Aims To increase the biodegradation of phenanthrene (PHE), pyrene (PYR) and fluoranthene (FLU) through mixed cultures of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria, using modified repeated batch fermentation. Methods and Results Novel bacterial strains of Pseudomonas putida, Pseudomonas sp. and Ralstonia eutropha were cultivated and the biodegradation and conversion of mixed PAH to medium-chain-length polyhydroxyalkanoates (MCL-PHA) was determined. The highest degradation of PAH (100%) and PHA production (50 center dot 0%) was obtained in medium containing 30 mmol l(-1) of mixed PAH after three cycles of repeated batch fermentation. The concentration of PAH in the reactor was increased from 30 to 90 mmol l(-1) with repeated additions of PAH, and bacteria were able to produce PHA at 40% of cell dry mass. The MCL-PHA were identified by gas chromatography/mass spectroscopy, with the 3-hydroxydecanoate (3-HD) monomer higher than 75 mol.%. Conclusions This study demonstrated that the biodegradation of PHE, PYR and FLU was enhanced by modified repeated batch fermentation using a mixed culture of bacteria. In addition, this fermentation strategy also increased the production of PHA, with an increase in monomer composition. Significance and Impact of the Study This was the first study to describe the enhancement of the degradation of mixed solutions of PHE, PYR and FLU, and PHA production, using novel mixed bacterial cultures and modified repeated batch fermentation. The MCL-PHA formed had uniquely high 3-HD content.