Detection and quantification of rumen methanogens using F ₄₂₀ autofluorescence profiling with spectral flow cytometry

Production of methane, a potent greenhouse gas, by methanogenic archaea in cattle, sheep, and other ruminants contributes around 16% of global methane emissions. Methane mitigation strategies are essential to respond effectively to the challenge of climate change, and many mitigations target rumen methanogens directly. In this study, we developed a method based on methanogen autofluorescence to identify and quantify methanogens within the complex rumen environment containing plant material that also fluoresces. The overlapping autofluorescence signals from methanogens and plant material can be resolved by applying spectral unmixing in spectral flow cytometry. This technique provides a rapid, practical approach for detecting and quantifying methanogens directly in rumen samples without the need for staining or additional fluorescent dyes or reagents. It provides a valuable tool to assess the impact of mitigation technologies. The method should also allow direct measurement of antibody binding to methanogens or determination of co-location of methanogens with other microbes.