Kinetic and Physiological Characterization of Acidophilic <i>Nitrobacter</i> spp. in a Nitrite-Oxidizing Culture

Novel acidophilic nitrite-oxidizing <i>Nitrobacter</i> spp. were enriched, with kinetic and physiological features characterized comprehensively. The acidophilic <i>Nitrobacter</i> 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 <i>Nitrobacter</i> genomes, which corresponded to previously unknown species within the <i>Nitrobacter</i> genus with average nucleotide identity (ANI) < 91% compared to the publicly available genomes. In contrast to the known NOB, the acidophilic <i>Nitrobacter</i> 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 <i>Nitrobacter</i> enrichment culture also exhibited a higher nitrite affinity (<i>K</i>_m = 0.19 ± 0.03 mg NO₂^--N/L) than all currently characterized <i>Nitrobacter</i> cultures. These characteristics define acidophilic <i>Nitrobacter</i> as both a <i>K</i>-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.