Nitrous oxide (N2O) is a potent greenhouse gas, and its stratospheric concentration is already 20% above the pre-industrial level. Over 70% of N2O is produced by microbial processes. Nitrous oxide reductase is the only enzyme able to reduce N2O to innocuous N2. The high abundance of organisms harbouring it and lacking the genetic potential to produce N2O, commonly known as specialist N2O-reducers, has only been recently disclosed in diverse ecosystems. In this work, we aim to understand which mechanisms select for them in open cultures and how N2O affects cellular metabolism. Two continuously-fed stirred-tank membrane reactors (CSTMR) were run at 20 °C, pH 7 and low dilution rate (0.14 d-1) under either acetate- or N2O-limiting conditions. Denitrifying activity could not be completely washed out from the reactors. Yet, a ten orders of magnitude increase in clade II nosZ gene abundance, often associated with specialists, was found under N2O limitation. Moreover, cultivation under N2O excess resulted in 30-50% lower biomass yields and a 25% higher maintenance coefficient as compared to N2O limitation. Lastly, polyhydroxyalkanoates (PHA) consistently accumulated (up to 20 wt%) under both acetate and N2O limitation, and the possible biochemical mechanisms are discussed. We conclude that (i) affinity for N2O was not selective enough to enrich for specialist N2O-reducers under the imposed conditions, and (ii) provide further evidence for the potential cytotoxicity of N2O on the cellular metabolism and, to the best of our knowledge, (iii) report the first evidence of PHA accumulation in N2O-respiring enrichments.