Isolation and characterization of caffeine degrading bacteria ... - NOPR

Indian Journal of Biotechnology Vol 11, January 2012, pp 86-91

Isolation and characterization of caffeine degrading bacteria from coffee pulp Sneha Nayak, M J Harshitha, Maithili, Charanya Sampath, H S Anilkumar and C Vaman Rao* Department of Biotechnology Engineering, NMAM Institute of Technology, Nitte 574 110, India Received 13 October 2010; revised August 2011; accepted 12 October 2011 Pure culture of a Gram-positive bacterium was isolated from the coffee pulp and maintained on standard conventional nutrient agar medium. The bacterium was identified as Brevibacterium sp. (MTCC 10313) by the Institute of Microbial Technology (IMTECH), Chandigarh, India. The bacterium was characterized with conventional tests and used to study the tolerance to different concentration of caffeine in both solid and liquid media. Brevibacterium sp. was grown in a liquid minimal medium containing 1-8 g/L caffeine with glucose and sucrose separately. The bacterium was able to tolerate up to 6 g/L of caffeine in solid medium and 4 g/L in liquid medium. From the bacterium, a plasmid of about 2500 bp mol wt was isolated. The isolated plasmid was used to transform Escherichia coli DH5α and the transformed colonies were inoculated in 1 to 8 g/L of caffeine containing minimal media to see whether the plasmid was involved in biodegradation of caffeine. It was observed that the plasmid biodegraded caffeine up to 2 g/L in minimal media, whereas non-transformed colonies could tolerate only up to 1 g/L caffein. Growth curves obtained in the minimal media showed that transformed cells of E.coli DH5α have greater ability to tolerate and degrade caffeine as compared to non-transformed cells. Keywords: Bacteria, caffeine, coffee pulp, Gram positive, plasmid, transformation

Introduction Caffeine (1,3,7-trimethylxanthine), a purine alkaloids naturally occurring in more than 60 plant species, finds greater commercial importance for its wide application in popular beverages like coffee, tea and various soft drinks, and also in pharmaceutical preparations1. Moreover, caffeine is also one of the major agroindustrial wastes generated from the coffee and tea processing plants and these wastes are often released into the water bodies2,3. Therefore, decaffeination of waste is very necessary from the point of view of environmental conservation4,5. Coffee pulp waste is generated in large quantities during wet method of coffee cherry processing6, which is known to contain 23-27% fermentable sugars on dry wt basis6,7. Most of the coffee pulp remains underutilized in many countries and a need exists for its treatment by appropriate biological waste treatment processes to overcome severe environmental pollution6. Being cost-intensive in nature, the treatment of the waste adversely affects the cost of production of coffee and hence it is generally dumped as a waste6,7. Caffeine is toxic to many microorganisms, while some microorganisms have the ability to grow in the

presence of caffeine and to degrade it. In literature, strains belonging to Pseudomonas, Serratia, Klebsiella, Rhodococcus, Alcalignes, Aspergillus, Penicillium, Fusarium and Stemphylium have been reported to be able to degrade caffeine8-12. Kurtzmann and Schwimmer13 first studied the degradation of this alkaloid using strains of Penicillium roqueforti and Stemphylium sp.. Later, Brand et al14 isolated a strain of Aspergillus niger from coffee husk, which was capable of degrading 90% of caffeine by solid state fermentation. Dash and Gummadi15 isolated Pseudomonas putida capable of degrading caffeine up to 5 g/L from the soil of coffee plantation. The present study was undertaken to isolate a bacterium from the coffee pulp obtained from coffee cherry pulping site of the coffee estate and to see its ability to degrade caffeine in different conditions of media. It was also undertaken to see whether the bacterium contains the plasmid and if so, whether the plasmid can bring about transformation of Escherichia coli DH5α strain in such a way that transformed bacteria can grow on caffeine containing media.

____________ *Author for correspondence: Tel: +91-8258-281248; Fax: +91-8258-281265 E-mail: [email protected]

Crystal violet, safranin stain and Gram’s iodine were procured from E Merck (India) Ltd., Mumbai. Nitrate reduction discs, oxidase discs, Kovac’s

Materials and Methods Chemicals, Reagents and Culture Media

NAYAK et al: CAFFEINE DEGRADING BREVIBACTERIUM SP. FROM COFFEE PULP

reagent, methyl red reagent, α-naphthol 5%, potassium hydroxide 40%, nutrient agar, Simmons citrate agar, mannitol motility media, SIM agar, skim milk agar, tryptone broth and urease broth were purchased from HiMedia Laboratories Pvt. Ltd., Mumbai. E. coli DH5α, LB agar and LB broth, molecular markers for DNA were obtained from Bangalore Genei. Sample Collection and Isolation of Bacteria

