Biochemical Engineering Journal 36 (2007) 288–293

Enhanced biodegradation of caffeine by Pseudomonas

sp. using response surface methodology
Swati Sucharita Dash, Sathyanarayana N. Gummadi ∗
Department of Biotechnology, Indian Institute of Technology-Madras, Chennai 600 036, India
Received 27 February 2006; received in revised form 2 February 2007; accepted 4 March 2007

Abstract
Pseudomonas sp. NCIM 5235 capable of degrading high concentrations of caffeine (>5 g/l) has been previously isolated from the soil of coffee
plantation area. The critical medium components viz., KH2 PO4 , Na2 HPO4 , caffeine and Fe2+ affecting the rate of caffeine degradation were
determined by Plackett–Burman design. These critical parameters were further optimized using response surface methodology. The optimum
concentrations of KH2 PO4 , Na2 HPO4 , caffeine and Fe2+ were found to be 3.4 g/l, 0.352 g/l, 6.4 g/l and 0.075% (w/v), respectively. Under optimal
conditions the caffeine degradation rate was increased to 0.18 g/(l h) from 0.1 g/(l h). Although the approach used for optimization is conventional,
the increase in the rate of degradation of caffeine is significant. This is the first report on a strain capable of growing at higher concentrations of
caffeine (>5 g/l) at maximum rate of degradation (0.18 g/(l h)).
© 2007 Elsevier B.V. All rights reserved.

Keywords: Caffeine degradation; Pseudomonas; Medium optimization; Plackett–Burman design; Central composite design

1. Introduction
Among the various alkaloids produced by plants, the purine
alkaloid caffeine (1,3,7-trimethylxanthine) finds greater com-
mercial importance due to its wide application in popular
beverages like coffee, tea, and various soft drinks and also in
pharmaceutical preparations. Although caffeine consumption
increases alertness and concentration by overcoming fatigue
[1], research on this compound has revealed many deleterious
effects that it may have on the human body. Prolonged caffeine
consumption not only leads to addiction and subsequent with-
drawal effects like headaches, nausea and drowsiness [2], it also
results in many clinical conditions like increase in blood pres-
sure [3,4], osteoporosis and cardiac arrhythmia [5,6]. The reason
for the increasing need for decaffeinated products can therefore
be well understood. In addition, the by-products (coffee pulp
and husk) of coffee processing that are rich in carbohydrate and
proteins [7,8] which can be used as animal feed or for the produc-
tion of organic fertilizers, single-cell protein (SCP) and biogas
[9–11] is otherwise discarded due to the presence of antinutri-
tional factors like tannins and caffeine. The wastes are generally
∗ Corresponding author. Tel.: +91 44 2257 4114; fax: +91 44 2257 4102.
E-mail address: gummadi@iitm.ac.in (S.N. Gummadi).
dumped into water bodies and xenobiotics released from them
then make the water and soil toxic. Hence decaffeination of the
by-products becomes a very necessary step in treatment of coffee
and tea wastes. Decaffeination is mostly carried out by chemi-
cal or physical techniques that are non-selective, expensive and
poses a threat to the environment in terms of release of toxic
compounds. Alternatively, microbial and enzymatic techniques
for caffeine removal have been found advantageous than other
methods [12].
In the literature, strains belonging to Pseudomonas, Serratia,
Klebsiella, Rhodococcus, Alcaligenes, Aspergillus, Penicillium
and Stemphylium have been reported to be able to degrade caf-
feine [13–21]. The highest rate of caffeine degradation among
these strains was observed for Pseudomonas sp. which was capa-
ble of growing at 5 g/l of caffeine with a degradation rate of
0.09 g/(l h) [15]. Keeping in view the high concentrations of
caffeine in effluents (∼10 g/l caffeine) and food products, the
strain for development of biodecaffeination process should be
capable to tolerate and degrade high concentrations of caffeine
at a faster rate. This has been one of the bottlenecks in the area
of biodecaffeination.
In this context, previously we had isolated a strain of Pseu-
domonas from the soil of coffee plantation area that is capable
of utilizing caffeine as sole sources of carbon and nitrogen.
This strain closely resembles Pseudomonas putida based on

1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2007.03.002
 S.S. Dash, S.N. Gummadi / Biochemical Engineering Journal 36 (2007) 288–293 289

