Precision Bioremediation of Chlorinated Ethenes in Heterogeneous Aquifers through Substrate Hydrological Coupling and High-Resolution Molecular Tracking

Abstract Applications of bioaugmentation in chlorinated solvent remediation are often hindered by unpredictable field performance and the inability to verify the colonization of inoculated strains in heterogeneous aquifers. This paper presents a field-validated engineering framework that addresses these limitations by integrating stress-tolerant strain selection, substrate hydrological coupling, and high-resolution molecular tracking. A robust Dehalococcoides mccartyi inoculum, dominated by the Victoria subgroup, was enriched in a membrane bioreactor to retain slow-growth microorganisms. A pilot-scale field study conducted in a heterogeneous aquifer revealed that soluble substrates were ineffective in high-hydraulic-conductivity zones because of rapid advective washout. Deploying an emulsified soybean oil-based substrate tailored to the site’s hydrogeology, combined with a Victoria-dominant consortium, established sustained reducing conditions and promoted complete dechlorination. Hierarchical oligonucleotide primer extension confirmed that the Victoria subgroup functionally replaced the metabolically restricted indigenous Dehalococcoides population, thereby overcoming the accumulation of dichloroethene and driving complete detoxification. In summary, this study provides environmental engineers with an effective protocol for matching substrate physics with hydrogeological constraints and for using lineage-specific tracking to ensure the reliability and efficiency of in situ bioremediation.