Coffee pulp was collected from coffee cherry processing site of the coffee estate in a sterilized container. Isolation of bacteria was carried out by spread plate method and pure cultures were obtained by streak plate method16. Bacterial Identification

Pure cultures were maintained on nutrient agar medium at 4oC and were sub-cultured at an interval of every 2 wk. Various morphological, physiological and biochemical tests were performed to identify the bacteria and identification was further authenticated as Brevibacterium sp. (MTCC 10313) by the Institute of Microbial Technology (IMTECH), Chandigarh, India. Preparation of Minimal Medium

The minimal medium was prepared by dissolving the following ingredients in distilled water (g L-1): KH2PO4, 3; Na2HPO4, 6; NaCl, 5; NH4Cl, 2; glucose or sucrose, 8; in the end, a solution of MgSO4 (0.1 g/L distilled water) was added and pH of the medium was adjusted to 6.7. After inoculation of the bacterium into the minimal medium, the temperature for culture condition was maintained at 37oC. Flask Culture Experiment for Growth Curve

The fresh (24 h) grown culture (10 µL) of nutrient broth was inoculated into 100 mL of the minimal meda containing 1-8 g/L caffeine and the control was without caffeine. A blank was also maintained for the purpose of taking optical density (OD) and growth of the bacteria was measured by taking the OD at 600 nm by using Spectronic 20 spectrophotometer at every 3 h interval from the time of inoculation1,15.

87

Transformation Experiment

Method of Mandel and Higa18, and Cohen et al19 with minor modification was used for bringing about transformation. Stab culture of E. coli DH5α obtained from Bangalore Genei was streaked on LB (Luria Bertini) agar plate in order to get single isolated colonies. Single colony was picked from the plate and inoculated into 5 mL of LB broth, which was incubated at 37oC overnight on shaker incubator maintained at 120 rpm. When the OD of the broth reached 0.6, culture was kept on ice for 10 min for cooling. 1 mL of culture was centrifuged at 5000 rpm in a refrigerated centrifuge maintained at 4oC. Supernatant was decanted and cell pellet was resuspended in autoclaved 0.1 M CaCl2 by gently inverting the eppendorf tubes and tapping the tube with index finger. Re-suspended cells were kept on ice for 10 min and centrifuged at 5000 rpm for 10 min. Supernatant was decanted, 0.5 mL of 0.1 M CaCl2 was added to the cell pellet and re-suspended by inverting and gently tapping the tube with index finger. The competent cells obtained were added to the vial containing isolated plasmid and the tube was inverted gently several times to mix the plasmid with competent cells. The tube containing competent cells with plasmid is kept on ice bath for 20 min and then transferred to a water bath maintained at 42oC for 90 sec. 500 µL of LB broth was added to the tube, mixed gently again and kept in an incubator at 37oC for 1 h. Screening for Transformed and Non-transformed E. coli DH5α

LB agar plates and minimal media plates containing different concentration of caffeine (1-8 g/L) and control with and without caffeine were kept ready. 10 µL of transformed and non transformed E. coli DH5α cells were spread on the plates. Culture plates were kept in an incubator at 37oC and growth of bacterium on control (with and without caffeine) and experimental were compared after 24 h. Colonies in each plate were counted by using colony counter and difference in the number of colonies at different concentration of caffeine (1-8 g/L) was taken an index for transformed and non-transformed E. coli DH5α. Growth Curve of Transformed and Non-transformed E. coli DH5α

Isolation of Plasmid from Bacterium

Birnboim and Doly17 alkaline hydrolysis method was adopted for the isolation of plasmid from 24 h culture of the bacterium. 20 µL of sample containing 0.2 mg/mL concentration of plasmid DNA suspended in Tris-EDTA buffer was loaded per well of 2% agarose gel.

Following the transformation experiment, shake flask culture studies were carried out in minimal media containing different concentrations of caffeine (1-8 g/L) for the transformed E. coli DH5α cells in order to obtain the growth curve. The growth curves, thus, obtained were compared with the nontransformed E. coli DH5α and Brevibacterium sp..