16S rRNA analysis [22]. The caffeine degrading ability of this
strain was also found to be better than the caffeine degrad-
ing activity of previously reported strains as it could bring
about complete degradation of caffeine at an initial concen-
tration of 5 g/l at the rate of 0.1 g/(l h). In the present study
an attempt was made to improve the caffeine degradation
rate by optimization of medium components using statistical
experiment design in order to enhance the rate of caffeine
degradation by this strain and make it competent for further
biotechnological studies such as immobilization and enzyme
production.
2. Material and methods
2.1. Microorganism
at 10,000 rpm for 10 min. The supernatant was used for analysis
of caffeine immediately, or stored at 4 ◦ C if immediate analysis
was not possible. The cell pellet was washed twice with Milli-Q
water and OD600 nm was measured for cell dry weight (OD600 nm
of 0.5 corresponds to 0.379 g dry weight/l).
2.4. Analytical determinations
Caffeine was estimated by HPLC (Agilent 1100 series) equip-
ment 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 (Merck) at 2 g/l was used as standard. The reten-
tion time of caffeine was found to be 4.9 min at a flow rate of
1 ml/min and at 28 ◦ C. Detection of caffeine was done at 254 nm
using UV detector.

Pseudomonas sp. NCIM 5235 was maintained at 4 ◦ C on
nutrient agar medium which had the following composition (g/l):
beef extract, 1; yeast extract, 2; peptone, 5; NaCl, 5; agar, 25 and
was subcultured every 2 weeks.
2.2. Media
The composition of basal medium (BM) was as follows (g/l):
Na2 HPO4 , 0.12; KH2 PO4 , 1.3; CaCl2 , 0.3; MgSO4 ·7H2 O, 0.3.
CAS medium consisted of BM supplemented with 5 g/l sucrose,
1.2 g/l caffeine and 0.02% (w/v) of Fe2+ . The initial pH of the
medium was 6. For solid medium, agar (25 g/l) was added to
CAS medium.
2.3. Flask culture experiments
Three loopfuls of 36 h grown culture from CAS plate was
transferred aseptically to 25 ml nutrient broth medium and incu-
bated to mid-log phase (OD600 nm ∼ 1.4) for 2.5 h on a rotary
shaker operating at 180 rpm at 30 ◦ C. Twenty-five milliliters of
sterile CAS medium in 100 ml Erlenmeyer flask, the compo-
sition of which was varied according to the Plackett–Burman
design or CCD (Tables 1 and 4) was inoculated with 6% (v/v)
of the culture and incubated for 4 days at 30 ◦ C and 180 rpm. At
regular time intervals 1 ml of culture was taken and centrifuged
2.5. Experimental designs and data analysis
2.5.1. Screening of the medium components using a
Plackett–Burman design
Plackett–Burman design [23], well established statistical
technique for medium component optimization [24,25] was
applied to screen the medium components critically affect-
ing degradation of caffeine by Pseudomonas sp. NCIM 5235.
Briefly, the medium components were screened for seven vari-
ables at two levels, maximum (+) and minimum (−). According
to the Plackett–Burman design, the number of positive signs (+)
is equal to (N + 1)/2 and the number of negative signs (−) is equal
to (N − 1)/2 in a row. A column should contain equal number of
positive and negative signs. The first row contains (N + 1)/2 posi-
tive signs and (N − 1)/2 negative signs and the choice of placing
the signs is arbitrary. The next (N − 1) rows are generated by
shifting cyclically one place (N − 1) times and the last row con-
tains all negative signs. The experimental design and levels of
each variable is shown in Table 1. The medium was formulated
as per the design and the flask culture experiments and analyti-
cal techniques were carried out as described in Sections 2.3 and
2.4. Response was calculated as the rate of caffeine degradation
expressed as g/(l h). All experiments were performed in tripli-
cates and the average of the rate of degradation was considered
as the response.

