Journal of Science, Ilamic Republic of Iran 24(4): 305-312 (2013) Univerity of Tehran, ISSN 1016-1104 http://jcience.ut.ac.ir Biodecaffeination by Peudomona peudoalcaligene TPS8, an Iolated Strain from Tea Plantation Soil Morahem Ahengroph *, Sajad Ababaf Department of Biology and Biotechnology, Faculty of Science, Univerity of Kurditan, P.O. Box 416, Sanandaj, Ilamic Republic of Iran Received: 28 September 2013 / Revied: 9 November 2013 / Accepted: 1 January 2014 Abtract Development of an environmental friendly and cot-effective proce for microbial degradation of caffeine to non-toxic compound are promiing to olve the problem of phyiochemical extraction of caffeine in the treatment the caffeine containing agroindutrial effluent. Thirteen bacterial train, iolated from tea plantation oil in the north region of Iran, were creened to how their abilitie in uing caffeine a the ole ource of carbon and nitrogen. The intrinic tolerance of the iolated train to the caffeine ubtrate wa meaured in a defined and complex medium by uing the agar dilution method. Baed on the tolerance efficiency, iolate TPS8 which howed maximum tolerance to caffeine wa elected and identified a Peudomona peudoalcaligene train TPS8 (GenBank acceion number KF414528) according to the cultural and phyiochemical characteritic and alo 16S rdna gene equencing. Growing cell of P. peudoalcaligene TPS8 were ued for the biodecaffeination experiment. The maximum removal of caffeine (80.2%) wa reached after a 72 h incubation uing 2.5 g/l of caffeine ubtrate without further optimization. Our reult how that growing cell of P. peudoalcaligeneae TPS8 can thu be efficiently ued a a imple and cheap proce for preparative decaffeination from agro-indutrial effluent. The preent urvey i the firt report on biodecaffeination uing Peudomona peudoalcaligene. Keyword: Caffeine; Microbial degradation; Tolerance profile; Peudomona peudoalcaligene train TPS8 Introduction Caffeine (C8H10N4O2) i a purine alkaloid preent in the bean, leave and fruit of more than ixty plant pecie, uch a tea (Camellia pecie), coffee (Coffea pecie), cocoa (Theobroma cacao) and the like [14]. Caffeine i widely ued in pharmaceutical preparation and beverage product and can be aociated with a variety of pharmacological effect. It i ued a a cardiac, neurological and repiratory timulant and alo a a diuretic compound [7]. Caffeine effect on leep, but thee effect are different in all individual. Caffeine rie alertne during awakene cycle, However, conumption on Caffein might lead to adiorder called * Correponding author: Tel/Fax: +988716624133; Email: m.ahengroph@uok.ac.ir 305
Vol. 24 No. 4 Autumn 2013 M. Ahengroph and S. Ababaf J. Sci. I. R. Iran Inomnia. [28]. The higher level of caffeine lead to higher rik of health problem uch a adrenal timulation, irregular mucular activity, cardiac arrhythmia, oteoporoi and heart output enhancement [13]. Exce caffeine i reported to caue mutation, inhibition of DNA repair and inhibition of adenoinemonophophodieterae [26, 30] and caue of micarriage in pregnancy period [9]. Regardle of the health effect, decaffeination proce i important from the view of environmental preervation [23]. Influx of coffee proceing indutrial effluent into lake can give rie to drinking water to be unuitable [3]. Caffeine in oil alo retrain eed grain germination and it growth [1]. Several method including organic olvent, water diffuion and uper critical carbon dioxide extraction have been applied to the removal of caffeine [22]. Conidering, thee procedure are hazardou to health, expenive, time conuming, intenive work and nonpecific for the removal of caffeine, alternative route for it removal including microbial biodecaffeination are being contantly explored. Caffeine i