Hydroxylation-mediated acesulfame degradation within a methane-fed biofilm: a novel and efficient biodegradation route

Acesulfame (ACE), a persistent organic pollutant, is frequently detected across diverse aquatic environments, yet its environmental remediation remains challenging due to the inherently slow biodegradation. Here, we unveil a highly efficient ACE removal strategy using a methane-fed biofilm, achieving degradation rates nearly six times higher than those of conventional activated sludge systems. Comprehensive analyses of transformation products (TPs) and toxicity profiles revealed that hydroxylation emerged as an important and effective biodegradation pathway, promoting deep ACE degradation. Microbial community analysis, methane interruption experiments and pure culture studies implicated aerobic methanotrophs Methylococcus sp. and Methylomonas sp. as key players in ACE degradation. Metagenomic and metatranscriptomic analyses demonstrated that these methanotrophs exhibited high expression levels of particulate methane monooxygenase (pMMO) and cytochrome P450 monooxygenase (CYP450) genes within the biofilm community. Enzyme inhibition assays, combined with TP profiling, confirmed that pMMO and CYP450 catalyze the hydroxylation-driven degradation of ACE. Scavenging experiments further suggested that hydroxyl radicals (•OH) generated through the catalytic activity of pMMO and CYP450 are crucial mediators in the ACE hydroxylation pathway. These findings provide the first evidence of hydroxylation-mediated ACE degradation in a methane-fed biofilm system, offering a promising and sustainable approach for the removal of recalcitrant organic contaminants from wastewater.