Kinetic Modeling of

Poly(-hydroxybutyrate) Production and

Consumption by Paracoccus
pantotrophus under Dynamic
Substrate Supply
M. A. van Aalst-van Leeuwen, M. A. Pot, M. C. M. van Loosdrecht,
J. J. Heijnen
Delft University of Technology, Kluyver Laboratory for Biotechnology,
Department of Biochemical Engineering, Julianalaan 67, NL-2628 BC Delft,
The Netherlands; fax: 31-1-527-82355; e-mail: mark.vl@stm.tudelft.nl
Received 6 March 1996; accepted 18 January 1997
Abstract: The objective of the research was to obtain
insights into the behavior of microorganisms under
feast/famine conditions as often occur in wastewater
treatment processes. The response of microorganisms to
such conditions is the accumulation of storage polymers
like poly(-hydroxybutyrate). The research was per-
formed using a pure culture of Paracoccus pantotrophus
LMD 94.21. A steady-state C-limited chemostat culture
was switched to batch mode and a pulse of acetate was
added. As long as external substrate (acetic acid) was
present, the organism grew and accumulated poly(-
hydroxybutyrate). After depletion of the external sub-
strate, the stored poly(-hydroxybutyrate) was used as
growth substrate. Poly(-hydroxybutyrate) accumulation
was found to be strongly dependent on the growth rate
of the organism before the pulse addition of acetate.
Poly(-hydroxybutyrate) accumulation was correlated to
the difference in maximum acetate uptake rate and the
acetate required for growth. Based on the interpretation
of the experimental results, a metabolically structured
model has been set up. This model adequately describes
the observed kinetics of the poly(-hydroxybutyrate) for-
mation and consumption. 1997 John Wiley & Sons, Inc.
Biotechnol Bioeng 55: 773782, 1997.
Keywords: PHB; poly(-hydroxybutyrate); Paracoccus
pantotrophus; dynamic growth; metabolic modeling;
polymers; activated sludge process
INTRODUCTION
Biological wastewater treatment processes usually occur
under dynamic conditions. The microorganisms involved
experience rapidly changing conditions of availability of
nutrients (feast/famine regime with respect to the carbon
substrate). This is due to variation of the influent flow as
well as to the specific process lay-out. Examples are the
biological phosphate removal process, treatment plants with
selectors for control of bulking sludge, and nitrogen re-
moval in single-sludge systems. This wastewater treatment
practice is in contrast with standard microbiological experi-
ments where it is customary to use continuous cultures. In
wastewater treatment processes volatile fatty acids form the
major soluble substrate. Regularly, it has been shown that
activated sludge organisms respond to feast/famine regimes
by the production of storage polymers (Van Loosdrecht et
al., 1997). Glycogen and poly--hydroxyalkanoates (PHA)
are the main reported bacterial storage polymers. Poly--
hydroxybutyrate (PHB) seems to be the more common stor-
age polymer under conditions of carbon source excess (van
den Eynde et al., 1984; Smolders et al., 1995). This can be
explained as follows: Under conditions of a periodic car-
bonsubstrate surplus, as under feast/famine conditions, the
substrate uptake rate will be larger than required for growth.
The uptake of substrate results in NADH
2
formation which
is consumed by oxydative phosphorylation, which leads to
ATP formation. If the consumption of ATP for growth pro-
cesses is limited, then ATP will accumulate, which in turn
results in an accumulation of NADH
2
. The production of a
more reduced storage polymer like PHB (which requires
NADH
2
) is then more likely than the production of glyco-
gen (which leads to formation of NADH
2
). In such a case
PHB does act as a NADH
2
sink. When glucose or other
sugars are the substrate, glycogen is expected to be the
substrate sink (Mino et al., 1995). This is because the rate of
glycolysis will be reduced when the NADH
2
level inside the
cell is increased.
The accumulation PHB can be used as an internal carbon
and energy source for growth when the external carbon
source is depleted (Zevenhuizen and Ebbink, 1974). For an
adequate description of activated sludge processes the dy-
namics of the production of, and growth on, reserve poly-
mers is therefore important.
This study was set up in order to study the phenomenon
Correspondence to: M. C. M. van Loosdrecht
Contact grant sponsors: Senter/Innovative Research Programme for En-
vironmental Biotechnology; Institute for Inland Water Management and
Waste Water Treatment; Foundation for Water Research/Future Treatment
Techniques for Municipal Waste Water
1997 John Wiley & Sons, Inc. CCC 0006-3592/97/050773-10
of production and consumption of PHB in more detail. For
this purpose an acetic-acid-limited continuous culture of
Paracoccus pantotrophus was used. This culture was turned
to the batch mode and a pulse of acetate was added (Pagni
et al., 1992; van Niel, 1995). Both the production of PHB
when acetate is present in excess and the consumption of
PHB when acetate is depleted were followed.
Most research on PHB production focuses on the indus-
trial production of biopolymers by pure cultures under non-
growth conditions (induced by, e.g., a N or P limitation).
The consumption of intracellularly stored PHB has not been
studied. Models developed based on PHB formation by
nongrowing cells cannot be used to describe the processes
during feast/famine regimes in activated sludge processes,
or other conditions where PHB production and growth oc-
cur simultaneously. Therefore a metabolically structured
model has been formulated which describes the metabolic
stoichiometry observed in this study. Also the kinetics of
accumulation and consumption of PHB is formulated based
on the experimentally observed rates of growth, acetate con-
sumption, PHB production, and PHB consumption.
MATERIALS AND METHODS
Organism
The organism used for all experiments was Paracoccus
pantotrophus (formerly known as Thiosphaera pantotro-
pha) LMD 94.21. It was obtained from the collection of the
Kluyver Laboratory (LMD). All cultures were checked for