INDIAN J BIOTECHNOL, JANUARY 2012

88

Results and Discussion From the morphology and conventional biochemical tests as given in Tables 1-3, it was confirmed that the bacterium isolated from coffee pulp was Brevibacterium sp., which was further confirmed by IMTECH and assigned authentication no. MTCC 10313. A survey of literature on the subject shows that it is the first report of this genus to be involved in caffeine biodegradation. A number of workers have shown that selective removal of caffeine in aqueous medium by P. putida is possible20,21. In the present study, the bacterium isolated from the coffee pulp was able to grow in the solid nutrient medium containing caffeine up to 6 g/L Table 1—Results of the morphology of bacterial isolate Tests performed

Characteristics of isolate

Cell Shape (Microscopy and Gram Cocci (Gram positive) staining) Cell shape Rod coccus cycle Size (microns) 0.5-1 Configuration Circular Margin Entire Elevation Raised Surface Moist Colony colour Cream Opacity Opaque Spore(s) Motility Non-motile Anaerobic growth Table 2—Results of the physiological tests performed to characterize the bacterial isolate Tests Growth at temperatures 4°C 18°C 25°C 30°C 37°C 42°C 55°C Growth at pH pH 6.8 pH 7.2 pH 8.0 pH 9.0 pH 10.5 Growth on NaCl (%) 2.0 5.0 6.0 8.0 10.0

Results + + + + + + + + + + + + +

(data not shown). Growth curve of the Brevibacterium sp. at different concentration of caffeine in minimal media containing glucose is shown in Fig 1a. It is evident from the growth curve that there was hardly any growth with the increasing concentration of caffeine beyond 4 g/L, whereas at 1-2 g/L concentration, the lag phase was prolonged to 24-40 h. One interesting aspect of this growth study is that Brevibacterium sp. could not grow in liquid minimal medium without caffeine and glucose as carbon source. Growth curve of the bacterium at different concentration of caffeine in minimal media containing sucrose is shown in Fig. 1b. It is evident from the growth curve that there was only growth in 1 and 2 g/L concentration of caffeine with sucrose, whereas no growth was observed at 4 and 8 g/L of caffeine with sucrose. Moreover, the lag phase at 1 and 2 g/L caffeine concentration was prolonged up to 65 h. In the control treatment, growth started at about 48 h after incubation and there was a steep rise in the population without a lag phase, which stabilized and entered into stationary phase after reaching 65 h (Fig. 1b). Table 3—Results of the biochemical tests performed to characterize the bacterial isolate Tests Methyl red test Voges Proskauer test H2S production Casein hydrolysis Citrate Indole Gelatin hydrolysis Starch hydrolysis Lysine Ornithine Catalase test Oxidase test Growth on McConkey Tween-40 Tween-60 Tween-80 Urease Arginine Acid production from Dextrose Galactose Maltose Mannitol Sorbitol Salicin Mannose Lactose Fructose Xylose

Results + + + + + + + + + + + -


Carbohydrate sources like glucose and sucrose in the minimal media showed minimal effect on growth pattern of bacteria at low concentration of caffeine, i.e., 1 and 2 g/L, except that the lag phase was much more prolonged in the sucrose containing media (65 h) as compared to glucose (24-48 h) at the same concentration of caffeine (Fig. 1a). Dash and Gummadi15 isolated the bacterium, Pseudomonas sp. NCIM 5235, from the soil of coffee estate, which was capable of degrading highest concentration of caffeine (10 g/L) at a maximum rate of 0.3 g/L/h as a whole cell biocatalyst without any growth. The strain was also known to tolerate high concentration of caffeine (∼20 g/L). In the bacterium, a plasmid was found, which had the ability to degrade the caffeine when E. coli DH5α cells were transformed by this plasmid15. In the present study also, a plasmid of approximately 2500 bp was isolated from the Brevibacterium sp. (Fig. 2). This plasmid was used for bringing about transformation in E. coli DH5α bacterium. Non-transformed E. coli DH5α cells could withstand up to 1 g/L caffeine in minimal media (Fig. 3a) with a

Fig. 1—Growth curve of Brevibacterium sp. isolated from coffee pulp at different concentration of caffeine in minimal media containing glucose (a) and sucrose (b)

89

Fig. 2—Profile of the plasmid DNA in agarose gel (Numbers on right indicate fragment size of the molecular marker of 1 Kb DNA. Fluorescent bands on the extreme right are the pre-cut fragments of DNA of different size as markers.)

