Wastewater-impacted Skagerrak Sea microbiomes anaerobically demethylate micropollutants

Methylated micropollutants such as naproxen and caffeine persist in wastewater effluents and accumulate in coastal sediments, including Hakefjorden, Skagerrak Sea, yet their anaerobic fate and role in methane emissions remain unresolved. In particular, it is unclear whether pollutant-derived methyl groups are routed mainly to CO₂ or can be transformed into CH₄ in sulfate-rich coastal sediments. Our primary objective was to resolve this routing by tracing the fate and microbiome responses to ¹³C-labeled naproxen and caffeine in sediment microcosms. We show that naproxen underwent rapid O-demethylation to desmethylnaproxen, with 90% ± 15.5% removed within 25 days, producing primarily ¹³CO₂ and some ¹³CH₄. Naproxen enriched methylotrophic and hydrogenotrophic Methanomicrobia, alongside Lokiarchaeia, Bathyarchaeia, and bacterial taxa like Eubacterium (Alkalibaculum A sporogenes) and Syntrophomonadaceae. Metagenomics revealed O-demethylation genes in enriched bacterial MAGs affiliated with uncultured Thermoanaerobaculia, indicating a bacterial demethylation potential. In contrast, caffeine was largely recalcitrant to degradation (∼85% ± 5% remaining), yet its ¹³C-labeled N-methyl groups fueled trace ¹³CH₄ production. These results show that methylated micropollutants can activate both bacterial and archaeal demethylation pathways in coastal sediment microbiomes.