Anaerobic biodegradation of phenanthrene (PHE) was investigated using an enrichment culture consists predominantly of sulfate-reducing bacteria (87 +/- 6%). Aqueous biodegradation experiments were designed using the rotatable central composite design with five levels. The designed concentrations were 2-50 mg L-1 for PHE, 480-3360 mg L-1 for sulfate, and 5-50 mg L-1 for initial biomass. Experimental results indicated that the biomass concentration was the most significant variable, followed by the sulfate and PHE concentrations. The desirability functions methodology (DFM) was applied to find out the maximum specific PHE removal rate (R-s). The maximum R-s of 9.0 mg g(-1) VSS d(-1) within the designed ranges was obtained when the initial PHE, sulfate and biomass concentrations were 18.5, 841 and 50 mg L-1, respectively. The R-s observed in the present study was higher than the values reported in the previous studies. Subsequently, a confirmation study was performed under the optimal conditions. and the results matched well with the R-s estimated using DFM. Samples collected during PHE biodegradation experiments inferred the formation of two novel metabolic intermediates, 2-methyl-5-hydroxybenzaldehyde and 1-propenyl-benzene, and subsequently degraded to p-cresol, phenol and hydrocarbons. (C) 2009 Elsevier B.V. All rights reserved.