In this study we focus on recovery and concentration of ortho-phenylphenol (OPP) from fermentation broth by aqueous two-phase flotation (ATPF). The effects of pH, ammonium sulfate concentration, OPP concentration, air flow-rate, flotation time and initial volume of organic phase were evaluated on process performance using multivariate statistical methodology. Under optimal conditions, the separation efficiency (E%), the concentration coefficient (alpha) and the distribution coefficient (D) for ATPF reached 57.55%, 4.23 and 9.86, respectively. When compared to others conventional techniques also used in this work, such as aqueous two-phase extraction, solvent sublation and liquid-liquid extraction, the ATPF showed better performance in terms of E%, alpha and D. Additionally, experiments carried out with fermentation broth derived from the biodesulfurization of 4-MDBT have shown promise in the recovery and concentration of OPP. In summary, the results indicated that the ATPF is an effective separation technique in the pretreatment of the OPP from bona fide mixtures. (C) 2016 Elsevier B.V. All rights reserved.