regarded a toxic for many microorganim at a concentration of 1 g/l. However, caffeine degradation ha been reported for a variety of microbial pecie belonging to Penicillium pp. [2, 27], Thermomonopora p. [29], Serratia p. [18], Kleiella and Rhodococcu p. [17], Alcaligene pp. [19], Trichoporon p. [15], Peudomona pp. [6, 8, 10, 11, 25, 31, 34], Brevibacterium p. [20], Apergillu pp. [12,21], Chryoporium keratinophilum [21]. We would like to link thi tudy to iolate and characterize native bacterial train to high-tolerance ability of Caffeine and a potential for efficient caffeine degradation under growing-cell condition. We creened 13 different bacteria train for their abilitie and hereby uing caffeine a the ole ource of carbon and nitrogen. Finally, a newly iolated train of Peudomona peudoalcaligene, deignated a TPS8, with high tolerance pattern a well a an efficient caffeine degradation wa howed over our experiment. The preent invetigation give the firt evidence for the biodecaffeination by P. Peudoalcaligene. Material and Method Chemical and Media Caffeine (> 99%) ued for caffeine degradation experiment wa from Sigma-Aldrich. The caffeine tock olution wa prepared in ditilled water and maintained at 4º C following terilization through a 0.22-μm microbiological filter. Nutrient agar (3 g/l beef extract, 5 g/l peptone and 20 g/l agar ph 7.4) wa purchaed from Kardanazma, Iran. All other chemical are of analytical grade. Enrichment culture and Iolation of caffeinedegrading bacteria Sample were collected randomly from tea plantation oil obtained in different area of Northern Iran and tored in the dark at 4º C until ue. Enrichment culture wa carried out by mixing one gram of collecting oil ample with 100 ml of autoclaved mineral M9 medium from Sambrook et al. [24] (7.5 g/l Na 2 HPO 4.2H 2 O, 3 g/l KH 2 PO 4, 0.5 g/l NaCl, 0.5 g/l MgSO 4.7H 2 O and 0.015 g/l CaCl 2 ph 7± 0.1) upplemented with 1 g/l of caffeine a the ole ource carbon and nitrogen ource in 250 ml Erlenmeyer flak and incubated at 28º C in a rotary haker et at 150 rpm for 7 day. The initial ph of the media wa adjuted to 7. After enrichment, ingle colonie were iolated on minimal M9-caffeine agar media by pread plate method and ubjected to further purification by treak plate method on the ame media. Morphological different colonie were elected a inocula for determining their caffeine tolerance profile uing the agar dilution method of Wahington and Sutter [33]. Caffeine tolerance the iolated bacterial train For the caffeine tolerance profile, 30 ml of melted Nutrient agar and Modified M9 defined minimal alt (glucoe 2g/l; NH 4 Cl 1g/l; MgSO 4.7H 2 O 0.5 g/l; CaCl 2 0.015 g/l; FeSO 4.7H 2 O 0.03 g/l; NaCl 0.5 g/l and Phophate buffer 0.1 M ph 7.4) agar media upplemented with different concentration of caffeine range from 2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20 g/l were prepared and poured into 10 cm plate. Each plate wa ubdivided into eight zone and 20 µl of 0.5 McFarland tandard of bacterial upenion (1.5 10 8 cfu/ml), prepared from bacteria culture grown overnight for 24 h, wa tranferred on each plate uing a ampler followed by incubation at 28º C for up to 5 day. The growth of bacteria train wa calculated viually. Each plate wa run in triplicate. The colony with the highet caffeine tolerance ability wa deignated a TPS8 and elected for further invetigation. Identification of the iolate TPS8 The iolate TPS8 i characterized by phyiologicalcultural and biochemical tet with 16S rdna gene equence analyi. Morphological and phyiological characteritic were performed according to the Cowan and Steel Manual for the Identification of Medical Bacteria [5]. The tet included gram reaction, colony hape, color on nutrient agar, preence of oxidae and catalae, motility, hydrolyi of tween 80, utilization of citrate (Simmon method), tyroine hydrolyi, ureae 306
