Background and Objectives: Microbial caffeine removal is a green solution for treatment of caffeinated products and agro-industrial effluents. We directed this investigation to optimizing a bio-decaffeination process with growing cultures of Pseudomonas pseudoalcaligenes through Taguchi methodology which is a structured statistical approach that can be lowered variations in a process through Design of Experiments (DOE). Material and Methods: Five parameters, i.e. initial fructose, tryptone, Zn+2 ion and caffeine concentrations and also incubation time selected and an L16 orthogonal array was applied to design experiments with four 4-level factors and one 3-level factor (44× 13). Data analysis was performed using the statistical analysis of variance (ANOVA) method. Furthermore, the optimal conditions were determined by combining the optimal levels of the significant factors and verified by a confirming experiment. Measurement of residual caffeine concentration in the reaction mixture was performed using high-performance liquid chromatography (HPLC). Results: Use of Taguchi methodology for optimization of design parameters resulted in about 86.14% reduction of caffeine in 48 h incubation when 5g/l fructose, 3 mM Zn+2 ion and 4.5 g/l of caffeine are present in the designed media. Under the optimized conditions, the yield of degradation of caffeine (4.5 g/l) by the native strain of Pseudomonas pseudoalcaligenes TPS8 has been increased from 15.8% to 86.14% which is 5.4 fold higher than the normal yield. Conclusion: According to the experimental results, Taguchi methodology provides a powerful methodology for identifying the favorable parameters on caffeine removal using strain TPS8 which suggests that the approach also has potential application with similar strains to improve the yield of caffeine removal from caffeine containing solutions.
