Biochemical Engineering Journal 44 (2009) 136–141

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Biochemical Engineering Journal

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Enhanced degradation of caffeine by immobilized cells of Pseudomonas sp. in

agar–agar matrix using statistical approach
Sathyanarayana N. Gummadi ∗ , K.B. Ganesh, Devarai Santhosh
Applied and Industrial Microbiology Laboratory, Department of Biotechnology, Indian Institute of Technology-Madras, Chennai 600 036, India

a r t i c l e i n f o a b s t r a c t

Article history: Previously, we isolated caffeine degrading Pseudomonas strain from soil of coffee plantation area, which
Received 5 August 2008 could utilize caffeine as sole carbon and nitrogen source and could tolerate caffeine up to 20 g/L. In
Received in revised form 21 October 2008 this study, caffeine degradation by immobilized cells of this strain was investigated. Various matrices
Accepted 15 November 2008
were considered and agar–agar was chosen based on degradation rate (0.08 g/(L h)), bead stability and
reusability. Further, immobilization parameters, viz., bead size (mm), agar–agar concentration % (w/v)
Keywords:
and cell concentration (g/L) were optimized using central composite design. The optimal conditions of
Biodegradation
cell concentration, agar–agar concentration and bead size were 7.8 g/L, 5% (w/v) and 6.2 mm. Under opti-
Immobilization
Immobilized cells
mal conditions, caffeine degradation rate was found to 0.15 g/(L h), which closely agrees with the model
Kinetic parameters predicted values. This is the first report on caffeine degradation at high concentrations (10 g/L) by immo-
Submerged culture bilized cells of Pseudomonas sp. Immobilization efficiency was 80%. Damköhler number is very much
Diffusion reaction higher than 1, suggesting that mass transfer is the rate limiting process.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction methods of caffeine removal are recommended [5]. Biodegradation

Caffeine (3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione), a
purine alkaloid, is a key component in most popular beverages
especially tea and coffee. Chronic intake of caffeine leads to adrenal
stimulation, irregular muscular activity, cardiac arrhythmias, osteo-
porosis and exhibits withdrawal symptoms such as headache and
fatigue [1]. Consumption of caffeine even results in malformation
of fetus and reduces fertility rates during pregnancy [2]. Apart from
negative implications, caffeine degradation is important from envi-
ronment point of view. Effluents from coffee processing industries
have high concentration of caffeine (∼10 g/L) and disposal of such
effluents into lakes makes drinking water non-potable [3]. In addi-
tion, processed coffee and tea pulps, though, rich in nutritional
compounds, such as carbohydrates and proteins cannot be used as
fodder directly because of presence of caffeine, tannins and other
toxic compounds [4]. Hence, from both health and environmen-
tal point of view, caffeine degradation is a major issue in food
processing industries in order to develop decaffeinated food prod-
ucts.
Till date, conventional methods such as solvent extraction and
supercritical fluid extraction have been employed to remove caf-
feine [5]. These conventional methods are expensive, toxic and
non-specific to caffeine. To overcome this, microbial and enzymatic
∗ Corresponding author. Tel.: +91 44 2257 4114 fax: +91 44 2257 4101.
E-mail address: gummadi@iitm.ac.in (S.N. Gummadi).
of caffeine using whole cells or enzymes is still an unresolved prob-
lem and requires lot of studies to commercialize the process. One
of the major bottle-necks in biodegradation of caffeine is a strain
that can tolerate high concentration and can degrade caffeine in a
shorter period.
For this purpose, we have isolated a bacterium that could
utilize caffeine as sole source of carbon and nitrogen in the pres-
ence of sucrose, which was not utilized but enhanced the rate of
caffeine degradation [6]. 16S rRNA analysis revealed that the bac-
terium resembles closely to Pseudomonas putida as indicated by 87%
sequence identity [7]. The strain was also known to tolerate high
concentrations of caffeine (∼20 g/L) [8]. Induced cells of this strain
could degrade high concentration of caffeine (10 g/L) at a maximum
rate of 0.3 g/(L h) as a whole cell biocatalyst without any growth
[9]. So far, this is the highest rate reported for caffeine degrada-
tion. These results suggest that the strain can be used to develop
a successful biological process for caffeine degradation. In order to
develop a successful bioprocess, it is also important to address cer-
tain important issues such as cell loading and reusability of cell.
For this purpose, immobilized cell system is more advantageous
than free cells. In this study, several matrices were experimented
in order to immobilize the cells and it was seen that agar–agar
was best in terms of caffeine degradation rates and reusability.
Further, immobilization parameters such as bead size, cell loading
and matrix concentration were optimized using response surface
methodology. Kinetics of free and immobilized cells was studied
under optimal conditions.

