Biological ammonium removal via heterotrophic nitrification/aerobic denitrification (HN/AD) presents several advantages in relation to conventional removal processes, but little is known about the microorganisms and metabolic pathways involved in this process. In this study, Pseudomonas stutzeri UFV5 was isolated from an activated sludge sample from oil wastewater treatment station and its ammonium removal via HN/AD was investigated by physicochemical and molecular approaches to better understand this process and optimize the biological ammonium removal in wastewater treatment plants. Results showed that P. stutzeri UFV5 removed all the ammonium in 48-72 hours using pyruvate, acetate, citrate or sodium succinate as carbon sources, C/N ratios 6, 8, 10 and 12, 3-6% salinities, pH 7-9 and temperatures of 20-40 °C. Comparative genomics and PCR revealed that genes encoding the enzymes involved in anaerobic denitrification process are present in P. stutzeri genome, but no gene that encodes enzymes involved in autotrophic nitrification was found. Furthermore, transcriptomics showed that none of the known enzymes of autotrophic nitrification and anaerobic denitrification had their expression differentiated and an upregulation of the biosynthesis machinery and protein translation was observed, besides several genes with unknown function, indicating a nonconventional mechanism involved in HN/AD process.Nitrogen is considered to be one of the main pollutants of industrial effluents, and its presence can cause the eutrophication of the environments where they are discharged 1,2 . Conventionally, biological ammonium removal from wastewater treatment stations occurs through two sequential and independent processes: autotrophic nitrification followed by anaerobic denitrification, where the first stage is carried out by autotrophic bacteria and/or archaeas in aerobiosis, and the second by heterotrophic bacteria in anaerobiosis 3-5 . Due to the differences in the physical and chemical conditions of the two stages, these processes usually occur at separate times in the effluent treatment plants, demanding a longer reaction time and more space. In addition, the autotrophic microorganisms involved in nitrification are of slow growth, making the detention time of the effluent in the reactor even longer.To overcome these limitations, academic studies have focused on new processes for biological nitrogen removal, and heterotrophic nitrification/aerobic denitrification (HN/AD) has been gaining prominence in the effluent treatment field. In this process, a single microorganism is able to transform ammonium into gaseous