The distillery wastewater (spent wash) contains dark brown colored recalcitrant organic compounds that are not amenable to conventional biological treatment. The characteristic recalcitrance to decolorization is due to the presence of brown melanoidin polymers. In the present study, feasibility of using Pseudomonas putida putida strain U for decolorization of spent wash was demonstrated. Batch cultures of P. putida decolourized spent wash by 24%, twofold higher decolorization was achieved following immobilization in calcium alginate beads. Glucose concentration was critical for decolourization, and improved color removal efficiency was obtained by periodic replenishment of glucose. Decolourization was also observed with lactose or whey as alternative carbon sources. The results of our study suggest that P.putida could be used for biological decolorization of molasses spent washes and that supplementation with whey (a by-product from cheese industry) can offer economical viability to the process.