Dual suppression of methanogenesis by dichloromethane and salinity: Competitive methyltransferase inhibition and energy-coupling disruption.

The pervasive co-occurrence of dichloromethane (DCM), a prevalent chlorinated solvent, with high salinity in industrial wastewater poses a poorly understood combined ecological stress on anaerobic microbial ecosystems. This study deciphered the molecular mechanisms underlying the dual inhibition of methanogenesis under such combined stress. Metagenomic analysis revealed that methanogens (particularly Methanobacterium and Methanothrix) were more sensitive to DCM and salinity stress, while fermentative bacteria and acetogens exhibited better resilience. Under salinity stress, methanogens primarily expelled extra Na+ was via the Na+ pump coupled with methyltransferase (Mtr). However, density functional theory (DFT) calculations demonstrated that DCM competitively bound to the cobamide cofactor in Mtr (ΔG = -10.5 kcal/mol for DCM vs. +14.6 kcal/mol for methylated carrier), thereby impairing sodium extrusion (58 % downregulation in mtrH gene abundance) and subsequent ATP synthesis. Concurrently, elevated Na+ levels forced a metabolic shift towards energy-intensive sodium extrusion pathways, as evidenced by the upregulation of mrpA (Na⁺/H⁺ antiporter, +20 %) and natA/natB (ABC sodium transporters, +162 %). These disruptions culminated in a severe bioenergetic crisis, indicated by decreased coenzyme F420 activity (31.11 ± 1.58 vs. 48.66 ± 2.09 U/L in control) and suppression of methane yield (22.31 ± 3.63 CH4/g COD vs. 178.91 ± 1.28 mL CH4/g COD in control). Our findings uncovered a novel dual inhibition mechanism, combining molecular-level competitive enzyme inhibition with cellular-scale energy uncoupling, providing critical insights into the microbial toxicological effects of co-occurring chlorinated solvents and salinity.