1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2008.11.010
 S.N. Gummadi et al. / Biochemical Engineering Journal 44 (2009) 136–141
2. Materials and methods
2.1. Microorganism and medium
Pseudomonas sp. was maintained on caffeine agar plates which
had the following composition (g/L): caffeine, 1.2; KH2 PO4 , 1.3;
Na2 HPO4 , 0.12; CaCl2 , 0.3; MgSO4 ·7H2 O, 0.3; sucrose, 5 and agar,
25 at pH 6.0. The strain was grown at 30 ◦ C in a BOD incubator and
subcultured once in a week.
2.2. Preparation of caffeine induced cells
Three loop full of actively growing cells from caffeine agar plates
were transferred to nutrient medium (seed culture) consisting of
1 g/L of beef extract, 2 g/L of yeast extract, 5 g/L of peptone and 5 g/L
of NaCl, in 100-mL Erlenmeyer flasks. The culture was incubated on
a rotary shaker at 180 rpm and 30 ◦ C till OD600 reached to ∼1.4–1.5.
After achieving desired OD, 6% (v/v) of seed culture was inocu-
lated into the CAS (Caffeine Associated Sucrose) medium, which
had the following composition: 6.4 g/L caffeine, 3.4 g/L KH2 PO4 ,
0.352 g/L Na2 HPO4 , 0.3 g/L CaCl2 , 0.3 g/L MgSO4 ·7H2 O, 0.075% (w/v)
FeSO4 ·7H2 O, and 5 g/L sucrose at pH 7.8 [10,11]. The cells were
grown on a rotatory shaker operating at 28 ◦ C and 180 rpm till caf-
feine degradation reached 90–95%. The cells were harvested by
centrifugation (at 10,000 × g for 10 min at 4 ◦ C) and washed with
10 mM potassium phosphate buffer at pH 7.0. After washing for
three times, the cells were resuspended in the same buffer such
that the initial concentration of the induced cell was ∼550 g/L [9].
2.3. Immobilization of Pseudomonas sp. by different matrices
Preliminary experiments were performed to screen the different
immobilization matrices for efficient caffeine degradation. During
the entire experiments final cell loading in the immobilized degra-
dation experiments was 8 g/L.
2.3.1. Calcium alginate
Sterile sodium alginate (3%, w/v) solution was prepared and
induced cells were suspended in this solution such that cell con-
centration was 8 g/L. The cell–alginate mixture was gently dropped
into 0.2 M calcium chloride solution. The beads were cured in 0.2 M
CaCl2 solution for 2 h and the beads were washed with sterile
water thrice such that calcium free beads were used for degradation
experiments. The measured average size of beads was 2.5 mm.
2.3.2. Agarose and agar–agar
Sterile agarose (3%, w/v) solution was prepared and melted by
heating. Induced cells were added to the agarose solution (cooled
to temperature ≤40 ◦ C) to achieve final cell concentration of 8 g/L.
Cell–agarose mixture was poured into sterile flat bottom 50-mm
diameter Petri plates coated with trace amount of refined sun-
flower oil (to facilitate the removal of solidified agarose block from
Petri plate) and allowed to solidify for 30 min at 4 ◦ C. The solidi-
fied agarose block was cut into equal size cubes (∼2 mm) such that
surface area obtained was equal to the surface area available for cal-
cium alginate beads. Agar–agar cubes were prepared as described
for agarose.
2.3.3. Polyacrylamide
Induced cells were added to acrylamide and bis-acrylamide
solution such that cell and acrylamide concentration was 8 g/L
and 3% (w/v) respectively. Polymerization was initiated by adding
0.12 ml of 5% ammonium persulfate (APS) and 0.06 ml of 5%
N,N,N ,N -tetramethylethylenediamine (TEMED) and allowed to
solidify at 4 ◦ C [12]. The solidified block was cut into equal size
137
Table 1
Coded and uncoded values of immobilization parameters used for central composite
design.
Variable Symbol Coded values
−˛ −1 0 +1 +˛
Cell concentration (g/L) X1 1.3 4 8 12 14.7
Matrix concentration, % (w/v) X2 0.6 2 4 6 7.4
Bead size (mm) X3 2.6 4 6 8 9.4
cubes (∼2 mm) such that surface area obtained is equal to the sur-
face area available for calcium alginate beads. Agar–agar cubes were
prepared as described for agarose.
2.3.4. Polyvinyl alcohol
To 3% (w/v) of polyvinyl alcohol, induced cells were added
such that cell concentration was 8 g/L. Polymer–cell mixture was
dropped slowly in to saturated boric acid and cured for 2 h at 4 ◦ C
[13]. After curing the beads were washed three times with ster-
ile water and immobilized beads free of boric acid were used for
caffeine degradation.
2.4. Caffeine degradation experiments with immobilized cells
The various immobilized beads were suspended in 250-mL
Erlenmeyer flasks containing reaction medium (1.2 g/L in 0.75 mM
phosphate buffer, pH 7.0). The degradation was carried out by incu-
bating the flasks on rotatory shaker operated at 180 rpm and 30 ◦ C.
At regular time intervals, samples were collected and analyzed for
caffeine degradation and cell leakage.
2.5. Analytical determinations
Caffeine was estimated by HPLC (JASCO series) equipment using
a HiQSil C-18 column with methanol and water (30:70, v/v) as
mobile phase. Pure caffeine (Merck) at 2 g/L was used as standard.
The retention time of caffeine was found to be 10 min at a flow rate
of 1 mL/min and at 30 ◦ C. Detection of caffeine was done at 273 nm
using UV detector.
2.6. Experimental design
Optimization of the three parameters of the matrix for degra-
dation by immobilized cells i.e. cell concentration (X1 ), matrix
concentration % (X2 ) and bead size (X3 ) was carried out using cen-
tral composite design (CCD) [14]. The interaction of the 3 variables
was designed by coded and non-coded values (Table 1). Accord-
ing to this design, the total number of treatment combinations is
2k + 2k + n0 where k is the number of independent variables and n0
the number of repetitions of the experiments at the center point.
For statistical calculation, the variables Xi have been coded as xi
according to the following transformation:
Xi − X0
xi = (1)
X
where xi is dimensionless coded value of the variable Xi , X0 the value
of the Xi at the center point, and X is the step change.
A 2k -factorial design with eight factorial points, six axial points
and six replicates at the center point. According to the CCD, the total
number of 20 experiment runs was carried out in duplicates and
the average degradation rates were used. The degradation experi-
ments were performed as described in Section 2.4 except that the
beads were prepared according to Table 2 and the caffeine concen-
tration used in the degradation medium was 5 g/L. The behavior of
138 S.N. Gummadi et al. / Biochemical Engineering Journal 44 (2009) 136–141

