Appl Microbiol Biotechnol (2011) 91:1007–1017
DOI 10.1007/s00253-011-3319-y
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Enhanced degradation of caffeine and caffeine demethylase
production by Pseudomonas sp. in bioreactors
under fed-batch mode
Sathyanarayana N. Gummadi & B. Bhavya
Received: 25 February 2011 / Revised: 7 April 2011 / Accepted: 7 April 2011 / Published online: 14 May 2011
# Springer-Verlag 2011
Abstract The growth of Pseudomonas sp. was studied in
fed-batch process with an aim to improve the caffeine
degradation rate and caffeine demethylase activity. The
effects of varying initial caffeine concentrations in the batch
mode, increase in the number of feeds, varying feed flow
rates, and added nutrients to the feed on the fed-batch process
were investigated. A maximum caffeine degradation rate of
0.82 g/L h and maximum caffeine demethylase activity of
2.6 U/mg were achieved using manual intermittent pulse feeds
of caffeine with substrate concentration as feedback parameter
for the fed batch started with an initial caffeine concentration
of 3 g/L. A slight increase in the caffeine degradation rate
(0.85 g/L h) and caffeine demethylase activity (3.4 U/mg) was
observed when the additional nutrients were added along with
caffeine in the feed. This is the first report showing complete
degradation of large magnitudes of caffeine amounting to
237 g in 75 h. These results show that the fed-batch conditions
achieved in this study using Pseudomonas sp. facilitate the
development of a sustainable bioprocess to degrade the high
concentrations of caffeine in industrial effluents.
Keywords Pseudomonas sp. . Caffeine degradation .
Caffeine demethylase . Bioreactor . Fed-batch
Introduction
Caffeine is a purine alkaloid widely used in several beverages
because of its action as a stimulant to the central nervous
S. N. Gummadi (*) : B. Bhavya
Applied and Industrial Microbiology Laboratory, Department
of Biotechnology, Indian Institute of Technology-Madras,
Chennai-36, India
e-mail: gummadi@iitm.ac.in
system. Excessive and prolonged consumption of it might
result in undesirable effects on health such as headache,
fatigue, apathy, adrenal stimulation, irregular muscular
activity, cardiac arrhythmias, and osteoporosis, and it also
has a deleterious effect on cardiac patients (Smith 2002;
Lorist and Tops 2003). Consumption of caffeine during
pregnancy causes malformation of the fetus and reduces
fertility rates (Eteng et al. 1997). Global production of coffee
was projected to grow by 0.5% annually from 1998–2010,
and the production output is expected to reach 7.0 million
tons by 2010. In addition, world consumption of coffee was
projected to increase by 0.4% annually from 6.7 million tons
in 1998–2000 to 6.9 million tons by 2010 (Murthy et al.
2004). This results in more solid wastes such as coffee husk
and tea waste, containing caffeine and other polyphenolics
which pollute the surrounding landmass by increasing
toxicity and bacterial contamination (Adams and Dougan
1981; Bressani 1987). The effluents from coffee and tea
processing industries has COD, BOD, and caffeine levels as
high as 5, 3, and 0.3 g/L, respectively (De Matos et al. 2001;
Weigel et al. 2004). Also, the caffeine content in black tea or
coffee is 1–2 g/L (Wanyika et al. 2010). Hence, there is a
growing need for decaffeination of both the consumables
and the wastes.
Several decaffeination processes like solvent extraction
and supercritical CO2 method are popular but have certain
disadvantages such as usage of toxic and expensive
solvents and non-specific extraction. Because of this,
flavor and aroma substances present in coffee and tea
are removed, and they are replaced by artificial com-
pounds (Gokulakrishnan et al. 2005). Among the current
methods, supercritical CO2 is efficient but expensive and
non-specific. This resulted in the development of biopro-
cess using microbes or microbial enzymes that can
selectively degrade caffeine which could be economic and
1008
eco-friendly. In addition, biodecaffeination technology can
be modified for the selective production of value-added
products like theobromine from caffeine present in waste
(Asano et al. 1993).
The bottleneck in the development of a viable bioprocess
for decaffeination is the availability of a strain which can
tolerate high caffeine concentrations as well as degrade it at
faster rates. Numerous strains like Pseudomonas, Klebsiella,
Serratia, Penicillium, Alcaligenes, and Aspergillus were
reported to utilize caffeine as the sole carbon and nitrogen
source (Yu et al. 2009; Beltrána et al. 2006; Woolfolk 1975;
Asano et al. 1993; Yamaoka-Yano and Mazzafera 1998;
Madhyastha and Sridhar 1998; Mazzafera et al. 1994;
Mohapatra et al. 2006; Hakil et al. 1999). Among these,
Pseudomonas putida is the most comprehensively studied
strain due to its ability to tolerate high concentrations of
caffeine. However, the strains reported so far are not capable
of degrading more than 5 g/L caffeine, and the degradation
rates are no more than 0.1 g/L h (Woolfolk 1975).
In this regard, we isolated Pseudomonas sp., which
could utilize caffeine as the sole carbon and nitrogen source
