Monitoring anaerobic digestion (AD) leachate for changes in acetate and propionate concentrations is essential for effective AD operation. In this paper the development of a novel propionate cell-based biosensor is described. A previously designed E. coli mutant (IMD Wldgy) that could selectively determine acetate concentrations in synthetic leachates, based on oxygen uptake measurements, was used as a starting point in the development of a propionate biosensor. However, the propionate-grown IMD Wldgy cells exhibited extremely low propionate:acetate O-2 consumption ratios (1:2.4). Screening for alternative propionate-grown E. coli strains naturally possessing a more favourable propionate:acetate O-2 consumption ratio identified strain IMD 1, which exhibited a positive ratio (1.6:1). To improve the selectivity of the strain, successive gene knockouts were performed generating the IMD 1 hldgyep mutant. However, propionate-grown IMD lhdlgyep's O-2 consumption ratio was deemed too low to be considered as a propionate detecting bio-element. It was reasoned that the mechanisms by which E. coil activates acetate had to be removed. Deleting acs (acetyl-CoA synthesase) and ackA (acetate kinase) from IMD Wldgyep, resulted in an E. coil IMD Wldgyepak knockout mutant that, when grown on propionate, produced a mean propionate:acetate O-2 consumption ratio of approx. 13:1. The resulting IMD Wldgyep and IMD Wldgyepak strains, which formed the acetate- and propionate-biosensor, respectively, were capable of detecting acetate and propionate concentrations ranging from 0.05 mM to 4.5 mM within two-phase AD synthetic leachates.