Fig. 3—Comparative study of the growth curves for non-transformed (a) and transformed (b) E. coli DH5α in minimal media

90

INDIAN J BIOTECHNOL, JANUARY 2012

very slow growth rate. However, the transformation brought about in E. coli DH5α by plasmid isolated from the Brevibacterium sp. showed that transformants could withstand up to 2 g/L caffeine concentration in the minimal medium (Fig. 3b). Growth rate of transformed and non-transformed E. coli DH5α in minimal media was significantly affected beyond 4 g/L concentration of caffeine. From these experiments, it is evident that the plasmid found in Brevibacterium sp. was indeed used by the bacterium for caffeine metabolism. From the comparative study of growth of Brevibacterium sp. in minimal medium with glucose or sucrose as carbohydrate source and different concentrations of caffeine (1-8 g/L), it is evident that the growth was almost the same for 1 g/L concentration of caffeine in both the medium, whereas the growth decreased in sucrose containing minimal medium at 2 g/L caffeine concentration (Table 4). Moreover, when the concentration of caffeine exceeded beyond 2 g/L in sucrose containing minimal medium, there was hardly any growth. Further, transformed and non-transformed DH5α E. coli showed lower growth rate in minimal media beyond 2 g/L caffeine concentration and up to 4 g/L as compared to Brevibacterium sp. (Figs 3a & b, Table 4). Transformed E. coli DH5α could tolerate up to 2 g/L caffeine concentration and showed lower growth rate in minimal medium beyond 2 g/L caffeine concentration and up to 4 g/L as compared to Brevibacterium sp. (Table 5). It is also clear from Table 5 that the doubling time for Brevibacterium sp. in minimal medium containing glucose or sucrose marginally differed by 0.6 h at 1 g/L of caffeine, whereas at 2 g/L, it was equal for both the carbohydrate sources. However, the doubling time for Brevibacterium sp. for the control medium was 0.6 h without caffeine but with sucrose as carbohydrate source. It was also observed that, with increase in concentration of caffeine in the medium, the doubling time also increased, which was significantly higher for non-transformed E. coli DH5α. There was also significant difference in doubling time for transformed and non-transformed DH5α E. coli in the control minimal medium without caffeine (Table 5). Pseudomonas sp. isolated by Gokulakrishnan et al1 from the soil of a coffee estate was able to tolerate caffeine up to 20 g/L. Further, Yamaoka-Yano and Mazzafera9 reported that Pseudomonas putida strain

Table 4—Comparative study of biomass of Brevibacterium sp. isolated from coffee pulp and transformed and non-transformed E. coli DH5α Slope values (biomass at different concentrations of caffeine) 1 mg/mL 2 mg/mL 4 mg/mL WCWO MM with glucose 0.086 (Brevibacterium sp.) MM with sucrose 0.086 (Brevibacterium sp.) MM with non-transformed 0.0053 E. coli DH5α MM with transformed 0.06 E. coli DH5α

0.055

~0

~0

0.013

~0

0.052

0.0033

~0

0.0088

0.04

0.0051

0.05

WCWO, Without caffeine with organism; MM, Minimal media Table 5—Comparative study for doubling time of Brevibacterium sp. isolated from coffee pulp and transformed and non-transformed E. coli DH5α Td (doubling time in h at different concentration of caffeine) 1 mg/mL 2 mg/mL 4 mg/mL WCWO MM with glucose (Brevibacterium sp.) MM with sucrose (Brevibacterium sp.) MM with non-transformed E. coli DH5α MM with transformed E. coli DH5α

1.8

3

-

-

1.2

3

-

0.6

33

69

-

23

6

6.6

28

21

WCWO, Without caffeine with organism; MM, Minimal media

isolated by them was capable of degrading caffeine up to 25 g/L in liquid medium and up to 50 g/L in solid medium. Compared to this, Brevibacterium sp. isolated from the coffee pulp and used in the present study was able to tolerate caffeine only up to 6 g/L in solid medium and 4 g/L in liquid medium. From the environmental perspective, it is important to explore new species of bacteria and fungi that are naturally capable of degrading caffeine so that these organisms could be exploited by genetic engineering to enhance the biodegradation of caffeine in the environment. Solid wastes, such as, coffee pulp and husk, are the major contributors of environmental pollution from the coffee estates5,22. The presence of caffeine in soil can also affect soil fertility as it inhibits seed germination and growth of seedlings23. Coffee pulp containing waste water is often discharged to the surrounding water bodies resulting in contamination of freshwater2,3. The ingestion of caffeine and its


