Temporal emergence of functional dark matter in microbial responses to PFAS revealed by materials-based cultivation

Microbial responses to xenobiotic compounds are difficult to resolve due to environmental complexity and limited functional annotation. Here, we establish a materials-based cultivation framework using macroporous elastomeric silicone foams (MESIF) to capture microbial adaptation across environmental contexts and timescales. Using glyphosate as a model compound and per- and polyfluoroalkyl substances (PFAS) as a poorly understood class, we show that responses differ depending on the availability of established metabolic pathways. Glyphosate exposure induced rapid, pathway-specific functional enrichment with minimal taxonomic change. In contrast, PFAS exposure did not yield consistent taxonomic or annotation-based signals but instead produced responses that emerged over time and were primarily detectable at protein and genome-resolved levels. LC-MS analyses revealed transformation dynamics, including formation of shorter-chain products. These responses were not explained by known degrader taxa but involved uncharacterized proteins and microbial populations, highlighting the extent of functional “dark matter” in microbial responses to persistent contaminants.