# Detection and quantification of rumen methanogens using F420 autofluorescence profiling with spectral flow cytometry

## TL;DR

A new method using spectral flow cytometry can quickly detect and count methane-producing microbes in cow and sheep stomachs, which could help reduce greenhouse gas emissions.

## Contribution

A novel spectral flow cytometry method for direct quantification of rumen methanogens based on F420 autofluorescence.

## Key findings

- Spectral unmixing distinguishes methanogens from other autofluorescent particles in rumen samples.
- The method quantifies methanogens over a wide concentration range without staining or dyes.
- Methanogen counts correlate with methane yields in animals treated with inhibitors.

## Abstract

Methanogenic archaea that reside in the rumen of sheep, cattle, and other ruminants generate 16% of global emissions of methane, a potent greenhouse gas. The majority of rumen methanogens belong to species that display readily observable autofluorescence due to their intracellular co-factor, F420. We developed a spectral flow cytometry method to directly quantify autofluorescent methanogens in the complex environment of the rumen. Rumen samples contain feed particles with natural autofluorescence signatures that overlapped those of F420-containing methanogens. Spectral unmixing using natural autofluorescence signatures allowed us to distinguish methanogens from other autofluorescent particles and to quantify both cultured methanogens in buffer and native methanogens in rumen content samples over a concentration range from 4 × 104 to 4 × 107 cells/mL. The methanogen signal was absent in microbial cultures known to lack F420 and in rumen content samples treated with sodium borohydride (NaBH4), which reduces F420 fluorescence. We showed a strong relationship between the number of autofluorescent methanogens in rumen content samples and methane yields in cattle and sheep treated with a methanogen inhibitor. We also assessed the impact of sample fixation on the spectral profiles of methanogen cells and showed that rumen samples stored at 4°C for up to 3 days remain suitable for enumeration. Our data thus demonstrate a new spectral flow cytometry method that can be used for rapid quantification of autofluorescent methanogens in rumen content samples.

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.