Biodecaffeiination by Pe eudomona p eudoalcaligen ne TPS8 Figure 1. UV V/vi aborptioon pectrum (A A) and contructtion of a calibrration curve (B) for determinaation of the con ncentration of caffeine in caffeine containing olution by y UV Analyi.. production, H2S productiion, nitrate reduction, r caein hydrolyi, llecithinae prooduction, gelaatin liquefactiion, and the prodduction of aciid from ugarr and growthh of 42º C. The iiolate wa further fu characterized baedd on molecular phhylogenetic analyi a a decribed d bel ow. Genomic DN NA wa extrracted from colonie c uinng a Genelute DN NA extractionn kit (Sigma) by following the recommendeed procedure of o the manufaacturer. Univeeral primer 8F ((5ʹ-agagtttgatccctggctcag-3ʹ) and 1541R (5ʹaaggaggtgatcccagccgca-3ʹ) were ued to amplify the partial 16S rdna gene [16]. The PC CR reaction w wa performed inn mixture conntained 0.6 µl µ of Smar T Taq Pfu DNA poolymerae (5 U/ µl); 5 µl PCR buffer (20 mm Tri-HC Cl (ph 7.9), 0.1 0 mm EDT TA, 5 mm 2M ME, added Stabillizer, glycerinn); 1 µl of each e primer (25 µm); 4 µl M MgCl2 (50 mm M); 0.6 µl dnt TP (10 mm) and 2 µl DNA tem mplate (180 ng/ n µl) in a fin nal volume off 50 µl with a thermal cyycler (Eppend dorf, Germanny). Amplification wa perform med according g to the follow wing time program m: 94 C for 10 min, follow wed by 30 cyccle of 94 C for 1 min, 56 C for f 1 min, and d 72 C for 2 m min, and a final exxtenion tep at 72 C for 10 0 min. Ampliffied PCR producct (5 µl) weere ubjected to tandard gel electrophorei by uing 1% agaroe gel and 1XT TAE buffer with a 1kb DNA A ladder (Feermentae) a a molecular weeight marker. The gel-puriffied PCR prodduct wa ent to Macrogen Company C (Seeoul, Korea) for equencing iin direction with w an autom mated equenncer. The equencce of 16S RDNA of the t iolate w were depoited inn GenBank affter analyzing g with regiteered STN equence by uing the tool of BLAS Phylogennetic (http://www..ncbi.nlm.nih.gov/blat). analyi wa performed with Molecu ular Evolutionnary Genetic Annalyi (MEG GA) oftware verion 4.0 by uing the Neighbor-Joininng method [32]. Caf affeine degrad dation experim ment A loop full of grown cultuure of P. Peud doalcaligene TP PS8 on nutrieent agar meddium fortified d with 1 g/ll cafffeine tranfe ferred to 2550-ml Erlenm meyer flak con ntaining 50--ml of minneral M9 medium m [24]] up pplemented with w variou cconcentration n of caffeinee (0.5-5 g/l) a the ole ouurce carbon and nitrogenn ou urce and incu ubated at 28º C for 120 h un nder agitationn 150 0 rpm on a ro otary haker. T Time-coure ample weree witthdrawn at diifferent intervval of time and a ubjectedd theem to analyi of cell growtth and reiduaal caffeine in a UV V Vi pectro ophotometer ((Specord 210 0, Carel Zei Technology, Geermany). Thee cell ma concentration c weere determined from tripliccate 2 ml am mple, whichh weere centrifuged for 10 minn at 5000 rpm m. The pellet weere wahed with ditilled waater, recentrifu fuged, dried att 75ºº C for 36 hour and weighted [4]. [ Caffeinee deg gradation in the t culture m media wa mo onitored by a deccreae in abo orbance in a U UV- viible Analytik A Jena' peectrophotometter with quarttz cuvette off 2 cm opticall patth according to t the Lakhm mi and Da [1 15] (Fig. 1A).. Th he cell were centrifuged c att 8000 rpm fo or 10 min at 4 C and the uperrnatant were ued for determination off thee reidual caaffeine in thee culture. Percentage P off cafffeine removaal wa calcullated a follo ow: caffeinee rem moval (%) = [(initial caffeiine concentraation- reiduall cafffeine concenttration) / (initiial caffeine co oncentration)]] 100 [31]. Calibrating ollution were prepared byy diluting tandarrd olution tto the follow wing caffeinee con ncentration in nterval 5 up tto 35 mg/l. Good G linearityy forr the concentration intervall examined by y the equationn and d the coefficient of determ mination for th he calibrationn plo ot a hown n in Fig.1B B. All experriment weree perrformed in triiplicate and thhe mean of three t eparatee exp periment wiith the tanddard deviation hown byy 307
