American Journal of Food Technology 2 (1): 21-29, 2007 ISSN 1557-4571 © Academie Jounals Ine, USA Optimization of Physical Parameters for Biodegradation of Caffeine by Pseudomonas sp.: A Statistical Approach Swati Shcharita Dash and Sathyanarayana N. Gummadi Department of Biotechnology, Indian Institute of Technology-Madkas, ‘Chennai 600 036, India Abstract: Pseulomonas sp. NCIM 5235 capable of degrading high concentrations of affine has been previously isolated frm the soil of coffee plantation area, The isolate was capable of degrading 64g L~initial concentration of caffeine at arate of 0.1 gL", In this study, the physical parameters viz., pH, temperature and shaking speed have been ‘optimized using central composite design. The optimum values of pH, temperature and shaking speed were found to be 78, 28°C and 190 zp, respectively, Under optimized condition of pH, temperature and shaking speed, the rate of degradation of caffeine has been. enhanced fom 0,18 0 0.29 g L-'h which is 16 fold higher than the normal rat, This is the frst report on degradation of high convention of caffeine at higher rates. Under ‘optimal conditions, the tain has also been found to degrade caffeine at 15 g L* initial concentration eflcienly within 48 h, This makes Pseudomonas sp. NCIM 5235 an atwactive candidate for development of biodecuffeination statics. Key words Caffeine degradation, Pseudomonas sp, statistical opimization, physical parameters Introduction Caffeine (1,3, rimethyixanthine), «purine alkaloid naturally oce sng in more than 60 plant species (Steffen, 2000), forms an assntial component ofa variety of beverages like ta coff2e and caffeinated soft drinks and numerous food products like chocolates and desserts as it increases alertness and concentration by overcoming fatigue (Nehlig, 1999), Apart from that caffeine is widely usedin pharmaceutical preparations as it enhances the effict of certain analgesics and antipyretic drugs, Caffeine is also used as a cardiac, neurological and respiratory stimulant and as a diuretic (Mazzafera, 2002), This makes cafeine one ofthe mest widely consumed pyyehoactive compounds with the global average consumption ranging fom 80 to 400 mg caffeine per person per day ‘Gokularishnan et a. 2605), Consequently, eaffine is also one ofthe major agro-industria wastes generated fiom coffee and fea processing plants. Caffeine containing wastes are offen released to the surrounding water hodies and it is detected in surface water, ground water and waste ‘water efTiuents often at a high concentzation (~ 10 g L~*) (Buetge eta, 2003, Weigel etal. 2004, Gassmeyer ofa, 2005), In order to fee the natural waters ftom this Nenobiati, asthe ingestion of cafTeine and its chvorinated byproducts (derived during chlorination of water) hus severe adverse effect on the physiological system (Gould and Hay, 1982; White and Rasmussen, 1998), decaffenation of the Dyproduets becomes a very necessary stap in treatment of coffee and tea wastes, Decuffination of ‘other food products and) beverages is also being recommended, keping in view the adverse effects ‘Corresponding Author: Satyararana Gananal, Deparment o isehnslog, Indl Instat of Tecnoloy-Maias, chen 600036, in Tek OL-H2RS7-N1N ase 9)-12387-108 2Am. J. Food Technol. 