The biodesulfurization (BDS) of dibenzothiophene following the 4S route by resting cells of Pseudomonas putida CECT5279, a genetically modified organism, is studied in this work. An enhancement of the BDS capacity by co-substrate addition is proposed to overcome the deceleration with respect to time observed in previous studies. Results show that it is possible to improve the yield of the BDS process by adding short chain organic acids, such as acetic, citric and succinic acids. The concentration levels of the co-substrates cited have been studied, and the best results are obtained at a co-substrate concentration of 1.5% using acetic acid, which is the co-substrate that yields the best results, mainly in fed-batch operations. The addition of this co-substrate provides higher intracellular concentrations of some key metabolites, such as NADH and ATP. Using cells of a single age as a biocatalyst, it is possible to enhance the yield of the process up to 140% in a batch process, and when employing the optimal mixture of cells, an improvement of 122% is achieved in a fed-batch process. A detailed analysis of the effect of the co-substrate on the reaction rates of the 4S route shows that the rates of all of the reactions were enhanced. (C) 2014 Elsevier Ltd. All rights reserved.