S,,,) was in-
dependently obtained from the batch tests with enriched
cultures of DPB. The 6, was 1.0 mol ATP/mol NADH,.
Smolders et al. (1994b) reported 1.85 under aerobic condi-
tions. Therefore, the energy (ATP) production in electron
transport phosphorylationwith nitrate is approximately40%
lower than oxygen. This is one of the exceptional cases in
which the in vivo efficiency of denitrification is measured.
John and Whatley (1970) measured the P/NADH, ratio in
vitro with nitrate or oxygen by using membrane prepara-
tions of Micrococcus denitrificans. They reported that the
P/NADH, ratio was 0.9 with nitrate, while it was 1.5 with
oxygen. The derived value for the S,,, of 1.0 and the 40%
lower efficiency compared with the PI0 ratio in this re-
search are within the same range.
Sensitivities of the HAc Loading Rate and the
P/NADH, Ratio on the PHB Content
In this research the power of metabolic modeling is shown.
The same model structure could be used under aerobic and
denitrifying conditions, since the metabolism under both
conditions is similar. The A, model, which was modified
from the N O model by using the measured 6, and with a
proper set of kinetic parameters (Table I), could predict the
concentration of all dissolved components in the A, SBR at
different SRT conditions. A problem in the simulations is

Table V. Metabolic reactions under anaerobic and anoxic conditions.

Anaerobic condition
HAc uptake -CHzO - 0.5 CH1,y6O5/6- 0.36 HPO3 + 1.33 CH1.500.5 + 0.17 COZ + 0.36 H,PO4
+ 0.059 H,O = 0
Maintenance -HPO3 - HZO + H3PO4 = 0
Anoxic condition
Biomass synthesis -1.63 CH1.500.5 - 0.57 HNO3 - 0.2 NH3 - 0.015 H,PO4
+ CH,~osOo,54No~zJ’o,o15
+ 0.28 N, + 0.63 CO, + 0.78 H,O = 0
Phosphorus uptake -0.46 CHl.500.5 - 0.41 HNO3 - H3PO4 + HPO, + 0.21 N, + 0.46 CO,
+ 1.55 H,O = 0
Glycogen synthesis -1.27 CHl,500,5 - 0.35 HNO3 + CH,o/605/6 + 0.17 N z + 0.27 CO, + 0.29 HZO = 0
Maintenance -CHl.500,5 - 0.9 HNO3 + 0.45 N, + COZ + 1.2 HZO = 0

690 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 52, NO. 6, DECEMBER 20, 1996
 -- I
20-

".
- ------I-

O
0
L
1
- .
2
. . - . . 5.
3 4
. I
6
0 4 . .
0 1
I s
2
.
3
.
4
- . 5- .
*

Time [hours] (a) Time [hours]

04
0
. 1. . 2. . 3. . 4. . 5. . 6I
Time [hours] (b) Time [hours]

--------..----
,

04
0
. 1. . 2. . 3. . 4. . 5 . .
Time [hours] (c) Time [hours]

a
O J . .
0 1
. 2. . 3. . 4. . 5. . 6I 0
0 1 2 3 4 5
1
Time [hours] (d) Time [hours]

Figure 1. Phosphate (U),nitrate (O), NH,+ (A),PHB (+), and glycogen (x) concentrations in batch test: (a) run 0, (b) run 1, (c) run 2, and (d) run 3.
Lines are simulation results.

KUBA ET AL.: METABOLIC MODEL OF DPB 69 1

 = 5
>

Y
f
€ 4
21 3
m-
0
= 2
t
c
8'
c
144 145 146 147 148 149 150
Time [hours] Time [hours]
(a) (a)

r 3 5
3
530
E
E 25
U

g20
0
0
2 15
a
6 10
B
o " 5
g o144 145 146 147 148 149 150
Time [hours]
(b) (b)
Figure 2. (a) Phosphate (W), nitrate (A),and NH4+ (0) concentrations Figure 3. (a) Phosphate (B), nitrate (A),and NH4+ (0) concentrations
during anaerobic and anoxic phases at 8 days SRT.(b) HAc (W), PHB (A), during anaerobic and anoxic phases at 14 days SRT.(b) HAc (W), PHB
and glycogen (0) concentrations during anaerobic and anoxic phases at 8 (A),and glycogen (0) concentrations during anaerobic and anoxic phases
days SRT.Lines are simulation results. at 14 days SRT. Lines are simulations results.