(Gokulakrishnan et al. 2007; Dash and Gummadi 2006).
Previously we have shown that this strain could tolerate and
degrade up to 20 g/L of caffeine (Gokulakrishnan and
Gummadi 2005; Gummadi and Devarai 2010). However,
growth rate was very low for caffeine concentrations
greater than 10 g/L and exhibits higher lag times (∼24–
60 h) (Gummadi et al. 2009). It also showed a rate of
caffeine degradation up to 0.29 g/L h after optimizing the
nutrients and physical parameters—pH, agitation, and
temperature (Dash and Gummadi 2007a, b). The enzyme
caffeine demethylase produced by this strain is found to be
responsible for caffeine degradation (Dash and Gummadi
2008). The production of this enzyme was studied in a
bioreactor, and a maximum of 18,762 U/g cell dry weight
was achieved by optimizing the batch fermentation conditions
(Gummadi et al. 2009). However, the degradation rate was
still 0.29 g/L h. To enhance caffeine degradation and also the
production of caffeine demethylase, an enzyme involved in
sequential demethylation of caffeine to xanthine, fed-batch
cultures were investigated in this study. This is the first study
to report complete degradation of high amounts of caffeine
with a competent degradation rate which makes this a
potential strain for caffeine effluent treatments and industrial
decaffeination.
Materials and methods
Chemicals
Pure caffeine (1,3,7 trimethylxanthine) was obtained from
Merck. NADH and dithiothreitol (DTT) were obtained
Appl Microbiol Biotechnol (2011) 91:1007–1017
from Sigma. All other reagents used in this study were of
analytical grade.
Microorganism and medium
Pseudomonas sp. NCIM 5235 was maintained on caffeine
associated sucrose (CAS) agar medium of composition (in
grams per liter): Na2HPO4, 0.12; KH2PO4, 1.3; CaCl2, 0.3;
MgSO4·7H2O, 0.3; sucrose, 5; caffeine, 1.2; and agar, 25
(pH adjusted to 6.0) at 30°C in a BOD incubator and sub-
cultured every alternate day. Reactor studies were done
with optimized CAS medium with the following composi-
tion (in grams per liter): Na2HPO4, 0.35; KH2PO4, 3.4;
CaCl2, 0.3; MgSO4·7H2O, 0.3; sucrose, 5; caffeine, 6.5;
and FeSO4, 0.075% (w/v) (Dash and Gummadi 2007a).
Batch bioreactor
The batch bioreactor experiments were performed in a
stirred 7.5-L bioreactor (Bioflo 110, New Brunswick
Scientific, USA) with 3.75 L as working volume. For the
seed culture, three loops of actively growing cells from
CAS agar plates were transferred to a 25-ml nutrient
medium containing (in grams per liter): beef extract, 1;
yeast extract, 2; peptone, 5; and NaCl, 5. When A600
reached 1.5, 6% (v/v) of this seed culture was transferred
aseptically to a bioreactor containing optimal CAS medium
(Dash and Gummadi 2007a). The conditions at which the
reactor was operated are: pH 7.0; temperature, 30°C;
aeration rate of 0.27 vvm; and agitation, 700 rpm (Gummadi
et al. 2009). The pH probe used in the reactor measures the
pH of the medium, and the feedback control mechanism of
the reactor automatically adjusts any change in pH by
feeding acid or base accordingly using a PID controller.
Dissolved oxygen content was measured by a DO probe.
Samples were collected and analyzed periodically for
growth, caffeine consumption, and caffeine demethylase
production.
Fed-batch studies
In this study, the fed-batch strategy was conducted based on
caffeine concentration as feedback control parameter using
manual intermittent feed mode, where the feed is given as
pulse inputs (repeated fed batch) or by aseptically pumping
it into the reactor. The whole reactor setup (along with the
medium) and the feed were sterilized by autoclaving.
Solubility of caffeine in water at 25°C is 21.7 g/L. The
final concentration of caffeine desired in the reactor at the
end of each feed is 6.5 g/L which is approximately 25 g of
caffeine. Hence, to feed 25 g of caffeine dissolved and
sterilized in water at 30°C, the minimum amount of water
required is 1 L. Hence, the volume of each feed was
Appl Microbiol Biotechnol (2011) 91:1007–1017
maintained at 1 L. The reactor was started in batch mode as
described earlier, and when caffeine was depleted com-
pletely, feed was added. In order to perform constant
volume fed-batch process, an equivalent amount of broth
was withdrawn from the reactor before every feed. In the
first set of experiments, the initial concentration of caffeine
in the batch mode was varied. In further sets, maximum
number of feeds which the cells can tolerate was assessed
with varying feed strategies, i.e., feeds were added either as
pulse inputs or at different flow rates (240, 300, 500 ml/h).
In the final set, feed composition was varied to investigate
the effect of added nutrients in the feed, and the revised
feed contained optimal CAS medium without sucrose.
Samples were collected at regular intervals for analyzing
the concentration of caffeine, biomass, and enzyme activity
in all the runs.
Taking these conditions into account, viz. repeated feeds
and feeds at different flow rates, a mathematical model can
be established by using the following differential equations.
For repeated fed batch with pulse feeds:
dX =dt ¼ mX ð1Þ

