Biodegradation of Caffeine by Trichosporon asahii Isolated from Caffeine Contaminated Soil

Biodegradation of Caffeine by Trichosporon asahii Isolated from Caffeine Contaminated Soil LAKSHMI V., NILANJANA DAS* Environmental Biotechnology Division School of Bio Sciences and Technology VIT University, Vellore-632014, Tamil Nadu, India-632 014 Abstract Studies were carried out on caffeine degradation using Trichosporon asahii, a yeast species isolated from caffeine contaminated soil. There was 100 % degradation of caffeine at 54 h by the yeast cells acclimated to the medium containing caffeine and sucrose both. Experiments with T. asahii growing on caffeine in the presence of 1 mm 1-aminobenzotriazole (ABT), an inhibitor of the cytochrome P-450 enzyme system, resulted inhibition of biomass production relative to positive control implicating the utilization of this enzyme system in caffeine degradation. The study of the enzymes responsible for caffeine degradation showed the enhanced activities of caffeine demethylases and xanthine oxidases. High Performance Liquid Chromatography (HPLC), Fourier Transform Infrared (FTIR) spectral analysis and Gas Chromatography - Mass Spectrometry (GC-MS) analysis of caffeine metabolites confirmed the biodegradation of caffeine by T. asahii. We propose the biodegradation pathway for caffeine which occurs via stepwise demethylation and oxidation process. Keywords Caffeine, T. asahii, Cytochrome P-450, Caffeine metabolites, Degradation pathway. 1. Introduction Caffeine (C 8 H 10 O 2 ) is an alkaloid whose basic structure is purine and exists widely in the leaves, seeds and fruits of a large number of plants. Among them, cocoa beans, tea, coffee, cola nut and guarana are the best known 1. It is extensively used in non-alcoholic beverages and also in pharmaceuticals because of its stimulant and muscle relaxant properties. The most dominant alkaloid in the purine compounds is caffeine 2. Although caffeine consumption increases alertness by overcoming fatigue, research on this compound revealed many deleterious effects that it may have on the human body 3. Prolonged consumption of caffeine results in headache, fatigue, apathy, adrenal stimulation, irregular muscular activity, cardiac arrhythmias and osteoporosis 4-8. The reason for the increasing need for decaffeinated products can therefore be well understood. Caffeine containing by-products and effluents generated from coffee and tea processing plants constitute a major part of the agro-industrial wastes in coffee producing nations 9. The presence of caffeine in soil affect soil fertility as it inhibits seed germination and growth of seedlings 10. Caffeine containing effluents are often discharged to the surrounding water bodies and subsequently, caffeine has been detected in surface water, ground water and waste water effluents at a high concentration (~10g caffeine/l) 11,12. The ingestion of caffeine has severe adverse effect on the physiological system 13. Thus caffeine degradation is a major issue in food processing industries. The conventional methods of caffeine removal (solvent, water and supercritical carbon dioxide extraction methods) are expensive, toxic and non-specific to caffeine 14. Hence from health, environment and economic point of view, it is necessary to find out the other mode of caffeine removal. Microbial methods of caffeine degradation are better alternative to this problem 15. There are reports on caffeine degradation using various microorganisms including bacteria 16, yeast and fungi 17. So far, there is no report on the mechanism of caffeine degradation using yeast. Therefore, the aim of the present investigation was to study the biodegradation of caffeine by the yeast T. asahii isolated from caffeine contaminated soil. 2. Materials and methods 2.1 Chemicals Caffeine (>99% pure) was purchased from Merck Limited, Mumbai, India. All other chemicals are of analytical grade procured from Himedia Limited, Mumbai, India and SRL Chemicals Limited, Mumbai, India. ISSN : 0975-5462 Vol. 3 No.11 November 2011 7988

