Prospector enables defined enriched consortium selection for PFAS degradation via defluorination and Chain-Shortening.

Per- and polyfluoroalkyl substances (PFAS) are recalcitrant environmental contaminants whose biodegradation remains challenging due to the exceptional stability of carbon-fluorine bonds. Here, we employed Prospector® nanowell array technology to isolate enriched bacterial consortia from cotton detritusphere capable of degrading perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) as selective growth-promoting substrates. Screening of 96,000 nanowells across four inoculum-loading regimes identified high-density loading (20-33 genera per community) as the sole condition that yielded functionally stable consortia; low-, mid-, and overload-density regimes failed to support serial passaging functional stabilization. Eight communities (A-H) were characterized for PFAS degradation, fluoride liberation, and taxonomic composition via full-length 16S rRNA sequencing. Community H achieved 87.8 ± 5.6% PFOS removal within 10 days with near-stoichiometric fluoride release (2.93 μM; 97.7% theoretical maximum fluoride yield from PFOS consumed), while Community E achieved the highest PFOA removal (44.9 ± 21.8%). PFOS and PFOA degradation capacities were inversely correlated across communities, with Atlantibacter and Stenotrophomonas predicting PFOS performance (r = 0.70 and 0.50) and Enterobacter predicting PFOA performance (r = 0.80). Bioreactor-scale validation of Community H demonstrated 91.5 ± 1.4% PFOS removal under aerobic and 62.6 ± 4.7% under anaerobic conditions over 14 days. LC-MS/MS detected perfluorohexane sulfonate (PFHxS) as a transient intermediate, peaking at 147.7 ng mL-1 (day 5, aerobic) before declining to < 5 ng mL-1 by day 13, confirming sequential C8 to C6 chain shortening. Shannon diversity correlated positively with degradation performance (r = 0.87 for PFOS), indicating that community complexity enhances transformation efficiency. This work establishes nanowell array-guided community assembly as a reproducible strategy for developing PFAS bioremediation consortia and provides evidence consistent with growth-associated defluorination without co-substrate amendment; the stoichiometric relationship between PFAS carbon and observed biomass warrants further investigation.