times with 50 mM potassium phosphate buffer (pH 8.0).
dS=dt ¼  1=YX =S dX =dt ð2Þ
where X is the biomass (gram per liter), t is the operation
time (hours), μ is the specific growth rate of cells (per
hour), S is the concentration of substrate caffeine (grams
per liter), and YX/S is the cellular yield coefficient (grams of
caffeine/grams of cells).
To maintain constant volume after each feed and to
account for dilution, the following equations were used in
addition to (1) and (2):When
Sk < 0:1; Skþ1 ¼ Sk þ 6:5 ð3Þ
Xkþ1 ¼ c»Xk ð4Þ
where Sk is the substrate concentration at the end of feed k,
Sk+1 is the substrate concentration at the beginning of feed
k+1, Xk+1 is the biomass concentration at the beginning of
the feed k+1, Xk is the biomass concentration at the end of
feed k, and c is the dilution factor.
For repeated fed batch with continuous feeds, the
following equations were used in addition to (3) and (4):
dX =dt ¼ mX

density at 600 nm. Cell dry weight for Pseudomonas sp.
dS=dt ¼  1=YX=S dX =dt þ ðF=V Þ»So ð5Þ
where F is the flow rate of the feed, and So is the substrate
concentration in the incoming feed. The value of μ is
adapted from the previous reports on the batch kinetics
1009
of this strain (Gummadi and Devarai 2010). Among the
best fits in the report, the Luong model closely resembles
the experimental data of this study. According to the
Luong model,
m ¼ ðmm  S Þ  ½f1  ðS=Sm Þgn =ðKS þ S Þ ð6Þ
where μm is the maximum specific growth rate, Sm is the
maximum substrate inhibitory concentration at which no
growth was observed, n is the constant which accounts for
the relationship between μ and S, and KS is the Monod
half-saturation constant. The parameter values used in this
study which are adopted from our earlier work are as
follows: mm ¼ 0:34; KS ¼ 4:21; Sm ¼ 23:41; and n ¼ 4:12
(Gummadi and Devarai 2010).
These theoretical equations are used to simulate substrate
and biomass profiles with the help of MATLAB 2009a
software. These are plotted against the experimental values
to compare and contrast the theoretical and practical
approaches for fed-batch studies.
Assay for caffeine demethylase activity
Pseudomonas sp. cells were harvested and washed three
The cells were then resuspended in lysis buffer, with cell
dry weight-to-buffer ratio 1:15 (grams per milliliter) and
lysed by sonication over ice. The lysis buffer contained
50 mM phosphate buffer at pH 8.0, 1 mM EDTA, 1 mM
DTT, 20% glycerol, and 10% ethanol. The supernatant was
then separated by centrifugation at 14,000 rpm at 4°C for
30 min and was used as the crude enzyme extract in the
assay. Enzyme activities were measured at 30°C in a
reaction mixture consisting of 7.5 mM caffeine in 50 mM
potassium phosphate buffer (pH 8.0), and 1 mM NADH.
Reaction was initiated by adding cell-free lysate containing
50 μg protein, and the reaction was stopped after 10 min by
the addition of 10% (w/v) trichloroacetic acid (TCA). The
reaction carried out with enzyme inactivated with TCA
prior to incubation served as blank for the assay. The
reaction mixture was then centrifuged at 14,000 rpm at 4°C
for 15 min, and the supernatant was analyzed by HPLC.
One unit of enzyme activity (U) was defined as the number
of micromoles of substrate (caffeine) degraded per minute
of reaction.
Analytical methods
Cell concentration was monitored by measuring optical
was calculated from A600 values (A600 of 1 corresponds to
0.75 g/L cell dry weight). Protein was estimated by
Lowry’s method (Lowry et al. 1951). Caffeine concentra-
tion was estimated by RP-HPLC (Jasco PU-2080 Plus
1010 Appl Microbiol Biotechnol (2011) 91:1007–1017

