An aerobic organic-aqueous system with forced aeration was shown to be inefficient in preventing significant volatile aromatic compounds loss in gassed systems since air sparging in n-hexadecane under abiotic conditions could reduce the toluene concentration from 2.1 g/L to about 0.5 g/L in 3 days with a gassing rate of 1VVM at 20 degrees C. However, the presence of such an organic phase was found to significantly reduce substrate loss in aerobic conditions in comparison to pure aqueous systems. It was thus decided to develop a new bioprocess based on an anaerobic microbial system operated in an organic-aqueous phase with nitrate respiration. The denitrifying bacterium used, Thauera aromatica K172, was produced by cultivation on sodium benzoate as carbon source under anaerobic conditions. This cultivated biomass (1.5 g/L) was shown to retain its ability to efficiently metabolize toluene in a biphasic medium without any significant loss of organic compound in the gas phase. Toluene biodegradation was thus performed in a biphasic system using a fed-batch technique involving sequential adding of both toluene and nitrate. The reaction rate with an initial concentration of toluene close to 14.5 g/L in hexadecane was found to be close to 0.5 g/L day and the molar stoichiometry of solute metabolization to nitrate reduction was close to 1:6. This work demonstrated that the denitrifying bacteria could efficiently degrade toluene in hexadecane-aqueous phase systems in which toxic compound release in the environment was prevented. (C) 2010 Elsevier Ltd. All rights reserved.