Enriching microbes capable of fluorotelomer acid defluorination: thermodynamic constraints and experimental challenges.

Fluorotelomer compounds are a major class of polyfluoroalkyl substances that can undergo partial microbial transformation via the one‑carbon removal pathway, which has the potential to achieve deep defluorination. However, the microorganisms and mechanisms driving this pathway remain elusive. We focus on 5:3 fluorotelomer carboxylic acid (5:3 FTCA) because, although its structure suggests susceptibility to biodegradation, it exhibits high environmental persistence, making it a useful model for probing the feasibility and constraints of the one‑carbon removal pathway. In this study, we combine density functional theory (DFT)‑based thermodynamic modeling with extensive experimental screening to evaluate the feasibility of enriching microorganisms capable of n:3 fluorotelomer acid (n:3 FTCA) defluorination. DFT calculations reveal that the overall pathway is energetically favorable under alkaline conditions, with dehydrogenation and hydroxylation, rather than defluorination, likely serving as rate‑limiting steps. Despite extensive screening of activated sludge and soils, pure bacterial cultures, and extreme-pH-tolerant enriched consortia, no individual degraders were identified, indicating that such microbes are rare and likely dependent on cooperative community interactions. Limited biotransformation of 5:3 FTCA to 4:3 FTCA was observed in mixed consortia containing a methylotroph, confirming the co‑metabolic nature of this process and demonstrating the limitations of high‑throughput assays for cultivating kinetically constrained degraders. To address these challenges, we propose a dual-stage enrichment strategy decoupling survival from functional selection: initial maintenance followed by targeted co-metabolic enrichment. Overall, this study clarifies the energetic basis and enrichment constraints of fluorotelomer degradation and provides a framework for cultivating microbes capable of the one-carbon removal pathway.