chlorinated byproducts (derived during chlorination of water) have severe adverse effects on the physiological system12. However, bio-decaffeination can be used effectively for the solid wastes like coffee husk and pulp, which can then be used as animal feed12,24. This can be achieved only through exploitation of naturally occurring organisms in the caffeine containing waste and by genetically engineering them to enhance their capacity of caffeine degradation. References 1 Gokulakrishnan S, Chandraraj K & Gummadi S N, Microbial and enzymatic methods for the removal of caffeine, Enzyme Microb Technol, 37 (2005) 225-232. 2 Buerge I J, Poiger T, Muller M D & Buser H R, Caffeine, an anthropogenic marker for wastewater contamination of surface water, Environ Sci Technol, 37 (2003) 691-700. 3 Glassmeyer S T, Furlong E T, Kolpin D W, Cahill J D, Zaugg S D et al, Transport of chemical and microbial compounds from known waste water discharge: Potential for use as indicators of human fecal contamination, Environ Sci Technol, 39 (2005) 5157-5169. 4 Roussos S, Hannibal L, Aquiahuatla M A, Hernandez T M R & Marakis S, Caffeine degradation by Penicillium verucosum in solid state fermentation of coffee pulp: Critical effect of additional inorganic and organic nitrogen sources, J Food Sci Technol, 31 (1994) 316-319. 5 Bressani R, Anti-physiological factors in coffee pulp, in Coffee pulp: Composition, technology and utilization, publication 108e, edited by J E Braham & R Bressani (International Development Research Centre, Ottawa, Canada) 1979, 83-88. 6 Schwimmer S, Kurtzmann R H & Heftmann E, Caffeine metabolism by Penicillium roqueforti, Arch Biochem Biophys, 147 (1971) 109-113. 7 Woolfolk C A, Metabolism of N-methylpurines by a Pseudomonas putida strain isolated by enrichment on caffeine as the sole source of carbon and nitrogen, J Bacteriol, 123 (1975) 1088-1106. 8 Mazzafera P, Olson O & Sandberg G, Degradation of caffeine and related methyl xanthines by Serratia marcescens isolated from soil under coffee cultivation, Microb Ecol, 31 (1994) 199-207. 9 Yamoka-Yano D M & Mazzafera P, Catabolism of caffeine and purification of xanthine oxidase responsible for methyluric acids production in Pseudomonas putida, Rev Microbiol, 30 (1999) 62-70.

91

10 Hakil M, Voisinet F, Gonzalez G V & Augur C, Caffeine degradation in solid state fermentation by Aspergillus tamari: Effects of additional nitrogen sources, Process Biochem, 35 (1999) 103-109. 11 Roussos S, Aquiahuatla M, Trejo-Hernandez M R, Perraud G I, Favela E et al, Biotechnological management of coffee pulp and isolation, screening, characterization, selection of caffeine degrading fungi and natural microflora present in coffee pulp and husk, Appl Microbiol Biotechnol, 42 (1995) 756-762. 12 Mohapatra B R, Harris N, Nordin R & Mazumder A, Purification and characterization of novel caffeine oxidase from Alcaligenes species, J Biotechnol, 125 (2006) 319-332. 13 Kurtzmann R H & Schwimmer S, Caffeine removal from growth media by microorganisms, Experientia, 127 (1971) 481-482. 14 Brand D, Pandey A, Roussos S & Soccol C R, Biological detoxification of coffee husk by filamentous fungi using a solid state fermentation system, Enzyme Microbial Technol, 27 (2000) 127-133. 15 Dash S S & Gummadi S N, Biodegradation of caffeine by Pseudomonas sp. NCIM 5235, Res J Microbiol, 1 (2006) 115-123. 16 Holt J G, Krieg N R, Sneath P H A, Stanley J T & Williams S T, Bergey’s manual of determinative bacteriology, 9th edn (Lippincott Williams and Wilkins Publishers, Philadelphia, USA) 2000, 23-110. 17 Birnboim H C & Doly J, A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Nucleic Acids Res, 7 (1979) 1513-1523. 18 Mandel M & Higa A, Calcium dependent bacteriophage DNA infection, J Mol Biol, 53 (1970) 159-162. 19 Cohen S N, Chang A C Y & Hsu L, Nonchromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by R-factor DNA, Proc Natl Acad Sci USA, 69 (1972) 2110-2114. 20 Middlehoven W J, Degradation of caffeine by immobilized cells of Pseudomonas putida strain C 3024, Appl Microbiol Biotechnol, 15 (1982) 214-217. 21 Sideso O, Marvier A, Katerelos N & Goodenough P, The characteristics and stabilization of a caffeine demethylase enzyme complex, Rev Microbiol, 36 (2001) 693-698. 22 Adams M R & Dougan J, Biological management of coffee processing, Trop Sci, 123 (1981) 178-196. 23 Friedman J & Waller G R, Caffeine hazards and their prevention in germinating seed of coffee, J Chem Ecol, 9 (1983) 1099-1106. 24 Mazzafera P, Degradation of caffeine by microorganisms and potential use of decaffeinated coffee husk and pulp in animal feeding, Sci Agric, 59 (2002) 815-821.