Extracellular DNA serves as a mesoscale cross-linker in aerobic granular sludge

Aerobic granular sludge (AGS) offers compact, energy-efficient wastewater treatment, but long-term structural instability limits its widespread application. The extracellular polymeric substance (EPS) matrix governs granule cohesion. Two decades of research have focused on proteins and polysaccharides as the primary structural scaffolds, while extracellular DNA (eDNA) has been systematically overlooked despite its established importance in clinical biofilms. This oversight is significant because eDNA carries one negative charge per nucleotide along its phosphodiester backbone, conferring inherent cross-linking potential through divalent cation coordination. However, its relative contribution compared with other EPS components, its spatial organisation, and the underlying bridging mechanism in AGS remain insufficiently characterised. A multi-scale evidence chain spanning from direct spatial observation to molecular mechanism is constructed herein. Confocal laser scanning microscopy (CLSM) with sequential propidium iodide (PI)/SYTO 9 staining reveals fluorescence patterns consistent with an eDNA network distributed throughout the intercellular matrix of intact granules. Selective enzymatic dissection coupled with Fourier-transform infrared spectroscopy (FTIR), ultraviolet-visible (UV–Vis), X-ray photoelectron spectroscopy (XPS), and differential scanning calorimetry (DSC) confirmed dose-dependent eDNA cleavage (A 260 /A 280 linear decline, R 2 = 0.97), reduced denaturation enthalpy by 14%, and decreased settling velocity by 12.9%, while leaving ζ-potential invariant. Scanning electron microscopy–energy dispersive X-ray spectroscopy (SEM-EDS) elemental mapping revealed spatial co-localisation of P, Ca, and Mg across the granule cross-section, and SEM morphology confirmed progressive matrix disruption upon DNase I treatment with structural recovery upon Ca 2+ re-supplementation. Ethylenediaminetetraacetic acid (EDTA) chelation reduced settling velocity by 17.2%; Ca 2+ /Mg 2+ re-supplementation fully restored it. Exogenous DNA supplementation demonstrated a finite retention capacity and dose-dependent settling recovery. These converging lines of evidence establish that eDNA does not constitute the primary scaffold of AGS. Instead, it operates as a mesoscale cross-linker whose phosphodiester backbone recruits divalent cations to bridge discrete EPS domains. This mechanism suggests that maintaining adequate Ca 2+ and Mg 2+ concentrations in the reactor influent may help preserve the eDNA-mediated cross-linking network, although validation under long-term, full-scale conditions is required.