International Journal of Food Microbiology 113 (2007) 346 – 350

www.elsevier.com/locate/ijfoodmicro

Short communication
A preliminary study of caffeine degradation by Pseudomonas sp. GSC 1182
S. Gokulakrishnan, K. Chandraraj, Sathyanarayana N. Gummadi ⁎
Department of Biotechnology, Indian Institute of Technology–Madras, Chennai 600 036, India
Received 12 February 2005; received in revised form 26 June 2006; accepted 10 July 2006

Abstract

Several microorganisms isolated from soil were tested for their ability to utilize caffeine as the sole carbon and nitrogen source. The isolate
identified as Pseudomonas sp. GSC 1182 showed 80% degradation of caffeine in 48 h when caffeine was used as the sole carbon and nitrogen
source. In the presence of sucrose (5 g/l), 100% degradation of caffeine was achieved within 36–40 h. The degradation rate was also found to
increase when fructose, lactose and galactose were used as carbon source. The isolate showed decreased level (b 10%) of caffeine degradation in
the presence of glucose. At an initial pH of 6.0, the complete degradation of caffeine was attained in 24 h. The addition of urea and ammonium
sulfate as external nitrogen source decreased the caffeine degradation to 35% and 70% respectively.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Pseudomonas; Caffeine; Microbial degradation; Submerged fermentation

