ABSTRACTThe feasibility of coupling photocatalysis and a biological treatment to remove a herbicide – 2,4-dichlorophenoxy acetic acid (2,4-D) – from pure water was examined using batch experiments following three protocols: aerated (A-BR) and non-aerated biodegradation (NA-BR) alone, and intimately combined photodegradation and biodegradation (P-B). In view of a subsequent biological treatment, 15 and 180 min irradiation times were chosen in accordance with spectrophotometric and LC-MS/MS results that indicated the decrease in the COD/TOC ratio during photocatalysis. Pre-treatment led to a quick decrease in concentration of 2,4-D and COD during the biological process: a 78.79 ± 0.30% COD removal and 38.23 ± 3.12% 2,4-D elimination was measured after 5760 min in A-BR, and 80.89 ± 0.81% COD and 81.36 ± 1.37% 2,4-D removal was achieved after 2880 min in P-B. For species identification using matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-TOF/MS equipment, Aeromonas eucrenophila, Stenotrophomonas acidaminiphila, Ralstonia pickettii, Sphingobacterium multivorum and Acinetobacter towneri were identified with high accuracy, and they play important roles in the degradation of 2,4-D.