A microbial biosensing system for the measurement of methane has been developed using an immobilized mixed culture of Pseudomonas aeruginosa and Klebsiella sp. together with a dissolved oxygen (O-2) sensor. When methane was applied to the biosensing system, the dissolved O-2 content decreased until a steady-state was reached. The biosensing system response depends linearly on methane concentration between 1.0 and 5.0% (v/v) with a detection limit of 0.3% (v/v) (S/N = 3) and a 100-s response time. Phosphate buffer (pH 7.0, 25 mM) and room temperature (20-25 degrees C) were chosen as the optimum working conditions. Some parameters including pH, temperature, operational and storage stability were studied in detail for characterization of the biosensing system. (C) 2008 Elsevier Inc. All rights reserved.