1. Introduction the caffeine degradation is dependent on external nitrogen

Caffeine (3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6 dione)
belongs to a group of compounds collectively known as purine
alkaloids and is a key component in many popular drinks
especially tea and coffee. The conventional methods of caffeine
removal are water decaffeination, solvent extraction and super-
critical carbon dioxide extraction (Dixon and Johnston, 1997).
An enzymatic degradation of caffeine could be desirable as the
decaffeinated coffee and tea pulp are rich in nutritional com-
pounds such as carbohydrates and proteins, and thus have a
good bioconversion potential (Pandey et al., 2000).
Bacterial strains of Serratia (Mazzafera et al., 1994) and
Pseudomonas (Asano et al., 1994; Yamoka-Yano and Mazza-
fera, 1998; Blecher and Lingens, 1977) are known to degrade
caffeine. In addition to bacteria, species of the genera Asper-
gillus (Hakil et al., 1998, 1999; Brand et al., 2000; Roussos et
al., 1995a,b), Penicillium (Hakil et al., 1998; Brand et al., 2000;
Roussos et al., 1994, 1995a,b), Stemphylium (Schwimmer et al.,
1971), Rhizopus and Phanerochaete (Brand et al., 2000) are
reported to degrade caffeine. However, studies have shown that
⁎ Corresponding author. Tel.: +91 44 2257 4114; fax: +91 44 2257 4102.
E-mail address: gummadi@iitm.ac.in (S.N. Gummadi).
0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2006.07.005
sources (Hakil et al., 1999; Roussos et al., 1994). In this study,
we have isolated a Pseudomonas sp. GSC 1182 from a coffee
cultivation soil that can degrade caffeine and conducted a pre-
liminary study on the effects of nutrients on caffeine degra-
dation. The ability to degrade caffeine by this isolate was highly
influenced by sugars, nitrogen source and pH.
2. Materials and methods
2.1. Chemicals
Caffeine (N99% pure) was purchased from Merck Limited,
Worli, Mumbai, India. All other chemicals were of analytical
grade procured from Himedia Limited, Mumbai, India and SRL
Chemicals Limited, Mumbai, India.
2.2. Media
The medium used contained (g/l): Na2HPO4, 0.12; KH2PO4,
1.3; CaCl2, 0.3; MgSO4·7H2O, 0.3. Caffeine medium consisted
of these salts supplemented with 1.2 g/l of caffeine. Carbon
sources if any added to the medium were sterilized separately
and then mixed to the medium under aseptic conditions. The pH
 S. Gokulakrishnan et al. / International Journal of Food Microbiology 113 (2007) 346–350
of the medium after autoclaving was 5.5. For solid medium,
agar (25 g/l) was added to the caffeine medium. The isolates
were maintained and sub-cultured in the caffeine agar medium.
2.3. Screening and identification of microorganisms degrading
caffeine
Soil samples collected at various depths (0, 15 and 30 cm
from the surface) from coffee and tea cultivation areas located in
Uthagamandalam (India) were used to obtain microorganisms
that degrade caffeine. About 1 g of soil sample (b 150 μm) was
suspended in 10 ml of 0.9% (w/v) saline and agitated in a vortex
shaker for 30 min. After centrifugation at 3000×g for 30 min,
100 μl of supernatant was spread on to caffeine-agar plates and
incubated at 30 °C. The isolates that utilized caffeine as sole
carbon and nitrogen source were picked and sub-cultured for
every two weeks. Further selection was made based on their
ability to utilize caffeine in submerged fermentation. The
promising strains were further examined for morphological,
physiological and biochemical characteristics as described in
Bergey's manual of determinative bacteriology (Buchanan and
Gibbsons, 1974).
2.4. Flask culture experiments
A single colony of the strain from caffeine agar plates was
transferred to 5 ml sterile caffeine medium. The tubes with cotton
plugs were aerated on a rotary shaker at 180 rpm and incubated for
24 h at 30 °C. About 2% (v/v) of the culture was transferred to
25 ml of the caffeine medium in 100 ml Erlenmeyer flasks with
cotton plugs and aerated on a rotary shaker at 180 rpm and
incubated for 48 h at 30 °C. Samples were collected at different
intervals of time and measured for cell growth and caffeine
degradation. In order to study the effect of carbon sources diffe-
rent carbon sources viz., sucrose, fructose, glucose, galactose and
lactose were added to the caffeine medium at 5 g/l and the time
course of cell growth, carbon source utilization and caffeine
degradation were measured. In order to study the effect of an
additional nitrogen source on caffeine degradation, caffeine
medium with 5 g/l sucrose was supplemented with inorganic
nitrogen source (ammonium sulfate 2.2 g/l) and organic nitrogen
source (urea 1 g/l). The amount of nitrogen in ammonium sulfate
and urea was calculated and added such that the amount of total
nitrogen content in the medium is the same. The effect of pH was
studied by varying the initial pH of the medium between 4 and 8.
All experiments were performed in triplicates under identical
conditions and the data presented are mean of triplicate
experiments.
2.5. Cell growth
Cell density in the medium was monitored by measuring the
optical density at 600 nm. In order to obtain the unknown cell
dry weight directly from the optical density readings, the
relationship between optical density and the corresponding cell
dry weight was established previously by calibration (OD600 of
0.5 corresponds to 0.379 g dry weight/l).
347
2.6. Analytical methods
Caffeine was estimated by HPLC (Agilent 1100 series from
Agilent technologies, Waldbronn, Germany, Product No.
G2170AA) equipment using a ZORBAX® SB-C18 column
(USA, Product No. 880975-902, Batch No. B03024) with
10 mM ammonium phosphate buffer (pH 2.5)/acetonitrile (4:1,
v/v) as mobile phase (with reference to specifications in the
HPLC user manual). Pure caffeine at 2 mg/ml 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 30 °C. Detection of caffeine was
done at 254 nm (detector sensitivity: 1 × 10− 14 absorbance unit).
Linear relationship between AU and caffeine concentration was
observed to exist between 0.5 mM and 10 mM of caffeine. The
concentrations of the carbon source in the medium were
estimated by the 3,5-dinitrosalicylic acid method (Miller, 1959).
It was observed to be sensitive in the range of 0.5 mM to 5 mM
of glucose.
3. Results and discussion
3.1. Screening and identification of microorganisms degrading
caffeine
Ten strains were isolated based on their ability to utilize
caffeine as the sole carbon and nitrogen source. Among the ten
isolates, isolate GSC 1182 degraded caffeine efficiently (Isolate
GSC 1182 utilized 100% of 1.2 g/l of caffeine in the medium in
40 h compared to other strains which were able to utilize less
than 10% of caffeine in the same time). The Gram negative,
non-spore forming, rod shaped isolate was identified as Pseu-
domonas sp. The isolate was positive for citrate utilization,
catalase, oxidase, lysine arginine test and motility. The isolate
was negative for indole production, H2S production, urease, and
triple sugar-iron test (red slant and red butt were observed hence
indicating that no carbohydrate fermentation had occurred and
instead peptones were catabolized). Morphological observa-
tions revealed that the colony was translucent and small with
raised elevation. These data suggest that the isolate GSC 1182 is
a Pseudomonas sp (Cappucinno and Sherman, 1999; Buchanan
and Gibbsons, 1974).
3.2. Time course of growth and caffeine degradation by strain
GSC 1182
The time course of caffeine degradation and growth in sub-
merged culture aerated by shaking and containing caffeine as
the sole carbon and nitrogen source is shown in Fig. 1 (Fig. 1a
and c respectively). Caffeine utilization was low at 24 h and
about 75% of initial caffeine was utilized in 40 h of growth
(Fig. 1a). In the presence of sucrose, the isolate degraded 100%
of caffeine (1.2 g/l) in 40 h (Fig. 1a). The cell growth was low
when caffeine was used as the sole carbon and nitrogen source
compared to the medium containing sucrose (Fig. 1c). These
results clearly showed that the presence of a carbon source in
the medium enhanced the caffeine degradation by Pseudomo-
nas sp. GSC 1182.
348 S. Gokulakrishnan et al. / International Journal of Food Microbiology 113 (2007) 346–350

Fig. 1. Caffeine degradation by Pseudomonas sp. GSC 1182 and the effect of different carbon sources on caffeine degradation by Pseudomonas sp. GSC 1182.
(a) Caffeine degradation, (b) carbon source utilization, (c) cell growth and (d) pH profile. The isolate was grown in 25 ml of a caffeine medium containing 1.2 g/l of caffeine
and 5 g/l of various carbon sources namely glucose, fructose, galactose, lactose and sucrose in 100 ml conical flasks at 180 rpm and 30 °C. 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.

