Stable-isotope probing identifies microorganisms actively degrading DMSP in anaerobic saltmarsh sediments

Billions of tons of dimethylsulfoniopropionate (DMSP) are produced every year in marine and coastal ecosystems such as saltmarshes and estuaries. DMSP has far-reaching roles in global carbon and sulfur cycling, also as an osmotolerant and signalling molecule. Furthermore, the microbial degradation of DMSP contributes significantly to the formation of dimethylsulfide (DMS) and methanethiol (MT), other abundant organosulfur compounds with ecological significance. Particularly, in anaerobic sediments, microbial DMS and MT degradation leads to the formation of methane, a powerful greenhouse gas. However, research to date has predominantly focused on aerobic settings, revealing diverse groups of microbes and enzymes mediating DMSP degradation. DMSP concentrations in anaerobic ecosystems and microbial populations underlying DMSP breakdown have never been studied, prohibiting improvements in our understanding of global carbon and sulfur cycles. To address this key knowledge gap, we applied stable-isotope probing combined with 16S rRNA sequencing to identify the active DMSP-degraders in anaerobic saltmarsh sediments. We collected sediments from a 5-10 cm depth of Medway Saltmarshes (UK) using 3.5cm Perspex corers. We transferred the samples to the laboratory and measured in situ DMSP concentrations of 7.7 (±0.5) μmol g−1 wet sediment. In line with the in situ concentrations, we set up replicated incubations anaerobically with 8 μmol g−1 13C- and 12C-labelled DMSP, and applied stable-isotope probing combined with 16S rRNA sequencing. We observed immediate degradation of DMSP in the sediment incubations and DMS production, suggesting the existence of a resident microbial community actively carrying out this process. A total of 48 μmol/g 13C- or 12C-DMSP was amended and a total of 34 (±3.2) μmol/g DMS was produced in the incubations over 12 days.  DNA was extracted and ultracentrifugation was applied to separate heavy and light DNA fractions for downstream analysis. 16S rRNA sequencing of the fractions from 13C and 12C-labelled DNA demonstrated significant enrichment of the family Nitrincolaceae within the order Oceanospirillales in 13C-heavy fractions compared to 13C- light and 12C-heavy fractions (P<0.05). Their relative abundance increased from 2% (±1.3) to 27.2% (±9.1). This demonstrates that they are the active DMSP degraders in anaerobic saltmarsh sediments. This is the first study quantifying significant concentrations of DMSP in anaerobic saltmarsh sediments and demonstrating Nitrincolaceae to be the active DMSP-degraders. Our findings not only broaden our understanding of microbial carbon and sulfur cycling but also highlight a previously overlooked route to methane formation in anaerobic saltmarsh sediments.  Our study underscores the need to identify microbial communities and pathways of DMSP breakdown across diverse anaerobic settings.