Introduction: Cows produce around 250-500 L of methane a day through enteric fermentation which takes place in the rumen and requires methanogenic microbes. Recent studies focus on ways of reducing methanogens in the rumen of cows by supplementing feeds with various factors. Current methods of detecting treatment success are challenging, time consuming and costly. We show a flow cytometer can quantify methanogenic microbes efficiently and simply.
Method: Initially methanogenic archaea were cultured in vitro and then treated with bromoform, an inhibitor of methanogenesis. Samples were run through the cytometer after dilution in PBS. The Cytek Aurora (4L-UV-V-B-R) was used to detect the expression of co-enzyme F420 in samples. Cow faecal matter was obtained from farms across Otago and mixed with PBS and they were then treated with bromoform and run through the cytometer. Finally cows from farms across NZ were treated with or without a methanogen inhibitor in their feed. Faecal samples were then sent to the University of Otago and run as before through the cytometer.
Results: The in vitro experiments showed that the fluorescence of co-enzyme F420 was clearly lost in a dose-dependent manner when treated with bromoform. This was also seen when treating cow faecal samples. We saw the same loss of F420 fluorescence in the in vivo experiments, but it was inconsistent.
Discussion: Co-enzyme F420 is essential for the methanogenesis process. The loss of F420 when treated with bromoform is promising. The inconsistent results from the in vivo experiments are thought to have come from the inhibitor instability in storage and will be repeated. The Cytek Aurora showed advantages over previous flow cytometric methods of quantifying methanogenic archaea using F420 including volumetric counting and full spectrum profiling. We no longer needed to use count beads thus reducing complexity, time and cost.