equipment) using a HiQSil C-18 column and water/ 6.5-g/L fed batches, owing to the differing concentrations
methanol 70:30 (v/v) as mobile phase at a flow rate of of the carbon source provided. The highest caffeine
1 ml/min and at 28°C. The retention time of caffeine was demethylase activity of 2.3 U/mg was observed at 23 h
observed to be at 8 min. Pure caffeine (Merck) at 2 g/L was with for the 3-g/L batch whereas 2.1 U/mg of activity was
used as standard. UV detection was at 254 nm (Dash and observed at 28 h for the 6.5-g/L batch (Fig. 1d). Caffeine
Gummadi 2007b). degradation rate values of the fed batches are as shown
(Fig. 1e). The rates of degradation of the 1st and 2nd feeds
are the highest for the fed batch started with 3-g/L initial
Results caffeine concentration among the three (1.1 g/L h), while
those of 6.5 g/L are slightly lower than them.
Effect of initial caffeine concentrations Fig. 2 shows the caffeine and biomass profiles of both
on the fed-batch process experimental and theoretical data. It was observed that the
fed batch with initial caffeine concentration of 1 g/L
Repeated fed batches with different initial concentrations of showed a decreasing trend as compared to the simulated
caffeine, viz. 1, 3, and 6.5 g/L in the batch were studied. theoretical values in both substrate and biomass profiles
The profiles of caffeine and biomass produced in all the after the first feed (Fig. 2a, b). However, the substrate and
three fed batches are as shown (Fig. 1a, b, c). It was biomass profiles are almost similar to the theoretical values
observed that the maximum cell dry weight for fed batch in the cases of 3- and 6.5-g/L fed batches (Fig. 2c–f) which
with 1 g/L was 1.1 g/L while it was 3.1 g/L for both 3- and is in accordance with the experimental observation.

Fig. 1 Effect of initial caffeine

concentration in the batch mode
(a) (b)
Biomass (g/L)/ Caffeine(g/L)

Caffeine CDW Caffeine CDW

Biomass (g/L)/ Caffeine(g/L)

on the fed batch process. 8 8
Caffeine degradation and
biomass profile of fed batch 6 6
started with a 1-, b 3-, and
c 6.5-g/L initial caffeine 4 4
concentrations. Comparison of
d caffeine demethylase activities 2 2
and e rates of caffeine
degradation in fed batches
0 0
started with caffeine at initial 0 6 12 18 24 30 36 0 6 12 18 24 30
concentrations of 1, 3,
Time (h) Time (h)
and 6.5 g/L
(c) (d)
3
1 g/L 3 g/L 6.5 g/L
Biomass (g/L)/ Caffeine(g/L)

Caffeine CDW
Caffeine demethylase

8
Activity (U/mg)

2
6

4
1
2

0 0
0 6 12 18 24 30 36 0 12 24 36
Time (h) Time (h)

(e)
1.5
1 g/L 3 g/L 6.5 g/L
Caffeine degradation
rate (g/L h)

1.0

0.5

0.0
Batch 1st feed 2nd feed
Appl Microbiol Biotechnol (2011) 91:1007–1017 1011

Fig. 2 Experimental and (a)

simulated values of fed (b)
Expt Sim
batches with varying initial 7.5 2 Sim
Expt
caffeine concentrations.
Substrate and biomass profiles

Caffeine (g/L)

Biomass (g/L)
of fed batches with initial 5.0
caffeine concentrations of a, b 1,
c, d 3, and e, f 6.5 g/L. Filled 1
blocks represent experimental 2.5
values, and dotted lines
represent theoretical profiles.
The arrow shows the onset of
delay in degradation of feeds 0.0 0
0 6 12 18 24 30 36 0 6 12 18 24 30 36
and also the corresponding
decrease in biomass for each Time (h) Time (h)
fed batch
(c) (d)
8 Expt Sim 3
Expt Sim