that a large difference between the measured and simulated
PHB and glycogen concentration was observed.
The underestimation of PHB concentration on the model
can be due to two possible reasons: a high sensitivity for (i)
deviating influent HAc or nitrate concentrations and (ii) the
6, obtained from independent batch tests. The main prob-
lem here is that a small error in the predicted PHB conver-
sion in one cycle (or batch tests) accumulates to a large error
in the steady-state calculation. In fact, the error is enhanced
by a factor equivalent to the number of cycles in one sludge
retention time (in these cases 32 or 56 times at 8 or 14 days
SRT), leading to large accumulation of such errors in the
estimation of storage components. This effect can be shown
by using slightly higher HAc loading rates in the simulation.
The results indicated that 2% and 5% differences of the
influent HAc concentration brought about approximately
15% and 35% differences on the PHB concentration under
both SRT conditions (Fig. 4). The 2%-5% higher HAc input
can be caused due to an error in preparing the media and
inaccurate flow rates of the influent pumps. The changed
influent HAc concentration had a large effect on the PHB
concentration and slight effect also on the glycogen con-
centration but little effect on the concentration of dissolved
components. The 5% higher influent HAc concentration re-
sulted in very good agreement on PHB between simulation
and experiment in both SRT conditions. This effect can be
understood as follows. According to the kinetic model of the
anoxic phase, the rate of growth, polyp synthesis, and gly-
cogen production stop if nitrate concentration is zero (Table
I). This means that the PHB consumption also stops [Eq. (3)

692 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 52, NO. 6, DECEMBER 20, 1996
 35 I
Anaerobic I Anoxlc et
were recalculated in the batch tests and in the cycle behav-
ior. The model with 10% lower or higher 6, still predicted

/I
Nitrate
the measured conversion of each component in the batch
~
tests, which indicates that 6, has little influence on the
L estimation of kinetic parameters. The model with 10%
lower 6, (0.9) resulted in very good agreement of the cycle
behavior of PHB and also glycogen between the simulation
and the experiment in both SRT conditions. Figure 5 shows
the results of PHB and glycogen in 14 days SRT.
It is clear that the model structure can simulate the be-
5- havior of soluble components very well. The prediction of
the steady-state PHB content is, however, extremely sensi-
04 I I I ! I I , I I I I ! I tive to a small change in HAc and nitrate loading into the
144 145 Time147
146 [hours] '41 150 system or the value of 6,. Since the measurements of HAc
and nitrate loading always have a certain margin of uncer-
(a)

35
30

5"
-
-
E
m
15
2 10
5
0
144 145 146 147 148 149 150
Time [hours]

144 145 146 147 148 149 1

Time [hours]
(b)
Figure 4. Comparison of measured (a) PHB and (b) glycogen concen-
trations during anaerobic and anoxic phases at 14 days SRT, with calcu-
lation using three different HAc loading rates. HAc concentration in media 35
was increased by 0% (12.5 mmol C L ) , 2% (12.75 mmol C L ) , and 5%
(13.13 mmol C L ) , on calculation.
30
T25

or (16)]. If there is a slight imbalance, within one cycle, Yi 2 0

between the PHB produced in the anaerobic phase and con- 15
sumed in the anoxic phase, this imbalance will accumulate
over many cycles. A small change in the effect on this 8 10
imbalance of PHB will lead to a significant effect on the B 5
ultimate steady-state PHB level. It should be noted that the
model is in the same way also very sensitive to small errors 0
144 145 146 147 148 149 1
in the nitrate loading rate.
Time [hours]
The second possible reason for the underestimation of
PHB concentration can be sensitivity for the 6,. In this (b)
research, 6, was independently found from the batch tests
Figure 5. Comparison of measured (a) PHB and (b) glycogen concen-
with the enriched DPB sludge, not by parameter estimation. trations during anaerobic and anoxic phases at 14 days SRT, with calcu-
By using the same kinetic parameters and somewhat lower lation using three different P/NADH, ratios (8,): 0.9, 1.0, and 1.1 mol
or higher 6, ( = 0.9 or 1.1, instead of 1.O), the conversions ATP/mol NADH,.

