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Modeling of growth kinetics for Pseudomonas spp. during benzene

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Article  in  Applied Microbiology and Biotechnology · January 2006

DOI: 10.1007/s00253-005-1997-z · Source: PubMed

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Appl Microbiol Biotechnol (2005) 69: 456–462
DOI 10.1007/s00253-005-1997-z
APPLIED MICRO BIAL AND CELL PHYSIOLOGY
D.-J. Kim . J.-W. Choi . N.-C. Choi . B. Mahendran .
C.-E. Lee
Modeling of growth kinetics for Pseudomonas spp.
during benzene degradation
Received: 22 February 2005 / Revised: 6 April 2005 / Accepted: 6 April 2005 / Published online: 23 April 2005
# Springer-Verlag 2005
Abstract A modeling study was conducted on growth
kinetics of three different strains of Pseudomonas spp.
(Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseu-
domonas putida) during benzene degradation to determine
optimum substrate concentrations for most efficient bio-
degradation. Batch tests were performed for eight different
initial substrate concentrations to observe cell growth and
associated substrate degradation using benzene-adapted cells.
Kinetic parameters of both inhibitory (Haldane–Andrews,
Aiba–Edwards) and noninhibitory (Monod) models were
fitted to the relationship between specific growth rate and
substrate concentration obtained from the growth curves.
Results showed that half-saturation constant of P. fluo-
rescens was the highest among the three strains, indicating
that this strain could grow well at high concentration, while
P. putida could grow best at low concentration. The in-
hibition constant of P. aeruginosa was the highest, implying
that it could tolerate high benzene concentration and there-
fore could grow at a wider concentration range. Estimated
specific growth rate of P. putida was lower, but half-sat-
uration constant was higher than those from literature study
due to high substrate concentration range used in this study.
These two kinetic parameters resulted in substantial dif-
ference between Monod- and Haldane-type models, indi-
cating that distinction should be made in applying those
models.
D.-J. Kim (*) . J.-W. Choi . N.-C. Choi .
B. Mahendran
Department of Earth and Environmental Sciences,
Korea University,
Seoul, 136-701, South Korea
e-mail: djkim@korea.ac.kr
Fax: +82-2-32903189
C.-E. Lee
Institute for Nano Science,
Korea University,
Seoul, 136-701,
South Korea
Introduction
In bioremediation of groundwater contaminated with ben-
zene, toluene, and p-xylene (BTX), selection of appropriate
bacteria as well as optimum contaminant concentrations on
which the specific bacteria can utilize as carbon and energy
source is essential. Microorganisms exhibit often different
responses for any external carbon sources unless they are
allowed to be adapted with that specific substrate. Practical
importance of optimum substrate metabolism by microor-
ganism is perhaps most obvious at bioremediation of aqui-
fer contaminated with BTX. Many studies performed on the
toxic component showed that even after extensive adapta-
tion microbial cells exhibited inhibitory growth kinetics
(Rozich et al. 1983, 1995; D’adamo et al. 1984). This toxic
(inhibitory), or Haldane kinetics, is characterized by an in-
crease in specific growth rate with increasing substrate con-
centration to a peak growth rate where growth rate declines
with further increases in substrate concentration afterward.
In contrast, Monod or noninhibitory (nontoxic) kinetics is
characterized by an increasing growth rate with increasing
substrate concentration with an asymptotic approach to a
maximum growth rate (Rozich and Colvin 1985).
To predict and evaluate the biodegradation rates of or-
ganic contaminants, researchers have developed mathe-
matical models that include substrate utilization, bacterial
growth and decay, and the utilization of electron acceptors.
In many cases, the kinetics of microbial growth and utili-
zation of substrate used in the models are described by the
Monod function (Monod 1949). Major problems arising from
obtaining reliable kinetic parameters are nonuniqueness
of fitted Monod kinetic parameters due to use of only a
single substrate depletion curve without microbial yield
data (Kelly et al. 1996), use of only one initial substrate con-
centration with microbial yield (Guha and Jaffe 1996), and
use of several initial substrate concentrations with microbial
yield but without significant increase in microbial popula-
tion (Alvarez et al. 1991). To overcome the influence of
microbial growth, several different initial substrate con-
centrations with microbial yield measurement were used
(Schirmer et al. 1999). In this approach, a dual Monod for-

