Fates of intracellular and extracellular antibiotic resistance genes during sludge anaerobic digestion with different pretreatments

Intracellular and extracellular antibiotic resistance genes (iARGs and eARGs) together constitute the resistome in sludge. However, the respective fates of iARGs and eARGs during sludge anaerobic digestion (AD) with pre -treatments are unclear. In this study, the removal effects of three kinds of pretreatment methods including alkaline pretreatments (ALP), thermal pretreatments (THP), and enzymatic pretreatments (ENP) on iARGs and eARGs removal in sludge were first compared. Then the variations and mechanisms of iARGs and eARGs during the subsequent 30-day batch AD were investigated. Quantification PCR was adopted to determine the intra-cellular and extracellular absolute abundances of seven ARGs (tet(A), tet(O), tet(X), sul1, sul2, blaSHV, and blaTEM). THP showed the most potent removal effects for iARGs and eARGs (1.3-5.1 log), followed by ALP (0.3-1.0 log), while ENP had a minor impact on their removal (0.05 log by protease pretreatment). In the subsequent AD, the absolute abundances of iARGs and eARGs rebounded during AD with THP. Intracellular tet(O) increased its abundances while the absolute abundances of other iARGs and all eARGs decreased during AD with ALP and ENP and without pretreatments. The removal amounts of eARGs were higher than those of iARGs. From the perspective of the whole pretreatment-AD process, no advantages were observed in the removal of both iARGs and eARGs than AD without pretreatments. Redundancy analysis and partial least squares path modelling results revealed that bacterial community composition directly influenced the iARGs variations. The variations in iARGs and sludge physicochemical properties together directly influenced the eARGs variations.