The effect of pH, aeration rate, and agitation rate on specific productivity of caffeine demethylase from Pseudomonas sp. was studied in a bioreactor. Maximum specific productivity of caffeine demethylase of 2,214 U g cell dry weight(-1) h(-1) was obtained at 0.27 vvm, 700 rpm, and pH 7.0. Under these conditions, volumetric oxygen transfer coefficient was 74.2 h(-1), indicating that caffeine demethylase production by Pseudomonas sp. was highly oxygen-dependent. Different metabolite formation at different agitation and aeration rates can be used as a strategy for recovery of pharmaceutically important metabolites from caffeine by manipulation of conditions in a bacterial culture. This is the first report on production of high levels of caffeine demethylase in bioreactors.

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Abstract:Decaffeination is an important process for the removal of caffeine from wastes generated by coffee and tea industries. Microbial degradation of caffeine is more useful than conventional chemical treatment because of its low cost and because it does not involve the use of toxic solvents. However, biodegradation of caffeine remains a problem because of the difficulty of finding a strain that can resist high concentration of caffeine in addition to be able to degrade caffeine at higher rates. In this study, we used the induced cells of Pseudomonas sp. for the degradation of caffeine. The induced cells (8 mg/ml) showed complete degradation of a initial concentration of caffeine of 1.2 g/l in 6 hours. The optimum pH was 7.0, the agitation rate was 180 rpm and the optimum temperature for degradation was 35• C.Under these conditions and in the presence of magnesium, complete degradation of 1.2 g/l of caffeine was accomplished in 4 hours. Additional trials determined that induced cells completely degraded an initial concentration of caffeine of 10 g/l in 26 hours. This is the first report on a strain that can degrade high concentrations of caffeine (e.g., 10 g/l) at the maximum rate of 0.385 g/l per hour. These results suggest that the strain can be used to successfully in developing a biological process for the degradation of caffeine.

The effect of various initial caffeine concentrations on growth and caffeine demethylase production by Pseudomonas sp. was studied in bioreactor. At initial concentration of 6.5 g l(-1) caffeine, Pseudomonas sp. showed a maximum specific growth rate of 0.2 h(-1), maximum degradation rate of 1.1 g h(-1), and caffeine demethylase activity of 18,762 U g CDW(-1) (CDW: cell dry weight). Caffeine degradation rate was 25 times higher in bioreactor than in shake flask. For the first time, we show highest degradation of 75 g caffeine (initial concentration 20 g l(-1)) in 120 h, suggesting that the tested strain has potential for successful bioprocess for caffeine degradation. Growth kinetics showed substrate inhibition phenomenon. Various substrate inhibition models were fitted to the kinetic data, amongst which the double-exponential (R(2) = 0.94), Luong (R(2) = 0.92), and Yano and Koga 2 (R(2) = 0.94) models were found to be the best. The Luedeking-Piret model showed that caffeine demethylase production kinetics was growth related. This is the first report on production of high levels of caffeine demethylase in batch bioreactor with faster degradation rate and high tolerance to caffeine, hence clearly suggesting that Pseudomonas sp. used in this study is a potential biocatalyst for industrial decaffeination.