• Caffeine , a purine alkaloids naturally occuring in more than 60 plant species which has a wide application and it is also one of the major agro industrial wastes.

• Coffee pulp and husk are major contributors of environmental pollution.• Caffeine in soil affects soil fertility.• Contamination of fresh water.• Effect on the physiological system.

• Pseudomonas, Serratia,Klebsiella, Rhodococcus, Alcalignes, Aspergillus,Penicillium, Fusarium and Stemphylium

• Kurtzmann and Schwimmer - Penicillium roqueforti and Stemphylium sp..

• Brand et al - Aspergillus niger from coffee husk

• Dash and Gummadi - Pseudomonas putida from the soil of coffee plantation

• To isolate bacterium from coffee pulp.

•Ability to degrade caffeine.

•Transformation of Escherichia coli DH5α strain.

• Crystal violet• Safranin stain

• Gram’s iodine

• Nitrate reduction discs• Oxidase discs

• Kovac’s reagent• Methyl red reagent

• α-naphthol 5%,• Potassium hydroxide 40%

• Nutrient agar• Simmons citrate agar• SIM agar• Skim milk agar• LB agar

• Tryptone broth • Urease broth• LB broth

• Mannitol motility media

• E. coli DH5α

• Molecular markers for DNA

• Pure cultures• Sub cultures• Various morphological , physiological and Biochemical tests.

• Bacterium was identified and authenticated as

Brevibacterium sp. (MTCC 10313) by the Institute of

Microbial Technology (IMTECH),Chandigarh ,

India.

• KH2PO4 3 g/L• Na2HPO4 6 g/L• NaCl 5 g/L• NH4Cl 2 g/L• glucose or sucrose 8 g/L• Distilled water 1L• In the end,a solution of MgSO4 (0.1 g/L distilled water) was added• pH 6.7

• Inoculate the bacterium .• Maintained at 37° C .

• 24h grown culture of Nutrient broth .

•10µl was inoculated into 100ml of minimal media ( 1- 8 g/L caffeine ) .

• Maintained control and blank .

• OD at 600nm at every 3h interval .

• Birnboim and Doly alkaline hydrolysis method .

• Isolation of plasmid .

• Plasmid DNA suspended in TE buffer .

• 2% agarose gel .

• Method of Mandel and Higa , and Cohen et al .

• Stab culture of E.Coli DH5α – LB Agar plate – LB broth – Over night incubation.

• 0.6 OD – Cooling – 1ml culture – 5000rpm – 4° C – Cell pellet – Autoclaved 0.1M CaCl2 .

• Repeat the step – 0.5ml of 0.1M CaCl2 .

• Competent cells – Isolated plasmid – Mix .

• Ice bath – Water bath – 500µl of LB broth – 37° C for 1h .

• LB Agar plates and Minimal media plates with different Caffeine concentrations ( 1-8 g/L ) .

• Control with and without caffeine .

• 10µl of transformed and non – transformed E.Coli DH5α cells were spread on the plates .

• Incubate at 37° C and compared after 24h .

• Counted colonies by using colony counter .

• Shake flask culture studies for the transformed E.Coli DH5α cells .

• Growth curves compared with non – transformed E.Coli DH5α and Brevibacterium sp..

• 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.