Kinetic and Physiological Characterization of Acidophilic Nitrobacter spp. in a Nitrite-Oxidizing Culture

Novel acidophilic nitrite-oxidizing Nitrobacter spp. were enriched, with kinetic and physiological features characterized comprehensively. The acidophilic Nitrobacter enrichment culture was cultivated in a membrane bioreactor at pH 4.6-5.5 for 500 days, with a relative abundance of 16S rRNA genes of 17.9 ± 0.5% during the characterization experiments. Metagenomic analysis recovered 2 Nitrobacter genomes, which corresponded to previously unknown species within the Nitrobacter genus with average nucleotide identity (ANI) < 91% compared to the publicly available genomes. In contrast to the known NOB, the acidophilic Nitrobacter enrichment culture exhibited strong tolerance to environmental stress, including low pH and free nitrous acid (FNA), and have low affinity for oxygen. Moreover, the acidophilic Nitrobacter enrichment culture also exhibited a higher nitrite affinity (K_m = 0.19 ± 0.03 mg NO₂^--N/L) than all currently characterized Nitrobacter cultures. These characteristics define acidophilic Nitrobacter as both a K-strategist and an adversity-strategist that tends to flourish in acidic activated sludge, where low pH (<5.5) and low nitrite levels suppress other nitrifiers. This study extends our understanding of nitrifiers growing in acidic environments and also provides a potential to remove nitrogen in acidic wastewater.