2 (D): 21-29, 2007 ‘of chronic caffeine consumption particularly on the cardiovascular system and health of women (Jonner era, 1988; Beraran ef a, 1990; Jee et al, 1999; Kalmar andl Cafarelli, 1999). In this aspect, ‘microbial and enzymatic techniques for decaffeinaton have been found advantagecus than other ‘conventional methods such as solvent extmetion or super critical carbon dioxide oxidation (Gokulaksishnan er af, 2005) Several microbial srains belonging to Pseudomonas, Serratia, Aspergilus, Penicillium and ‘Stemphynan bave been reported to be able to degrade caffeine (Selwimmer era. 1971, Woolfolk, 1975; Bletcher and Lingens, 1977; Mazzaferra etal, 1994; Asano er af, 1994; Hakil eral 1999; Yamaoka-Yana and Mazzaferra, 1999), However, these stains were not capable of degrading a higher concentrations of caffeine, except for Pseudomonas sp. capable of growing at 5g L~* of ‘affaine with a degradation sate of 09 g L-" h~ Previously we have isolated a stain of Pseudomonas fiom the soil of coffee plantation area thats capable of uilizing caffeine as sole scutee oF carbon and trogen. This strain closely resembles Pseudomonas putida based on 16S RNA. analysis (Desh and Gutman, 2006). Kinetic stues on this bacteria shows tht i follows substrate inhiaition “inates for caffeine degradation and cafeine concerration of 20 g L~ was found to be inhibitory for srowth (Gokulaksshnan and Gud, 2006), The degrading ability ofthis strain was also found to ‘behets ast could bring about complete degradation of caffeine at an inital concentration of 5 g L~* attherate of0.1 g Lh Opkinization of mein components resulted in farterinerease in the rate of dzgradation to 0.18 g L~'h (unpublished data), Tn the present study. physical parameters like pH, temperature and shaking speed were ‘optimized using response surice mstheloogy fer studying the interaction effets among the variables and further inereasing the rate of degradation of eaffene by this strain Materials and Methods Chemicals Pure caffeine was procured from Merck, India. All other reagents were of analytical grade procured in India, Microorganism Pseudomonas sp. NCTM 5235 was maintained cn nutrient agar meviuum which had the following, ‘composition (g L->: bee? extuet 1; yeust extmct 2; peptone, 5; NaCl, $ and agar, 25 and was subcultured every two weeks. Media ‘The composition of CAS medium was as follows (gL: NaHPO,, 0.12; KH,PO, 1.3; CaCl, 03; MgS0,, 7 H,0, 03, sueros, 5 eatin, 1.2 and agar, 25."The inital pH ofthe medium was 6, For flask culture experiments optimized CAS medium with the following composition was used (gL) Na.HPO,, 0.352, KH,PO,, 3.4, CuCl, 0.3; MgSO, 7 H,0, 0.3 suerose, 5 eafTeine, 6.4 anu 0.07 (Wis) of Fe". The initial pH of the medium was adjusted us per experimental design (Table 2}. ‘Flask Culnure Experiments Three loopfil of 36h grown culture fromm CAS plate was transfered aseptically to 25 mi nutrient ‘broth medium and incubated to midelog phase (ODya~1-4) for 2.5 h ona rotaty shaker operating at 180 rpm al 30°C. Twenty five milter of sterile optimized CAS medium was inoculated with 6% (viv) ofthe cule andl incubated foe four days at conditions specified y the experimental design (Table 2). Ategular time inervals | mi of culture was taken and centrifuged at 10000 yp for 10min. ‘The supernatant was used for analysis of caffeine, The cll pellet was Washed twice with MIKO water al OD gag, Was measured for cell dry weight nAm. J. Food Technol. 2 (D): 21-29, 2007 Caffeine Estimation Caffeine was estimated by HPLC (Agilent 1100 series) equipment using a ZORBAX C-18 column wi 10mnM ammonium phosphute buffer (pH 2 SVcetotile (4-1, ww) as motile phase, Pure caffeine (Merck) at 2 gL” was used as standard. The retention ime of caffeine was found to be 49 minat low rate of | mL min and t 28°C, Detection of caffeine was done at 254 nm using UV detector. Optimization of Physical Parameters Using a Ceniral Composite Design ‘The physical parameters viz. pH, temperature and shaking speed were optimizes using central ‘compesite design (Box and Wilson, 1951; Box and Hunter, 1957). According to this design, the total ‘numberof treatment combinations is + 2k +n, where Kis the number of independent variables and 1, the numberof