In permeable (sandy) sediments, eutrophication is intensifying emissions of nitrous oxide (N2O), a potent greenhouse gas with approximately 300-time higher radiative forcing than carbon dioxide. Permeable sediments habour diverse microbial communities that are metabolically flexible and well adapted to frequent transitions between oxic and anoxic redox states. However, little is known about how N2O production and consumption is regulated by these microorganisms under dynamic redox transitions of the system. In this study, we combined isolation, genomics and physiological characterizations to validate the metabolic capacity of key N2O emission regulators, guided by previous metagenomic investigations. Microorganisms were sampled from permeable sediments of Port Phillip Bay (Australia) , from which a total of 35 strains were isolated. Eight strains within the Gammaproteobacteria were isolated using an enrichment method using either glucose, acetate or starch as sources of carbon and electrons and nitrate as the electron acceptor. Profiles of microbial communities prior and post enrichment was investigated via 16S rRNA gene amplicon sequencing. Other isolates belonging to the orders Flavobacteriales (Class Flavobacteriia), Rhodobacteriales (Class Alphaproteobacteria) and Micrococcales (Class Actinomyceta) were obtained from the direct plating approach. The whole genome sequencing of these isolates revealed that some isolates encoded genes to reduce nitrate to N2O (e.g. NapA, NirK, and norB) whereas others contained the nosZ gene encoding the nitrous oxide reductase responsible for converting N2O to N2. Biochemical assays are being performed to verify the metabolic pathways mediating N2O fluxes of the isolates. Microorganisms obtained from this study are promising candidates to investigate microbial N2O emission and to inform novel N2O mitigation strategies.