mulation consisting of two governing equations for sub-
strate degradation and microbial growth, where the Haldane
inhibition term (Haldane 1930; Andrews 1968) was intro-
duced to account for a slower substrate utilization rate at
high substrate concentrations due to toxicity, was used.
However, in the study of Schirmer et al. (1999), Monod ki-
netic parameters were estimated for only substrate degra-
dation curve with microbial yield coefficient obtained from
only two measured data points of microbial growth at initial
and after complete degradation. Although the problem of
nonuniqueness in kinetic parameters during parameteriza-
tion could be solved by the supplement of microbial yield
coefficient, it is questionable whether the specific growth
rate vs substrate concentration curve actually followed the
Haldane type since with only four initial substrate concen-
trations, it would not suffice to construct such a curve.
Reardon et al. (2000) used Monod model to obtain kinetic
parameters using single substrate consumption and biomass
concentration data as follows:
1 dX
max S

¼ ¼ (1)
X dt KS þ S
where μ is specific growth rate (h−1), μmax is maximum
specific growth rate (h−1), KS is half-saturation constant (mg/l),
and X, S, and t are microbial cell, initial substrate concen-
trations (mg/l), and time, respectively. For a given initial
microbial cell Xo, the microbial cell concentration X at time
t can be given by
X ¼ Xo e
t
which describes an exponential growth with time. In the
study of Reardon et al. (2000), Eq. (2) was used to obtain
the two kinetic parameters by fitting to the observed cell
growth curve. However, problem arises in fitting Eq. (2) to
microbial growth since the measured growth curve showed
a stationary phase after exponential growth. Although
modeling of cell growth using Eq. (2) given in the form of
exponential growth is not impossible, it may lead to an
incomplete fitting of the measured data. A more reliable
way of estimating kinetic parameters would be to construct
μ–S relationship from the measured growth curves for
several different initial substrate concentrations. Construc-
tion of such curve will offer additional benefit of checking
whether the specific growth of a microbial cell actually
follows Monod or Haldane type. This approach was used
by Abuhamed et al. (2004), who studied the growth
kinetics of Pseudomonas putida for benzene, toluene, and
phenol degradation.
For inhibitory type, Haldane–Andrews model (Haldane
1930; Andrews 1968) with the additional kinetic param-
eter, i.e., inhibition constant KI (mg/l)
S cultured in 250 ml Erlenmeyer flasks containing 100 ml

¼
max
KS þ S þ SKI
2
457
was used to account for inhibitory effect of substrate con-
centration on microbial growth. Another model of in-
hibitory kinetics was proposed by Aiba et al. (1968) and
was adapted by Edwards (1970). This model is a modi-
fied version (Aiba–Edwards model) of Monod equation,
which describes the Haldane-type growth kinetics similar
to Haldane–Andrews model as follows:
S

¼
max expð
S=KI Þ (4)
KS þ S
The abilities of the equations (Eqs. 3 and 4) mentioned
above to represent experimental data were discussed by a
number of authors. As pointed out by Tan et al. (1996), the
goodness of these kinetic parameters by a certain model
will largely depend on the microorganisms and environ-
ment conditions such as history of microbial adaptation
and substrate concentration range used for the experiment.
More research is therefore necessary to understand the
fundamental mechanisms of growth kinetics subject to
different initial substrate concentration in the microbial
degradation of benzene using different strains of Pseudo-
monas spp.
This study focuses on the determination of growth ki-
netics in terms of optimum (KS) and toxic (KI) substrate
concentration for microbial activity using single culture of
Pseudomonas spp. (Pseudomonas aeruginosa, Pseudomo-
nas fluorescens, P. putida) by conducting batch tests on
cell growth for various initial benzene concentrations. The
(2) results of cell growth vs substrate concentration data were
used to determine appropriate biokinetic parameters using
three growth models of Monod, Haldane–Andrews, and
Aiba–Edwards, depending on the substrate concentration
range used.
Materials and methods
Organisms and culture preparation
Bacterial strains P. aeruginosa KCCM-40269 and P. fluo-
rescens KCCM-11362 were obtained from Korea Culture
Center for Microorganisms, and P. putida KCTC-1769
was obtained from Korean Center for Type Cultures,
Seoul, South Korea. All glassware and materials used for
the study were sterilized in the autoclave (twice at 121°C
for 15 min) to prevent any influence by other microorgan-
isms. Prior to the experiments, freeze-dried bacteria were
revived in 250 ml shake flasks containing 100 ml of LB
medium (tryptone 10 g, yeast extract 5 g, NaCl 5 g/l of
distilled water with a pH of 7.0) over a period of 2 days.
Subsequently, cultures were streaked onto LB agar plates
and grown at 30°C for 2 days then stored at 4°C. Agar
plates were renewed every 2 weeks. The strains were pre-
(3)
of LB medium (pH 7.0), at 30°C overnight in an orbital
shaker at 140 rpm. The volume sufficient to give an initial
cell concentration of 1.0 optical density at 600 nm (OD600)
458