repetitions of the experiments at the eenter point. For statistical calculation, the variables X, have been coded as x according to the following transformation: ao ax ‘where xis dimensionless cade value of the variable XX, the value of the X, athe centerpoint and |X the step change, A 2fuctorial design with six axial points und six replicates atthe center point ‘with a total number of 20 experiments was employed for ptimizing the medium components. ‘The behavice ofthe system was explained by the following quadkatic equstion: Y= Bet BB+ BBX? BBN, o where Y is the predicted response, B,,is the intercept term, B, isthe linear effect, iis the squared ffect and Bis the intraction effect. The repression equation (Eq. 2) was optimized foe maximum value to obtain te optimum conditions using MATL.AB® version 7.0 (Mathyveeks Inc, Natick, Massachusetts, USA). Results and Discussion ‘The interaction between the physical parameters viz, pH, temperature and shaking speed in ‘lation to caffeine degradation was staied by response surface methodology. central composite dlosign was usod for studying tho infraction of those variables within a ange of -1.68 to +168 in relation to caffeine degradation (Table 1), The experimental plan is given in Table 2, The results ‘obtained from the cantal composite design experiments wet fitted to a second order polynomial ‘equation to explain the dependence of caffeine degradation on the medium compenents Experiments were performed with varicus combinations of pH, temperature and shakina speed asgivenin Table 2. The rte of degradation of caffeine was highest (026 g L-* hr) when pH, ‘Table 1: Coded and Uncoed values of psc parnetrs used feel composite desig, Coded aes a ss vibes “L ° a 45 6 as 452 Tanperaure °C) 20 so sso seat ‘Shing speed pa) 1500 1) 2100 au6 2aAm. J. Food Technol. 2 (D): 21-29, 2007 ‘uble2: Central onasdesin fr pinzing pyc prance for cfsine degen by Padma. NCD 55 ‘Catsinedegaton rte @ LH) Temperate Stakingapend uno Black RPM Eyes pe et “r ‘a0 008 1 4 a 26 026 1 oa 4 a2 arr 1 ° ° 022 ieee a 008 2 0 4 a a 001 2004 Hn a one 2 ° ° ° om 2 ° ° ° on 3 he oo ° 028 3 ° Ls ° ois 3 ° he ° 008 3 ° ° 68 ors 3 ° ° ° om 3 © © © om "00,9576 R= 096. Espermeial values re anenge ripeates wie cwhacd temperature and shaking speed were at +1, -1 and +1 level, respectively (Run # 2), Caffeine dopradation was completely inhibited in all cases where the pH was at-I or -1.68leveli.e, at 4S of 3.4 indicating inhibition of bacterial growth at lower pH. The rate of degradation was however not much different in eases of where temperature and shaking speed were at loves indicating that the negative effect of one ofthe vatable was compensated by the oer, Wren all tt three variables were ar-+1 level (Run # 10}, the rate of degradation was not much diferent from that of center points (Run # 5,6, 11, 12, 19, 20) ‘The rate of degradation was lowest when the temperature -1.68 level and the cher parameters at O levels (Run# 15). Ths shows thatthe bacteria can survive at a temperature lower but the enzymes response for bringing out degradation were not optimally active at tat temperature, Similar results were also obianed when the shaking spocd was at -1,68 level and the other parameters at 0 levels (Run 17). In conditions of slower shaking speed, the oxygen avallbilityis considerably lower and ‘his in tum affects the degradation process which is supposed to be oxidative, Interesting results were obtained in ease ofthe shaking speed ut + 1.68 level and the olher two parameters at O levels (Run# 18) Tt was noted that at high shaking speed, caffeine degradation proceeded up to 80% within 24 hand then remained constant, Growth was also noted to be inhibited a hat ime pont, Accumulation of