Optimum relative frequency and fluctuating substrate selection in reinforcing anammox-mediated anabolic adaptation

Adaptation to substrate fluctuations is a life actuality of microbes in global municipal wastewater treatment plants (WWTPs). Yet there remains a lack of definite information on how influent changes with different alternation frequencies shape the stability of anammox consortia and the metabolic regulations they feedback. According to human rhythmic activity, day-fluctuant fed (every 6 h, alternating between 50 and 100 mg NH₄⁺-N/L) substantially diminished the robustness of nitrogen removal efficiency (NRE; 84.1 ± 7.0%, left-skewed distribution [R² = 0.87]) and shock-resistance ability (>30% effluent variability). Unexpectedly, the anammox ecosystem under week-fluctuant mode (every 6 d) displayed adapted growth (NRE 86.6 ± 3.1%, normal distribution [R² = 0.97]), higher extracellular polymeric substances (EPS) yields, and superior tolerance (juggling the shortest recovery time and highest NRE, tightest protein secondary structure facing long-term load shocks) than steady-state (75 mg NH₄⁺-N/L). 16S sequencing showed that the influent disturbance led to increased levels of bacterial diversity, however, a similar microbiota composition between week-fluctuant and steady systems was detected. Notably, K strategist Candidatus Kuenenia was more sensitive to substrate fluctuations, with the lower relative abundance at day-fluctuant (23.4 ± 5.1%) and week-fluctuant (39.5 ± 4.3%) than at steady-state community (47.5 ± 4.2%). Conversely, Candidatus Jettenia had higher relative abundance at day-fluctuant (i.e., 1.3 ± 0.1%) compared to that at week-fluctuant (0.2 ± 0.04%) and steady-state (0.05 ± 0.03%). Importantly, untargeted metabolomics revealed that week-fluctuant grown anammox microbiota increased protein synthesis and transporter expression while decreasing expression of catabolic pathways (citric acid cycle and bypass) as a strategy for efficient substrate uptake and utilization, which clearly different to day-fluctuant and steady-state survival ways. Overall, we predictively reported an "anabolic adaptation growth state" for the anammox consortia and put forward the associated reinforcement control strategy.