Vol. 24 No. 4 Autumn 20 013 M. Ahe engroph and S. S Ababaf J. Sci. I. R. Iran n Figure 2. Caffeine tolerancce profile of baccterial train. vertical bar.. pro oduced catalae and oxidaae. The colon nie appearedd rou und, mooth and a not formeed a pigment. On the bai of cultural and morphologica m al characteritic a well a phy yiochemical characteriticc, the iolate i i preliminaryy plaaced in the genu g of the Peudomona a. Microbiall chaaracteritic of o train SHL L1 a tudied are lited inn Table 1. The ap pproximate fuull-length 16S S rdna genee (15 502 pb) from train TPPS8 wa am mplified andd eq quenced. 16S RDNA genee equence of train TPS88 R Reult Screening foor high caffein ne-tolerant ba acteria A electivve creening method baed on enrichm ment technique w wa adapted for f the electtion of bacteerial culture on tthe bai of growing g on caaffeine in a w welldefined liquuid media a decribed in n Material and method ecttion. Thirteen bacterial traain were iolaated from collectiing oil amplle of tea plan ntation (northhern Iran), which were deignated a iolatte TPS1-TPS S13. In order to reeach efficient biodecaffeinaation, it i neeeded to apply an eefficient bacteerial train which can tolerrate high caffeinee concentratioon. Keeping in view, caffeeine wa added to nutrient agar and y ynthetic defiined minimal alt media at concentration ran nging from 2.55 to 20 g/l and thhe intrinic tollerance of thee bacterial traain to caffeine w wa evaluatedd (Fig. 2). It i i clear from the reult that iolate TPS S8 have maaximum caffeeine tolerance w with a tolerancce pattern 10 and a 15 g/l in the defined andd complex media, m repecctively (Fig. 2). Becaue of it maximum m tolerance, train TPS8 w wa elected, for characterizedd and examined e biodecaffeinaation experim ment. Table T 1. Phenotypic characterritic of caffeine-degrading train TPS8 Ch haracteritic Strain TPS8 Sh hape Rod Grram reaction Gram-negativee Ox xidae Caatalae Niitrate reduced Sim mmon citrate Urreae Geelatinae producction H2S from TSI Caaein hydrolyi Tw ween 80 hydroly yi Ty yroine hydrolyi Leecithinae produ uction Accid production from: f glu ucoe araabinoe fru uctoe maaltoe maanitol uccroe xy yloe eth hanol gly ycerol Grrowth in 42º C Characterittic of the train TPS8 with poten ntial caffeine degrradation The iollate TPS8 identified by combining it morphologiccal and biocchemical charracteritic w with information obtained from fr it 16S rdna ggene equence. S Strain TPS8 wa hown to be a Graamnegative, noon-porulatingg, trictly aero obic bacilli, and 308
Biodecaffeination by Peudomona peudoalcaligene TPS8 caffeine removal by P. peudoalcaligene TPS8 wa monitored during 144 h incubation period (Fig. 4A and B). A hown by b the determination of thee dry bioma level at different caffeine upplementation (Fig. 4A), the growth of TPS8 wa increaed (cell dry weight 0.51 g/l) when the initial caffeine concentrationn increaed to 2.5 g/l along with w a rie incubation time for 72 h. However, higher amount of caffeine gradually decreaed the growth of train TPS8, probably becauee of caffeine inhibition [10]. When the train grew to the late-exponential of growth phae (72 h growth with celll dry weight 0.51 g/l), maximum caffeine removal (80.2%, from 2.52 g/l to 0.495 g/l) wa attained and decreaed ignificantly whenn the