Process Biochemistry 41 (2006) 1417–1421

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Short communication
Kinetics of cell growth and caffeine utilization by
Pseudomonas sp. GSC 1182
S. Gokulakrishnan, Sathyanarayana N. Gummadi *
Department of Biotechnology, Indian Institute of Technology-Madras, Chennai 600 036, India
Received 9 June 2005; received in revised form 3 October 2005; accepted 19 December 2005

Abstract
Caffeine in coffee pulp prevents the utilization of nutrition rich coffee pulp effectively. Microbial methods of caffeine degradation can help in
detoxifying the pulp and it prove as a better alternative to the conventional methods of environmental unfriendly solvent extraction process of
decaffeination. Previously Pseudomonas sp. GSC 1182 was isolated from the soil samples of coffee cultivation area to degrade caffeine. In this
report, the cell growth kinetics of Pseudomonas sp. GSC 1182 at various concentration of caffeine ranging from 0.05 to 20 g/l was studied. The
kinetic data showed substrate inhibition kinetics and maximum growth rate was obtained when the isolate was grown in medium containing 2.5 g/l
of initial concentration of caffeine. The results also showed that the isolate was able to grow at high concentrations of caffeine (10 g/l). Different
substrate inhibition models were fitted to the kinetic data and found the Luong model was best. The model predicted kinetic parameters were in
agreement with the experimental findings. The kinetic parameters were: Sm = 20.2 g/l; KS = 0.59 g/l; mm = 0.24 h
1 and n = 2. This is the first
detailed report on the cell growth kinetics of microorganism using caffeine as the substrate.
# 2005 Elsevier Ltd. All rights reserved.

Keywords: Caffeine degradation; Cell growth; Substrate inhibition kinetics; Luong model; Biomass yield; Pseudomonas sp

1. Introduction
Caffeine (3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6 dione),
a purine alkaloid is a key component in many popular drinks
especially tea and coffee. Coffee pulp is rich in carbohydrates,
proteins, etc., but the presence of anti-nutritional factors such
as caffeine, tannins and poly phenols restrict its use as a
domestic fodder and its dumping causes environmental
pollution [1]. There is an increasing demand for decaffeinated
beverages because of the growing belief that ingestion of
caffeine can have adverse effect on health. The conventional
methods of caffeine removal (solvent, water and supercritical
carbon dioxide extraction methods) are either costly or it
involves the usage of toxic organic chemicals. Hence, from
health, environment and economic point of view, it is necessary
to resort to other methods of caffeine removal. Microbial
method of caffeine degradation is a better alternative to this
problem [2].
Several bacterial and fungal strains were reported to degrade
caffeine [3–7]. Caffeine is toxic to bacteria and caffeine
* Corresponding author. Tel.: +91 44 2257 4114; fax: +91 44 2257 0509.
E-mail address: gummadi@iitm.ac.in (S.N. Gummadi).
concentration greater than 2.5 g/l in the medium inhibited the
growth of many bacterial species [8,9]. Among the reported
strains, Pseudomonas sp. isolated by enrichment on caffeine as
the sole source of carbon and nitrogen from soil was able to
grow in medium containing 5 g/l of caffeine [10]. In
microorganisms, caffeine was degraded either through
demethylation or oxidative pathway [2]. Strains with high
degradation efficiency and ability to tolerate high concentra-
tions of caffeine are needed for efficient caffeine degradation.
In order to develop an economical bioprocess, the kinetic data
of cell growth and caffeine utilization is very important. So far,
no reports are available on the kinetics of cell growth and
caffeine utilization.
Previously we isolated Pseudomonas sp. GSC 1182 from
soil of coffee and tea cultivation area capable of utilizing
caffeine as the sole carbon and nitrogen source (Gokulakrish-
nan et al., 2005 currently under review). The caffeine
degradation rate by the isolate was enhanced in the presence
of sucrose in the medium, which was not consumed. The isolate
could utilize fructose as carbon source (100% consumption),
yielding higher biomass and caffeine degradation. However, the
presence of glucose in the medium completely inhibited the
growth and caffeine degradation. Based on these results, it has
been hypothesized that sucrose somehow induces the caffeine

1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2005.12.018
1418 S. Gokulakrishnan, S.N. Gummadi / Process Biochemistry 41 (2006) 1417–1421

degradation enzymes (Gokulakrishnan et al., 2005 currently region non-linear least square algorithm. The following constraint was given to
mm and KS in all models while estimating the parameters
under review). In this study, kinetics of cell growth in medium
containing various concentrations of caffeine was studied and mmax  mm  3mmax ; KS > 0 (2)
the kinetic data were fitted to different models available in the Here mmax is the maximum specific growth rate, which would be obtained if
literature. The results showed that the isolate was able to grow the growth was not substrate inhibited and was calculated by fitting the data of
at a very high initial concentration of caffeine (10 g/l). uninhibited region to Monod’s equation.
The yield YX/S (g dry weight/g substrate) was calculated by
ðXM
X0 Þ
YX=S ¼ (3)
2. Materials and methods ðS0
SM Þ