purity once a day by plating on a non-selective plate count
agar incubated at 37C.
Culture Conditions
Standard 2-L chemostats (Applikon Schiedam, The Nether-
lands) were inoculated with 50 mL of overnight batch cul-
ture. A desired dilution rate (between 0.05 and 0.2 h
1
) was
set by controlling the liquid flow to the reactor. A level
controller was used for controlling the liquid level in the
reactor (Noorman et al., 1991). The temperature was con-
trolled at 37C using a thermostat bath, the pH was con-
trolled at 8.0 by adding 2 N sulfuric acid or 2 N sodium
hydroxide solution. The well-aerated reactors were operated
with an air flow of 1 NL-min
1
and stirred with two stan-
dard geometry six-blade turbines at 1000 rpm. The dis-
solved oxygen concentration (DO) during the steady states
was always above 80% air saturation. The criterion for
reaching steady state was when a constant biomass concen-
tration, measured as total organic carbon (TOC), was de-
tected, normally within 35 days. All steady-state cultures
were acetate limited.
Growth Media
Batch medium was according to Taylor and Hoare (1969),
trace element solution was according to Vishniac and Santer
(1975). The a-selective plate count agar (PCA) contained
tryptone, 5 g L
1
; yeast extract, 2.5 g L
1
; glucose, 1
g L
1
; and Difco Bacto Agar, 18 g L
1
(pH 7.0). Chemo-
stat medium was adapted from the chemostat medium for P.
pantotrophus (Robertson et al., 1988), but with a higher
acetate concentration to allow for higher steady-state bio-
mass concentrations. It contained NH
4
Cl, 1.6 g L
1
;
K
2
HPO
4
, 0. 8 g L
1
; KH
2
PO
4
, 0. 3 g L
1
;
CH
3
COONa 3H
2
O, 10.9 g L
1
; MgSO
4
7H
2
O, 0.4
g L
1
; 0.1 mL L
1
of silicone antifoam (BHD, England);
and 4 mL L
1
of trace elements solution. All chemicals
were of analytical grade.
Pulse Experiments
Pulse experiments were started by adding 50 mL of con-
centrated sodium acetate solution to the steady-state culture
with a syringe. The flow of growth medium to the reactor
and of spent liquid from the reactor was stopped during the
pulse experiment. This made the system into a batch ex-
periment. During the pulse experiments the DO was always
between 80% and 10% of air saturation. Samples were taken
at preset intervals and analyzed for TOC, ammonium, and
PHB content.
Analysis
The supernatant was separated from the bacterial cells by
centrifugation in a Sorval RC-5B for 15 min at 9000 rpm at
4C. The ammonium concentration of the supernatant was
measured with an ion-selective electrode (Metrohm, model
6.0506.010) and the acetate concentration by total organic
carbon (TOC) (Dohrmann, DC-190), correcting for the eth-
ylenediaminetetraacetic acid (EDTA) from the trace ele-
ments solution. Biomass concentration was measured by
subtracting the TOC of the supernatant from the TOC of the
bacterial cell suspension. The exhaust gas from the reactor
was dried over a membrane (Perma Pure Dryer, PD-625-
12PP) and its carbon dioxide concentration was measured
on-line with an infrared analyzer (Rosemount, model 870).
The oxygen concentration was measured with a paramag-
netic analyzer (Servomex, series 1100). The PHB content of
the washed and dried biomass was determined by extrac-
tion, hydrolyzation, and esterification in a mixture of hy-
drochloric acid, 1-propanol, and dichloroethane at 100C.
The resulting organic phase was extracted with water to
remove any free acids. The propylesters were analyzed by
gas chromatography. Benzoic acid was used as an internal
standard throughout the procedure. Ash content was deter-
mined according to Dutch standard method NEN6621
(NNI, 1982). The elemental composition on the biomass
was determined with a Carbo-Erba Instruments CHNS-O
EA1108 elemental analyzer. Respiration measurements
were done with a Yellow Springs Instruments model 53
biological oxygen monitor (BOM). For the respiration mea-
surements, 10-mL samples of the batch were aerated while
stirring until the maximum oxygen concentration was
774 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 55, NO. 5, SEPTEMBER 5, 1997
reached (in less than 1 min). After that the aeration was
stopped and the oxygen concentration was recorded.
Composition of the Biomass
Bacterial biomass is assumed to consist of two compart-
ments (Roels, 1983): (i) an active biomass compartment and
(ii) a poly(-hydroxybutyrate) compartment. The active
biomass is capable of reproduction and growth; the PHB is
used as storage for carbon and energy. The fraction of PHB
in the total biomass is called f
phb
. The fraction of active
biomass in the total biomass is 1-f
phb
.
Calculations of Balances and Rates
From the measured concentrations conversion rates were
calculated in mol (m
3
h)
1
. Subsequently all rates of con-
sumption or production were expressed as specific rates per
unit of active biomass. Elemental balances were calculated
to check the validity of the experiments. The Macrobal soft-
ware for estimation and balancing of conversion rates
(Hellinga, 1992) was used to calculate the error in both the
degree of reduction balance and the carbon balance.
Parameter Estimation
The parameters were determined in the spreadsheet program
Quattro Pro for DOS 5.0 (Borland International Inc., 1993),
by simultaneously solving all equations for all the experi-
mental data while minimizing the residual sum of squares.
METABOLIC MODEL FOR PHB FORMATION
AND CONSUMPTION
The aerobic metabolism of a bacterium capable of storing
PHB growing on ammonia as sole nitrogen source and ei-
ther acetate or PHB as carbon and energy source can be
described by the seven internal reactions described below. A
schematic representation of the model structure is given in
Figure 1. We have used the C-mole convention (Roels,
1983) throughout this article.
Reaction 1. Synthesis of Acetyl-CoA from Acetate
Acetate is taken up by the cell by means of active transport
and activated to form acetyl-P
i
. This is further converted
into acetyl-CoA. At pH 7 this transport and activation will
require 2 ATP per mol acetate (Stouthamer, 1973).

