Zoned Bioremediation of Trichloroethene and Toluene Co-Contamination Using Immobilized Anaerobic Consortia in Saturated Porous Media

Trichloroethene (TCE) and toluene are ubiquitous co-contaminants in groundwater and soil, yet effective strategies for their concurrent remediation remain challenging due to inhibitory interactions and substrate competition. In this study, we obtained two anaerobic enrichment cultures capable of detoxifying TCE and toluene with degradation rates of 141.7 ± 57.5 and 317.6 ± 1.3 µmol/(L·d), respectively. The 16S rRNA gene amplicon sequencing and quantitative PCR revealed that Dehalococcoides spp. dechlorinated TCE to ethene, while Azoarcus - Aromatoleum spp. mediated the toluene degradation under nitrate-reducing condition, with growth yields of 2.5 ± 1.3 × 10 8 copies/µmol Cl − released and 1.3 ± 0.2 × 10 9 copies/µmol toluene consumed, respectively. The enrichment cultures were successfully immobilized, maintaining degradation efficiencies comparable to those of free cells when individually exposed to respective contaminants. Under co-contamination scenario, complete TCE dechlorination was achieved only with immobilized cultures, as the immobilization matrix mitigated the toxicity from denitrification intermediates. When incorporated into sand columns to construct permeable reactive barriers (PRBs) simulating the groundwater flow system, the immobilized cultures completely degraded TCE and toluene (up to 547 μM) individually within 17 days. A hydrodynamically zoned bioremediation strategy was implemented for co-contamination removal, achieving 91.2% upstream TCE dechlorination and 78.98% downstream toluene oxidation. Elevated TCE loading (338 μM) without sufficient hydraulic retention time caused accumulation of dechlorination intermediates, whereas subsequent reduced TCE (169 μM) restored complete dechlorination to ethene. Toluene degradation remained suboptimal due to inhibitory effects from upstream TCE and its dechlorination intermediates. This study provides valuable technical insights and practical guidance for designing and implementing zoned bio-PRBs to address challenging groundwater co-contamination problems.