2.2 Microorganism and growth medium T. asahii was isolated from soil samples obtained from coffee cultivation area of Coffee Board, Yercaud, Tamilnadu, India. The yeast was identified to the species level using VITEK 2 compact yeast card reader with the software version: 03.01 from Council for Food Research and Development (CFRD), Kerala, India. Caffeine Liquid Medium (CLM) containing (g l -1 ): K 2 HPO 4, 0.8; KH 2 PO 4, 0.2; MgSO 4.7H 2 O, 0.2; CaCl 2.2H 2 O, 0.1; FeSO 4.7H 2 O, 0.005; yeast extract 0.2 and caffeine, 2.0 was used as the growth medium, for enrichment and screening. YEPD (Yeast Extract Peptone Dextrose) as well as CLM were used for induction of the organisms by varying caffeine concentration. Caffeine liquid medium containing sucrose (2 g l -1 ) was used for the degradation studies. For solid medium, agar (20 g l -1 ) was added to caffeine liquid medium. The initial ph of the medium was adjusted to 6.5 and and the temperature was maintained at 28 C. 2.3 Enrichment and development of caffeine degrading culture A loop full of actively growing culture of the yeast was transferred to 100 ml of YEPD broth containing 1 g l -1 caffeine and incubated at 28 C in an orbital shaker for 48 h. About 5 % (v/v) of the 48 h grown pre-inoculum was transferred to 100 ml of YEPD broth containing 3 g l -1 caffeine and grown under the same conditions. Samples were drawn at known intervals of time for the measurement of cell growth. Biomass accumulated after 48 h was harvested by centrifuging at 20,000 g for 5 minutes at 0-4 C to form a pellet. The biomass pellet was aseptically transferred into 250 ml flask containing 100 ml of caffeine liquid medium containing 5 g l -1 caffeine and incubated at 28 C in an orbital shaker for 4 days for inducing cells to degrade caffeine. These induced cells were harvested by centrifugation as before. The cells were washed several times with phosphate buffer to remove residues of caffeine. Three grams of these induced cells were suspended in phosphate buffer, which were used for caffeine degradation experiments. 2.4 Degradation of caffeine in the presence of sucrose Three different inocula of T. asahii viz (1) acclimated to medium containing caffeine alone (2) acclimated to medium containing both caffeine and sucrose and (3) acclimated to medium containing only sucrose were used to study the caffeine degradation in presence of sucrose. For acclimating the culture, they were grown for three cycles in CLM containing respective substrates (each 2 g l -1 initial concentration) prior to their use as inocula. To study the effect of acclimation condition of inocula on caffeine degradation, following experiments were performed. The inoculum which was acclimated to caffeine alone was grown in two different media, one containing both caffeine and sucrose and the other medium with caffeine alone. The other two inocula were grown in the medium containing both caffeine and sucrose. Sucrose and caffeine removal percentage as well as yeast growth were monitored periodically. 2.5 Cytochrome P-450 enzyme assay Involvement of Cytochrome P-450 monoxygenase enzyme system in caffeine degrading yeast cells was detected indirectly in the presence of Cytochrome P-450 monoxygenase enzyme system inhibitor,1-aminobenzotriazole following the method of Kanaly and Hur 18. Biomass inhibition was studied by culturing the yeast cells in caffeine liquid medium containing 0.1 mm 1-aminobenzotriazole (ABT) and 2 g l -1 caffeine. Medium without inhibitor was kept as control. The activity of Cytochrome P-450 in microsomal fraction of the yeast species grown in CLM was quantified in UV-visible spectrophotometer (HITACHI- U-2800, Japan) as described by Choi et al. 19. 2.6 Preparation of enzymes and enzyme assays Cytosolic fractions of the yeast species were obtained by growing them in caffeine liquid medium with and without caffeine for 48 h. The cells were pelleted by centrifugation and suspended in phosphate buffer of ph 7.0. The cell suspensions were disrupted by ultrasonicator keeping sonifier output at 40 amps and maintaining temperature below 4 C, giving eight strokes of 5 s each with 2 min interval. Microsomal fractions of the yeast cultures were isolated by culturing the yeasts in caffeine liquid medium with and without caffeine for 48 h. Pellets were isolated by centrifugation and suspended in phosphate buffer of ph 7.0. The cell suspensions were disrupted by ultrasonication and the resultant suspensions were again centrifuged at 12000 g for 15 min and at 25000 g for another 15 min. The supernatants of the resultant solutions were precipitated by 16 mm CaCl 2 and used as microsomal fraction 20. All enzyme assays were performed with reference blanks containing all components except the enzyme. 