Chemical probes reveal individualized gut microbiome biotransformation capacity and the impacts of ex vivo fermentation conditions

The gut microbiome transforms endogenous and exogenous chemicals, contributing to bioactivation or detoxification via the formation of metabolites with altered bioactivity. Most high content microbiome assays infer function from genetic composition rather than direct assessment of biotransformation activity, and it remains difficult to predict functional consequences of environmental factors and experimental variation. Therefore, we developed an anaerobic fecal fermentation workflow that couples targeted LC-MS/MS quantification of dynamic profiles of 20 chemical probes with untargeted metabolomics to profile human microbiome biotransformation capacity and used it to assess the impact of experimental conditions on biotransformation profiles. Across five donors and 240 fermentations, inoculum density and growth medium composition strongly influenced probe transformation rates, whereas the biotransformation capacities of fecal slurries frozen at −80°C did not differ from fresh fecal samples. Individual donors could be uniquely stratified on the basis of biotransformation profile data in a way that was not recapitulated by 16S rRNA taxonomic structure or predicted functional pathways. Finally, expected biotransformation products and metabolic trends could be confirmed with untargeted metabolomics characterization. This scalable platform directly profiles gut microbial biotransformation activity, supporting wider applications of standardized microbiome functional phenotyping in humans and quantitative models of microbiome-competent biokinetics assessment in pharmacology and toxicology.