Biomass (g/L)
6
Caffeine (g/L)

1
2

0 0
0 6 12 18 24 30 0 6 12 18 24 30
Time (h) Time (h)

(e) (f)
8 3
Expt Sim Sim
Expt
Biomass (g/L)

6
Caffeine (g/L)

1
2

0 0
0 6 12 18 24 30 36 0 6 12 18 24 30 36
Time (h) Time (h)

Effect of increase in the number of feeds
Batches started with 3 and 6.5 g/L gave similar results in
terms of rates of degradation and activity of caffeine
demethylase. Hence, fed batches with increasing number
of feeds were studied to assess the efficacy of cells. The
profiles of caffeine and biomass produced are as shown
(Fig. 3a, b). The highest cell dry weight reached in both the
cases was around 3 g/L. The maximum caffeine demethy-
lase activity of 2.6 U/mg was achieved for the 3-g/L fed
batch whereas only 2.3 U/mg was observed for the 6.5-g/L
fed batch (Fig. 3c). Caffeine degradation rate profiles
showed that caffeine degradation rate was maintained
higher (1 g/L h) for a longer time when the batch was
started with 3 g/L caffeine initially (Fig. 3d). The fed batch
with 3 g/L initial caffeine could degrade nine feeds while
the fed batch with 6.5 g/L initial caffeine degraded only
eight. This implies that 237 g of caffeine was degraded in
75 h by the former with an overall degradation rate of
0.82 g/L h while 225 g of caffeine was degraded in 85 h by
the latter with an overall degradation rate of 0.69 g/L h.
Theoretical profiles obtained from the mathematical
equations show a constant increase in biomass which in
turn leads to degradation of an infinite number of feeds with a
considerable decrease in the time taken for degradation of
each feed (Fig. 4). Experimental values followed the
theoretical model for the first few feeds in corroboration
with the report on batch kinetics for this strain (Gummadi
and Devarai 2010). However, they show a decreasing trend
in the biomass after a few feeds and thus delay in the
degradation of feeds. In the case of the 3-g/L fed batch, an
increase in the time taken for degradation of each feed as
1012 Appl Microbiol Biotechnol (2011) 91:1007–1017

Fig. 3 Effect of increase in (a) (b)

the number of feeds on the
8 Caffeine CDW

Biomass (g/L)/Caffeine (g/L)

8 CDW

Biomass (g/L)/Caffeine (g/L)

degradation efficiency of the Caffeine

cells. Caffeine and biomass

profiles of fed batches started 6 6
with a 3- and b 6.5-g/L initial
caffeine concentrations. A 4 4
comparison of the c caffeine
demethylase activities and d 2 2
rates of caffeine degradation
in fed batches started with
0 0
caffeine at initial concentrations
0 12 24 36 48 60 72 84 96 0 12 24 36 48 60 72 84 96 108
of 3 and 6.5 g/L
Time (h) Time (h)

(c) (d)
3 6.5 g/L
3 g/L
1.5
Caafeinedemethylase

3 g/L 6.5 g/L

Caffeine degradation
Activity (U/mg)

rate (g/L h)
2
1.0

1 0.5

0 0.0
0 12 24 36 48 60 72 84 96 108 0 12 24 36 48 60 72 84 96 108
Time (h) Time (h)

compared to the simulated values was observed after 33 h biomass profiles can be observed for the fed batch with a
(Fig. 4a). This corresponds to the negative deviation in 6.5-g/L initial caffeine concentration (Fig. 4c, d). Biomass
biomass profile from the theoretical values after three feeds showed a declining trend after 48 h which corresponds to the
at around 34 h (Fig. 4b). Similar trends in substrate and delayed degradation in feeds after the fourth feed.