2.6.1 Caffeine demethylase Caffeine demethylase activity was determined following the method of Gummadi et al. 21. Enzyme activities were measured in 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 0.1 ml enzyme solution and reaction was stopped after 10 min by addition of 10% (w/v) trichloroacetic acid (TCA). Reaction carried out with enzyme inactivated with ISSN : 0975-5462 Vol. 3 No.11 November 2011 7989

TCA prior to incubation served as blank for the assay. The reaction mixture was then centrifuged at 20,0009 g and 4 C for 15 min and the supernatant was analyzed by HPLC. A similar procedure was followed for other methylxanthines, caffeine being replaced by the other methylxanthines (1 mm). One unit of caffeine demethylase activity (U) was defined as the number of µmol of substrate (caffeine or other methylxanthines) degraded per minute of reaction. 2.6.2 Xanthine oxidase Xanthine oxidase activity was determined following the method of Yu et al. 22. To assay xanthine-oxidizing enzyme activity, a 1 ml reaction mixture consisting of 50 mm potassium phosphate buffer (ph 7.5), an appropriate amount of enzyme solution, 0.5 mm xanthine, 0.5 mm NAD + as the electron acceptor was incubated at 28 C. Enzyme activity was determined by monitoring the increase in absorbance at 340 nm due to NADH production. One unit of enzyme activity was defined as µmol of NAD + reduced per minute per ml of enzyme. Aliquots were removed from the reaction mixture periodically to monitor the formation of metabolites by HPLC. 2.6.3 Caffeine oxidase Caffeine oxidase activity was assayed following the method of Madyastha et al. 23 (1999) with minor modifications. The standard assay mixture contained 0.5 ml of 0.2 M phosphate buffer (ph 7.5), 0.2 ml of 0.055 mm caffeine in the same buffer, 0.1 ml of 1.1 mm 2,6-dichlorophenolindophenol (DCIP) and 0.2 ml of 0.55 mm phenazine methosulfate (PMS). The reaction was started by the addition of 0.1 ml of enzyme solution in a total volume of 1.1 ml, and the rate of decrease in the absorbance at 600 nm was followed against a blank in which double distilled water was added in place of enzyme. One unit (U) of caffeine oxidase activity was defined as the µmol of DCIP reduced per minute per milliliter of enzyme solution. The caffeine oxidase was also assayed with molecular oxygen (dissolved O 2 ) as electron acceptor instead of PMS coupled DCIP by measuring the absorbance of the assay at 300 nm. 2.7 Extraction and analysis of degradation products Caffeine degradation was monitored by High Performance Liquid Chromatography (HPLC) and Fourier Transform Infrared spectroscopy (FTIR). Identification of metabolites was carried out by GC MS. The metabolites produced during biodegradation of caffeine were extracted with equal volume of chloroform from clear supernatant. The extracts were dried over anhydrous Na 2 SO 4 and evaporated to dryness. The crystals obtained were used for FTIR analysis. The crystals dissolved in a small volume of HPLC-grade methanol were used for HPLC and GC-MS analysis. HPLC analysis was carried out on a Waters instrument equipped with a dual λuv VIS detector and a C18 column. The mobile phase used was water : methanol (70:30) at a flow rate of 1 ml/min for 10 min. FTIR analysis was done in the mid IR region of 4000 400 cm-1 with 50 scan speed, following the method of Paradkar and Irudayaraj 24 with minor modification. The GC/MS analysis of caffeine and its metabolites were carried out using Agilent 6890 GC equipped with Agilent 5973 N mass selective detector following the method of Shrivas and Wu 25 with some modification. The mass spectrometer was operated in the electron impact mode with an electron current of 70 ev. Aliquots of 1 µl were injected automatically with an auto sampler (AUC20i) in splitless mode via a GC inlet (injector temperature 250 C). A HP-5 MS capillary column (30 m 0.25 mm ID, 0.25 µm film thickness) was connected directly to the ion source of the mass spectrometer. The following temperature program was maintained during separation of caffeine. One hundred and fifty degree Celsius for 1min; 20 C /min to 230 C for 2.0 min and total time of analysis was 7 min. The trap, transfer line and manifold temperature were set at 200, 280 and 50 C respectively. The mass range of scan spectra was 50-250 Da. The biodegradation products were identified by comparison of retention time and fragmentation pattern, as well as with mass spectra in the NIST spectral library. 