Resolving metabolic interdependencies during chlorobenzene dechlorination in a Dehalobacter-containing community

Chlorinated benzenes are persistent groundwater contaminants whose anaerobic transformation can depend on organohalide-respiring microorganisms embedded within metabolically interconnected communities. Here, we asked which microorganisms and metabolic functions characterize a Dehalobacter -containing dechlorinating community, and whether bioinformatics and genome-resolved metabolic modeling could identify candidate interactions supporting organohalide respiration. To address these questions, we integrated dechlorination data from related Dehalobacter -containing enrichment lineages with genome-resolved metagenomics and constraint-based modeling. Transformation of 1,2,4-trichlorobenzene (TCB) to dichlorobenzenes and monochlorobenzene (MCB) was observed in one enrichment lineage, whereas MCB transformation to benzene was observed in a related lineage; in the latter, benzene increased from 0 to 62.3 μ mol bottle −1 as MCB decreased from 88.3 to 22.0 μ mol bottle −1 over 120 days. These experiments comprised one observation unit per lineage rather than independent biological reactor replicates and therefore do not establish continuous TCB-to-benzene conversion within a single culture. Genome-resolved reconstruction identified two Dehalobacter populations among 12 community members selected for metabolic modeling. Following structural, phenotypic, and thermodynamic quality control, pairwise random flux sampling predicted a metabolically differentiated exchange network: Methanobacterium consumed H 2 in all 11 pairwise models (median model-normalized flux, 22.36 mmol gDW −1 h −1 ), whereas Methanothrix consumed acetate in all 11 (10.16 mmol gDW −1 h −1 ) and released CH 4 (9.51 mmol gDW −1 h −1 ), distinguishing hydrogenotrophic and acetoclastic methanogenic functions. Model-guided community reduction across 39 sensitivity scenarios produced objective-minimal candidate communities in 34 scenarios, predominantly bicultures with condition-dependent recruitment of a third member. A Dehalobacter member occurred in every candidate community and Methanothrix recurred across all evaluated scenarios, defining a compact model-predicted Dehalobacter –methanogen backbone rather than demonstrating biological indispensability. Together, these results distinguish observed dechlorination from genome-inferred metabolic potential and model-predicted interactions, providing experimentally testable hypotheses for metabolic dependencies that could inform more predictable anaerobic bioremediation strategies.