was transferred to 100 ml of fresh LB medium to ensure
high cell concentrations and used for the study.
Culture conditions in growth and degradation
experiments
All enrichments of three strains (P. aeruginosa, P. fluo-
rescens, P. putida) were done in a mineral salt medium
(MSM) (Vrionis et al. 2002) with slight modifications that
contain the following constituents per liter of distilled
water: K2HPO4, 6 g; KH2PO4, 4 g; (NH4)2SO4, 2 g;
MgCl2, 4.95 g; CaCl2, 1.25 g; H3BO3, 0.225 g; ZnSO4·7-
H2O, 0.6 g; NiSO4, 0.18 g; (NH4)6Mo7O24·4H2O, 0.134 g;
CuSO4·5H2O, 0.0225 g; MnSO4, 0.375 g; CoCl2, 0.14 g;
FeCl3, 0.03 g. A 1 ml of culture grown on LB medium was
transferred onto MSM (200 ml) containing 5 mg/ml
glucose in 250-ml flasks and incubated at 30°C in an
orbital shaker at 140 rpm. Addition of 5 mg/ml glucose to
MSM was necessary in order to achieve optimum cell
growth and benzene degradation in our previous study
(Kim et al. 2005). The cells in late exponential growth
phase were harvested by centrifugation at 10,000 rpm and
4°C for 15 min and resuspended in fresh MSM and
washed twice with MSM. After this, the cells were again
centrifuged and resuspended in MSM to a final concen-
tration of 1.0 OD600 and used as a source of inoculum for
the experiments.
Growth kinetics and biodegradation experiments
Batch experiments were conducted to investigate the ef-
fect of initial benzene concentrations on the growth kinet-
ics of Pseudomonas spp. (P. aeruginosa, P. fluorescens, P.
putida) and corresponding benzene biodegradation. Eight
different concentrations (30, 60, 90, 120, 150, 200, 300,
and 400 mg/l) of benzene were prepared in 250-ml flasks

1.2 1.2
a b
1.0 1.0
Cell growth (OD600)
Cell growth (OD600)

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
0 2 4 6 8 0 2 4 6 8
Time (day) Time (day)

1.2
c
1.0
Cell growth (OD600)

0.8

0.6
30(mg/l)
60(mg/l)
0.4 90(mg/l)
120(mg/l)
150(mg/l)
0.2 200(mg/l)
300(mg/l)
400(mg/l)

0.0
0 2 4 6 8
Time (day)

Fig. 1 Growth of Pseudomonas spp. for different initial substrate concentrations: a P. aeruginosa, b P. putida, c P. fluorescens
 459

containing 200 ml of MSM amended with 5 mg/ml glu-
cose. Bacterial concentration corresponding to 0.02 OD600
of each strain, which had been adapted to a varying and
increasing range (20–500 mg/l) of aqueous benzene, was
inoculated into the medium. The culture flasks were closed
using Teflon cap and sealed with Para film to prevent
volatilization of benzene. All culturing was done in sealed
flasks with 34.6% (v/v) of headspace. The flasks were
shaken at 140 rpm and at a temperature of 30°C in the
shaking incubator until the experiment was completed.
The suspension was sampled at regular intervals using a
sterilized air-tight glass syringe to determine cell growth and
benzene degradation. A sterile control experiment was car-
ried out to check the volatility of aqueous benzene in cul-
ture medium. All batch experiments were run in duplicate.
Analytical methods
Bacterial concentrations were determined by UV–visible
spectrophotometer (Heyios β, Thermo-Electron Corpora-
tion) at 600 nm by measuring the absorbance of the cell
solutions. Aqueous benzene was quantified using high-per-
formance liquid chromatography (Young Lin, Seoul, South
Korea) equipped with a fluorescence detector (M720),
M925 pump, Rheodyne injector, and C18 column (150×
4.6 mm; Phenomenex, Torrance, CA, USA) at an excita-
tion wavelength of 254 nm. The elution gradient was 50%
cell peaks for initial substrate concentrations of 30 to 120
mg/l.
Cell growth and degradation kinetics
Examples of cell growth and associated benzene degrada-
tion are shown in Fig. 2 for the case of initial substrate
concentration of 90 mg/l (Fig. 2a) and 400 mg/l (Fig. 2b).
For substrate concentration of 90 mg/l, cells of all the
strains started immediately growing with formation of a
mirror between the two curves, indicating that Pseudomo-
nas spp. could degrade benzene as a carbon and energy
source. P. aeruginosa showed that the highest cell growth
with corresponding substrate decline among three strains.
After cell growth reached the exponential phase, a sta-
tionary phase was followed, during which consumption of
aqueous benzene was significantly reduced. Substrate deg-
radation patterns for the case of 400 mg/l, where strong
inhibition to cell growth was observed, showed almost an
identical curve between strains during the initial lag phase.
However, degradation of substrate for P. putida and P.
fluorescens increased consistently with the increasing cell
growth during early stage of exponential growth, whereas
substrate degradation by P. aeruginosa was insignificant.
1.2 1.2
a
1.0 1.0
Benzene concentration (C/C0)

