Nitrate Conversion and Sludge Characteristics with Glycerol–Sodium Acetate Co-Feeding at Low Temperature

Low temperatures constrain biological denitrification and increase the importance of carbon-source selection. This study examined partial replacement of sodium acetate with glycerol during sequential sludge cultivation at 24, 12 and 8 °C. Glucose, sodium acetate, glycerol and their three binary mixtures were compared; each component supplied 50% of the COD in a mixed feed. Nitrogen-species profiles were evaluated alongside sludge concentration, settling, extracellular polymeric substances (EPSs), reductase activity potentials and community composition. In the 8 °C batch profiles, glycerol–acetate showed a steeper nitrate decline during the first hour than acetate alone. Its transient nitrite peak reached 22.6 mg N L−1 at 2 h, compared with 18.4 mg N L−1 at 3 h for acetate; acetate subsequently reached low residual nitrate sooner. The glycerol–acetate reactor also showed steadier mixed liquor suspended solids and sludge volume index trajectories and increasing CER-extractable EPSs. After 8 °C cultivation, this sludge retained 64.7% of its nitrate-reductase potential and 29.7% of its nitrite-reductase potential, measured at a common assay temperature of 22 °C. Along the cultivation sequence, 50% acetate-COD substitution accompanied sustained early nitrate conversion, steadier MLSS/SVI and increasing extractable EPSs, together with greater transient nitrite accumulation.