1
2
Acetic acid
1
2
H-S-CoA 1ATP
+
1
2
Acetyl-S-CoA +
1
2
H
2
O = 0 (1)
Reaction: 2. Synthesis of Biomass Monomers
from Acetyl-CoA
Anabolism, the synthesis of active biomass, involves two
distinct types. First the biomass monomers are synthesized
(r
2
), and thereafter the biomass precursors are polymerized
into active biomass (r
3
). The biomass composition given in
Eq. (2) is the measured average composition of aerobic P.
pantotrophus chemostat cultures without intracellular stor-
age polymers (Pot, 1995):

1
2
1 +
x
Acetyl-S-CoA NH
3

m
ATP
+ 1CH
1.73
O
0.44
N
0.24
+
x
CO
2
+ H
2
O
+

1 +
x
4.00 4.13
2

NADH
2
= 0 (2)
The theoretical amount of ATP needed for the synthesis of
biomass precursors from acetyl-CoA is 0.66 mol ATP per
C-mole of biomass (Stouthamer, 1973), so
m
0.66
[mol/C-mol]. In the synthesis of 1 C-mol of biomass pre-
cursors with acetate as the carbon source of 0.267 mol CO
2
is produced (Gommers et al., 1988), so
x
0.267 [C-
mol/C-mol].
0.6335 Acetyl-S-CoA 0.24 NH
3
0.66 ATP
+ 1 CH
1.73
O
0.44
N
0.24
+ 0.267 CO
2
0.207 H
2
O + 0.469 NADH
2
0 (2a)
Reaction 3. Polymerization of Biomass Precursors
and Maintenance
1 CH
1.73
O
0.44
N
0.24