Table 2
Central composite design for optimizing immobilization parameters for caffeine degradation by cells of Pseudomonas sp. immobilized in agar–agar matrix.

Run no. Block Cell concentration (g/L) Matrix concentration (%w/v) Bead size (mm) Rate of caffeine degradation (g/(L h))

Y (experimental)a Y (model)

1 1 −1 −1 −1 0.0851 0.0804
2 1 1 −1 1 0.1057 0.0974
3 1 −1 1 1 0.1408 0.1634
4 1 1 1 −1 0.1403 0.1624
5 1 0 0 0 0.1429 0.143
6 1 0 0 0 0.1429 0.143
7 2 −1 −1 1 0.1326 0.1004
8 2 1 −1 −1 0.1712 0.1374
9 2 −1 1 −1 0.1361 0.1354
10 2 1 1 1 0.1358 0.1304
11 2 0 0 0 0.1429 0.143
12 2 0 0 0 0.1429 0.143
13 3 −1.68 0 0 0.1277 0.1346
14 3 1.68 0 0 0.1414 0.1548
15 3 0 −1.68 0 0 0.0439
16 3 0 1.68 0 0.1404 0.1179
17 3 0 0 −1.68 0.1511 0.1602
18 3 0 0 1.68 0.1397 0.1501
19 3 0 0 0 0.1429 0.143
20 3 0 0 0 0.1429 0.143

R2 = 0.81; R = 0.9.
a
Experimental values are average of duplicates within ±5% standard error.

