Combating oxygen toxicity through antioxidants during anammox granular sludge preservation and mechanistic insights

The successful preservation of high-quality anammox granular sludge (AnGS) is critical for the wide application of anammox biotechnology. However, AnGS is prone to the stress of oxygen toxicity under starvation conditions during long-term preservation, resulting in activity decay and cell death. Therefore, this study investigated typical antioxidants, including calcium ascorbate (Vc-Ca), oligomeric proanthocyanidins (OPC), methionine (Met), epigallocatechin gallate (EGCG), and melatonin (MT), and demonstrated their roles in combating oxygen toxicity during AnGS preservation. The results showed that all the antioxidants had a positive effect on alleviating the oxygen toxicity to AnGS, while the magnitudes were diverse. To evaluate the efficacy of antioxidants in combating oxygen toxicity, a multidimensional evaluation system based on microbial activity, live cell ratio, anammox bacteria (AnAOB) quantity, and operating expense of specific anammox activity (SAA) and AnAOB quantity was established. Met was assessed as the preferable antioxidant. Specially, in the Met addition group, the DO concentration and ORP were 21.01% and 24.35% lower during the initial stage, while SAA, AnAOB activity, AnAOB quantity, and live cell ratio of AnGS reached 45.75%, 219.55%, 52.78%, and 18.85% higher than the control group after long-term preservation, respectively. Moving forward, a multi-level blocking mechanism, involving extracellularly decreasing DO concentration, intracellularly promoting cysteine metabolism and enhancing CAT activity, was proposed for Met to scavenge the reactive oxygen species during AnGS preservation. The findings of this study contribute to innovate AnGS preservation methodology, paving the way for the reliable supply of high-quality AnGS and promoting the sustainable development of anammox biotechnology.