In this work, a differential biofiltration reactor was used to explore the potential of metabolic uncouplers to improve pollutant (toluene) degradation rates. Metabolic uncouplers were reported to reduce the cell mass in activated sludge systems, but are untested in biofilters and the current work is the first to report the impact of different metabolic uncouplers in a biofilter. Initially soil was used as a biofilter bed and later experiments were conducted in pure cultures in a biofilm reactor. A simple diffusion system was developed to generate the desired concentration of toluene to the system. Gas chromatography and a carbon dioxide analyzer were connected online to the reactor which improved the precision of the data collected and also the robustness of the measurements. Preliminary experiments including effect of substrate concentration, different nutrients and temperature were done to optimize the conditions before starting the metabolic uncoupler screening studies in soil. Based on the results, inlet toluene concentration between 180 ppm and 250 ppm was used throughout the studies. Also it was found that the toluene degraders were nitrogen limited. Temperature studies showed that the elimination capacity (EC) increased with increasing temperature, from 34 ± 1.4 g.m-3.h-1 to 49.8 ± 2.6 g.m-3.h-1 for temperatures of 20 to 45 oC, respectively. Nine potential metabolic uncouplers were screened in batch serum bottles. The nine uncouplers tested were dinitrophenol (dNP), p-nitrophenol (pNP), benzoic acid (BA), carbonylcyanide p-trifluoromethoxy phenylhydrazone (FCCP), carbonylcyanide m-chloromethoxy phenylhydrazone (CCCP), pentachlorophenol (PCP), malonic acid (MA), m-chlorophenol (mCP) and 2, 4, 6-trichlorophenol (TCP). Other than dNP and pNP (nitrogen containing uncouplers), seven other uncouplers were further tested in the differential biofilter reactor. Only PCP and TCP increased the toluene degradation rate significantly. PCP increased the toluene degradation rate by 35% at 140 µM, whereas 4051 µM TCP increased the rate by 18%. Though FCCP behaved as a classical uncoupler when compared with others, the EC increase was not significant. Five toluene degraders were isolated from soil subjected to toluene and were identified using 16s rDNA/18s rDNA analysis. Out of five, two potential toluene degraders, Stenotrophomonas maltophilia and Pseudomonas putida were used to develop a biofilm reactor. PCP, TCP and CCCP were tested in the biofilm reactors and found that PCP increased the surface elimination capacity (SEC) by 85% at 140 µM in S. maltophilia biofilm reactor and CCCP increased the SEC by 27% at 1 µM in P. putida biofilm reactor. Finally a simple model was developed to calculate the energy uncoupling coefficient for non-growth systems like ours to quantitatively represent the uncoupling mechanism.