Table 1
The experimental design using the Plackett–Burman method for screening of medium components affecting caffeine degradation by Pseudomonas sp. NCIM 5235a

Run X1 X2 X3 X4 X5 X6 X7 Caffeine degradation rates (g/(l h))b

1 −1 +1 +1 −1 −1 +1 +1 0.100
2 +1 −1 +1 +1 −1 −1 +1 0.125
3 +1 +1 −1 +1 +1 −1 −1 0.120
4 −1 +1 +1 −1 +1 +1 −1 0.154
5 −1 −1 +1 +1 −1 +1 +1 0.120
6 +1 −1 −1 +1 +1 −1 +1 0.142
7 +1 +1 −1 −1 +1 +1 −1 0.098
8 −1 −1 −1 −1 −1 −1 −1 0.167
a X , KH PO at a high concentration of 2.6 g/l and low concentration of 1.3 g/l; X , Na HPO at a high concentration of 0.24 g/l and low concentration of 0.12 g/l;
1 2 4 2 2 4
X3 , MgSO4 ·7H2 O at a high concentration of 0.6 g/l and low concentration of 0.3 g/l; X4 , CaCl2 ·2H2 O at a high concentration of 0.6 g/l and low concentration of
0.3 g/l; X5 , sucrose at a high concentration of 10 g/l and low concentration of 5 g/l; X6 , caffeine at a high concentration of 10 g/l and low concentration of 5 g/l; X7 ,
Fe2+ at a high concentration of 0.04 % (w/v) and low concentration of 0.02 % (w/v); +1 and −1, the higher and lower concentration of variable, respectively.
b Experimental values are average of triplicates within ±5% standard error.
290 S.S. Dash, S.N. Gummadi / Biochemical Engineering Journal 36 (2007) 288–293
The effect of each variable was calculated using the following
equation:
 rates obtained in the screening experimentsa
Mi+ − Mi−
Exi = , (1)
N
where Exi is the effect of the tested variable, Mi+ and Mi− the
rates of caffeine degradation from trials in which the variables
being measured were added to the medium at their maximum and
minimum level respectively and N is the number of experiments
carried out, which is 8 in this case, as the number of variables to
be tested is 7.
The standard error (S.E.) of the variables was the square root
of variance and the significance level (P value) of each variable
is calculated by using the Student’s t-test:
Exi
t= , (2)
S.E.
where Exi is the effect of the tested variable. The variables
with higher confidence levels were considered to influence the
response or output variable.
2.5.2. Optimization of medium components using a central
composite design
The screened medium components affecting caffeine degra-
dation were optimized using central composite design [26,27].
According to this design, the total number of treatment combi-
nations 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 = (3)
␦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 axial points and six replicates
at the center point with a total number of 30 experiments was
employed for optimizing the medium components.
The behavior of the system was explained by the following
quadratic equation:
  
