Chronic anticancer-drug exposure reorganizes the functional microbiome and resistome of aerobic granular sludge

Although anticancer drugs are increasingly detected in hospital wastewaters, it remains unclear whether chronic exposure primarily alters microbial community composition or reorganizes microbial functional potential and antibiotic resistance reservoirs. This study investigated the effects of environmentally relevant concentrations of cyclophosphamide, methotrexate, and tamoxifen on the functional microbiome and resistome of an aerobic granular sludge sequential batch reactor during 90 days. Cytotoxic exposure reduced inorganic nitrogen removal to approximately 25–35%, compared with 35–50% in the control, and decreased biomass concentration from 2825 to 1837 mg L⁻¹ , mean granule diameter from approximately 11.5–6.0 mm, and settling velocity from 107 to 83 m h⁻¹ , whereas COD removal remained above 90% from day 10 onward. Apparent drug removal was compound-dependent, reaching 51.9–67.5% for cyclophosphamide, 49.2–69.4% for methotrexate, and 14.6–26.2% for tamoxifen, which showed preferential association with granular biomass. Despite comparatively stable microbial diversity and overall community composition, pronounced changes occurred in microbial functional potential and resistome structure. Resistome composition differed significantly between treatments, with stronger separation for the taxonomy-based resistome (ANOSIM R = 0.852) than for the functionality-based resistome (R = 0.556); treatment-associated changes involved 21 versus 8 antibiotic families and 19 versus 9 drug classes, respectively. Functional responses differed among biogeochemical processes, with nitrogen-related potential showing stronger and more persistent changes than carbon-fixation potential. Multivariate analyses associated functional variation primarily with biomass- and solids-related parameters. Overall, chronic anticancer-drug exposure reorganized microbial functional potential and resistance reservoirs without extensive taxonomic turnover.