Sulfur content in fossil fuels is known to be the most important anthropogenic cause of sulfur oxide emissions to the atmosphere. In order to avoid health, environmental and technical problems caused by this compound, legislation imposes restrictive limitations to fuel sulfur content. Biodesulfurization (BDS) can become a complementary technology to hydrodesulfurization (HDS) to face this situation. Pseudomonas putida CECT 5279 is a genetically modified microorganism which can act as a desulfurizing biocatalyst. This microorganism has the ability of performing the metabolic 4S pathway from Rhodococcus erythropolis IGTS8, in order to clesulfurize DBT, as a model compound. Maximum in vivo activities of monooxygenase enzymes (DszA and DszC) are shown when late exponential growth phase is reached (23 h), while desulfinase enzyme DszB presents a maximum activity during the early exponential growth phase (5 h), as previously reported Ill. Also, it has been proved that the combined utilization of these two cell ages yields excellent results when used as biocatalyst for desulfurization in The aim of this work is to optimize the ratio and total biomass concentration of both 5 h and 23 h growth time cells in a complex biocatalyst for desulfurization by performing resting cells biodesulfurization assays using dibenzothiophene (DBT) as sulfur model compound. The best combination of cells was determined aiming for the highest clesulfurization in the shortest time of operation while investing the minimum concentration of biomass. A particular cell mixture, containing 66.7% of 23 h growth time cells, was found to work as the most effective desulfurization biocatalyst. (C) 2011 Elsevier Ltd. All rights reserved.