2.1. Materials
Caffeine (>99% pure) was purchased from Merck Limited. All other
chemicals were purchased from SRL Chemicals, India.
2.2. Microorganism
Pseudomonas sp. GSC 1182 was isolated from soil of coffee cultivation area
was maintained and sub-cultured once in every week in caffeine medium (CAS)
containing (g/l): Na2HPO4, 0.12; KH2PO4, 1.3; CaCl2, 0.3; MgSO47H2O, 0.3;
caffeine, 1.2; sucrose, 5; FeSO47H2O, 0.001 at pH 5.5. For solidification, CAS
medium was supplemented with 25 g/l of agar. The strain did not show any
change in adaptation during sub-culture since the initial existence of the isolate
was in caffeine rich natural environment.
where XM and X0 are the maximum and initial dry cell concentrations
and SM and S0 are substrate concentration at maximum and initial dry cell
concentrations.
2.5. Analytic methods
Cell growth was measured by monitoring the optical density at 600 nm
(OD600 of 0.5 corresponds to 0.379 g dry weight/l). Caffeine was estimated by
HPLC (Agilent 1100 series) equipment using a ZORBAX C-18 column with
10 mM ammonium phosphate buffer (pH 2.5)/acetonitrile (4:1, v/v) as mobile
phase. Pure caffeine at 2 g/l was used as the standard. Retention time of caffeine
was found to be 5.1 min at a flow rate of 1 ml/min and at 28.5 8C.
3. Results and discussion

2.3. Kinetic experiments 3.1. Time course of growth of Pseudomonas sp. GSC 1182
at various concentrations of caffeine
A single colony of the strain from CAS-agar plates was transferred to 25 ml
sterile nutrient medium in 100 ml Erlenmeyer flask. The nutrient medium had The isolate Pseudomonas sp. GSC 1182 was capable of
the following composition (g/l): beef extract, 1; yeast extract, 2; peptone, 5;
NaCl, 5. The flask was incubated for 10 h at 30 8C and 180 rpm. About 4% (v/v)
utilizing caffeine as the sole carbon and nitrogen source. The
of the seed culture was transferred to 25 ml of CAS medium containing various presence of sucrose in the medium enhances the caffeine
initial caffeine concentrations ranging from 0.05 to 20 g/l in 100 ml Erlenmeyer degradation but the isolate was not able to utilize sucrose
flasks and incubated on a rotary shaker at 180 rpm and at 30 8C. Samples were (Gokulakrishnan et al., 2005 under review).1 The cell growth
collected at different intervals of time and measured for cell growth and caffeine (X) increased with increase in initial substrate (caffeine)
degradation. For each initial concentration of caffeine, specific growth rate was
calculated in exponential phase. The specific growth rate (m) in exponential
concentration up to 4 g/l. Further increase in S, the cell growth
phase is calculated by the following equation: decreased and no growth was observed at 20 g/l. The strain
ðX2 =X1 Þ
exhibited shorter lag periods until the substrate concentration
m ¼ ln (1) was 2.5 g/l and longer lag was observed at higher concentra-
ðt2
t1 Þ
tions of caffeine. It has been reported that the growth of
where X1 and X2 are the cell dry weight obtained at time t1 and t2, respectively. microorganisms was inhibited when the initial caffeine
All experiments were performed in triplicates under identical conditions and all
results had a standard deviation 5% to 10% about the mean.
concentration was greater than 2.5 g/l [8,9]. It was also
reported that after several years of maintenance on 1 g/l
2.4. Kinetic models caffeine Pseudomonas sp. have gained the ability to grow on
high caffeine concentration (5 g/l) after appreciable delay
The substrate inhibition models considered to explain the cell growth [10]. However, after several years of maintenance on 5 g/l of
kinetics are Haldane, Double exponential, Edwards, Luong, Webb, Moser caffeine the isolate was reported to develop more resistance to
and Teissier (Tables 1 and 2). The parameters of different growth models
5 g/l of caffeine. But the isolate could not develop resistance
considered were estimated using MATLAB1 software. Since the models had
non-linear coefficients, the parameters were estimated iteratively with trust for 7.5 g/l of caffeine [10]. In contrast, the present strain
was able to grow in medium containing 10 g/l caffeine
Table 1 concentration without acclimatization to this concentration in
Estimated parameters of various substrate inhibition models explaining only lab because the isolate was naturally adapted to high
uninhibited region concentrations of caffeine. The complete utilization of
Model Equation Parameter R2 caffeine was observed within 48 h when the initial concen-
S n tration was up to 4 g/l. At higher substrate concentration
Moser m ¼ KmSmþS n
n = 0.6735, KS = 0.8302, 0.9404
mm = 0.219 (5–10 g/l) 50% caffeine was consumed in 72 h. The caffeine
h
i degradation was very low (10% in 72 h) when the initial
Tessier m ¼ mm 1
exp
S KS = 0.5262, mm = 0.16 0.9178
KS