train entered e in the tationary phae. During thee period, the caffeinee removal percentage in the medium decreaed to 76.5%, 78.2% and 77.7% after 96, 120 and 144 h, repectively (Fig. 4B). Dicuion Figure 3. Phylogenetic tree of train TPS8 and the related taxa baed on amplification of 16S rdna gene. Boottrap value (expreed a percentage of 1000 replication) greater than 50% are hown at the branch point. Acinetobacter p. wa ued a outgroup. Scale bar repreent 0.01 ubtitution per nucleotide poition. GenBank acceion number are given in parenthee. were depoited in the GenBank and acceion number i KF414528. Phylogenetic analyi implied the train wa a member of genu of Peudomona p., and the cloet phylogenetic relative wa P. peudoalcaligene (Gene Bank acceion no HQ407234; identity over 99%). In Figure 3, Phylogenetic tree obtained by neighbor- the joining analyi of 16S rdna gene equence how poition of the train TPS8 within the genu Peudomona. Baed on thee obtained reult, the iolate wa identified a Peudomona peudoalcaligene train TPS8. Reult of biodecaffeination experiment Peudomona peudoalcaligene train TPS8 grew in M9 broth medium when caffeine at the initial concentration 0.5 to 5 g/l wa provided a the ole carbon and nitrogen ource. The time coure of Attempt forr the removal toxic caffeine from the polluted effluent through chemical and microbiologicall procee have been tried. The advantagee of microbial method i that caffeine removal from the polluted environment i o fat andd improve the nutritional value of the coffee pulp. Due to high conumption of caffeine and related methylxanthine preent in food, beverage pharmaceutical andd agronomy indutrie, thi i not much urpriing thatt ome caffeine-degradingg microorganim have beenn creened for in the environment. The T initial report on the caffeinee degradation by microorganim were in early 1970 [27]. Caffeine degradation i catalyzed by everal microorganim with potential of caffeine degradaingg belonged to bacterial and fungal pecie. In bacteria, Peudomona pecie and in fungi Apergillu and penicillium pecie are efficient in removal of caffeine. In the current tudy, enrichment of the caffeine- degrading bacteria were performed by adding oil ample, obtained from different area of the t north part of Iran, into M99 mineral alt medium containing 1 g/l caffeine a the ole ource of carbon and nitrogen. 13 Colonie with different kind of morphology weree iolated from M9-caffeine M agar plate. Among them, train TPS8 ha been hownn to have thee tremendou caffeine tolerance ability and d a potential to degrade thi compound under ambient condition. By combining morphological and phyiological characteritic with 16S rdna gene equence analyi, thi iolate wa identified a Peudomona P peudoalcaligene train TPS8 (GeneBank acceion no. KF414528). Thi report preent data for the firt time on ability of the novel 309
Vol. 24 No. 4 Autumn 20 013 0.5 0.64 1..5 M. Ahe engroph and S. S Ababaf 2.5 3.5 d a the olee cafffeine in 48 h when caffeeine wa ued carrbon and nitro ogen ource. A train of Peudomona P tu utzeri Gr21Z ZF ha beenn reported for caffeinee deg gradation whiich i able to ddegrade 59% of caffeine att con ncentration of o 1.2 g/l affter 24 h (8 8). Gutiérrez-Sán nchez et al. developed a proce fo or tudie off cafffeine degradaation with Appergillu tama ari. At a rangee of 2 to 4 g/l of initial i caffeinee concentratio on, after 96 h of fermentation n, 41-51 % off the initial caffeine wa deg graded (12). There T are manny attempt to o improve thee cafffeine degradation uing ooptimization procee byy varrying the en nvironmental parameter and nutrientt con ndition (addition of exterrnal carbon and nitrogenn ou urce for example). Maddyatha and Sridhar (17)) rep ported a procee