Fate of intracellular, extracellular polymeric substances-associated, and cell-free antibiotic resistance genes in anaerobic digestion of thermally hydrolyzed sludge

Thermal hydrolysis of sludge is a promising approach to mitigate antibiotic resistance genes (ARGs) propagation in an-aerobic digestion (AD). Although ARGs in sludge may be fractioned into intracellular, extracellular polymeric sub-stance (EPS)-associated, and cell-free ARGs, the fate of these different fractions in AD has never been investigated. This study presents a detailed characterization of intracellular and extracellular ARGs in AD of sludge thermally hydro-lyzed at 90 degrees C and 140 degrees C. EPS-associated ARGs represented the major fraction of the total extracellular ARGs in all samples, while its lowest abundance was observed for thermal hydrolysis at 140 degrees C along with the lowest EPS levels. The results suggested a positive correlation between EPS-associated ARGs with intracellular and cell-free ARGs. Fur-thermore, various EPS components, such as proteins and e-DNA, were positively correlated with beta-lactam resistance genes. sul1 dominated all samples as an EPS-associated resistance gene. These results provide new insights into the sig-nificance of different ARGs fractions in their overall dissemination in AD integrated with thermal hydrolysis.