S½1þðS=Ki Þ
Webb m ¼ KmmþSþðS 2 =K Þ
Ki = 1167, KS = 0.3386, 0.9213 1
S i
mm = 0.1664 The revised manuscript was currently under review in International Journal
of Food Microbiology.
 S. Gokulakrishnan, S.N. Gummadi / Process Biochemistry 41 (2006) 1417–1421 1419

Table 2
Estimated parameters of various substrate inhibition models explaining the entire data
Model Equation Parameter R2
Haldane m ¼ mm S=ðKS þ SÞð1 þ ðS=Ki ÞÞ mm = 0.478, Ki = 1.139, KS =1.137 0.8546
Edwards mm S ð
S=Ki Þ Ki = 4.496, KS = 0.9288, mm = 0.322 0.9484
m¼ KS þS exp
h

i
Double exponential m ¼ mm exp
S
S Ki = 5.701, KS = 0.701, mm = 0.2373 0.9449
Ki
exp KS

n
Luong S
m ¼ KmSmþS 1
SSm Sm = 20.17, KS = 0.5995, mm = 0.2407, n = 2.962 0.9425

caffeine was up to 15 g/l. The growth was completely 3.3. Fitting of kinetic data
inhibited when the concentration of caffeine was 20 g/l.
This is the first report on microorganisms growing at very Various substrate inhibition models were used to model the
high concentration of caffeine (10 g/l). In order to obtain a cell growth kinetics and the model parameters were estimated
stronger resistance to higher concentrations of caffeine, the
isolate can be adapted by maintaining the isolate in medium
containing high concentrations of caffeine as reported earlier
[10].

3.2. Effect of initial caffeine concentration on specific

growth rate

Specific growth rate (m) for each initial caffeine concentra-

tion (S) were calculated from the plot of ln (X) versus time
in logarithmic phase as discussed in Section 2. The slope of
the line during the exponential phase gives the specific growth
rate. Specific growth rate increased with increase in substrate
concentration till 2.5 g/l and then decreases with increase
in substrate concentration suggesting substrate inhibition
kinetics (Fig. 1).

Fig. 1. Plot of specific growth rate (m) vs. initial substrate concentration (S).
The isolate was grown in 25 ml of CAS medium (pH 6) with various initial
caffeine concentration viz. (g/l) 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 4.0, 5.0, 7.5, 10.0,
15.0 and 20.0 in 100 ml conical flasks at 180 rpm and 30 8C. Samples were
taken at various time points to measure cell mass produced. m for each initial
caffeine concentration was calculated as discussed in Section 2. The experi-
ments were performed in triplicates under identical conditions and the average
result had a standard deviation varying between 5% and 10% about the
mean.
Fig. 2. Simulated specific growth rates by various substrate inhibition models.
The parameters of different growth models considered were estimated using
MATLAB1 software. The parameters were estimated as described in Section 2.
Here mmax is the maximum specific growth rate which would be obtained if the
growth is not substrate inhibited and was calculated by fitting the data of
uninhibited region to Monod’s equation. The experiments were performed in
triplicates under identical conditions and the average result had a standard
deviation varying between 5% and 10% about the mean. (a) Models that
were fitted only to uninhibited data. (b) Models that were fitted to entire data.
1420 S. Gokulakrishnan, S.N. Gummadi / Process Biochemistry 41 (2006) 1417–1421

Fig. 3. Parity plot for the predictions of specific growth rate by various substrate
inhibition models that fitted for the entire data. Standard deviation was
calculated for experiments performed in triplicates under identical conditions.

using MATLAB (Tables 1 and 2). Certain substrate inhibition

models are able to fit only the uninhibited data (Table 1,
Fig. 2a) and certain models fit to the entire data (Table 2,
Fig. 2b). Parity plot showing the estimated specific growth
rate by different models that fit to the entire data versus
experimental specific growth rate is shown in Fig. 3. Even
though, the simulated specific growth rates by Edwards and
Double exponential model lie within 1s line (Fig. 3), the
kinetic parameters (mm, KS) estimated did not match with
experimental values. Among all the tested models, Luong
model explained the growth kinetics better than other models
(Table 2, Fig. 2b). Luong model describes the substrate

