Advancing anaerobic microbial studies with in situ Raman spectroscopy: Methanogenic archaea as a model.

Methanogenic archaea play a critical role in methane (CH4) production and the global carbon cycle, yet accurately monitoring their gas metabolism under anaerobic conditions remains a technical challenge. In this study, we developed a Raman spectroscopy-based gas quantification model, achieving high-precision monitoring of CO2-N2-CH4 ternary gas mixtures over a temperature range of 12-52 °C. The model exhibited strong linear correlations between the Raman peak area ratios and gas molar ratios, which were further validated against gas chromatography, revealing no significant differences (p > 0.05). This confirms the reliability and accuracy of the approach.. Building upon this model, we conducted real-time monitoring of the gas metabolism of methylotrophic methanogenic archaea under anaerobic conditions. The results demonstrated that methanol concentration significantly influenced the gas production kinetics. At a methanol concentration of 10 μL/mL, the highest CH4 yield (59.97 %) was achieved, along with stable metabolic activity. In contrast, higher concentrations caused substrate saturation effects, leading to decreased metabolic efficiency. Furthermore, by integrating Raman spectroscopy with high-precision pressure monitoring, this study successfully achieved real-time molar quantification of CH4 and CO2 during methanogen cultivation. This approach provided detailed insights into gas production dynamics and substrate utilization patterns. Compared to traditional methods, this non-destructive, real-time monitoring platform offers a novel tool for anaerobic metabolism research and lays a solid foundation for applications in biogas optimization, industrial fermentation, and renewable energy development.