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Partial nitritation providing a suitable effluent for subsequent anammox is a critical step in a two-stage autotrophic nitrogen removal system. This study demonstrates an innovative approach for attaining partial nitritation in an acidic bioreactor operating at a slightly low pH (i.e., 5–6). This approach is based on our hypothesis in this study that acid-tolerant ammonia-oxidizing bacteria (AOB) can produce nitrite and protons to self-sustain free nitrous acid (FNA, NO2 – + H+ ↔ HNO2) at a parts per million level, as an inhibitor of nitrite-oxidizing bacteria (NOB). With influent nitrogen of about 200 mg/L and operating conditions of high dissolved oxygen, long sludge retention time, and moderate temperature, a lab-scale acidic bioreactor with FNA up to 2 mg of HNO2-N/L successfully established stable nitrite accumulation in the effluent for 200 days, with an average ratio [NO2 –/(NO2 – + NO3 –)] exceeding 95%. A 16S rRNA amplicon sequencing analysis showed that Nitrosospira was the dominant AOB in the biomass of the bioreactor, while Nitrosomonas and Nitrospira, two typical nitrifying genera in neutral wastewater treatment, both disappeared after the startup of partial nitritation. Kinetic characterization revealed that Nitrosospira had a substrate affinity of 11.4–16.5 mg of total ammonia (NH4 + + NH3)/L. It also revealed that less than 3.5 mg of HNO2-N/L FNA did not inhibit AOB activity significantly. Acidic operation is economically attractive because it can be achieved via acidophilic ammonia oxidation without adding chemical acid. However, hazardous gas, nitric oxide (NO), should be removed from gas produced by acidic nitrifying bioreactors.