the system was explained by the following quadratic equation:
   Similarly, degradation experiments were performed under iden-
Y = ˇ0 + ˇi xi + ˇii xi2 + ˇij xi xj
where Y is the predicted response, ˇ0 the intercept term, ˇi the
linear effect, ˇii the squared effect, and ˇij is the interaction effect.
The model results were tested by comparing the value obtained
from the regression equation with experimental results obtained,
in duplicates, by setting the variables to their optimum values as
determined by the CCD.
2.7. Optimization of immobilization parameters using statistical
experimental design
Induced cells were prepared according to Section 2.2. Degrada-
tion experiments were conducted under various combinations of
cell loading, matrix composition and bead size in 25 mL of sterile
reaction medium consisting of 5 g/L caffeine in 0.75 mM potassium
phosphate buffer in 100-mL Erlenmeyer flasks. Samples were col-
lected at regular intervals of time and analyzed for caffeine. The
time profile of caffeine concentration was plotted and the caffeine
degradation rate was calculated as the initial slope of the curve.
2.8. Statistical analysis
Regression analysis of the data obtained was performed using
Design Expert package (Version 7, Stat-ease Inc., Minneapolis, MN,
USA, 2001). The coefficients of the regression equation were also
estimated using the same software. The statistical significance of
the model was determined by the application of Fischer’s F-test. The
fit between the model and the experimental data was evaluated by
ANOVA (Analysis of Variance). All the experiments were performed
twice and each measurement was done in duplicates.
2.9. Kinetics of caffeine degradation by immobilized cells
Immobilized cells were prepared according to the optimal con-
ditions (cell loading, matrix concentration, bead size) obtained from
studies performed in Section 2.6. Degradation experiments were
performed as described earlier in 25 ml of reaction medium in
which caffeine concentration was varied between 0.1 and 10 g/L in
0.75 mM potassium phosphate buffer in 100-ml Erlenmeyer flasks.
(2) tical conditions of caffeine concentration and cell loading under
free cell conditions. The time profile of caffeine concentration was
plotted and the caffeine degradation rate was calculated as the ini-
tial slope of the curve for both immobilized cells and free cells.
Immobilization efficiency was calculated as the ratio of the rate of
caffeine degradation by immobilized cells to degradation rate by
free cells under optimized conditions. Using the rates of caffeine
degradation calculated at different caffeine concentrations, recip-
rocal Lineweaver–Burk plots were used to determine the kinetic
parameters, Km and Vmax . Damköhler number, which is defined
as the ratio of the maximum reaction rate to the maximum mass
transfer rate, was calculated as follows:
rmax
NDa = (3)
(De /ı)S
where NDa is the Damköhler number, rmax is the maximum reac-
tion rate without any limitations imposed by external mass transfer
(mass transfer of caffeine from bulk liquid to cells) which was
calculated as rate of caffeine degradation under free cell condi-
tions (i.e. the maximum possible rate that can be obtained when
there is no external mass transfer), De is the effective diffusivity
of caffeine through the matrix, ı is the area available for diffusion
and S is the bulk substrate concentration. In this study, we used
De as 0.46 × 10−7 cm2 s−1 , which is the diffusivity of caffeine from
guarana seeds to supercritical carbon dioxide [15].
3. Results and discussion
3.1. Screening of different matrices for immobilization of
Pseudomonas sp.
In this study, we used Pseudomonas sp. a strain isolated from
soil of coffee plantation area, which was reported to have highest
degradation rate (0.3 g/(L h) up to 15 g/L) and can degrade high con-
centrations of caffeine (up to 20 g/L) [11]. In our earlier studies, we
reported that the cell free extract of this strain could degrade caf-
feine and its metabolites, viz., theobromine, 7-methylxanthine and
xanthine. Since the enzyme(s) was found to be highly unstable, we