3. Results and Discussions 3.1 Effect of sucrose on caffeine degradation Caffeine acclimated yeast cells were grown in Caffeine Liquid Medium (CLM) containing both caffeine and sucrose maintained at ph 6.5, temperature 28 C in an orbital shaker at 120 rpm and growth of caffeine acclimated yeast cells is shown in Fig. 1a. Sucrose and caffeine were utilized simultaneously and the degradation rates of both these substrates were similar. Fig. 1b presents the observations with only caffeine as growth substrate where rate of caffeine degradation and yeast growth showed no significant difference. In our previous study, an increased biomass production as well as caffeine degradation by T. asahii was noted when sucrose was supplemented in the liquid medium containing caffeine 26. ISSN : 0975-5462 Vol. 3 No.11 November 2011 7990

Fig. 1a Growth of caffeine acclimated yeast culture showing sucrose and caffeine utilization. Fig. 1b Growth of caffeine acclimated yeast culture showing caffeine utilization. In case of sucrose and caffeine acclimated culture, both the substrates were utilized simultaneously but sucrose utilization rate was higher than that of caffeine (Fig. 2a). In contrast, when yeast culture was acclimated to only sucrose, caffeine degradation was observed only after the complete utilization of sucrose (Fig. 2b). Typical diauxic growth pattern was observed. Initial growth phase was associated with sucrose utilization. This was followed by a long lag phase of 18 h and a second exponential growth phase was associated with caffeine utilization. There was 100 % degradation of caffeine at 54 h by the yeast cells acclimated to caffeine and sucrose containing medium whereas the culture acclimated to sucrose alone and caffeine alone, degradation was delayed. Therefore, the present study showed that the substrate removal pattern exhibited by caffeine degrading T. asahii was significantly influenced by the acclimation characteristics of the culture. Fig. 2a Growth of caffeine and sucrose acclimated culture showing sucrose and caffeine utilization. ISSN : 0975-5462 Vol. 3 No.11 November 2011 7991

Fig. 2b Growth of sucrose acclimated culture showing sucrose and caffeine utilization. 3.2 Cytochrome P-450 enzyme assay In the present study, involvement of Cytochrome P-450 monoxygenase enzyme system in yeast cells grown in caffeine liquid medium (CLM) was detected in the presence of inhibitor, 1-aminobenzotriazole (ABT). The results showed that the production of biomass was inhibited in the presence of Cytochrome P-450 inhibitor compared to the control (Fig. 3) confirming the involvement of the Cytochrome P-450 monoxygenase enzyme system in caffeine degradation. Similar results were reported in case of other filamentous 27 and white rot fungi 28,18. The capacity of human Cytochrome P-450 monooxygenase to metabolize caffeine yielding trimethyl uric acids, paraxanthine and minor amounts of theobromine has been reported by Tanaka et al. 29. Further the activity of Cytochrome P-450 in yeast cells grown in the presence and absence of caffeine were assayed. The results showed that Cytochrome P-450 enzyme activity of 0.437±0.11 nmol (mg protein) -1 was present in yeast species grown in caffeine and no activity was detected in the absence of caffeine confirming that Cytochrome P- 450 enzymes in yeasts are involved in caffeine degradation. Cell dry weight (g/l) 2.5 2 1.5 1 0.5 0 0 20 40 60 Time (h) Fig. 3 Cytochrome P-450 enzyme inhibition assay. Without ABT With ABT 3.3 Enzyme activities during caffeine degradation The data shown in Table 1 represents the enzyme activities present in cytosolic and microsomal