x
+
m
ATP

ATP
+
1
n
CH
1.73
O
0.44
N
0.24

n
= 0 (3)
The amount of ATP needed for polymerization of biomass
precursors to active biomass is 1.5 mol ATP per C-mole of
biomass (Verduyn et al., 1991), so
x
1.5 [C-mol/C-mol].
The specific ATP consumption due to maintenance pro-
cesses m
ATP
can be calculated from the measured specific
acetate consumption due to maintenance processes m
s
(see
below).
Figure 1. Schematic representation of the metabolism of an organism
capable of producing and consuming PHB.
VAN AALST-VAN LEEUWEN ET AL.: POLY(-HYDROXYBUTYRATE) PRODUCTION AND CONSUMPTION 775
Reaction 4. Carbon Source Catabolism
The catabolism can be written as (Stouthamer, 1973)
0.25 Acetyl-S-CoA 0.75 H
2
O + 0.5 CO
2
+ 1 NADH
2
0 (4)
Reaction 5. Oxidative Phosphorylation
In oxidative phosphorylation ATP is produced from
NADH
2
. The efficiency of this process, the amount of ATP
produced per molecule of NADH
2
oxidized, can be ex-
pressed by the P/O ratio, or (Roels, 1983):
1 NADH
2
0.5 O
2
+ H
2
O + ATP 0 (5)
In this research the P/O ratio is considered independent of
the growth rates used (0.050.2 h
1
).
Reaction 6. Synthesis of the Storage Product PHB
from Acetyl-CoA
The substrate is used not only for synthesis of biomass and
for catabolism but also for the synthesis of PHB. No decar-
boxylation occurs during the production of PHB (Doi,
1990). From 2 mol acetyl-CoA 1 mol of -hydroxybutyrate
can be made, which is polymerized into PHB. For the for-
mation of PHB from acetyl-CoA no additional ATP is
needed (Doi, 1990). This results in (Smolders et al., 1994).
0.5 Acetyl-S-CoA + 1 CH
1.5
O
0.5
0.25 NADH
2
0
(6)
Reaction 7. Synthesis of acetyl-CoA from PHB
In the absence of acetate, the microorganisms utilize the
intracellularly accumulated PHB as carbon and energy
source. The storage polymer is hydrolyzed and converted
into acetyl-CoA. One mole of ATP has to be invested for
every mole of -hydroxybutyric acid (HB) converted into
acetyl-CoA (Dawes and Senior, 1973):
1 CH
1.5
O
0.5
0.25 ATP + 0.5 Acetyl-S-CoA
+ 0.25 NADH
2
0 (7)
Linear Equations for Acetate Uptake, Growth,
and PHB Formation
The internal reactions can be related to observable conver-
sion rates (Roels, 1983). The following six conversion rates
can be measured experimentally: acetate, oxygen, carbon
dioxide, ammonia, biomass, and PHB. An equation relating
acetate uptake, growth, and PHB formation can be defined
by using six internal reactions, namely reaction 1, 2, 3, 4, 5,
and 6. Equation 7 is not included since it does not occur
under conditions of acetate surplus. Assuming there is no
net production or consumption of biomass precursors [Eq.
(8a)], NADH
2
[Eq. (8b)], ATP [Ea. (8c)], and acetyl-CoA
[Eq. (8d)], one obtains the following four relations.
0 = r
2
r
3
(8a)
0 = 0.469 r
2
+ r
4
r
5
0.25 r
6
(8b)
0 = r
1
0.66r
2


1.50 +
m
ATP

r
3
+ r
5
(8c)
0 = 0.5 r
1
0.6335 r
2
0.25 r
4
0.5 r
6
(8d)
From the reaction stoichiometry it is clear that
(r
s
) r
1
(9a)
r
p
r
6
(9b)
and from the overall degree of reduction balance follows:
4(r
s
) + (4)(r
o
2
) 4.50 r
p
+ 4.13 r
x
or 2(r
s
)
2.065 r
x
2.25 r
p
2 r
o
2
r
5
(9c)
solving Equations (8a)(9c) with r
x
and r
p
as unknown rates
gives, for r
s
,
r
s
=
1
Y
SX
max
r
x
+
1
Y
SP
max
r
p
+ m
s
C
x
(10)
with
Y
SX
max
=
4 2
4.13 + 4.32 (11a)
Y
SP
max
=
4 2
4.5 (11b)
m
S
=
m
ATP
2 1 (11c)
Substitution of r
x
( C
x
), r
s
= (q
s
C
x
), and r
p