KUBA ET AL.: METABOLIC MODEL OF DPB 693

tainty, the accurate prediction of PHB contents will always
be cumbersome in the A, process.
Comparing A2 and A/O Model
The simulation results showed that the metabolic model
structure previously derived for the aerobic phosphorus re-
moval process can be used also for the denitrifying dephos-
phatation process. However, several model parameters in
each process obtained a strongly different value; see Table
I (qsmaX,k, kpp). From the aspect of modeling the waste-
water treatment process, this makes the model complex,
because two sets of kinetic parameters are required for the
phosphorus removal under anaerobic-aerobic and anaero-
bic-anoxic processes. From a physiological point of view,
the kinetic parameters could be expected to be similar in
both processes, since the metabolism is essentially the same.
Microbiologically, it could be that the kinetic parameters
deviate because the A, sludge contained a population dif-
ferent from the A/O sludge. In that case indeed the stoichi-
ometry and kinetic structure should not be affected, but the
kinetic parameters can change strongly between different
enriched cultures.
At present it is difficult to state whether the differences
are due to different microbial populations present or an
intrinsic problem in the model structure. Future research is
needed to resolve this question.
Overall Conversions during Anaerobic and
Anoxic Phases
The overall stoichiometry of the A, phosphorus removal
process at 8 days SRT is shown in Figure 6. With 1 mol C
HAc, 1.33 mol C PHB is synthesized and 0.5 mol C gly-
cogen is anaerobically converted, while 0.36 mol P phos-
phate is released. In the anoxic phase, 0.18 mol C PHB is
used for the synthesis of polyp and 0.70 mol C PHB is used
for the synthesis of glycogen. Including maintenance, 0.40
mol C PHB is used for biomass synthesis. In each cycle 0.16
mol C active biomass, 0.05 mol C glycogen, and 0.05 mol
C PHB are removed as excess organic matter. The con-
sumed amount of nitrate for the synthesis of polyp is 0.16
mol, for glycogen 0.19 mol, for biomass 0.09 mol, and for
maintenance 0.13 mol.
The reported output values of the conventional A/O phos-
phorus removal process (Smolders et al., 1994b, 1995b) are
also shown in Figure 6. The A/O process had the same input
and SRT as the A, process. The biomass (MLVSS; the sum
of active biomass, PHB, and glycogen) produced in a cycle
is 0.26 mol C in the A, process, while it is 0.41 mol C in the
A/O process. Thus, in the A, process the overall biomass
yield is much lower than the A/O process, indicating less
energy requirement for the excess sludge treatment process.
Since the A, process does not require oxygen for phospho-
rus removal, clearly, energy for aeration can be saved in the
A, process (nitrification then has to occur in a separate

Anaerobic In
1.OO mol-C HAc
0.04 mot-P PO4

II
\

out NO
0.16 blomass 0.34
lywgen degradation ? 0.04polyp 0.04
0.05 glycogen 0.06
0.05 PHB 0.01
0.75 C02 0.59
0.58 NO3 NO3
tf 0.29 N2 ------ N2
--I--

---- 02 0.55 0 2
/

0.15 maintenance

Anoxic
Figure 6. Conversions, expressed in moles, during the anaerobic and anoxic phase of the A, phosphorus removal process (8 days SRT) after addition
of 1 mol C HAc. Reported output values of the anaerobic-aerobic ( N O ) process (Smolders et al., 1994b, 1995b) (8 days SRT) are shown in parentheses.

694 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 52, NO. 6, DECEMBER 20, 1996
phase). Moreover, in the A, process, 0.58 mol nitrogedmol
C HAc can be simultaneously removed from wastewater
due to denitrification.
CONCLUSIONS
A metabolic model has been established for denitrifying
dephosphatation, based on the previous aerobic phosphorus
removal model of Smolders et al. The form of the kinetic
equations used was the same, but a major difference is the
P/NADH, ratio. The measured PNADH, ratio in electron
transport phosphorylation with nitrate was approximately
1.0 mol ATP/mol NADH,. This value indicates that the
ATP production efficiency with nitrate is approximately
40% lower than with oxygen. This is one of the first times
the relative efficiency of the oxidative phosphorylation with
different electron acceptors could be established in vivo.
Results of the simulation indicate that this model can
predict the conversion of each dissolved as well as poly-
meric component in the anaerobic-anoxic SBR operation
under different SRT conditions using a proper set of kinetic
parameters. However, partly different sets of kinetic param-
eters for denitrifying and aerobic phosphorus removal pro-
cesses are obtained.
The authors thank S. van Hateren, C. Ras, D. C. Reuvers, G. van
der Steen, and M. Zomerdijk for the analytical work and P.
Kroon and S. Lispet for technical assistance in the experimental
setup. This research was financially supported by NOVEM
(351230/1110) and STOWA (RWZI 2000-3234/5). One of us
(E.M., University of Agriculture Wien) participated in the re-
search due to a grant from the EC-ERASMUS program.
NOMENCLATURE
ATP required for uptake of HAc (mol ATP/mol C)
ATP required for polyphosphate synthesis (mol ATPhol P)
P/NADH, ratio (mol ATPhol NADH,)
energy for transport of phosphate (mol P h o l NADH,)
biomass formation and polymerizationconstant (mol ATPhol C)
concentration of component i (mourn3)
intracellular fraction of component i (mol/mol)
saturation constant of component i (moum3)
rate constant of component i (h-')
maintenance (mol/mol C h)
specific conversion rate of HAc (mol C h o l C * h)
conversion rate of component i (mol/m3 * h)
yield coefficient (moumol)
Subscripts and superscripts
anaerobic
ATP
poly-P-hydroxybutyrate
glycogen
maximum
anoxic
ammonia
nitrate
p phosphate
pp polyphosphate
s HAC
x active biomass
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KUBA ET AL.: METABOLIC MODEL OF DPB 695