Demethylation of Naproxen and Caffeine by Marine Sedimentary communities

Naproxen and caffeine may enter marine environments through discharge of wastewater, subsequently accumulating in marine sediments at nanogram per gram (ng/g) levels. These compounds, containing methyl groups linked to heteroatoms, may serve as potential substrates for microbial communities, including methanogens.  Methanogens could utilize these methyl groups directly for methylotrophic methanogenesis, or indirectly for hydrogenotrophic or acetoclastic methanogenesis. Alternatively, some other microorganisms are capable solely of demethylation. Our study investigates the capacity of marine sedimentary communities to demethylate naproxen and caffeine, potentially leading to methane production in marine sediments.

To elucidate the biotransformation pathways and potential methane production from these compounds, we employed a multiple line of evidence approach, including microbial incubation with surface marine sediments, stable isotope analysis, concentration analysis, and 16S rRNA amplicon sequencing. Sediments were collected from Hakefjorden, near the town of Stenungsund on the Swedish west coast, adjacent to the Strävliden WWTP's discharge outflow. Sediments were characterized to contain 4 and 7 ng/g of caffeine and naproxen, respectively. The top sediment layer (2-10 cm) was used for microbial incubation experiments with ¹³C-labeled methyl group substrates, including naproxen, caffeine, and standard methanogenesis substrates such as acetate, methylamine, and carbonate. This enabled tracing of the microbial transformation of the methyl groups within these compounds. Results indicate that both naproxen and caffeine act as precursors to ¹³C-methane production, with naproxen additionally leading to ¹³C-carbon dioxide formation. The presence of these compounds enriched specific microbial populations, including methanogens (Methanomicrobiaceae), other Archaeal groups (Lokiarchaeia), various fermentative bacteria, and sulfate-reducing bacteria.

The transformation of naproxen and caffeine stemming from wastewater into methane and carbon dioxide highlights alternative substrates for greenhouse gas production in marine sediments, an area of concern considering these and other anthropogenic compounds' presence in the sediment. This underscores the need for detailed research into the interactions between marine microbes and methylated pharmaceuticals, given their potential impact on greenhouse gas emissions.