Isolation and characterization of caffeine degrading bacteria from coffee pulp

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 DH5 alpha 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 DH5 alpha have greater ability to tolerate and degrade caffeine as compared to non-transformed cells.