used whole cells to degrade caffeine [7,16]. In order to develop a suc-
 S.N. Gummadi et al. / Biochemical Engineering Journal 44 (2009) 136–141
cessful bioprocess, we immobilized the cells to degrade caffeine and
tested them for their reusability. In this attempt, we used several
matrices such as calcium alginate, agarose, agar–agar, polyacry-
lamide and polyvinyl alcohol to immobilize Pseudomonas sp. In all
these studies, cell concentration was kept constant (8 g/L). Among
all tested matrices, polyvinylalcohol was found to be very fragile
with the beads tending to disintegrate immediately after addition to
the reaction medium (Fig. 1). Calcium alginate, one of the most com-
monly used immobilization matrix [17,18] in bioprocess showed
100% degradation of caffeine in this study (Fig. 1). Cells entrapped
in agarose and agar–agar matrices also brought about 100% degra-
dation of caffeine. However, cells immobilized in agar–agar matrix
showed highest degradation rate (0.08 g/(L h)). The rates of degra-
dation of caffeine for calcium alginate, agarose and polyacrylamide
were 0.056, 0.054 and 0.014 g/(L h) respectively. Agar–agar beads
could also be successfully reused at least for three times with-
out appreciable change in the degradation rate (data not shown).
Apart from that, it was seen that there was no appreciable decrease
in caffeine concentration (<1%) when agar beads without bacte-
rial cells and agar beads immobilized with heat inactivated cells
were used. Very few reports are available on degradation of caf-
feine by immobilized cells. It has been reported that P. putida strain
immobilized in agar matrix could degrade 99% of 0.13 g/L of caf-
feine [19]. Pseudomonas alcaligenes was known to degrade 90% of
1 g/L caffeine in 80 h using 100 g/L of cell debris [20]. In compar-
ison to these reports, Pseudomonas sp. used in this study showed
much higher degradation rate with much lower cell loading. Since
the results are promising, we optimized the parameters influencing
the immobilization to enhance degradation rate.
3.2. Optimization of immobilization parameters
Preliminary experiments were conducted to study the variables
and their levels affecting the caffeine degradation by immobilized
cells. It has been found that cell loading, matrix concentration
and immobilized bead size affects the degradation rate critically
(data not shown). The effect of caffeine (substrate) concentration
on degradation rate was studied. It has been found that degradation
rate increased with caffeine concentration and reached a maximum
between 5 and 10 g/L. Hence, caffeine concentration was fixed at
5 g/L during optimization studies.
Response surface methodology (RSM) is a collection of statistical
techniques that describe the effect of independent variables, alone
Fig. 1. Effect of different matrices used to immobilize Pseudomonas sp. for caffeine
degradation. The cell loading was constant at 8 g/L and initial concentration of caf-
feine was 1.2 g/L.
139
Table 3
ANOVA table for caffeine degradation rate by immobilized cells of Pseudomonas sp.:
effect of cell concentration, matrix concentration and bead size.
Source Sum of Degrees of Mean square P > F
F = MSR/MSE
squares (SS) freedom (DF) (MS = SS/DF)
Blocks 0.00161 2
Model 0.0171 9 0.0019 3.2 0.0532
Error 0.0046 8 0.0006
Corrected 0.02331 19
total
or in combination, on a process using limited number of experi-
ments. RSM has been successfully applied in optimizing conditions
in many bioprocesses [21–23]. The three variables viz., cell con-
centration, matrix concentration and bead size, which affect the
rate of degradation of caffeine by immobilized cells of Pseudomonas
sp. were optimized by response surface methodology. A central
composite design was used for studying the interaction of these
variables within a range of −1.68 to +1.68 in relation to caffeine
degradation (Table 1). Degradation experiments were performed as
per Table 2. At center point (run nos. 5, 6, 11, 12, 19, 20) degradation
rate of 0.14 g/(L h) was observed (Table 2). Degradation rate reduced
