Glycerol is commonly employed for denitrification purposes in full-scale wastewater treatment. In non-acclimatized biomass, the glycerol is very inefficient resulting in a high C/N ratio and low-standard denitrification rates. The acclimatization is driven by the microbial enrichment of Saccharimonadales and Propionibacteriales as found in different sampled municipal sludges flanking the dominant presence of Burkholderiales. The selective strategy is based on a very efficient process in terms of C/N ratios and standard denitrification rates, but it leads to nitrite accumulation. As a result, severe and unexpected nitrous oxide emissions were found in full-scale with emission factors up to 2.5% kgN2O (kgKJNremoved)-1. Simultaneous dosage of isobutirate in a full-scale experiment could counter the nitrous oxide emissions. As nitrous oxide emissions were found proportional to the dosed glycerol-based COD, the authors suggest that, in case of acclimatization of biomass to glycerol, an emission factor based on the dosed COD should substitute the general nitrous oxide emission factors based on incoming or removed nitrogen to the plant.