fractions of the yeast cells grown in the presence and absence of caffeine. Caffeine demethylase and xanthine oxidase activities were observed in microsomal and cytosolic fractions respectively. Caffeine oxidase activity was observed in both the fractions. Significant induction in the activities of all the three enzymes were noted compared to control. However, the activity of caffeine oxidase is less than caffeine demethylase and xanthine oxidase activities. Significantly increased activity of caffeine demethylase was observed for caffeine, theophylline, paraxanthine, 1, 3 and 7 -methylxanthines comparing to theobromine. Similar caffeine demethylating activity was demonstrated in the crude extracts of P. putida 30-32. ISSN : 0975-5462 Vol. 3 No.11 November 2011 7992

Table 1 Enzyme activities during caffeine degradation. Enzymes Substrate Enzyme activity a Microsomal fraction Cytosolic fraction Caffeine demethylase 1 Control b NA NA Caffeine 0.566 ± 0.012 NA Theophyline 0.302 ± 0.011 NA Theobromine NA NA Paraxanthine 0.311 ± 0.012 NA 1-methylxanthine 0.512 ± 0.014 NA 3-methylxanthine 0.531 ± 0.015 NA 7-methylxanthine 0.526 ± 0.011 NA Xanthine oxidase 2 Control b NA NA Xanthine NA 0.431 ± 0.011 Caffeine oxidase Control NA NA Caffeine NA NA a All the measurements were performed thrice and expressed as mean ± standard deviation. b Control culture was grown in the caffeine liquid medium without caffeine. 1 Enzyme activity- µmol substrate degraded min -1. 2 Enzyme activity- µmol NAD + reduced ml -1 min -1.NA- no activity. 3.4 Analysis of degradation products Metabolites resulting from caffeine degradation were analyzed by HPLC and FTIR. The products were identified by GC-MS. HPLC analysis of caffeine showed only one major peak with retention time 3.49 min (Fig. 4a). Compared to control, HPLC analysis of degradation products showed additional peaks at different retention time 3.719 (theophylline), 4.201(paraxanthine), 2.017 (1-methylxanthine), 2.319 (xanthine), 2.923 (uric acid) min, at 24 h (Fig. 4b) and 3.719 (theophylline), 4.201(paraxanthine), 2.017 (1-methylxanthine), 2.453 (3-methylxanthine), 2.709 (7-methylxanthine), 2.319 (xanthine), 2.923 (uric acid) min at 48 h (Fig. 4c). HPLC analysis of samples collected at 54 h showed no peaks in the chromatographs indicating complete degradation of caffeine (Fig. 4d). (a) (b) (c) (d) ISSN : 0975-5462 Vol. 3 No.11 November 2011 7993

Fig. 4 HPLC analysis of control, caffeine (a) and its degradation products at 24 h (b) and 48 h (c) and complete degradation at 54 h (d). FTIR spectra of control caffeine (Fig. 5a) showed the specific peaks in fingerprint region (3500 500 cm -1 ). The peaks at 3338.42 cm-1 supports for H-C-H stretching in methane group. The peak at 3100.74 cm -1 corresponds to aromatic -CH stretching vibrations. The peak at 2850.27 cm -1 indicates -CH stretch. The peak at 1700.75 cm -1 corresponds to C=0 stretch of ketones. The peaks at 1637.81 and 1483.83 cm -1 corresponds to C=C and C-C stretches respectively. The other peaks at 1547.00 cm -1 and below 1483.83 cm -1 represents C-N stretches. FTIR spectra of degradation products (Fig. 5b) showed different peaks at 3423.56 cm -1 for the presence of N H stretching and 3111.16 cm -1 for NH 3 + stretching. Absence of peaks at 3338.42 and 3100.74 cm -1 indicated the demethylation of caffeine. The FTIR spectra data supported the complete degradation of caffeine by T. asahii. (a) (b) Fig. 5 FTIR spectral analysis of control, caffeine (a) and its degradation products (b). GC MS data of caffeine showed retention time 11.5 min (Fig. 6a) with molecular weight 194.1. Gas chromatogram of the degradation products obtained at 48 h showed the existence of four intermediates (Fig. 6b). ISSN : 0975-5462 Vol. 3 No.11 November 2011 7994