(q
p
C
x
) followed by division by the biomass concentration
C
x
gives
q
s
=
1
Y
SX
max
+
1
Y
SP
max
q
p
+ m
S
(12)
From earlier experiments with acetate-limited chemostat
cultures of P. pantotrophus (Pot, 1995) it was observed that
Y
SX
max
0.45 0.02 [C-mol/C-mol] and m
S
0.038 0.004
[C-mol/(C-mol h)]. Using Eqs. (11a) and (11c) the P/O
ratio 1.84 [mol ATP/mol NADH
2
] and the ATP main-
tenance coefficient m
ATP
0.102 [mol ATP/(C-mol h)]
were calculated. It is assumed here that these values can be
considered constant within the experimental range. Using
Eq. (11b) the maximum yield of PHB on acetate was cal-
culated, Y
SP
max
0.648 [C-mol/C-mol]. Substituting these
yield coefficients in Eq. (12) gives
q
s
2.22 + 1.544 q
p
+ 0.038 (13)
which is a linear equation describing substrate uptake,
growth, and PHB formation by P. pantotrophus. Here, Y
SP
max
cannot be measured independently, since this has to be done
under nongrowth conditions, e.g., absence of nitrogen. Un-
der these conditions a complete change in metabolism oc-
curs, which makes the results irrelevant for the present case
776 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 55, NO. 5, SEPTEMBER 5, 1997
of actively growing cells with an excess of both carbon and
nitrogen.
From the carbon balance and the degree of reduction
balance relations for the specific carbon dioxide production
rate (q
C
) and for the specific oxygen consumption rate (q
O
)
can be derived as a function of and q
P
:
q
C
(q
s
) q
p
1.22 + 0.544q
p
+ 0.038 (14)
(q
O
) 1.188 + 0.419q
p
+ 0.038 (15)
These metabolic linear equations [(13)(15)] provide the
stoichiometry upon which a kinetic model for the PHB pro-
duction phase can be based.
Linear Equation for Growth with PHB
as Substrate
An equation describing growth and consumption of PHB in
the absence of acetate (r
1
0) can be defined by using five
internal reactions, namely reaction 2, 3, 4, 5, and 7. Assum-
ing that there is no net production or consumption of bio-
mass precursors, NADH
2
, ATP, or acetyl-CoA, an equation
for the specific PHB consumption rate can be derived as a
function of the observable specific conversion rate :
q
p
=
1
Y
PX
max
+ m
P
(16)
with
Y
PX
max
=
4.50 0.50
4.13 + 4.32
(17a)
m
P
=
m
ATP
4.5 0.50
(17b)
With the above-mentioned values for m
ATP
and we ob-
tained Y
PX
max
0.653 [C-mol biomass/C-mol PHB] and m
P
0.0131 [C-mol/(C-mol h)]. The linear equation describ-
ing PHB consumption and growth by P. pantotrophus now
reads:
(q
p
) 1.532 + 0.0131 (18)
From the carbon balance and the degree of reduction bal-
ance, relations for the specific carbon dioxide production
rate (q
C
) and the specific oxygen consumption rate (q
O
) can
be derived:
q
C
0.532 + 0.0131 (19)
(q
O
) 0.693 + 0.0147 (20)
These metabolic linear equations [(18)(20)] provide the
stoichiometry upon which a kinetic model for the PHB con-
sumption phase can be based.
It is noted that one can compare the direct growth of
biomass on acetate (Y
SX
max
0.45 C-mol biomass/C-mol
acetate) with the indirect growth on the PHB cycle, where
acetate is converted into PHB and PHB is converted into
biomass: (Y
SX
max
)
PHB
Y
SP
max
Y
PX
max
0.648 0.653 0.42
C-mol biomass/C-mol acetate
This PHB storage/growth mechanism only leads to 6%
lower biomass yield compared to direct acetate use. Growth
on acetate with PHB as intermediate appears to be quite
efficient from an energetic point of view.
RESULTS
Description of PHB Production and Consumption
The PHB production by a steady-state culture of P. panto-
trophus after a pulse addition of substrate was measured.
Also growth on the internally accumulated storage polymer
after depletion of the added substrate was studied. During
the pulse experiment two distinct phases could be observed,
as shown in Figure 2. First is a rapid uptake of substrate,
which is used for growth and PHB production (090 min).
Next, after the depletion of the substrate, growth on the
accumulated storage polymer occurs (90200 min).
To facilitate mathematical modeling, the results of all
pulse experiments are divided into two phases: (i) the PHB
production phase where acetate is the sole energy and car-
bon source for growth and PHB production and (ii) the PHB
consumption phase where PHB is the sole carbon and en-
ergy source for growth. For both phases q
S
, q
P
, q
N
, q
O
, q
C
,
and q
X
are determined from the measured concentration
curves as a function of time.
Conversions during the PHB Production Phase
The converted amounts of acetate, ammonium, oxygen, ac-
tive biomass, PHB, and carbon dioxide are given in Table I.
At initial growth rates 0.05 h
1
and 0.10 h
1
much of the consumed HAc is converted to PHB (25%
30%). At the high growth rate 0.20 h
1
still 20% of
Figure 2. Typical example of PHB production and subsequent PHB con-
sumption by P. pantotrophus after an acetate pulse was added to a steady-
state chemostat (D 0.05 h
1
) and the flow through the reactor stopped
(, acetate; , active biomass; , PHB; , ammonium).
VAN AALST-VAN LEEUWEN ET AL.: POLY(-HYDROXYBUTYRATE) PRODUCTION AND CONSUMPTION 777
HAc is converted to PHB. As a result of this the PHB
fraction of the cells increases from 3% to around 30% (20%
at the high initial growth rate).
As an indication of the accuracy of the measurements the
results of the carbon balances and the degree of reduction
balances are also shown in Table 1. The accuracy of the
measurements is thought to be adequate to be used as a basis
for the modeling of processes during the PHB production
phase. The only serious deviation is the first experiment at
growth rate 0.10 1/h which has an error of 25% in the degree
of reduction balance.
Kinetic Model for the PHB Production Phase
The experiments showed that during the PHB production
phase acetate is taken up with a constant specific uptake rate
per unit of active biomass. An almost identical result for the
maximum specific acetate uptake rate by P. pantotrophus
was obtained by van Niel 1995:
q
s
q
S
max
(21)
Our observations are related to experiments at relatively
high growth rates. How bacteria subjected to a very low
growth rate respond requires a further study.
It is assumed that the rate of production of PHB during
the pulse experiment can be described as a function of the
fraction of PHB of the total biomass. It was clear that the
rate was declining when f
PHB
increased. It was decided to
choose the most simple form of (black box) kinetic equation
which would still describe the experimental data. This re-
sulted in Eq. (22):
q
p
= q
P
max