Y = β0 + β i xi + βii xi2 + βij xi xj (4)
where Y is the predicted response, β0 the intercept term, βi the
linear effect, βii the squared effect, and βij is the interaction
effect. The regression equation (Eq. (2)) was optimized for max-
imum value to obtain the optimum conditions using MATLAB®
version 7.0 (Mathworks Inc., Natick, MA, USA).
3. Results and discussion
3.1. Screening of medium constituents affecting caffeine
degradation rate by Pseudomonas sp. NCIM 5235
A total of seven medium components were analyzed for
their effect on caffeine degradation rates by Pseudomonas sp.
NCIM 5235 using the Plackett–Burman design. The experimen-
tal design and corresponding responses (caffeine degradation
Table 2
Coefficients, effects, and t-values of calculated from the caffeine degradation
P > |t|
Variable Coefficients Effect t-Value
X1 −0.007 −0.014 −1.34875 0.15
X2 −0.012 −0.0204 −2.31214 0.05
X3 −0.003 −0.006 −0.57803 0.29
X4 −0.0015 −0.003 −0.28902 0.34
X5 +0.0002 +0.0004 0.038536 0.39
X6 −0.012 −0.0204 −2.31214 0.05
X7 −0.0065 −0.013 −1.25241 0.15
a Variables coded are the same as mentioned in Table 1.
rates) are shown in Table 1. Each row of the table represents an
experiment involving all the seven independent variables. The
signs + and − represent the higher and lower concentrations of
the independent variables under study. The rate of degradation
of caffeine for each experimental run is also shown in Table 1.
Based on this data, the coefficient and effect of each variable
was calculated and the significant levels of the variables were
estimated by t-test (Table 2). The t-values for four variables,
i.e. KH2 PO4, Na2 HPO4, caffeine and Fe2+ were observed to
be higher as compared to the other three variables (sucrose,
MgSO4 ·7H2 O and CaCl2 ·2H2 O) and represented a confidence
level ≥85%. Thus the analysis showed that KH2 PO4 , Na2 HPO4 ,
caffeine and Fe2+ have a significant effect on the degradation of
caffeine by Pseudomonas sp. The four variables thus screened
were further analyzed for their optimal combinations by a central
composite design and the interactions between these variables
were also studied.
Originally, degradation studies were done at initial caffeine
concentration of 1.2 g/l. With repeated subculturing and manip-
ulation of seed age it was observed that Pseudomonas sp. NCIM
5235 was able to completely degrade caffeine at an initial con-
centration of 5 g/l at a rate of 0.1 g/(l h), which was higher than
the values reported so far [15,18]. It was therefore of inter-
est to see if change in the medium component has any effect
on the rate of degradation. The results of Plackett–Burman
screening design seem to be in agreement with experimental
findings. MgSO4 ·7H2 O and CaCl2 ·2H2 O serve as micronutri-
ents for the bacteria and increase in their level would hardly
affect the degradation rate, as indicated from the analysis. Pre-
vious studies had shown that Pseudomonas sp. used in the
study did not utilize sucrose in the medium but the presence of
sucrose enhanced the degradation of caffeine. The concentration
of sucrose in the range of 0.5–5 g/l did not show any effect on the
degradation of caffeine [28]. Plackett–Burman screening design
analysis also confirmed that sucrose has no substantial effect on
caffeine degradation rates compared to KH2 PO4 , Na2 HPO4 , caf-
feine and Fe2+ . As caffeine undergoes conversion into simpler
compounds, the metabolites accumulate in the culture medium
bringing about an increase in pH [28]. These changes in pH
from acidic to alkaline results in inhibition of bacterial growth
and caffeine degradation. KH2 PO4 and Na2 HPO4 are known
to confer buffering capacity to the medium and change in any
of these two parameters would affect the caffeine degradation
rate, which is largely dependent on pH of the medium [28]. Pre-
 S.S. Dash, S.N. Gummadi / Biochemical Engineering Journal 36 (2007) 288–293
Table 3
Coded and uncoded values of medium components used for central composite
design
Medium component Coded values
−2 −1 0 +1
KH2 PO4 (g/l) 1.95 2.6 3.25 3.9
Na2 HPO4 (g/l) 0.18 0.24 0.30 0.36
Caffeine (g/l) 5 7.5 10 12.5
Fe2+ (%, w/v) 0.015 0.03 0.045 0.06
liminary results showed that the growth of the isolate ceased
after some time in a medium with 10 g/l caffeine due to the
accumulation of metabolites which resulted in pH changed to
9.0. At this increased pH, the expression of caffeine degrad-
ing enzymes was possibly repressed, as observed in studies on
Klebsiella and Rhodococcus and Alcaligenes sp. [16,17], which