optimizatioon for caffein ne degradationn witth Klebiella and Rhodococccu p. by ad dding glucoee and d reported 10 00% caffeine degradation after 10 h off inccubation time with initial caaffeine concen ntration of 0.55 g/l. Syed Bakeer and co-woorker (31) reported thatt ind duced cell of a train of Peudom mona p. S7,, io olated from Co offee Arabica L, howed decaffeination off 98.61% under optimization proce by varying thee env vironmental parameter p uuch a ph, haking peedd and d time incubaation. El-Mchhed et al. (8) developed a pro oce optimizzation for ccaffeine degrradation withh Peeudomona tutzeri Gr211ZF in the preence off uccroe (5 g/l) which 80.1% % caffeine deg gradation wa obttained in 48 hr. The cafffeine removaal by iolatedd traain P. peudoalcaligene TPS8 wa comparable too tho oe previoully reported for caffeine degradationn (Taabel 2). Comp pared with prreviou reportt, our reult ho ow that growing cell of P P. peudoalca aligene TPS88 can n thu be efficciently ued aa a imple, aafe and cheapp pro oce for preparative decaaffeination fro om indutriall efffluent. It h hould be higghlighted thaat thi yieldd (80 0.2%) wa achieved a withhout further optimization.. Th hi reult clearrly uggetedd the great po otential of thee io olate TPS8 forr microbial deggradation of caffeine. c 5 Cell dry weight (g/l) 0.56 0.48 0.4 0.32 0.24 0.16 0.08 0 0 24 48 72 96 Time in hourr 120 J. Sci. I. R. Iran n 144 Time coure of o caffeine degradation by P P. Figure 4. T peudoalcalligene TPS8 (A A) Curve growtth of train TPS S8 in the MM99 medium withh addition of different d caffeinne concentratioon. (B) Effect of initial caffeiine concentratioon on it degraadation by traiin TPS8 in thee MM9 medium m. The reult repreent thhe mean of three eparaate experiment, and deviation bar indicated.. iolated traiin Peudomonna peudoalca aligene TPS88 in the developm ment of an ennvironmental friendly procce for microbbial caffeinne degradattion. The P. peudoalcaliigene TPS8 being reportted here how wed 80.2% of 22.5 g/l caffe feine removed in 72 hoour incubation w without furthher optimizaation. The ffirt caffeine biodegradation wa w reported with train of m p. (27). B But, Penicillium roqueforti annd Stemphylum the train were capablle of caffein ne degrading in concentrationn of 0.19 g/l after 29 h. Mazzafera M and coworker (18) developed a proce for caffeeine degradation uuing Serratiaa marcecen which i ablee to degrade 1000% of 0.6 g/ll of caffeine after 72 h. T The induced baccterial cell of o Peudomo ona alcaligeene CFR1708 w were found too be capablee of complettely degrading caffeine (1 g/ll) from olu ution containning caffeine in 6 h (25). Gokuulakrihnan et al. (11) reporrted the biodegraadation of caaffeine by Peudomona p. P train GSC11182 which howed 80% % degradationn of Concluion We have decribed here th the detail of the creeningg nattive Iranian bacterial trainn in being ab ble to perform m bio odecaffeination proce. A novel io olated train,, Peeudomona peudoalcalige p ene TPS8, wa w creenedd fro om tea plantaation oil, wh which ha hig gh ability forr cafffeine tolerancce (up to 15g//l) and caffein ne degradationn (80 0.2 %) without any externnal carbon/nittrogen ourcee add dition and further procce optimizaation, whichh inccreae the co ot of decaffe feination procce. Altoughh maany Peudomona pp. (P P. putida, P. tutzeri, P. 310