3.3. Effect of different carbon sources on caffeine degradation
Since the presence of sucrose in a caffeine medium enhanced
caffeine degradation, experiments were performed to study the
effect of different carbon sources. Among all the tested carbon
sources, sucrose was found to be the best (Fig. 1a). The caffeine
degradation rate by the isolate was the same up to 24 h when
grown in a medium containing sucrose and fructose. After 24 h,
the degradation rate was higher in a medium containing sucrose
than fructose (Fig. 1a). The presence of glucose in the caffeine
medium inhibited the caffeine degradation (b 10%), which was
lower than the control experiment i.e. caffeine medium without
any carbon source. In the presence of fructose, the caffeine
degradation was close to 100% in 48 h and the isolate utilized
more than 95% of fructose (Fig. 1b), which resulted in higher
biomass (Fig. 1c). But in the case of glucose, lower caffeine
degradation, glucose consumption and biomass were observed
(Fig. 1a–c). These obtained results indicate that the caffeine
degradation in the presence of fructose is directly correlated to
the biomass production. However, the caffeine degradation was
100% in 48 h when sucrose was used in the medium, whereas
only 15% of the sucrose was utilized (Fig. 1b) and the biomass
produced was close to the control experiment. Based on these
results, it can be hypothesized that the presence of sucrose in the
medium somehow induces the caffeine degrading enzymes.
This is reflected by very low consumption of sucrose, low
biomass and higher caffeine degradation rates (Fig. 1a–c). It
was also found that the negative effect of glucose was masked
when sucrose was added.
Similar results are observed when comparing lactose, galactose
and glucose. Lactose, a disaccharide of galactose and glucose,
showed appreciable caffeine degradation (N 90%), less consump-
tion (b15%) and correspondingly lower cell dry weight (Fig. 1). In
the presence of galactose, the isolate showed a similar caffeine
degradation profile as that of lactose, whereas the consumption of
galactose was more than 90%, which resulted in higher biomass
(Fig. 1a–c). Similarly, the negative effect of glucose was masked
when lactose was used in the medium (Fig. 1a–c). In all the studies,
 S. Gokulakrishnan et al. / International Journal of Food Microbiology 113 (2007) 346–350 349

Table 1
Effect of initial pH and external nitrogen sources on caffeine degradation
Time Amount of caffeine degraded (g/l) ± standard deviation about the mean Cell dry weight (g cell dry weight /l) ± standard deviation about the mean
(h)
Effect of various initial pH on caffeine degradation
pH 4 pH 5.5 pH 6 pH 8 pH 4 pH 5.5 pH 6 pH 8
0 0 0 0 0 0.08 ± 0.04 0.1 ± 0.02 0.1 ± 0.002 0.14 ± 0.004
18 0.006 ± 0.006 0.707 ± 0.039 0.995 ± 0.033 0.035 ± 0.051 0.11 ± 0.05 0.15 ± 0.01 0.25 ± 0.004 0.18 ± 0.002
24 0.314 ± 0.012 1.068 ± 0.073 1.19 ± 0.002 0.827 ± 0.106 0.11 ± 0.05 0.2 ± 0.02 0.3 ± 0.003 0.2 ± 0.001

Effect of various external nitrogen sources on caffeine degradation

Control Ammonium sulfate (A) Urea (U) Both A and U Control Ammonium sulfate (A) Urea (U) Both A and U
0 0 0 0 0 0.13 ± 0.02 0.16 ± 0.02 0.16 ± 0.02 0.15 ± 0.02
18 1.024 ± 0.012 0.574 ± 0.005 0.247 ± 0.017 0.487 ± 0.087 0.26 ± 0.03 0.28 ± 0.02 0.18 ± 0.03 0.27 ± 0.03
24 1.169 ± 0.014 0.806 ± 0.015 0.412 ± 0.001 0.766 ± 0.005 0.3 ± 0.02 0.32 ± 0.01 0.22 ± 0.005 0.33 ± 0.04
In order to study the effect of pH on caffeine degradation, the isolate was grown in 25 ml of caffeine medium (with 1.2 g/l of initial caffeine) whose initial pH varied
between 4 and 8, with sucrose (5 g/l) in 100 ml conical flasks at 180 rpm and 30 °C. To study the effect of external nitrogen sources on caffeine degradation, the isolate
was grown in 25 ml of caffeine medium (initial pH 6) with different nitrogen sources viz., urea, ammonium sulfate and both ammonium sulfate and urea along with a
control (without any external nitrogen source) such that the amount of total nitrogen content in the medium is the same in 100 ml conical flasks at 180 rpm and 30 °C.
Standard deviations were calculated for the results of experiments performed in triplicates under identical conditions.

