Biological nutrient removal from wastewater to reach acceptable levels is needed to protect water resources and avoid eutrophication. The start-up of an anaerobic ammonium oxidation (anammox) process from scratch was investigated in a 20 L sequence batch reactor (SBR) inoculated with a mixture of aerobic and anaerobic sludge at 30 ± 0.5 °C with a hydraulic retention time (HRT) of 2–3 days. The use of NH
4Cl, NaNO
2, and reject water as nitrogen sources created different salinity periods, in which the anammox process performance was assessed: low (<0.2 g of Cl
−/L), high (18.2 g of Cl
−/L), or optimum salinity (0.5–2 g of Cl
−/L). Reject water feeding gave the optimum salinity, with an average nitrogen removal efficiency of 80%, and a TNRR of 0.08 kg N/m
3/d being achieved after 193 days. The main aim was to show the effect of a hydrazine addition on the specific anammox activity (SAA) and denitrification activity in the start-up process to boost the autotrophic nitrogen removal from scratch. The effect of the anammox intermediate hydrazine addition was tested to assess its concentration effect (range of 2–12.5 mg of N
2H
4/L) on diminishing denitrifier activity and accelerating anammox activity at the same time. Heterotrophic denitrifiers’ activity was diminished by all hydrazine additions compared to the control; 5 mg of N
2H
4/L added enhanced SAA compared to the control, achieving an SAA of 0.72 (±0.01) mg N/g MLSS/h, while the test with 7.5 mg of N
2H
4/L reached the highest overall SAA of 0.98 (±0.09) mg N g/MLSS/h. The addition of trace amounts of hydrazine for 6 h was also able to enhance SAA after inhibition by organic carbon source sodium acetate addition at a high C/N ratio of 10/1. The start-up of anammox bacteria from the aerobic–anaerobic suspended biomass was successful, with hydrazine significantly accelerating anammox activity and decreasing denitrifier activity, making the method applicable for side-stream as well as mainstream treatment.
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