Fig. 4 Experimental and

simulated values of fed batches (a) (b)
showing the effect of increasing 10.0 4 Sim
Expt Sim Expt
number of feeds. Substrate
and biomass profiles of fed
Caffeine (g/L)

7.5 3
Biomass (g/L)

batches with initial caffeine

concentrations of a, b 3 and c,
d 6.5 g/L. Filled blocks 5.0 2
represent experimental values,
and dotted lines represent 2.5 1
theoretical profiles. The arrow
shows the onset of delay in
degradation of feeds and also 0.0 0
the corresponding decrease in 0 12 24 36 48 60 72 0 12 24 36 48 60 72
biomass for each fed batch Time (h) Time (h)

(c) (d)
7.5 Expt Sim 4
Expt Sim
Biomass (g/L)

3
Caffeine (g/L)

5.0

2.5
1

0.0 0
0 12 24 36 48 60 72 84 96 0 12 24 36 48 60 72 84 96
Time (h) Time (h)
Appl Microbiol Biotechnol (2011) 91:1007–1017 1013

Effect of varying flow rates of the feed could degrade was lesser than that of the repeated fed
batches. The maximum overall degradation rates achieved
As the drop in the caffeine demethylase activity in the case were only 0.66, 0.74, and 0.64 g/L h for 240-, 300- and
of the fed batch with 3-g/L initial caffeine concentration is 500-ml/h feed flow rates, respectively (Fig. 5e).
due to sudden influx of caffeine, the feed was pumped Fig. 6 shows the substrate and biomass profiles of both
aseptically into the reactor at different flow rates (240, 300, experimental and theoretical values which follow similar
and 500 ml/h) to decrease this effect. The profiles of trends shown in Fig. 4. The simulated profiles in Fig. 6a, c,
caffeine and biomass for all the three feed flow rates are as e show that the time required for degradation of each feed is
shown (Fig. 5a, b, c). Though the biomass shows an in accordance with the experimental values till the first few
increasing trend in all the three cases, it could not be feeds. However, later, there is a considerable decrease in the
maintained for longer at 3 g/L unlike repeated fed batches. time taken for degradation in the simulations as compared
In the case of the 300-ml/h feed flow rate, caffeine to experimental values. This is due to the correspondingly
demethylase activity of 2.9 U/mg was achieved whereas increasing biomass in the theoretical profiles as opposed to
fed batches with 240- and 500-ml/h feed flow rates gave the experimental values which follow a decreasing trend after
lesser caffeine demethylase activities of 2.5 and 2.8 U/mg, a few feeds (Fig. 6b, d, f). In the case of the 240-ml/h feed
respectively (Fig. 5d). Unlike the repeated fed batches, flow rate, the degradation slowed down after the third feed
feeds at varying flow rates could not be degraded with high which corresponds to the decrease in biomass after 36 h
degradation rates. Also, the number of feeds that the cells (Fig. 6a, b). Similarly, delays in degradation of feeds were

Fig. 5 Effect of varying feed (b)

(a)
flow rates on the caffeine Caffeine CDW 5 Caffeine CDW
Biomass (g/L)/Caffeine (g/L)

Biomass (g/L)/Caffeine (g/L)

degradation rate and caffeine
demethylase activity. Caffeine 4
and biomass profiles of fed 4
batches started with 3-g/L 3
3
caffeine concentration with
feed flow rates of a 240, b 300, 2
2
and c 500 ml/h. A comparison
of the d caffeine demethylase 1 1
activities and e rates of
caffeine degradation in fed 0 0
batches with varying feed 0 12 24 36 48 60 72 84 0 20 40 60 80
flow rates of the substrate—240, Time (h) Time (h)
300, and 500 ml/h
(c) (d)
4
CDW
Biomass (g/L)/Caffeine (g/L)

5 Caffeine 240 ml/h 300 ml/h 500 ml/h

Caffeine demethylase
Activity (U/mg)

4 3

3
2
2
1
1

0 0
0 12 24 36 48 60 72 84 96 0 12 24 36 48 60 72 84 96
Time (h) Time (h)

(e)
2
Caffeine degradation

240 ml/h
rate (g/L h)

300 ml/h
500 ml/h
1

0
0 12 24 36 48 60 72 84 96
Time (h)
1014 Appl Microbiol Biotechnol (2011) 91:1007–1017

Fig. 6 Experimental and (a) (b)

simulated values of fed batches 7.5 4 Sim
Expt
with varying feed flow rates. Expt Sim
Substrate and biomass profiles
3

Caffeine (g/L)

Biomass (g/L)
of fed batches with feed flow 5.0
rates of a, b 240, c, d 300, and
2
e, f 500 ml/h. Filled blocks
represent experimental values, 2.5
1
and dotted lines represent
theoretical profiles. The arrow
shows the onset of delay in 0.0 0
0 12 24 36 48 60 72 0 12 24 36 48 60 72
degradation of feeds and also
Time (h) Time (h)
the corresponding decrease in
biomass for each fed batch (c) (d)
5 4 Expt Sim
Expt Sim
4

Biomass (g/L)
Caffeine (g/L)