when cell concentration was at −1.68 (run no. 13) than at +1.68
(run no. 14). Matrix concentration and bead size influenced caffeine
degradation more than cell concentration. When matrix concentra-
tion was −1.68 (run no. 15) caffeine degradation rate was zero. The
maximum rate of degradation (0.1712 g/(L h)) was observed when
cell concentration was at +1 level with matrix concentration and
bead size at −1 level (run no. 8). Interestingly run no. 2 (where
conditions are same as that of run no. 8 except that bead size was
at +1 level) showed low degradation rate of 0.1 g/(L h) (Table 2).
This can be explained by the fact that smaller bead size (high sur-
face area) decreases the total mass transfer resistance due to the
decrease of contribution of intraparticle resistance. Similarly, intra-
particle resistance increases with bead size, which in turn increases
the total mass transfer resistance. This suggests that bead size has
prominent effect on degradation of caffeine. Lowest degradation
rate was observed in run no. 1 (0.085 g/(L h)). In run no. 7 (where
conditions are same as that of run no. 1 except that bead size was
at −1 level), degradation rate was 0.13 g/(L h). This shows that there
exists strong interaction effect between the variables and study of
this interaction effect is very important and cannot be neglected.
The results obtained from the central composite design experi-
ments were fitted to a second order polynomial equation (Eq. (3)) to
explain the dependence of caffeine degradation on the parameters
of immobilization.
Y = 0.143 + 0.006x1 + 0.022x2 − 0.003x3 + 0.006x12 − 0.022x22
+0.004x32 − 0.0075x1 x2 − 0.015x1 x3 + 0.002x2 x3 (4)
where Y is the predicted response, x1 the coded value of X1 (cell con-
centration), x2 the coded value of X2 (matrix concentration %) and
x3 is the coded value of X3 (bead size). The equation was optimized
by using the ‘Solver Tool’ of Microsoft Excel. The optimum values of
cell concentration, matrix concentration and bead size were 7.8 g/L,
5% (w/v) and 6.2 mm respectively. The interaction effect discussed
above can be observed from the quadratic equation (4). For exam-
ple, the coefficient of x1 x3 was greater than the coefficient of x1
(cell concentration) and x3 (bead size) in the order of magnitude.
This clearly suggests that there exists a strong interaction effect
between the variables and the interaction effect was accounted
using response surface methodology. The experimental values of
degradation rates were compared with those predicted by the mod-
els (Table 2). The coefficient of determination (R2 ) and coefficient
of correlation (R) was 0.81 and 0.9 respectively. By ANOVA anal-
ysis, we found that calculated F value was 3.2, which was several
140 S.N. Gummadi et al. / Biochemical Engineering Journal 44 (2009) 136–141
Fig. 2. Response surface plot showing the effect of cell concentration and matrix
concentration at optimal matrix concentration on caffeine degradation rate by
immobilized cells of Pseudomonas sp.
times greater than tabulated F8,9 at the probability level of 0.0532
(Table 3).
Surface plots of caffeine degradation rates over independent
variables, cell concentration, matrix concentration and bead size
are shown in Figs. 2–4 respectively. The response surface showed
maximum activity at the optimal points determined below, sug-
gesting that the optimal conditions determined were correct. It is
also clear from Fig. 3 that the optimal point obtained from the model
is clearly a saddle point, which lies within the selected range of
the parameters. It can be seen that the degradation rate actually
dips to a minimum around the optimal point and rises observably
in increasing direction of x1 and x3 outward from the point. Such
behavior was observed during the optimization of several biological
processes using central composite design [24].
Under the optimal conditions of variables, a maximum degrada-
tion rate of 0.148 g/(L h) was obtained from the quadratic equation.
In order to check whether the model predicted value was correct,
experiments were performed in triplicate under optimal conditions
