Decay kinetics-based precise feeding enables long-term preservation of anammox granular sludge.

Long-term preservation of anammox granular sludge (AnGS) is crucial for the development of anammox technology. Although conventional constant substrate feeding strategy (in short, constant feeding) can alleviate‌ the starvation stress of AnGS during preservation, it risks the toxic substrate inhibition due to the decay of microbial activity over time. In this study, a Decay Kinetics-based Precise Feeding strategy (in short, DKPF) was proposed and its feasibility was verified through a 240-day preservation at 4 °C. Results showed that DKPF was an efficient preservation technology of AnGS, retaining 57.5% nitrogen removal activity after preservation for 240 days, which was 41.0% and 45.8% higher than that of starvation control (CK) and constant feeding, respectively. Residual NH4+-N and NO2--N remained at 6.37 ± 1.06 and 3.73 ± 1.74 mg N/L on day 240, whereas both accumulated under constant feeding. DKPF coordinated the substrate supply with the theoretical maintenance demand, maintained higher ATP and glycogen levels, and retained 30.6% and 44.6% more AnAOB than CK and constant feeding, respectively. DKPF also relieved the decoupling effect of nitrite on oxidative phosphorylation, sustaining the transcriptional level of hzsA gene in dormant AnAOB. These findings provide a decay-informed feeding principle for improving the long-term preservation of AnGS.