Sustained organic loading perturbation favors nitrite accumulation in bioreactors with variable resistance and resilience of nitrification and nitrifiers

Sustained perturbations are of interest in environmental biotechnology as they can lead systems to alternative stable states that may not be reversible. Studies assessing these concerns are scarce as robust replication is required. Here, we tested the effect of sustained organic loading variations (food-to-biomass ratio, F:M; carbon-to-nitrogen ratio, C:N) on both structure and function of activated sludge bacterial communities, focusing on nitrification and nitrifiers. Two sets of replicate 5-liter sequencing batch reactors were operated at two different, low (n = 4) and high (n = 3), F:M (0.19 and 0.36 mg COD/mg TSS/d) and C:N (3.5 and 6.3 mg COD/mg TKN) conditions for a period of 74 days, following 53 days of sludge acclimation at low F:M and C:N (henceforth referred to as F:M-C:N). The inoculum was taken from a full-scale treatment plant. Resilience was tested during the last 14 days by operating all seven reactors at low F:M-C:N. Samples were analyzed using metagenomics, 16S rRNA gene amplicon sequencing, and effluent characterization. High F:M-C:N reactors exhibited different ecosystem functions and nitrifier abundance compared to the ones at low F:M-C:N. Perturbed high F:M-C:N reactors displayed quantifiable and initially variable functional resistance. Stable nitrite accumulation (77%) was achieved through high F:M-C:N loading with concurrent suppression of Nitrospira, revealing a new partial nitrification strategy for nitritation-denitritation systems. Subsequently, only two of the three reactors experiencing a switch back from high to low F:M-C:N recovered the nitrification function, with an increase in Nitrobacter (r-strategist) abundance as the predominant NOB replacing the niche initially occupied by Nitrospira (K-strategist). Overall, the AOB community was more diverse and resilient than the NOB community. We showed that functional resistance and resilience can vary across replicate reactors in a closed system, and that nitrification resilience need not coincide with a return to the initial nitrifying community structure.