and caffeine degradation rates were determined. It has been found
that experimental values (0.15 g/(L h)) agree very closely with the
model. This suggests that response surface methodology can be suc-
cessfully used to optimize the immobilization parameters affecting
the caffeine degradation.
Fig. 3. Response surface plot showing the effect of bead size and cell concentration
at optimal matrix concentration on caffeine degradation rate by immobilized cells
of Pseudomonas sp.
Fig. 4. Response surface plot showing the effect of bead size and matrix concentra-
tion at optimal cell concentration on caffeine degradation rate by immobilized cells
of Pseudomonas sp.
3.3. Caffeine degradation kinetics by immobilized and free cells
Immobilized cells of Pseudomonas sp. was prepared according
to the optimal conditions obtained in this study (cell concentra-
tion: 7.8 g/L; matrix concentration: 5% (w/v) and bead size: 6.2 mm).
Under the same cell loading, degradation experiments were per-
formed by varying the initial concentration of caffeine from 0.1 to
10 g/L in the reaction medium. Experiments were also performed
with free cells of Pseudomonas sp. at a cell concentration of 7.8 g/L.
The kinetic results are shown in Fig. 5 for free and immobilized
cells. It has been observed that the degradation rates under immobi-
lized conditions are lower than the rates under free cells conditions.
Immobilization efficiency with agar–agar matrix was found to be
80% ± 7 suggesting that agar–agar matrix was best. It has been
shown that induced cells of Pseudomonas sp. acts as biocatalyst
(no growth) in caffeine degradation medium (containing only caf-
feine in buffer) [9], the kinetic data were fitted to Michaelis–Menten
kinetics and the kinetic parameters were estimated (Table 4). Free
cells showed lower Km value of 0.66 g/L compared to 0.78 g/L for
immobilized cells, with corresponding R2 values for the curve fit
are 0.98 and 0.91. These values of Km indicate that the substrate,
caffeine, is more accessible to free cells than immobilized cells. This
Fig. 5. Kinetics of caffeine degradation by free and immobilized cells of Pseudomonas
sp.
 S.N. Gummadi et al. / Biochemical Engineering Journal 44 (2009) 136–141
Table 4
Kinetic parameters for caffeine degradation by immobilized and free cells.
Cell culture Vmax (g/(L h)) Km (g/L)
Immobilized 0.16 0.78
Free 0.18 0.66
also suggests that external mass transfer (caffeine from bulk to cell
in immobilized matrix) resistance affects degradation of caffeine
by immobilized cells and could be the limiting step in comparison
to the reaction rate. This was further confirmed by calculating the
values of Damköhler number at different caffeine concentrations,
which were very much higher than 1. These values suggest that
mass transfer is the rate limiting process during the degradation of
caffeine by immobilized cells of Pseudomonas sp.
4. Conclusion
This work studied the degradation of caffeine by immobilized
cells of Pseudomonas sp. Different matrices were compared and
agar–agar was observed to be the best in terms of degradation
rate as well as reusability. Optimization of the matrix parame-
ters affecting degradation rate, namely cell concentration, matrix
concentration and bead size, was carried out using response sur-
face methodology. Central composite design was used to conduct
the experiments and the results showed that there is a strong
interaction between cell concentration and bead size. This work is
significant as it is the first to report degradation of caffeine at high
concentrations of up to 10 g/L by immobilized cells. Comparison of
the rates showed that the degradation of caffeine by immobilized
cells was rate limited primarily by mass transfer of the substrate.
The results of this study could lead to practical application of Pseu-
domonas sp. for commercial decaffeination.
Acknowledgements
This work is supported by research grant from Department of
Science and Technology. Authors acknowledge Swati for her help
during the manuscript preparation.
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