3
2
2

1
1

0 0
0 12 24 36 48 60 72 0 12 24 36 48 60 72
Time (h) Time (h)

(e) (f)
7.5 4 Expt Sim

Expt Sim

Biomass (g/L)
Caffeine (g/L)

3
5.0

2.5
1

0.0 0
0 12 24 36 48 60 72 84 0 12 24 36 48 60 72 84
Time (h) Time (h)

observed after the fourth and third feeds for fed batches with
300- and 500-ml/h feed flow rates, respectively (Fig. 6c, e).
These corroborate with the negative deviations in their
biomass profiles (Fig. 6d, f).
Effect of additional nutrients in the feed composition
Among the different feed strategies (repeated fed batch,
feed with varying flow rates—240, 300, and 500 ml/h), the
repeated fed batch with 3-g/L initial caffeine concentration
resulted in high caffeine degradation rate and better caffeine
demethylase activities. To assess the effect of added nutrients
to the feed, the repeated fed batch with 3-g/L initial caffeine
concentration was studied with revised feed which contained
optimal CAS medium without sucrose. The profiles of
caffeine and biomass are depicted in Fig. 7a, b which are
similar in both the cases. The biomass shows an increasing
trend and reached a maximum of around 3 g/L in both the
cases, and the number of feeds degraded was also the same.
Maximum caffeine demethylase activity of 3.4 U/mg was
observed which was more than 2.6 U/mg achieved in the
3-g/L repeated fed batch (Fig. 7c). The degradation rate
profiles with respect to time are similar in both the cases
(Fig. 7d). Only a slight increase in overall degradation rate
was observed (0.85 g/L h) for the fed batch with revised
feed as compared to that of the repeated fed batch with
earlier feed.
Hence, among all the fed-batch runs, the repeated fed
batch with initial caffeine concentration of 3 g/L gave
maximum overall degradation rate of 0.82 g/L h which was
the highest caffeine degradation rate reported so far. As for
enzyme activity, the repeated fed batch with initial caffeine
concentration of 3 g/L using revised feeds gave the highest
value of 3.2 U/mg.
Discussion
Fed batch cultures are often better alternatives to batch
cultures for substrates which are toxic to the cells. They
give higher biomass concentration and productivity of
growth-associated products. As caffeine demethylase was
Appl Microbiol Biotechnol (2011) 91:1007–1017 1015

Fig. 7 Effect of added (a) (b)

Caffeine CDW Caffeine CDW
nutrients in the feed on 8 8

Biomass (g/L)/Caffeine (g/L)

Biomass (g/L)/Caffeine (g/L)
caffeine degradation rate and
caffeine demethylase activity.
6 6
Caffeine and biomass profiles
of fed batches with 3-g/L
initial caffeine concentration 4 4
a without and b with added
nutrients in the feed. A 2 2
comparison of the c caffeine
demethylase activities and
d rates of caffeine degradation 0 0
0 12 24 36 48 60 72 84 96 0 12 24 36 48 60 72 84 96
of the fed batches with and
without added nutrients in Time (h) Time (h)
the feeds
(c) (d)
4 1.5
Caffeine
Caffeine demethylase

Caffeine+nutrients Caffeine Caffeine+nutrients

Caffeine degradation
Activity (U/mg)

rate (g/L h)
1.0

0.5
1

0 0.0
0 12 24 36 48 60 72 84 0 12 24 36 48 60 72 84 96
Time (h) Time (h)