accounts for the termination of bacterial growth. Hence these
two variables are significant and this has been also established
by analysis of Plackett–Burman design. The significance of caf-
feine concentration is also indicated by the high coefficient and
t-value for caffeine obtained from analysis of data obtained from
Plackett–Burman design (Table 2). The fourth variable, i.e. Fe2+
also plays a crucial role in caffeine degradation by Pseudomonas
sp. NCIM 5235. It has been shown in previous studies with caf-
feine degrading enzymes that Fe2+ has a role in electron transfer
and is therefore necessary for activity of the enzymes which
are basically metalloproteins [16,29]. Also, earlier studies with
metal ions have shown that caffeine degradation is enhanced in
the presence of Fe2+ as compared to other metal ions, which is
due to the increase in cell dry weight [28].
3.2. Optimization of medium components using central
composite design
The critical nutrient components viz., KH2 PO4 , Na2 HPO4 ,
caffeine and Fe2+ affecting the caffeine degradation rates were
selected for further optimization by response surface method-
ology. A central composite design was used for studying the
interaction of these variables within a range of −2 to +2 in rela-
tion to caffeine degradation (Table 3). The experimental plan is
given in Table 4. The results obtained from the central compos-
ite design experiments were fitted to a second order polynomial
equation to explain the dependence of caffeine degradation on
the medium components1 :
Y = 0.175 − 0.002708x1 − 0.001458x2 − 0.014375x3
−0.003542x4 − 0.017031x12 − 0.015781x22
−0.025781x32 − 0.0008281x42 − 0.0010313x1 x2
−0.009687x1 x3 + 0.002813x1 x4 − 0.015312x2 x3
+0.004687x2 x4 − 0.022188x3 x4
1 Standard error value of x1 , x2 , x3 and x4 coefficients is 0.0061; standard error
value of x12 , x22 , x32 and x42 coefficients is 0.00576; standard error value of x1 x2 ,
x1 x3 , x1 x4 , x2 x3 , x2 x4 and x3 x4 coefficients is 0.00754.
291
+2
4.55
0.42
15
0.075
Fig. 1. Response surface plot showing the effect of caffeine and Fe2+ at optimal
KH2 PO4 and Na2 HPO4 on caffeine degradation rate.
where Y is the predicted response, x1 the coded value of X1
(KH2 PO4 ), x2 the coded value of X2 (Na2 HPO4 ), x3 the coded
value of X3 (caffeine) and x4 is the coded value of X4 (Fe2+ ).
The analysis of variance of the quadratic regression model (Eq.
(5)) suggested that the model is very significant as indicated by
a low probability value [(Pmodel > F) = 0.01] by the Fisher’s sta-
tistical test for ANOVA (Table 5). The values of R2 (0.8225),
adjusted R2 (0.6) and R (0.91) also indicated that the model
predicted values are in perfect agreement with the experimental
values. MATLAB software was used to generate optimal val-
ues for the four variables described by the model equation. The
optimal values were 3.4 g/l, 0.352 g/l, 6.4 g/l and 0.075 % (w/v)
for KH2 PO4 , Na2 HPO4 , caffeine and Fe2+ , respectively. The
response surface plot showing the dependency of caffeine degra-
dation rate on X3 (caffeine) and X4 (Fe2+ ) when X1 (KH2 PO4 )
and X2 (Na2 HPO4 ) were kept at optimal levels (Fig. 1). The
peak corresponds to the maximum rate of degradation of caf-
feine and similar plots were also constructed between X1 and
X3 and so on (results not shown). The model predicted value
of caffeine degradation rate at optimal values of X1 , X2 , X3 and
X4 was found to be 0.18 g/(l h). Four sets of experiments were
carried out at the optimal values of X1 , X2 , X3 and X4 . The
experimental value of caffeine degradation rate was observed to
be 0.179 ± 0.013 g/(l h) which agreed with the model predicted
value of caffeine degradation rate.
Analysis of the data obtained from the central composite
design experiments clearly indicates the interaction between the
selected nutrients which affects the rate of degradation of caf-
feine. The degradation rates for caffeine at initial concentrations
of 7.5 g/l and 12.5 g/l were not the same in all cases. The degra-
dation rate was high (0.18 g/(l h)) when caffeine was at +1 level
(12.5 g/l) and all other variables at −1 level (Run #5). The lowest
(5) degradation rate (0.014 g/(l h)) was when all the components are
at +1 level (Run #18). This indicates that the initial caffeine con-
centration alone does not affect the rate of degradation because
two extremes in the rates were observed for the same initial con-
centration of caffeine (12.5 g/l) with different conditions of other
292 S.S. Dash, S.N. Gummadi / Biochemical Engineering Journal 36 (2007) 288–293