acetonitrile/50% water with a flow rate of 1.0 ml/min. The

samples collected using the 1 ml glass syringe were trans-

Cell growth (OD600)

0.8 0.8
ferred to a 0.5-ml microtube, centrifuged at 3,000 rpm for
1 min, and 2 μl of aliquots of supernatant were injected to 0.6 0.6
HPLC.
0.4 0.4

Results 0.2 0.2

Effect of initial substrate concentration on cell growth 0.0 0.0

0 2 4 6 8
Time (day)
Growth curves of Pseudomonas spp. (P. aeruginosa, P.
fluorescens, P. putida) for different initial substrate con- 1.2 1.2

centrations are presented in Fig. 1. For all strains, cells b

Benzene concentration (C/C0)

1.0 1.0
were growing rather well for initial substrate concentra-
tions less than 200 mg/l with OD600 of about 0.8 at the end
Cell growth (OD600)
0.8 0.8
of exponential phase followed by a stationary phase, but
inhibition of growth appeared from 300 mg/l especially for 0.6 0.6
P. putida and P. fluorescens, and all three stains showed a
long lag phase from 400 mg/l. The severe inhibition to cell 0.4 0.4
growth was most pronounced in P. fluorescens. It is ob-
vious that aqueous benzene higher than 300 to 400 mg/l 0.2 0.2
can be toxic or inhibitory to Pseudomonas spp., although
these strains were previously adapted up to 500 mg/l of 0.0 0.0
0 2 4 6 8
benzene concentrations. The time required for exponential
Time (day)
growth was the lowest for P. aeruginosa followed by P.
putida and P. fluorescens, indicating that P. aeruginosa Fig. 2 Cell growth and substrate degradation of Pseudomonas
could grow well at the concentration range up to 300 mg/l spp. for initial substrate concentrations of a 90 and b 400 mg/l.
Open rectangles, triangles, and circles represent cell growth of
without any inhibition. No clear trend was found between P. aeruginosa, P. putida, and P. fluorescens, and filled rectangles,
cell peaks and initial substrate concentrations because mi- triangles, and circles represent degradation of benzene by P.
crobial growth of all three strains showed relatively similar aeruginosa, P. putida, and P. fluorescens, respectively
460
0.18
Cell growth and subsequent substrate degradation data for
other concentration ranges (30–200 mg/l), which are not
0.15 shown here, were similar to the case of 90 mg/l.
Specific growth rate(hr )
-1

0.12
Growth kinetics of single cultures
obs - P. aeruginosa
0.09 obs - P. putida
obs - P. fluorescens Based on the growth curves of Pseudomonas spp., the
fitted - P. aeruginosa
0.06 fitted - P. putida
specific growth rate (μ) was obtained for each initial
fitted - P. fluorescens substrate concentration (S) using the following relation-
ship given in Eq. (1):
0.03

¼ ln ðX2 =X1 Þ=ðt2
t1 Þ (5)

0.00
0 50 100 150 200 250

Substrate concentration(mg/l)
where X2, X1 are the cell concentrations at maximum and
initial growth time t2, t1 during exponential growth. The
Fig. 3 Observed data of specific growth rate versus substrate observed and fitted relationship between μ and S is plotted
concentration and model fit using Monod model. Open rectangles,
triangles, and circles represent the specific growth rates of P. in Figs. 3 and 4 for Monod and Haldane types, re-
aeruginosa, P. putida, and P. fluorescens, respectively spectively, by assuming that substrate concentrations
higher than 200 mg/l become inhibitory. For Monod
type, P. putida showed the fastest and highest specific

