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The present study describes the microbial production of polyhydroxyalkanoates (PHA) from thermally pre-treated sewage sludge at pilot scale level, investigating for the first time the effect of the organic loading rate (OLR) under oxygen limitation on biomass storage properties and kinetics. Polymer characteristics have been also evaluated. The selection/enrichment of PHA-storing biomass was successfully achieved in a Sequencing Batch Reactor (SBR) under short hydraulic retention time (HRT; 2 days). Low OLR (2.05 g COD/L d) was ideal for the selection of an efficient PHA-producing consortium cultivated under limited oxygen availability. In the fed-batch accumulation conducted under high DO regime, such biomass was characterized by 51% of PHA content on cell dry weight, with a related storage yield (YP/Sbatch) of 0.61 CODPHA/CODS. On the contrary, medium OLR (4.56 g COD/L d) was not technically feasible to sustain the required consortium's selection under low DO regime. The PHA produced by biomass cultivated under low DO regime was characterized higher thermal stability and crystalline domain compared to PHA traditionally produced under high DO regime. The mass balance assessment highlighted a global yield of 51 g PHA/kg VS (volatile solids of thickened sludge), which was 9% lower than yield obtained under high DO regime, in the face of a realistic reduction of the energy cost of the process.