The four intermediates with retention time 7.11, 8.29, 8.45 and 9.58 min were identified as xanthine with molecular weight 152.11, methylxanthine with molecular weight 166.137, uric acid with molecular weight 168.11 and dimethylxanthine with molecular weight 180.64 respectively. Gas chromatogram (Fig. 6c) of degradation products at 54 h did not show the existence of any compound which confirmed the complete degradation of caffeine T. asahii. (a) (b) (c) Fig. 6 Gas chromatogram of control, caffeine (a) and its degradation products at 48 h (b) and 54 h (c). Based on our results of enzyme activities and analysis of caffeine metabolites, we propose the major degradation pathway of caffeine by T. asahii as shown in Fig. 7. According to our proposal, degradation of caffeine occurs via stepwise demethylation and oxidation processes. The sequence of products formed during caffeine degradation is as follows: 1,7-dimethylxanthine, 1,3-dimethylxanthine, 1, 3 and 7-methylxanthines, xanthine and uric acid. Studies have shown that in case of Pseudomonas putida, metabolites such as paraxanthine, theobromine, 7-methylxanthine, 7-methyluric acid and xanthine were formed from demethylation ISSN : 0975-5462 Vol. 3 No.11 November 2011 7995

and oxidation of caffeine 33. In case of filamentous fungi, caffeine was first demethylated to form theophylline which was demethylated to give 3-methylxanthine and then xanthine 34. Fig. 7 Proposed biodegradation pathway of caffeine by T. asahii. 4. Conclusion Yeast cells acclimated to medium containing both caffeine and sucrose showed enhanced caffeine degradation pattern in the caffeine liquid medium (CLM). Enhancement in the activities of enzymes viz. caffeine demethylase and xanthine oxidase during caffeine degradation indicated the involvement of these enzymes in degradation. Based on the degradation products detected by HPLC, FTIR and GC-MS, it is proposed that degradation of caffeine followed stepwise demethylation and oxidation to form uric acid which was then completely degraded. Based on these results, it can be concluded that T. asahii can serve as promising microorganism for developing decaffeination process in food industries. This microbiological method can also be a better alternative to the existing environmental unfriendly conventional methods for caffeine removal. Acknowledgments We thank CFRD, Kerala, India, for helping us for identification of the isolated yeast species. We also wish to thank VIT University for providing laboratory facilities. References [1] Wang X, Wan X, Shuxia Hu, Caiyuan P. (2008) Study on increase mechanism of the caffeine content during the fermentation of tea with microorganisms. Food chem., 107:1086-109. [2] Guru M, Icen H. (2004) Obtaining of caffeine from Turkish tea fiber and stalk wastes. Bioresour Technol, 94:17 19. [3] Nehlig A. (1999) Are we dependent upon coffee and caffeine? A review on human and animal data. Neurosci Biobehav Rev, 23:563 576. [4] Smith A. (2002) Effects of caffeine on human behavior. Food Chem Toxicol, 40:1243 1255. [5] Jee S.H, Jiang H, Whelton P.K, Suh I, Klag M.J. (1999) The effect of chronic coffee drinking on blood pressure: A meta-analysis of controlled clinical trials. Hypertension, 33:647 652. [6] Jenner D.A, Puddey I.B, Beilin L.J, Vandongen R. (1988) Lifestyle and occupation related change in blood pressure over a six-year period in a cohort of working men. J Hypertens Suppl, 6:605 607. [7] Kalmar J.M, Cafarelli E. (1999) Effects of caffeine on neuromuscular function. J. Appl Physiol, 87:801 808. [8] Lorist M.M, Tops M. (2003) Caffeine, fatigue, and cognition. Brain Cogn, 53:82 94. [9] Adams M.R, Dougan J. (1981) Biological management of coffee processing. Trop Sci, 123:178 196. [10] Friedman J, Waller G.R. (1983) Caffeine hazards and their prevention in germinating seed of coffee. J Chem Ecol, 9:1099 1106. [11] Buerge I.J, Poiger T, Muller M.D, Buser H.R. (2003) Caffeine, an anthropogenic marker for wastewater contamination of surface waters. Environ Sci Technol, 37:691 700. [12] Glassmeyer S.T, Furlong E.T, Kolpin D.W, Cahill J.D, Zaugg S.D, Werner S.L, Meyer M.T, Kryak D.D. (2005) Transport of chemical and microbial compounds from known wastewater discharges: potential for use as indicators of human fecal contamination. Environ Sci Technol, 39:5157 5169. [13] White P.A, Rasmussen J.B. (1998) The genotoxic hazards of domestic wastes in surface waters. Mutat Res, 410:223 236. [14] Gokulakrishnan S, Gummadi S.N. (2006) Kinetics of cell growth and caffeine utilization by Pseudomonas sp. GSC 1182. Process biochem, 41:1417-1421. ISSN : 0975-5462 Vol. 3 No.11 November 2011 7996

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