Intermittent glycerol feeding shapes a glycogen-storing phenotype for enhanced nitrite accumulation via sequential...

Achieving stable and efficient denitratation is a promising strategy to supply nitrite for mainstream anammox processes, yet the underlying microbial mechanisms remain poorly understood, especially with alternative carbon sources like glycerol. Here, we hypothesized that intermittent feeding regimes could drive higher nitrite accumulation by selecting for specific metabolic phenotypes. To test this, an intermittently-fed sequencing batch reactor (SBR) and a continuously-fed up-flow anaerobic sludge blanket (UASB) reactor were operated under identical carbon-restricted conditions (COD/NO₃⁻-N = 3.5). The SBR achieved a significantly higher and stable nitrite accumulation efficiency (NiAE) of 43.0 ± 4.1 % compared to the UASB (18.9 ± 5.4 %). Genome-resolved metagenomics revealed that the SBR community was enriched in species with glycogen-storing potential, leading to a 45.8 % greater overall abundance of genes related to glycogen metabolism compared to the UASB. Phenotypic analyses confirmed that the SBR sludge contained significantly more intracellular glycogen and displayed a nitrate reductase (NAR) to nitrite reductase (NIR) activity ratio that was an order of magnitude higher than the UASB sludge. This kinetic imbalance underpinned a "sequential denitrification" phenotype, where nitrate was preferentially reduced to nitrite. Critically, the UASB-enriched community, when subjected to batch-fed conditions, also exhibited a high denitratation potential close to the SBR culture, demonstrating that this phenotype is a plastic trait regulated by the glycerol feeding strategy rather than a fixed attribute of the community structure. This study uncovers a fundamental mechanism where intermittent glycerol availability directs metabolic flux towards glycogen storage, which in turn regulates electron partitioning between NAR and NIR. This highlights a significant divergence between genotypic potential and realized phenotype, offering a new paradigm for controlling nitrogen transformation pathways in engineered ecosystems.