Deciphering acetate stress responses in methanogen using D₂O-Labeled Single-Cell Raman spectroscopy.

Area of Science:

• Microbiology
• Biochemistry
• Environmental Science

Background:

• Volatile fatty acid accumulation is a challenge in anaerobic digestion.
• Methanosarcina barkeri exhibits high acetate stress tolerance, but underlying mechanisms remain unclear at the single-cell level.

Purpose of the Study:

• To investigate the physiological and metabolic responses of Methanosarcina barkeri to varying acetate concentrations using single-cell Raman spectroscopy.
• To understand the single-cell mechanisms of acetate stress tolerance in M. barkeri.

Main Methods:

• Utilized heavy water-labeled single-cell Raman spectroscopy (D2O-SCRS) to analyze M. barkeri.
• Examined responses across different acetate concentration gradients (50, 100, and 200 mM).
• Applied multivariate analysis, including principal component-linear discriminant analysis, to interpret Raman spectral data.

Main Results:

• High acetate concentrations (200 mM) significantly inhibited methane production and microbial growth.
• D2O-SCRS showed a concentration-dependent decrease in metabolic activity (C-D ratio) with increasing acetate levels.
• Multivariate analysis identified distinct metabolic reprogramming and phenotypic transitions, with specific biomarkers indicating disruption of nucleic acid, protein, and lipid functions.

Conclusions:

• Acetate stress significantly impacts M. barkeri's metabolism and cellular functions at the single-cell level.
• D2O-SCRS is a valuable tool for elucidating microbial adaptive strategies in anaerobic digestion.
• Findings provide insights into the mechanisms of archaeal stress response in complex ecosystems.