0.18 0.18
a observed
b observed
Haldane-Andrews model Haldane-Andrews model
0.15 Aiba-Edwards model 0.15 Aiba-Edwards model
Specific growth rate(hr-1)
Specific growth rate(hr-1)

0.12 0.12

0.09 0.09

0.06 0.06

0.03 0.03

0.00 0.00
0 100 200 300 400 0 100 200 300 400

Substrate concentration(mg/l) Substrate concentration(mg/l)

0.18
c observed
Haldane-Andrews model
0.15 Aiba-Edwards model
Specific growth rate(hr-1)

0.12

0.09

0.06

0.03

0.00
0 100 200 300 400

Substrate concentration(mg/l)

Fig. 4 Observed data of specific growth rate versus substrate concentration and model fit using two Haldane-type models of Haldane–
Andrews and Aiba–Edwards. a P. aeruginosa, b P. putida, c P. fluorescens
 461
Table 1 Growth kinetic parameters of Monod, Haldane–Andrews, and Aiba–Edwards model
Microorganism Monod Haldane–Andrews Aiba–Edwards
μmax Ks μmax/Ks SSE μmax Ks KI μmax/Ks SSE μmax Ks KI μmax/Ks SSE
(h−1) (mg/l) (h−1) (mg/l) (mg/l) (h−1) (mg/l) (mg/l)

P. aeruginosa 0.14 4.5 0.031 0.0007 0.3 30 130 0.010 0.0054 0.3 30 240 0.010 0.0049
P. fluorescens 0.19 47.1 0.004 0.0012 0.4 110 80 0.004 0.0066 0.7 170 155 0.004 0.0034
P. putida 0.16 4.7 0.034 0.0003 0.3 30 100 0.010 0.0028 0.4 40 150 0.010 0.0008
0.73a 0.12a 6.083a – 0.62b 1.65b 180b 0.376b – – – – – –
0.44c 3.36c 0.376c – – – – – – – – – – –
a
From Reardon et al. (2000)
b
From Abuhamed et al. (2004)
c
From Oh et al. (1994)