the final pH of the medium increased to 8 from an initial pH of 5.5
except in the case of glucose where the pH was decreased to 4.0
(Fig. 1d). This prompted us to further investigate the effect of initial
pH on caffeine degradation. In further studies, the caffeine medium
was supplemented with sucrose (CAS medium).
It has been reported that some strains of Aspergillus and Peni-
cillium sp. degraded caffeine when sucrose was supplemented as
the carbon source (Hakil et al., 1998). In the case of bacterial
systems, Serratia marcescens and Pseudomonas putida degraded
caffeine in the absence of glucose or sucrose or any other carbo-
hydrates in the medium (Mazzafera et al., 1994; Woolfolk, 1975).
However, it has been reported that certain Pseudomonas species
and other bacteria degraded caffeine in the presence of a carbon
source (Madyastha and Sridhar, 1998; Asano et al., 1993). In
Pseudomonas sp. fructose and glucose were shown to enhance the
degradation of caffeine to theobromine by Pseudomonas sp. In
contrast to these studies, the present study revealed that the presence
of glucose inhibited caffeine degradation and the disaccharides
sucrose and lactose enhanced caffeine degradation with very little
consumption of the disaccharides.
3.4. Effect of initial pH on caffeine degradation
In order to study the effect of pH on caffeine degradation,
the initial pH of the CAS medium was varied between 4 and
8 (Table 1). At an optimal pH 6.0, complete degradation of
caffeine was attained within 24 h and resulted in higher biomass
(Table 1). About 70% degradation was attained at pH 8 and less
than 15% degradation was attained at pH 4. In control (un-
adjusted pH ∼ 5.5), 90% degradation was observed in 24 h. In all
these cases, the final pH of the medium increased to about 8. In
the case of the medium at pH 4, there was less than 15%
degradation at the end of 24 h and there was no change in pH.
These results suggest that optimum pH for caffeine degradation
is 6.0 and at this pH, the time required for complete degradation
of caffeine was reduced from 48 h to 24 h. In further studies, the
initial pH of the medium was adjusted to 6.0.
3.5. Effect of additional nitrogen sources on caffeine
degradation
The addition of external nitrogen sources (both organic and
inorganic) inhibited the caffeine degradation (Table 1). The
inhibitory effect was stronger for urea than ammonium sulfate.
The presence of both urea and ammonium sulfate in CAS
medium did not have a cumulative effect in inhibiting caffeine
degradation. The amount of biomass produced was 0.3 g/l in
control and in medium containing ammonium sulfate and
ammonium sulfate and urea (Table 1). These results showed that
the presence of the above mentioned additional nitrogen sources
in the CAS medium inhibited caffeine degradation. Similar
results were reported for Penicillium (Roussos et al., 1994) and
Aspergillus (Hakil et al., 1999) species but not for bacteria.
It has been reported that an external supply of the nitrogen
source improved the degradation of caffeine in Pseudomonas
sp. Tryptone at 0.5% (w/v) was found to be the most effective
nitrogen source (Asano et al., 1993). In contrast, for the present
strain Pseudomonas sp. GSC1182, caffeine degradation was
inhibited by the presence of external nitrogen sources consi-
dered in this study (ammonium sulfate and urea).
In this report, we isolated a new bacterial strain and showed its
ability to degrade caffeine. The isolate degraded caffeine more
efficiently when sucrose was the carbon source and in addition,
the degradation was inhibited in the presence of glucose. The
result that disaccharides lactose and sucrose were promotive
without substantial consumption warrants further study in terms
of their regulatory roles. In order to develop a successful process
for decaffeination, the enzymes involved in the degradation of
caffeine by isolate need to be identified and characterized. Further
studies on these aspects are under progress.
Acknowledgements
SNG & KCR acknowledge IC and SR, IIT-Madras for
financial assistance. Authors acknowledge The King Institute,
350 S. Gokulakrishnan et al. / International Journal of Food Microbiology 113 (2007) 346–350
Chennai and Sundaram Medical Foundation, Chennai for their
help in identifying the bacterium. Dr. Manoj is acknowledged
for critical reading of the manuscript. SNG acknowledges Ram
and Shyam for their strong support and stimulation.
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