Vitamin B₁₂ effects on chlorinated methanes-degrading microcosms: Dual isotope and metabolically active microbial...

Field-derived anoxic microcosms were used to characterize chloroform (CF) and carbon tetrachloride (CT) natural attenuation to compare it with biostimulation scenarios in which vitamin B₁₂ was added (B₁₂/pollutant ratio of 0.01 and 0.1) by means of by-products, carbon and chlorine compound-specific stable-isotope analysis, and the active microbial community through 16S rRNA MiSeq high-throughput sequencing. Autoclaved slurry controls discarded abiotic degradation processes. B₁₂ catalyzed CF and CT biodegradation without the accumulation of dichloromethane, carbon disulphide, or CF. The carbon isotopic fractionation value of CF (ƐC_CF) with B₁₂ was -14±4‰, and the value for chlorine (ƐCl_CF) was -2.4±0.4‰. The carbon isotopic fractionation values of CT (ƐC_CT) were -16±6 with B₁₂, and -13±2‰ without B₁₂; and the chlorine isotopic fractionation values of CT (ƐCl_CT) were -6±3 and -4±2‰, respectively. Acidovorax, Ancylobacter, and Pseudomonas were the most metabolically active genera, whereas Dehalobacter and Desulfitobacterium were below 0.1% of relative abundance. The dual C-Cl element isotope slope (Λ=Δδ¹³C/Δδ³⁷Cl) for CF biodegradation (only detected with B₁₂, 7±1) was similar to that reported for CF reduction by Fe(0) (8±2). Several reductive pathways might be competing in the tested CT scenarios, as evidenced by the lack of CF accumulation when B₁₂ was added, which might be linked to a major activity of Pseudomonas stutzeri; by different chlorine apparent kinetic isotope effect values and Λ which was statistically different with and without B₁₂ (5±1 vs 6.1±0.5), respectively. Thus, positive B₁₂ effects such as CT and CF degradation catalyst were quantified for the first time in isotopic terms, and confirmed with the major activity of species potentially capable of their degradation. Moreover, the indirect benefits of B₁₂ on the degradation of chlorinated ethenes were proved, creating a basis for remediation strategies in multi-contaminant polluted sites.