growth at low substrate concentration, whereas P. fluo-
rescens showed gradual increase.
The observed specific growth rates showed an increas-
ing tendency with increased substrate concentration up to
a certain range, and after this range, the specific growth
rate tended to decrease rather sharply (Fig. 4), implying
that the Haldane type can be applied. The decrease in
specific growth rate seems to be strain-dependent; P.
putida showed the decrease at 200 mg/l, P. fluorescens at
300 mg/l, and P. aeruginosa at 400 mg/l. The increase in
this critical concentration is directly related with the
Haldane inhibition concentration (KI). Two different in-
hibitory models of Haldane–Andrews (Eq. 3) and Aiba–
Edwards (Eq. 4) were applied to describe the observed
growth kinetics. It is noted that Aiba–Edwards model gave
a better fit to the observed data than Haldane–Andrews
model. The distinct improvement in terms of sum of
squared errors (SSE) in fitting the observed data (as also
shown in Table 1) by Aiba–Edwards model indicates that
this model is more suitable to our data than the Haldane–
Andrews model. A rather poor fit by Haldane–Andrews
model is attributed to the inability of the model for
describing specific growth data with strong inhibition at
high substrate concentrations.
Discussion
The results of model fit to the observed data of μ–S are
shown in Table 1 together with the literature values. For
Haldane–Andrews model, KS and KI of P. aeruginosa
were found 30 and 130 mg/l, respectively, indicating that
this strain could grow at the widest substrate concentration
range among the three strains. P. fluorescens showed the
highest KS (110 mg/l), indicating that this strain can grow
well at a rather high substrate concentration compared to
other two strains. Similar results were found for Aiba–
Edwards model, which showed the highest KI (240 mg/l)
for P. aeruginosa and the highest KS (170 mg/l) for P.
fluorescens.
For Monod type, the estimated growth parameters of P.
putida were μmax=0.16 h−1 and KS=4.7 mg/l, which was
slightly higher than μmax=0.14 h−1 and KS=4.5 mg/l of P.
aeruginosa, whereas those of P. fluorescens were μmax=
0.19 h−1, KS=47.1 mg/l. Higher estimation for μmax of P.
fluorescens, although the specific growth rate was much
lower at the low substrate concentration range than the
other two strains, is attributed to the comparatively high
KS estimated. It is likely that the increase in KS leads to the
increase in μmax and vice versa. This indicates that there is
a correlation between these two parameters. Owing to this
dependence, comparison of the parameters between strains
does not offer reliable evaluation. For instance, a small
decrease in μmax from 0.16 to 0.14 h−1 resulted in a small
change in KS from 4.7 to 4.5 mg/l for two strains of P.
putida and P. aeruginosa. For this reason, μmax/KS ratio
was proposed as a better parameter to assess the advan-
tage in competition for a nutrient at low concentrations
(Kovarova-Kovar and Egli 1998). According to this ratio,
P. putida showed the highest value, an indication of high-
est specific growth per substrate concentration, which is
reasonably well depicted by experimental data (Fig. 3).
The μmax of P. aeruginosa and P. putida estimated by
Haldane–Andrews and Aiba–Edwards model was almost
similar between strains with μmax=0.3–0.4 h−1, but μmax of
P. fluorescens increased from 0.4 h−1 (Haldane–Andrews)
to 0.7 h−1 (Aiba–Edwards). This increase in μmax resulted
in the increase in KS as was shown for Monod model. It is
remarkable that the ratio of μmax/KS for all strains is
identical between Haldane–Andrews and Aiba–Edwards
model. Similar to Monod model, μmax/KS (0.01) of P.
aeruginosa and P. putida estimated by two inhibitory mod-
els was higher than that of P. fluorescens (0.004), indi-
cating that these two strains grow better at low benzene
concentrations.
Estimated parameter values were compared with those
from literature studies for P. putida grown in benzene
(Table 1). The μmax=0.3 h−1 of Haldane–Andrews model
was lower than μmax=0.62 h−1, but the KS=30 mg/l was
much higher than the literature value of 1.65 mg/l
(Abuhamed et al. 2004). The μmax and KS of P. putida
by Monod model were 0.16 h−1 and 4.7 mg/l. These
values for P. putida reported in the literature studies were
μmax=0.73 h−1, KS=0.12 mg/l (Reardon et al. 2000) and
μmax=0.44 h−1, KS=3.36 mg/l (Oh et al. 1994). Regardless
of the model type, the lower μmax and higher KS of this
study are attributed to the higher substrate concentration
range used in the batch experiment than those (43 mg/l by
462
Reardon et al. 2000 and 70 mg/l by Oh et al. 1994) of the
literature studies. Another possible explanation is found
from the fact that in this study, cells were adapted up to a
substrate concentration of 500 mg/l, while cells in the
literature study (Abuhamed et al. 2004) were adapted to
the substrate concentration of 90 mg/l.
On the other hand, both μmax and KS parameters in two
models of Monod type (Monod model) and Haldane type
(Haldane–Andrews, Aiba–Edwards model) resulted in
substantial difference in their values (μmax and KS of
Monod model were 2–3.5 times and 4–6 times less with
those of two inhibitory models, respectively), implying
that distinction should be made in applying those models
since the two models are developed with different as-
sumptions on growth kinetics: Monod type for noninhib-
itory condition and Haldane type for inhibitory condition.
Nevertheless, the ratio of μmax/KS showed consistent val-
ues between two inhibitory models for each strain, im-
plying that it can provide a better reference for microbial
growth than the values of μmax and KS.
It should be noted that the use of two different models
(inhibitory vs noninhibitory) does not differ in the inter-
pretation of the experimental results in terms of qualitative
aspects since for both models the same conclusions can be
drawn such that (1) μmax and KS of P. fluorescens were the
highest among the three strains and (2) KS of P. putida and
P. aeruginosa was the lowest, indicating that these two
strains could grow well at lower substrate concentrations.
However, they differ in quantitative aspects since the val-
ues of kinetic parameters, such as μmax, KS, were substan-
tially different between two models. These results indicate
that care should be taken for selection of appropriate
growth models and their kinetic parameters when sim-
ulating the fate and transport of BTX in aquifers. These
findings would provide important information on the se-
lection of microorganism and on optimum environment
for bioaugmentation techniques to be used in aquifer sys-
tems contaminated with relatively high concentrations of
hydrocarbons.
Acknowledgements This work was supported by the Korea Re-
search Foundation (grant no. KRF-2004-005-C00060).
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