Table 4
Central composite design for optimizing medium components for caffeine degradation by Pseudomonas sp. NCIM 5235a
Run no. Block KH2 PO4 Na2 HPO4 Caffeine Fe2+ Rate of caffeine degradation (g/(l h))

Y (experimental) Y (model predicted)

1 1 −1 −1 −1 +1 0.115 0.105
2 1 +1 −1 −1 −1 0.14 0.111
3 1 −1 +1 −1 −1 0.125 0.121
4 1 +1 +1 −1 +1 0.13 0.161
5 1 −1 −1 +1 −1 0.18 0.148
6 1 +1 −1 +1 +1 0.08 0.083
7 1 −1 +1 +1 +1 0.05 0.078
8 1 +1 +1 +1 −1 0.08 0.075
9 1 0 0 0 0 0.175 0.177
10 1 0 0 0 0 0.175 0.175
11 2 −1 −1 −1 −1 0.05 0.06
12 2 +1 −1 −1 +1 0.135 0.122
13 2 −1 +1 −1 +1 0.145 0.14
14 2 +1 +1 −1 −1 0.105 0.086
15 2 −1 −1 +1 +1 0.03 0.059
16 2 +1 −1 +1 −1 0.10 0.11
17 2 −1 +1 +1 −1 0.08 0.10
18 2 +1 +1 +1 +1 0.014 0.016
19 2 0 0 0 0 0.175 0.155
20 2 0 0 0 0 0.175 0.155
21 3 −2 0 0 0 0.15 0.131
22 3 +2 0 0 0 0.11 0.12
23 3 0 −2 0 0 0.12 0.133
24 3 0 +2 0 0 0.15 0.127
25 3 0 0 −2 0 0.1 0.12
26 3 0 0 +2 0 0.095 0.062
27 3 0 0 0 −2 0.14 0.167
28 3 0 0 0 +2 0.19 0.153
29 3 0 0 0 0 0.175 0.193
30 3 0 0 0 0 0.175 0.193
a Experimental values are average of triplicates within ±5% standard error.

nutrients. It can be also observed that when there is a change in Fe2+ (x2 x4 ) indicating these are the two factors the interaction
only one variable with all other variable at 0 level, the rate of of which affects caffeine degradation the most. This is again in
degradation is lower as compared to the center points (Run #21 accordance with experimental results. The optimal values for the
to Run #28), except for Run #28 where the rate of degradation is variables as predicted by MATLAB were found to be within the
higher (0.19 g/(l h)) due to higher content of Fe2+ (at +2 level). design region except for that of Fe2+ which was found to be at
The rate of degradation for initial caffeine concentration of 5 g/l the extreme higher point. This showed that the model correctly
(Run #25) is lower than that of the center points where the ini- explains the influence of the chosen variables on the degrada-
tial concentration is at 10 g/l. Also the rate of degradation is tion of caffeine by Pseudomonas sp. NCIM 5235. The rate of
low when caffeine is at +2 levels indicating that caffeine can- degradation of caffeine by this bacteria achieved using the opti-
not be metabolized at such high concentrations even with the mized condition is 0.18 g/(l h) which is higher as compared to
change in nutrient composition and this finding agrees with the other bacterial species like Klebsiella and Rhodococcus with a
results obtained from kinetic studies done on the isolate where degradation rate of 0.05 g/(l h) [31] or P. putida with a degrada-
the minimum inhibitory concentration of caffeine was found to
be 20 g/l [30]. The value of responses indicates that the ranges
of variables chosen are appropriate. Table 5
ANOVA table for optimizing medium components for caffeine degradation by
Table 4 also reflects that the interactions between the vari- Pseudomonas sp. NCIM 5235a
ables seem significant in deciding the rate of degradation of
caffeine. The degradation rates were observed to be high in Source Sum of Degrees of Mean F P>F
squares freedom square
most cases where caffeine concentration was 7.5 g/l initially.
The values of coefficients obtained by the model equation (Eq. Blocks 0.007665 2 – – –
(5)) reflect the interactions of the chosen parameters in relation Model 0.047201 14 0.003372 3.701 0.0119
Error 0.011841 13 0.000911 – –
to the rate of degradation. The value of coefficient for interac-
tion terms is always greater than that of linear terms indicating Corrected total 0.066708 29 – – –
that the effect of variables is more when two factors interact. a R2 = 0.8225; R = 0.91; adjusted R2 = 0.6; average absolute devia-

The coefficient is highest for the combination of caffeine and tion = 0.03011.
 S.S. Dash, S.N. Gummadi / Biochemical Engineering Journal 36 (2007) 288–293
tion rate of 0.095 g/(l h) [15]. Therefore Pseudomonas sp. NCIM
5235 can prove useful in development of improved process of
decaffeination.
4. Conclusion
Optimization results show that with some modification in few
chosen medium components the degradation of a higher concen-
tration of caffeine could be achieved along with the enhancement
of the rate of degradation of caffeine. The degradation of caf-
feine under optimized conditions can prove useful in generation
of important metabolites like theobromine and other methylx-
anthines or can be used to recover enzymes for development of
biological methods of decaffeination. Although a conventional
technique, response surface methodology can be successfully
used to optimize the nutrient conditions and study the interaction
effect among the nutrients on caffeine degradation rate.
Acknowledgement
This work was supported by grant from Department of Sci-
ence and Technology, India.
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