reported to be a growth-associated enzyme (Gummadi et al. caffeine concentration and could degrade a greater number
2009), fed-batch cultures are appropriate for the production of feeds due to lesser stress in terms of caffeine degraded.
of the enzyme and also for enhancing the degradation rate. This explains the high degradation rates being maintained
Hence, different strategies were adopted in order to for almost all the feeds for the fed batch with 3-g/L initial
optimize the fed-batch conditions. caffeine concentration as opposed to that of 6.5 g/L. The
Previous reports show that caffeine is required for the effect of acclimatization is also shown by the caffeine
induction of caffeine demethylase enzyme production in demethylase activity profile (Fig. 3c). Activity was high at
this Pseudomonas strain. According to those reports, 3 g/L the end of the 3-g/L batch and drops suddenly for the two
of caffeine is optimum for maximum growth rate subsequent feeds owing to the change in the caffeine
whereas 6.5 g/L caffeine gives optimum degradation rate concentration from 3 to 6.5 g/L. From this point, activity
(Gokulakrishnan and Gummadi 2005; Gummadi and steadily increases indicating the acclimatization of the cells.
Devarai 2010). Hence, the effect of initial caffeine However, in the case of the fed batch with 6.5-g/L initial
concentration on the fed batch process was studied. The caffeine concentration, though the activity shows a slight
results corroborate with the previous reports as the highest decrease for the first two feeds, it steadily increases. As the
degradation rate in the batch was achieved with 6.5-g/L cells begin to lose their efficacy due to stress induced by
initial caffeine concentration. However, the repeated fed high amounts of caffeine, the activity starts to decrease
batch with 3 g/L gave the highest rates of degradation for (Fig. 3c).
the first and second feeds (1 g/L h) and caffeine When feed was aseptically pumped at different feed flow
demethylase activity indicating that 3 g/L of caffeine rates to decrease the effect of sudden influx of caffeine on
yields better growth rates and hence, better degradation for growth and caffeine demethylase production, the 300-ml/h feed
the subsequent feeds (Fig. 1d, e). In the case of 1 g/L fed flow rate gave optimal results. However, the highest overall
batch, cells were poorly induced and suddenly over- degradation rate observed was only 0.74 g/L h. In the case of
whelmed with the feed containing more than six times the 240-ml/h feed flow rate, the cells were starved due to the
the initial caffeine concentration. Hence, the degradation slower rate at which the substrate was provided and hence gave
took considerably longer time (0.4 g/L h). lower degradation rates and caffeine demethylase activities. At
In the second set, fed batches with increasing number of the 500-ml/h feed flow rate, the degradation rate and the
feeds were run with both 3 and 6.5 g/L of initial caffeine enzyme activity were lowered due to substrate inhibition as
concentration to inspect the maximum feeds of caffeine that caffeine is fed at a faster rate.
the cells can withstand. Hence, during the subsequent feeds, In the final set of experiments, the enzyme activity in the
cells in the former were acclimatized to the incoming case of feeds with additional nutrients is greater indicating
1016
that the additional nutrients in the feed enhance the caffeine
demethylase activity in agreement with the previous report
that sucrose enhances the caffeine demethylase activity
(Dash and Gummadi 2007a) (Fig. 7c). The degradation
rates in both the cases were similar (Fig. 7d) with only a
slight increase in the presence of additional nutrients.
Therefore, feeds without nutrients are economical for high
degradation rates, as there is no significant difference in the
results between both types of feeds. However, feeds with
additional nutrients should be incorporated when high
enzyme activities are desired.
The remarkable feature in all the simulated theoretical
profiles is that there is a constant increase in biomass which
in turn leads to degradation of an infinite number of feeds
with a considerable decrease in the time taken for
degradation of each feed (Figs. 2, 4, 6). Experimental
values followed the theoretical model for the first few
feeds but show a decreasing trend in the biomass after
that, which in turn lead to delay in the degradation of
feeds. This can be attributed to the fact that caffeine is
toxic for many microbes, and caffeine beyond 10 g/L
inhibits the growth of the strain used in this study
(Gummadi et al. 2009). Also, this may be due to the stress
that the cells undergo after a few feeds as caffeine is known
to cause DNA damage (Zhou et al. 2000). Though the
substrate inhibition profiles used for simulations quantify
this effect to a certain extent, the actual series of complex
biochemical reactions on which the growth of the microor-
ganism proceeds cannot be completely quantified. There-
fore, the observations in this study show how the cells
deviate from the ideal behavior and thus render a more
practical approach in developing large-scale processes for
caffeine degradation.
Also, the experimental results corroborate with the
reports showing improved efficiency in degradation of
xenobiotics or toxic compounds by fed-batch cultures over
other methods (Saravanan et al. 2008; Piakong 2006; Ivan
et al. 2006). Hence, it can be concluded that a sustainable
bioprocess can be developed from the fed-batch culture of
this strain to degrade caffeine efficiently. For achieving
high degradation rates, repeated fed-batch cultures with 3-g/L
initial caffeine concentration and manual intermittent
pulse feeds with substrate concentration as the feedback
control parameter can be undertaken, whereas the revised
feed can be used to achieve high enzyme activities. Also,
caffeine as high as 237 g can be completely degraded in
only 75 h which makes the Pseudomonas sp. used in this
study a potential strain for caffeine effluent treatment and
industrial decaffeination.
Acknowledgments This work was supported by a financial grant
from the Indian Institute of Technology, Madras.
Appl Microbiol Biotechnol (2011) 91:1007–1017
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