metabolites, particularly xine, was also rote at this eomition. ‘pH was also observed to increase upto 9 which vas probably due to the accumulation of metabolites. ‘These fiwingsinicate the possibilty of Feed buck inkibition mechanism cecurring with the caffeine degradation phenomena. Atinoreased aeration with high caffeine concentration, the rate of eonversion of eaffeine to its metabolites is very rapid as compared to the rate of assimilation by the cells. This results in the accumulation of metabolits in th culture medium, which in turn inhibit the eaffeine degrading enzymes ane bring about a change in pH to alkaline resting in growth inhibition. Inhibition ‘of gronth and eafsine degradation by Pseudomonas sp. NCIM 3235 at higher pH has been neted in 24Am. J. Food Technol. 2 (D): 21-29, 2007 ‘able: ANOVA table for catTeine desadaton fT pH, tanger an shakin speed Sauce Sum of ques a ‘Mean sqae E BE, Blocks ‘ooinie 2 Mosel ai9s012 ° omnia 1 ocons Bre 012352 8 omens al fa2ng220 1s cation experiments with the strain (data not shown). So also inition of caffeine degradation at higher inal concentration of caffeine has been reported ealir for Pseudomonas putida (Woolfolk, 1975). More studies on this eed back inhibition mechanism wil prove useful in developing strategies of obtaining higher concentrations of metabolites of caffsine degradation pathway that have many applications, ‘The second order regression equation (Eq, 3) shows the dependence of caffeine degradation on DH, temperature and shaking speed. Multiple regression analysis ofthe experimental data generated ‘the parameters ofthe equation which i represented as follows y 3468 + 0.097644, -0.026060x, + 0,004659%, -0,0386P8x,-0.051050%.! - 0.0139853,2-0.008750x.x, + 0.003750 ¥,x,-0.0012503.x, 8 ‘where Y is the predicted response, x, s the coded value of X; (PE), x, is the coded value of X, lemperature) and, x; i the coded value of X, (shaking speed). Statistical testing ofthe model was performed by the Fisher's statistical test for ANOVA (Table 3). The P-value, which i the ratio of mean squae dc o regression tothe mean squared to error, was ealculated to be 13.34, The analysis ‘of variance of the quadkatc regression model also suggests hat the model is very significant as indicated by « low protubility value ((P,..°F)~ 0.0006]. Soalso the values of RE (0.8518) and R (0.9376) also indiested that the model predicted values are in perfect agreement with the ‘experimental valuss as shown in Table 2, Equation 3 also predicts the interaction of variables affecting caffeine degradation. Coeflisien’s of imeruction terms Were significant as compared to linear terms indicating that the interaction between the variables cannot be neglected ‘The made! equation was solved using MATLAB 7.0 software to generate the optimal values for the above three variables. The optimum values of pH, temperature and shaking speed were found to ‘be 78, 28°C and 190 mpm, respectively. The model predicted value of rate of degradation of eafleine twas determined to be 0.29 8 Lh which is 1.6 fold higher than the rate of degradation under _unoptimized conditions, Sufuee plots of de response using Eq, (3) when one ofthe Variables is fixed at optimum value and the other two are allowed to vary are show in Fig, 1 a-c, It was seen that with ‘there sen increase in the rate of easing degradation with the increase in pH within the design region (Fig. 1a and b,Inall the three cases the optimum rate of degradation predicted by the plots matched ‘with the experimentally predicted value for opium rate of caffeine degradation, In order to verify the optimal conditions obtained using central composite design experiments were performed under the opti cooitions and compared with dhe center