Stability of microbial functionality in anammox sludge adaptation to various salt concentrations and different salt-adding steps

The stability of community functioning in anaerobic ammonia oxidation (anammox) sludge adaptation to various salinity changes are concerned but not fully explored. In this study, two anammox reactors were designed in response to different salt levels and salt-adding methods. The reactor PI, run with small stepwise salt increments (0.5%–1.0%), removed >90% of nitrite and ammonium in the influent over the range of 0%–4% salt. By contrast, the reactor SI, run with a sharp salt increment (>2.5%), exhibited a reduced performance (by up to 44%) over the same salt range with a new steady state. The observed resilience times after salt perturbations indicated that the PI reactor recovered substantially and rapidly at all imposed salt levels. Principal coordinates analysis of 16S rRNA gene amplicon sequences revealed that bacterial community structures of the anammox sludge altered conspicuously in response to the salinity changes. However, quantitative PCR analysis showed that the shift in copy number of studied nitrogen-converting genes encoding hydrazine synthase (hzsA), bacterial and archaeal ammonia monooxygenases (amoA), nitrite oxidoreductase (nxrB), nitrite reductase (nirK), and nitrous oxide reductase (nosZ) was not significant (p > 0.05) in anammox sludge across the salt levels of 0.5%–4%, which suggests the stability of microbial community functioning in the osmoadaptation processes. The freshwater anammox Ca. Kuenenia showed high osmoadaptation by potentially adopting both high-salt-in and low-salt-in strategies to dominate in both reactors. The quantitative transcript analysis showed that the active anammox bacteria represented by hzsA transcripts in the SI reactor were approximately two orders of magnitude lower than those in the PI reactor during the long-term exposure to 4% salinity, manifesting the influence by the salt-increasing methods. These results provided new insight into osmo-adaptation of the anammox microbiome and will be useful for managing salinity effects on nitrogen removal processes.