Biodecaffeination by Peudomona peudoalcaligene TPS8 Tabel 2. Variou microorganim ued for the caffeine degradation Microorganim Caffeine concentration (g/l) Incubation time (h) Optimization approach (Ye a /No b ) % Caffeine removal Reference Peudomona peudoalcaligene TPS8 2.5 72 No 80.2 [Current tudy] Peudomona tutzeri Gr21ZF 1.2 24 No 59 48 Ye 86 [8] Peudomona p. S7 5 24 Ye 98.6 [31] Peudomona putida CT25 2 72 Ye 50 [10] Trichoporon aahii 2 96 Ye 100 [15] Peudomona putida CBB5 2.5 192 No 100 20 Ye 100 [35] Peudomona p. GSC 1182 1.2 48 No 80 [11] Mix cultre of Klebiella/ Rhodococcu p. 0.5 10 Ye 100 [17] Serratia marcecen 0.6 72 No 100 [18] Peudomona putida 5 50 No 95 [34] Stemphyllium p. 0.19 29 Ye 100 [27] a : Caffeine degradation experiment were performed under optimal condition (addition of external carbon and nitrogen ource and/or optimize environmental culture condition). b : Caffeine degradation experiment were performed in the preence of caffeine a ole carbon and nitrogen ource and caffeine removal yield were achieved without further optimization. aeruginoa and P. alcaligene ) train have been reported for the caffeine degradatiobn, no report i available on caffeine removal uing P. peudoalcaligene. Comparing the reult obtained at thi tudy with earlier one encourage u to conclude that the growing cell of P. Peudoalcaligene TPS8 can be efficiently ued a a cot- effective biocatalyt for the preparative treatment of the caffeine containing olution. Further tudie on thi way to obtain higher yield of caffeine removal are in progre. Reference 1. Batih D.R., Singh H.P., Kaur M., Kohli R.K., Yadav S.S. Caffeine affect adventitiou rooting and caue biochemical change in the hypocotyl cutting of mung bean (Phaeolu aureu Roxb.). Acta. Phyiol. Plant. 30: 401-405 (2008). 2. Brand D., Pandey A., Rouo S., Soccol C.R. Biological detoxification of coffee huk by filamentou fungi uing a olid tate fermentation ytem. Enz. Microb. Technol. 27: 127-133 (2000). 3. Buerge I.J., Poiger T., Muller M.D., Buer H.R. Caffeine, an anthropogenic marker for watewater contamination of urface water. Environ. Sci. Technol. 37: 691-700 (2003). 4. Choi G.W., Um H.J., Kim Y., Kang H.W., Kim M., Chung B.W., Kim Y.H. Iolation and characterization of two oil derived yeat for bioethanol production on Caava tarch. Bioma. Bioenerg. 34: 1223-1231 (2010). 5. Cowan S.T., Steel K.J. Manual for the Identification of Medical Bacteria, 2nd Ed., Univerity Pre, London, pp. 94-150 (1993). 6. Dah S.S., Gummadi S.N. Catabolic pathway and biotechnological application of microbial caffeine degradation. Biotechnol. Lett. 28: 1993-2002 (2006). 7. Dah S.S., Gummadi S.N. Degradation kinetic of caffeine and related methylxanthine by induced cell of Peudomona p. Curr. Microbiol. 55: 56-60 (2007). 8. El-Mched F., Olama Z., Holail H. Optimization of the environmental and phyiological factor affecting microbial caffeine degradation and it application in caffeinated product. Baic. Re. J. Microbiol. 1: 17-27 (2013). 9. Erato M.S., Laura C., Ambra P., Guido M. Maternal caffeine conumption and ine caua recurrent micarriage. Eur. J. Obtet. Gynaecol. Reprod. Biol. 158: 220-224 (2011). 10. Fan F.Y., Xu Y., Liang Y.R., Zheng X.Q., Borthakur D., Lu J.L. Iolation and characterization of high caffeinetolerant bacterium train from the oil of tea garden. Afr. J. Microbiol. Re. 5: 2278-2286 (2011). 11. Gokulakrihnan S., Chandraraj K., Gummadi S.N. A preliminary tudy of caffeine degradation by Peudomona p. GSC1182. Int. J. Food. Microbiol. 113: 346-350 (2007). 12. Gutiérrez-Sánchez G., Rouo S., Augur C. Effect of caffeine concentration on bioma production, caffeine degradation, and morphology of Apergillu tamarii. Folia. Microbiol. 58: 195-200 (2013). 13. Halltrom T., Wolk A., Glynn A., Michaelon K. Coffee, tea and caffeine conumption in relation to oteoporotic fracture rik in a cohort of Swedih women. Oteoporo. Int. 17: 1055-1064 (2006). 14. Heckman M.A., Weil J., De Mejia E.G. Caffeine (1, 3, 7- trimethylxanthine) in food: A comprehenive review on conumption, functionality, afety, and regulatory matter. J. Food. Sci. 75: 77-87 (2010). 15. Lakhmi V., Da N. Caffeine degradation by yeat iolated from caffeine contaminated ample. Int. J. Sci. Nat. 1: 47-52 (2010). 311