points (Fig. 2). As seen fiom Fig, 2 the experimental results match with the model predicted one, proving thatthe optimal values of the physical parameters obtained were ideal, Such a condition may prove usefl in studying the metabolism of caffeine by the stain of Pseudomonas in order to develop methods fer biclogical decaifeination ‘The botlneek in biodecafleination of effete containing cBiluntss th lack of a microorganism capil oF tolerating and degrading ealzine at high coneentaions, as the concentrations of calling in cfflunts can be as high as 10 gL“! The next objective, therefore, was to study the degradation of 2sBr J Boo Toby 2 (Ne 21-29, 07 i qi 7 : pie te tie opti) © Fig. 1 Reponse sitio plas toning effet of pH, eeperanne end staking speed on rate of (silane dapradation ty Pema cp. NCIRESZ5 (@) lft of tmpeatune and pa cptisal aking speed corteof cafes deg, ©) ee. of pH se cheking speed at Cptraal lexperaure cn mle of eafere degadstin, (0) eflet of duking wea) ad ‘Srpentize a cplal pAfonraleof affine degradation 6Fig. 2 Fig.» Am. J. Food Technol. 2 (D): 21-29, 2007 2 Rat ofateine dering 1"2°) ‘cents ‘Opsieat Odin pant (Experineta) (Model) Comparison of caffeine degradation by Pseudomonas sp. at optimized and unoptimized conditions of physical parameters. Experiments were performed at center point conditions as specified by CCD table (Table 2) ant at optimal conditions obtained affer solving Eq, 3 ‘Samples were drain at 6 interval and caffeine was estimated by RP-HPLC on @ C-18 column ‘vith 10:mM ammonium phosphate buffer (pH 2.5yscetonitile (4:1, viv) as motile phase Tee) ‘Degradation of high initial concentrations of eaffeine et optimal conditions of pl, temperature and shaking speed by Pseudomonas sp. NCIM 5235, The inital caffeine concentration was varied from 6.4 to 20 g L’ and fermentation was earied out at optimal conditions of pH, ‘emperature and shaking speed es predicted from Eg. 3. Samples were dean at 6 interval ad caffeine was estimated by RP-HPLC on a C-18 column with 10 mM ammonitam phosphate buffer (pH2.SYacetonitrle (41, viv) as mobile phase be metabolized completely within 24,36 and 48 h sespectively (ig. 3). Under optimized conditions ‘he inhibition at high concentrations as depicted by kinetie studies onthe strain (Gokulakishnan and ‘Gummadi, 2006) could be overcome and it could also degrade caffeine at inital concentrations of 20 g L~ but at lower rates, This makes Pseudomonas sp. NCIM 5235 an exvellent candidate for developing methods for efllvet treatment asthe need for dilution of cflluent to bring caffine to lower ‘concentrations can be done sway’ with thereby reducing the eost of operation,Am. J. Food Technol. 2 (D): 21-29, 2007 Conclusion Optimization results show that with some modification in physical parameters viz, pH, ‘emperahure and shaking speed the degradation of a higher concentration of caffeine could be achieved and o als the rate of degradation could be enhanced. The degradatien of caffeine under optimized conditions can prove useful in generation of important metabolites ike theobromine and ofber ‘methylxanthines or can be wsed to recover enzymes for development of biological methods of decaffeination. Response surfioe methodology can be suecessflly used to optimize the physical parameters and stuly the interaction effect among the same cn caffeine degradation rate, References, Asano, ¥., T. Komeda and #, Yamada, 1994, Enzymes involved in thecbromine production from caffeine by a Pseudomonas putida No. 352. Biosci. Biotechnol. Biochem. 