[P(CH₂OH)₄]Cl induced natural shift of methanogenic pathway through disrupting bacterial disulfide bonds and...

Ionic liquids (ILs), which are expanding produced and applied as alternatives to volatile organic solvents, have shown the ability to deteriorate the anaerobic biotransformation of organics. It is unclear, nevertheless, how ILs affect different functional anaerobes during anaerobic digestion, leaving a knowledge gap in the environmental risks of ILs. Here, we revealed that the differences of Gram-staining bacteria probably were the part drivers of a shift in methanogenic pathway from acetoclastic to hydrogenotrophic methanogenesis in anaerobic microcosms exposed to a typical IL (Tetrakis (hydroxymethyl) phosphonium chloride, [P(CH₂OH)₄]Cl). The results showed that 0.1-4 mg/L [P(CH₂OH)₄]Cl respectively decreased methane production rate and carbon-use efficiency by 4.43-43.90 % and 0.52-57.23 % during anaerobic digestion. Microbial community and microscopic examination analysis indicated that most Gram-positive bacteria were more likely to survive in the [P(CH₂OH)₄]Cl-present environment than Gram-negative bacteria. Mechanistically, [P(CH₂OH)₄]Cl distorted cell walls of anaerobes, and then perturbed protein homeostasis in the periplasm by breaking disulfide bonds and disrupting disulfide-bond-forming pathways. Moreover, Gram-positive bacteria exhibited a higher tolerance than Gram-negative bacteria, potentially due to their thicker peptidoglycan structures and reliance less on disulfide bonds to stabilize proteins, leading to the remodeling of microbiome function and carbon-transport pathway. This study is the first to reveal the differential impact of [P(CH₂OH)₄]Cl on Gram-positive vs. Gram-negative anaerobes during methanogenesis, providing new insights into the ecological risks of ILs and contributing to their optimal design.