Vol. 24 No. 4 Autumn 2013 M. Ahengroph and S. Ababaf J. Sci. I. R. Iran 16. Löffler F.E., Sun Q., Li J., Tiedje J.M. 16S rrna genebaed detection of tetrachloroethene-dechlorinating Deulfuromona and Dehalococcoide pecie. Appl. Microbiol. Biotechnol. 66: 1369-1374 (2000). 17. Madyatha K.M., Sridhar G.R. A novel pathway for the metabolim of caffeine by a mixed culture conortium. Biochem. Biophy. Re. Commun. 249: 178-181 (1998). 18. Mazzafera P., Olon O., Sandberg G. Degradation of caffeine and related methylxanthine by Serratia marcecen iolated from oil under coffee cultivation. Microb. Ecol. 31: 199-207 (1996). 19. Mohapatra B.R., Harri N., Nordin R., Mazumdar A. Purification and characterization of a novel caffeine oxidae from Alcaligene pecie. J. Biotechnol. 125: 319-327 (2006). 20. Nayak S., Harhitha M.J., Sampath M.C., Anilkumar H.S., Rao C.V. Iolation and characterization of caffeine degrading bacteria from coffee pulp. Indian. J. Biotechnol. 11: 86-91 (2012). 21. Pai P.V., Pai A., Pai S., Devadiga S.Y., Nayak V., Rao C.V. Effect of glucoe and nitrogen ource on caffeine degradation by four filamentou fungi. Indian. J. Biotechnol. 12: 432-434 (2013). 22. Park H.S., Im N.G., Kim K.H. Extraction behavior of caffeine and chlorophyll in upercritical decaffeination of green tea leave. LWT Food. Sci. Technol. 45: 73-78 (2012). 23. Rouo S., Hannibal L., Aquiahuatl M.A., Hernandez M.R.T., Maraki S. Caffeine degradation by Penicillium verrucoum in olid-tate fermentation of coffee pulp critical effect of additional inorganic and organic nitrogen ource. J. Food. Sci. Tech. My. 31: 316-319 (1994). 24. Sambrook J., Fritch E.F., Maniati T. Molecular cloning: a laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y (1989). 25. Sarath Babu V.R., Patra S., Thakur M.S., Karanth N.G., Varadaraj M.C. Degradation of caffeine by Peudomona alcaligene CFR 1708. Enzyme. Microb. Technol. 37: 617-624 (2005). 26. Sarath Babua V.R., Patraa S., Karantha N.G., Kumarb M.A., Thakur M.S. Development of a bioenor for caffeine. Anal. Chim. Acta. 582: 329-334 (2007). 27. Schwimmer S., Kurtzman R.H. Caffeine metabolim by Penicillium roqueforti. Arch. Biochem. Biophy. 147: 109-113 (1971). 28. Snel J., Lorit M.M. Effect of caffeine on leep and cognition. Prog. Brain. Re. 190: 105-117 (2011). 29. Srivatava K.C. Propertie of thermotable hemicellulolytic enzyme from Thermomonopora train 29 grown in olid tate fermentation on coffee proceing olid wate. Biotechnol. Adv. 11: 441-65 (1993). 30. Swati S.D., Sathyanarayana N.G. Inhibitory effect of caffeine on growth of variou bacterial train. Re. J. Microbiol. 3: 457-465 (2008). 31. Syed Baker., Sahana S., Rakhith D., Kavitha H.U., Kavitha K.S., Satih S. Biodecaffeination by endophytic Peudomona p. iolated from Coffee arabica L. J. Pharm. Re. 5: 3654-3657 (2012). 32. Tamura K., Dudley J., NeiM., Kumar S. Molecular Evolutionary Genetic Analyi (MEGA) oftware verion 4.0. Mol. Biol. Evol. 24: 1596 1599 (2007). 33. Wahington J.A., Sutter V.L. Dilution uceptibility tet: agar and macro-broth dilution procedure. In: Lennette EH, Balow A, Hauler JR, WJTruant, J. (Ed.). Manual of Clinical Microbiology, 3nd Ed., American Society for Microbiology, Wahington DC, pp. 453-458 (1980). 34. Woolfolk C.A. Metabolim of N-methylpurine by a Peudomona putida train iolated by enrichment on caffeine a the ole ource of carbon and nitrogen. J. Bacteriol. 123: 1088-1106 (1975). 35. Yu C.L., Louie T.M., Summer R., Kale Y., Gopihetty S., Subramanian M. Two ditinct pathway for metabolim of theophylline and caffeine are coexpreed in Peudomona putida CBB5. J. Bacteriol. 191: 4624-4632 (2009). 312