58: 2303-2304. Beqgman, EA, LK. Massey, KJ. Wise and DJ Sherrard, 1990, Effects of dietary caffeine on renal ‘handling of minerals in adult vionnen, Life Sei, 47: 357-564, Blecher, R. and F. Lingens, 1977. The metabolism of eaffeine by « Pseudomonas putida strain. Hoppe-Seyler’sZ. Physiol. Chem., 388: 807-817. Box, GE. and KB, Wilson, 19S1, On the experimental designs for exploring response surfaces. Ann. Math, Stat, 13: 1-45, ox, GEP. aed .S, Hust, 1957, Multistctor experimental designs for exploring response surfaces ‘Ann, Math, Stat, 28: 195-241 Buerge, IJ... Poiger, M.D, Muller and H.R, Buser, 2003, Caffeine, an anthropogenic marker for ‘wastewater contamination of surface waters. Environ. Sci, Technol, 37: 691-700. Dash, SS, and S.N, Gummadi, 2006, Biodegradation of caffeine by Pseudomonas sp. NCIM 5235, Res. J. Microbiol. 1: 115-123. Glessieyer, ST, ET. Furlong, D.W. Kolpin, JD. Cahill, 1D. Zauge, SL. Wemer, M-T. Meyer and D.D. Kryak, 2005, Transport of chemical and microbial compounds frm known wastewater discharges: Potential for use as indicators of human facal contamination. Environ. Sci. Teehnel, 30, 5157-5169, Golulalsishnan, S, K Chandraraj and S1N. Gummadi, 2005, Microbial and enaymatie methods for the removal of eaffine, Enzyme Microbiol. Technel 37: 225-232, Golulakrishnan, 8. and SN. Gummadi, 2006. Kinetics of cell growth and eaffeine utilization by Pseudomonas sp. GSC 1182. Process Bioehem., AI: 1417-1421 Gould, J.P, and TR. Hay, 1982, The nature of the reactions between chlorine and purine and Pyrimidine bases: Products and kinetics. Water Sei. Technol., 14: 629-640. Hakil, M, F.Voisinet, G. Vinicgra-Gonzilez and C. Augut, 1999, Caffsne degradation in slid state femnentatioa by cspergiliu tamarit Effets of addtional nitrogen scusces. Process Biochem, 35, 103-109, Jee, SH. H. ang, PX. Whelton, I Suh and MJ. Klag, 1999, The Effet of Chionie Coffee Denking ‘on Blood Pressure: A Meta-Analysis of Controlled Clinical Trials. Hypertension, 33: 647-652 Jenner, DA, LB. Puddey, LJ. Beilin and R. Vandongen, 1988, Lifestyle and occupation related change in blood presse over a six-year perio in a cohort of working men. J. Hypertens, Suppl, 6: 605-607. Kalmar, JM. and B. Cafarelli, Physiol, 87: 801-808, Mazzatere, P.O. Olssonand G. Sandberg, 1994. Degradation of caffeine and related methylxanthines by Serrania marcescens isolated from soil under coffee ciation. Micrebicl, Eel, 31: 199-207 fleets of caffsine on neuromuscular function. J. Applied 28Am. J. Food Technol. 2 (D): 21-29, 2007 Mazzafera, P,, 2002. Degradation of caffeine by microorganisms and potential use of decaffeinated coffee husk and pulp in animal feeding. Sci. Agrical., 59: 815-821 Neblig, A, 1999. Are we dependent upon coffee and caffeine? A review on human and animal data. ‘NeutoseiBiobebiv. Rev , 23: 563-576. Schwimmer, S, RH. Kurtzmana and E. Heftmann, 1971. Caffeine metabolism by Penicilion roqueford. Asch, Biochem. Biophys, 147- 109-113, Stefen, DG, 2000. Chenssty and health benefits ofealzinated beverages: symposium overview. In Caffeinated Boverages, Health Bonefits, Physiological Effeets and Chemisty, Ser. 54.ACS, Parliament, TH., CT, Ho and P, Schieber (Eds). Washington DC. pp: 2-8 ‘Weigel, SU. Berger, Ensen, R Kallenbom, H. Thoresen and H, Hilierfuss, 2004, Detemnination ‘of selected pharmsccuticals and caffeine in sewage and seawater fom TromsoNorway with emphasis on ibuprofen andits metabolites. Chemosphere, 56: 583-592, White, PA, and J.B, Rasmussen, 998. The genotoxic hazards of damesti wastes in surface waters Mutat. Res, 410: 223-236, ‘Woolfalk, .A., 1975. Metabolism of N-methylpurines by a Pseudomonas putida strain isolated by ‘nsichment on caffeine asthe sole souree of carbon and aitrogen, J. Bacteriol, 123, 1088-1106. ‘Yamoks-Yano, D.M. and P. Mazzafera, 1999, Catabolism of caffeine and purification of 4 xanthine ‘oxidase response for methylurc scids production in Pseudomonas putida L. Rev. Microbiol, 30: 62-70, »