Aerobic batch biodegradation experiments and respirometric analysis were performed in order to investigate the effect of S-0/X-0 (substrate/biomass) ratio and preliminary acclimation on bi-solute biodegradation of phenolic compounds. It was shown that 2-chlorophenol (2-CP) and 2-nitrophenol (2-NP) could be cometabolically biodegraded only with acclimated biomass in the presence of phenol as growth substrate. Acclimation resulted in domination of phenol oxidizing bacteria which could induce the necessary enzymes for cometabolic transformation of 2-CP and 2-NP. Biodegradation of the cometabolic compounds occurred even after depletion of phenol at resting cell conditions. Both compounds could be successfully biodegraded by the acclimated biomass at initial substrate concentrations as high as 300 mg/L Respirometric analysis showed that the optimum S-0/X-0 ratio ranged between 1.5 and 5.5 mg CODeq/mg MLSS for cometabolic substrates 2-CP and 2-NP, whereas it was as high as 8.5 mg CODeq/mg MLSS for phenol which corresponds to a phenol concentration of about 1500 mg/L (C) 2012 Elsevier Ltd. All rights reserved.