1
f
PHB
f
PHB
max

(22)
The two kinetic equations combined with Eq. (14) leave
only three unknown parameters, q
S
max
, f
max
PHB
, and q
P
max
. These
parameters can be determined by simultaneously solving all
equations for all the unbalanced experimental data while
minimizing the residual sum of squares. The results are in
Table II. It appears that the maximum specific acetate up-
take rate q
S
max
is similar for all experiments and independent
of the initial growth rate:
q
S
max
0.96 0.14 (23)
The maximum specific PHB production rate q
P
max
also ap-
pears to be similar for all experiments (initial growth rates
0.050.2 h
1
):
q
P
max
0.58 0.12 (24)
The maximum obtainable PHB fraction in the cell, f
max
PHB
,
appears to be a linear function of the steady-state growth
rate (Fig. 3). It is observed that f
max
PHB
decreases as
increases.
Conversions during the PHB Consumption Phase
The overall conversions of acetate, ammonium, oxygen, ac-
tive biomass, PHB, and carbon dioxide are shown in Table
Table I. Conversion and balances for the PHB production phase.
Growth rate
before pulse
addition h
1
Time elapsed
for full HAc
uptake (h)
Acetate
consumed
(C-mmol/L)
Ammonium
consumed
(mmol/L)
Oxygen
consumed
(mmol/L)
PHB
produced
(C-mmol/L)
Active
biomass
produced
(C-mmol/L)
Carbon
dioxide
produced
(mmol/L)
Deviation
in degree
of reduction
balance (%)
Deviation
in carbon
balance (%)
0.05 1.438 65.54 5.08 34.54 16.02 12.24 26.59 0.5 16
0.05 1.403 67.05 5.54 37.18 20.03 17.75 28.56 16 1.0
0.10 0.803 43.15 3.74 26.89 16.64 8.02 23.74 25 12
0.10 1.104 68.51 5.56 27.69 19.49 16.44 22.55 2.8 15
0.20 0.822 61.30 5.63 29.46 11.54 18.20 24.43 0.1 12
0.20 0.899 66.95 6.88 26.46 13.81 20.11 31.65 6.3 2.1
Table II. Biokinetic parameters for PHB production phase.
Growth rate
before pulse
addition (h
1
)
q
S
max
[C-mol (C-mol h)
1
]
q
P
max
[C-mol (C-mol h)
1
]
f
max
PHB
[C-mol (C-mol)
1
]
0.05 0.865 0.550 0.3186
0.05 0.957 0.619 0.3658
0.10 0.875 0.730 0.3189
0.10 0.919 0.654 0.3056
0.20 1.218 0.539 0.2074
0.20 0.920 0.409 0.2459
Average 0.96 0.14 0.58 0.12
778 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 55, NO. 5, SEPTEMBER 5, 1997
III. Also the time elapsed until the given amount of PHB
was consumed is shown. The oxygen consumption was
measured with both gas phase measurements and biological
oxygen monitor (BOM) measurements. The results of both
methods were almost identical.
As an indication of the accuracy of the measurements, the
results of the carbon balances and the degree of reduction
balances are also shown in Table III. The accuracy of the
measurements is adequate to describe what happens during
the PHB consumption phase. There is only a serious devia-
tion at the first experiment at growth rate 0.05 h
1
with an
error of 21% in the degree of reduction balance and 16% in
the carbon balance.
Kinetic Model for the PHB Consumption Phase
For the growth on internal storage compounds no proper
theoretical mathematical expression is available in the lit-
erature. Therefore we used here the most simple expression
capable of describing the relation between PHB content and
observed PHB consumption rate, namely, a first-order rela-
tion:
q
p
= K
P
f
PHB
f
PHB
min
(25)
This black box kinetic equation combined with Eq. (18)
leaves only two unknown parameters, f
min
PHB
and K
P
. The
minimum PHB fraction of the total biomass is introduced,
since carbon-limited chemostat biomass always contained
some PHB (Pot, 1995). The average f
min
PHB
from steady state
chemostat cultures was found to be:
f
min
PHB
0.0434 (26)
The K
P
can be fitted from the experimental data, as shown
in Table IV. The rate constant for PHB consumption K
P
is
found to be a linear function of the steady-state growth rate
, as shown in Figure 4. The rate of PHB consumption
seemingly increases with the growth rate under which the
cells were cultivated.
DISCUSSION
A dynamic model describing the response of a steady-state
culture of P. pantotrophus to a pulse addition of carbon
substrate was constructed. From the experimental and
model results it has become clear that immediately after the
pulse addition the substrate uptake rate reaches its maxi-
mum value. However, the growth rate only slowly relanates
to the maximal growth rate. In the transient period the ex-
cess of substrate taken up is stored in the cell as PHB. After
depletion of the external substrate the stored PHB is used as
growth substrate. The change in growth rate after the pulse
addition of HAc is shown in Figure 5. The discontinuity in
the specific growth rate at 1.5 h after addition of the pulse
was easily observed experimentally from a sudden increase
in the dissolved oxygen concentration in the reactor (not
shown). During growth of PHB the growth rate slowly de-
creases, probably due to the decreasing PHB content.
It can be discussed whether the cells maintain a maximal