Fig. 4. Determination of YX/S values during the growth of Pseudomonas sp.
GSC 1182 on caffeine. YX/S = (XM
X0)/(S0
SM). X0 is the cell mass at time
t = 0; XM is the maximum cell mass obtained; S0 is the substrate concentration at
time t = 0; SM is the substrate concentration at XM. The yield factor was
calculated only in the region where there was 100% utilization of the substrate
(till S = 4 g/l) with in 72 h. Results are the average values of experiments
performed in triplicates under identical conditions whose standard deviation
varying between 5% and 10% about the mean.
Fig. 5. The effect of medium conditions on specific growth rate (m). The isolate
was grown in 25 ml of CAS medium with 1.2 g/l caffeine in 100 ml conical
flasks. In order to study the effect of pH on m, the initial pH of medium was
varied between 4 and 8 and the strain was grown at 180 rpm and 30 8C. To study
the effect of temperature on m, the strain was grown at 180 rpm with various
incubation temperatures (8C) viz., 25, 30, 35, 40 and 45. Similarly the isolate
was grown at 30 8C in CAS media at various shaking speeds (rpm) viz., 100, 180
and 250 to study the effect of shaking speed on m. The experiments were
performed in duplicates under identical conditions and the average result had a
standard deviation varying between 5% and 10% about the mean. (a) Effect
of initial pH of medium on m. (b) Effect temperature on m. (c) Effect of shaking
speed on m.
 S. Gokulakrishnan, S.N. Gummadi / Process Biochemistry 41 (2006) 1417–1421
inhibition kinetics of cell growth with single limiting
substrate and the model is as follows:
 n
m S S caffeine concentration on the growth of Pseudomonas sp. GSC
m¼ m 1

KS þ S Sm
mm is the maximum specific growth rate, m the specific growth
rate, S the limiting substrate concentration (caffeine in this
study), KS the Monod half saturation constant, SM the max-
imum substrate inhibitory concentration at which no growth
was observed and n is the constant which accounts the
relationship between m and S [11]. The value of Sm predicted
by Luong model (20.2 g/l) was close to the experimental
observed value (20 g/l). The constant ‘n’ estimated by Luong
model was 2.9 which was greater than 1 suggesting that the
non-linear relationship (concavity upward) between m and S
exist during inhibition [11]. Hence a rapid initial drop was
followed by a slow decrease to zero. The parameters mm and
KS estimated by Luong model (0.24 1/h and 0.59 g/l, respec-
tively) were closer to the experimentally obtained mm and KS
values (0.17 1/h and 0.34 g/l, respectively). These results
suggest that Luong model explains the kinetic data better
than other models.
A plot of (Xm
X0) versus (S0
SM) was made in the region
where complete utilization of substrate (up to 4 g/l) occurred
within 72 h. The slope of the line gives yield, which was found
to be 0.2621 g dry weight/g substrate consumed (Fig. 4). It
should be noted that the yield obtained in this study is low
because of probable usage of substrate for cell maintenance at
higher concentrations of caffeine. In addition, the yield will
also depend on the extent of reduction of initial substrate used
[12].
3.4. Effect of medium conditions on specific growth rate
The effect of medium conditions such as pH, temperature
and shaking speed on specific growth rate was studied. For
this purpose, the isolate was grown at an initial concentration
of caffeine of 1 g/l since the maximum m was obtained
between 1 and 2.5 g/l. When the initial pH of medium was 4,
specific growth rate was nearly 2.5 times less than the
optimum pH of 6.0 for caffeine degradation (Fig. 5a). It was
observed that specific growth rate increased with increase in
temperature and it decreased sharply beyond 35 8C (Fig. 5b).
It was also found that shaking speed in the range 100–
250 rpm did not have pronounced effect of specific growth
rate (Fig. 5c).
1421
4. Conclusions
The main aim of this study is to understand the effect of
(4)
1182. This is the first report on the kinetics of cell growth on
medium containing caffeine as the limiting nutrient. The kinetic
studies revealed that the growth of Pseudomonas sp. GSC 1182
was inhibited by substrate concentration greater that 2.5 g/l.
But the isolate was able to grow even at high concentrations of
caffeine (10 g/l) with low specific growth rate. These results
indicated that this strain was found to be best strain
withstanding higher concentrations of caffeine reported so
far. The specific growth rate of this isolate at substrate
concentration can be increased by acclimatizing the strain by
adapting it to grow by enrichment in medium containing higher
concentration of caffeine. Among the various models con-
sidered, Luong model was able to explain the cell growth
kinetics and its kinetic parameters are Sm = 20.2 g/l, KS =
0.59 g/l, mm = 0.24 1/h and n = 2.9.
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