substrate uptake rate at lower growth rates (approaching
zero) than studied here. In a chemostat, according to the
standard theory, the substrate uptake rate is equal to the
maximal uptake rate multiplied by the Monod factor for the
substrate affinity. This implicates that the organisms will
induce a maximal level of substrate uptake enzymes,
whereas the enzyme system for cell growth will (as in these
Figure 3. Biokinetic parameter f
max
PHB
for the PHB production phase as a
function of the initial steady-state growth rate of P. pantotrophus. Equation
for the regression line: f
max
PHB
0.38 0.78 .
Table III. Conversions and balances for the PHB consumption phase.
Growth rate
before pulse
addition (h
1
)
Time elapsed
for full HAc
uptake (h)
Acetate
consumed
(C-mmol/L)
Ammonium
consumed
(mmol/L)
Oxygen
consumed
(mmol/L)
PHB
consumed
(C-mmol/L)
Active
biomass
produced
(C-mmol/L)
Carbon
dioxide
produced
(mmol/L)
Deviation
in degree
of reduction
balance (%)
Deviation
in carbon
balance (%)
0.05 2.060 2.16 1.56 12.18 11.03 5.20 10.11 21 16
0.05 3.032 2.34 1.76 16.14 13.78 3.39 11.15 10 9.8
0.10 1.835 2.10 1.18 nm 18.32 10.42 nm nm nm
0.10 3.003 4.27 1.96 14.15 13.37 6.12 9.47 16 12
0.20 2.036 3.33 1.37 8.29 12.77 9.77 6.72 3.8 2.4
0.20 2.006 8.30 1.09 10.81 11.10 4.68 14.78 25 0.3
nm Not measured.
VAN AALST-VAN LEEUWEN ET AL.: POLY(-HYDROXYBUTYRATE) PRODUCTION AND CONSUMPTION 779
experiments) probably not be fully induced. This could in-
dicate that even if the cells are cultivated close to a growth
rate of zero the maximal substrate uptake activity will be
maintained. This point requires more detailed study. Under
feast/famine conditions in wastewater treatment processes
the conditions are strongly different from a chemostat: Short
periods of high substrate concentration are followed by pe-
riods of no substrate present. It can be argued that under
these conditions those organisms that have a fully induced
substrate uptake system accumulate more substrate and out-
compete organisms that do have lower substrate uptake
rates.
The derived model for PHB production under non-
growth-limiting conditions and growth on PHB under sub-
strate-limiting conditions adequately describes the changes
in concentration of active biomass, PHB, and ammonium
during the experiment (see Figs. 6 and 7). Also the oxygen
and carbon dioxide conversion rates were correctly pre-
dicted (not shown here). A linear regression function be-
tween f
max
PHB
and yields
f
max
PHB
0.783 + 0.39 (27)
The maximum specific growth rate for P. pantotrophus was
measured to be 0.43 h
1
. If the regression function is ex-
trapolated to this growth rate, the resulting f
max
PHB
equals
0.047 [C-mol (C-mol)
1
] which is almost identical to the
measured f
min
PHB
[Eq. (26)]. In other words no PHB accumu-
lation occurs if a pulse of additional substrate is added to a
continuous culture with a dilution rate close to the maxi-
mum specific growth rate. This is in accordance with the
expectations. If the specific growth rate reaches zero, the
maximum obtainable PHB content f
max
PHB
becomes 0.39
[C-mol (C-mol)
1
].
Many authors have indicated that the maximal substrate
uptake rate of an organism is often independent of the actual
growth rate of that organism (Roels, 1983). However, the
fate of the substrate under these conditions has rarely been
studied. Pagni et al. (1992) and van Niel et al. (1995) stud-
ied this phenomenon and also found a correlation between
lower growth rate before addition of the substrate pulse and
higher PHB accumulation after the substrate pulse. The ex-
act mechanism behind these observations was not de-
scribed. We propose that PHB is generally used as a buffer
for the substrate taken up but not directly needed for growth.
With a certain relaxation time the specific growth rate will
increase (Fig. 5) and simultaneously the PHB production
decreases. An initially faster growing organism needs less
Figure 4. Biokinetic parameter K
P
for the PHB consumption phase as a
function of the initial steady-state growth rate of P. pantotrophus. Equation
for the regression line: K
P
0.50 + 4.65 .
Figure 5. Specific growth rate during a pulse experiment as calculated by
the model.
Figure 6. Measured and calculated concentrations for the PHB produc-
tion phase for the experiment in which the steady state was obtained at D
0.05 h
1
(points indicate measured concentrations, lines are calculated
by the model: , acetate; , active biomass; , PHB).
Table IV. Biokinetic parameter for PHB consumption phase.
Growth rate
before pulse
addition (h
1
)
K
P
[C-mol (C-mol h)
1
]
0.05 0.699
0.05 0.788
0.10 0.902
0.10 1.058
0.20 1.406
0.20 1.454
780 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 55, NO. 5, SEPTEMBER 5, 1997
time to reach
max
and accumulates less PHB. This is in-
corporated in our simplified black box approach by the cor-
relation between f
max
PHB
and .
PHB accumulation has been modeled as a two-stage pro-
cess (Kurja et al., 1994), a granule formation stage followed
by a granule growth stage. The rate of polymerization of
HB-monomer to PHB is constant during this second stage
because both the monomer concentration and the number of
granules are constant. Electron microscopy of P. pantotro-
phus biomass shows that small PHB granules are already
present in the cell during chemostat operation (Robertson et
al., 1988; Vishniac and Santer, 1975). It can be assumed that
during the PHB production experiment only granule growth
will be of importance. The maximum specific PHB produc-
tion rate q
P
max
as used in this article could then be related to
the maximum HB-monomer polymerization rate as de-
scribed by Kurja et al. 1994).
From this and other research (van den Eynde et al., 1984;
van Loosdrecht et al., 1997) the picture becomes clear that
storage polymers can play a crucial role in microbial growth
under dynamic conditions. PHB accumulation has been re-
ported in several hundred laboratory stains (Doi, 1990) as
well as in activated sludge systems (van Loosdrecht et al.,
1997). PHB is formed directly from a central intermediate
(acetyl-CoA) of the cell metabolism. Cells subjected to
changes in substrate concentrations use PHB to efficiently
balance the difference between substrate uptake rate and
substrate requirements for growth. Such a mechanism is not
only important for the cell to balance its internal metabo-
lism, but it also gives a competitive advantage over organ-
isms incapable of producing storage polymers. This is es-
pecially important in wastewater treatment processes where
the substrate often becomes available in a highly dynamic
manner. Storage of substrate and balanced growth is then
probably a better competitive strategy than periods of fast
growth alternating with periods of starvation. Using only
conventional Monod-type kinetics to predict competition is
obviously not enough to accurately describe what happens
in wastewater treatment processes.
The here proposed model can be used to evaluate the
growth on internally accumulated PHB in activated sludge
cultures. The parameters K
P
and f
max
PHB
are properties of the
sludge and will therefore strongly depend on the process
conditions used (type of substrates and organisms, tempera-
ture, pH). If the sludge age is known and the PHB levels
during the process are measured, the maximum PHB frac-
tion (f
PHB
) and the rate constant K
P
for growth on PHB can
be determined directly. Future experiments with activated
sludge should give an indication how far the proposed
model structure can be used to improve activated sludge
models.
We are indebted to the analytical team of the Department of
Biochemical Engineering for their analytical assistance. The re-
viewers are acknowledged for their critical remarks, which
greatly improved the article.
NOMENCLATURE
D dilution rat (h
1
)
DO dissolved oxygen concentration (mmol O
2
m
3
)
C
S
substrate concentration (C-mol m
3
)
C
X
biomass concentration (C-mol m
3
)
Y
SX
macroscopic yield of biomass on acetate [C-mol (C-mol)
1
]
Y
SX
max
maximum yield of biomass on acetate [C-mol (C-mol)
1
]
Y
SP
max
maximum yield of PHB on acetate [C-mol (C-mol)
1
]
Y
PX
max
maximum yield of biomass on PHB [C-mol (C-mol)
1
]
r
i
internal reaction rate number i (C-mol c
3
h
1
)
q
i
specific internal reaction rate number i [C-mol (C-mol h)
1
]
r
Z
conversion rate of compound Z (C-mol m
3
h
1
)
q
Z
specific conversion rate of compound Z [C-mol (C-
mol h)
1
]
m
S
maintenance coefficient for growth on acetate [C-mol (C-
mol h)
1
]
m
P
maintenance coefficient for growth on PHB [C-mol (C-
mol h)
1
]
m
ATP
maintenance coefficient based on ATP [mol (C-mol h)
1
]
f
PHB
fraction of PHB of the total biomass (C-mol C-mol
1
)
(1-f
PHB
) fraction of active biomass of the total biomass (C-mol C-
mol
1
)
f
min
PHB
minimum PHB content of the biomass (C-mol C-mol
1
)
f
max
PHB
maximum PHB content of the biomass (C-mol C-mol
1
)
q
S
max
maximum acetate uptake rate [C-mol (C-mol h)
1
]
q
P
max
maximum PHB uptake rate [C-mol (C-mol h)
1
]
K
P
rate constant for growth on PHB
Greek Symbols
P/ O rat i o, ATP produced per NAHDH
2
oxi di zed
(mol mol
1
)
Yi degree of reduction of compound i
specific growth rate (h
1
)

max
maximum specific growth rate (h
1
)
Subscripts
C carbon dioxide
N ammonia
O oxygen
P PHB, poly--hydroxybutyrate
S substrate, acetate
X biomass
Figure 7. Measured and calculated concentrations for the PHB consump-
tion phase for the experiment in which the steady state was obtained at D
0.05 h
1
(points indicate measured concentrations, lines are calculated
by the model: , PHB; , active biomass).
VAN AALST-VAN LEEUWEN ET AL.: POLY(-HYDROXYBUTYRATE) PRODUCTION AND CONSUMPTION 781
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