Warming the globe with methane burps? Eat more linseed and nitrate.

It’s no secret that the cattle industry is a major source of methane, a potent greenhouse gas that contributes significantly to global warming. But cows can’t make methane alone– in order to pull nutrients out of plants, animals like cattle, sheep, and deer leverage a unique gut microbiome that includes methane-producing archaea. One strategy to reduce methane emissions is to modify the materials provided to the rumen gut microbes. Basically, scientists want to research and implement a special diet for cows that lowers the methane content of their burps.

To make methane, the archaea consume hydrogen and carbon dioxide, of which hydrogen is the limiting factor for methanogenesis. The hydrogen and carbon dioxide come from other microbes like bacteria, protozoa, and fungi as they ferment plant matter. This means that dietary changes affecting these microbes also affect methane production. By analyzing how the microbe species react to changes in diet, scientists can make better suggestions about how to feed cows in an environmentally optimal manner.

With the goal of identifying changes in the microbiota in response to dietary supplements known to affect methane production, Dr. Milka Popova and her colleagues at the French National Institute of Agricultural Research studied the effect of three diet additives– nitrate, linseed, and saponin– on the relative abundance of relevant microbes in the rumen microbiome. Their paper was published this February in the American Society for Microbiology’s Applied and Environmental Biology journal. Dr. Popova and her team had two primary goals for this set of experiments: first, to understand how the microbial ecosystem in the cows’ guts responds to these supplements; and second, to compile conclusive data across 2 different studies, as data from prior studies on the subject was often inconsistent.

The first additive, nitrate, had been shown to decrease methane emissions, potentially by promoting the activity of ammonia-producing microbes and thus competing for hydrogen. Recall that hydrogen is the limiting starting material in methane production. The idea is that the hydrogen that would otherwise be used to make methane is siphoned off by other microbes to make ammonia instead, thereby decreasing methane output.

The second additive, linseed oil, is high in fatty acids. In the normal cow diet, hydrogen-producing fermentation reactions occur to break down cellulose and release digestible carbohydrates for energy. Supplementing the diet with the linseed fatty acids provides easy-access carbs so that decreased fermentation occurs and less hydrogen is available for methanogenesis.

Finally, saponins have been shown to be toxic to protozoan fermenters. Hypothetically, by killing protozoan fermenters, saponin supplementation could decrease methane emissions by way of hydrogen limitation. However, as other microbes in the cow gut can inactivate saponins, this approach was questionable from the outset.

Dr. Popova and her colleagues hypothesized that linseed and saponins would affect methane production by affecting hydrogen producers and that nitrogen would do so by diverting hydrogen away from methanogenesis. But the data told a slightly different story!

How does one even determine what the microbe populations look like inside a cow gut? The authors set up two studies. In the first, cows were fed control, linseed-supplemented, nitrate-supplemented, or linseed plus nitrate-supplemented diets. In the second, saponins were substituted for linseed. All supplemented diets except saponin alone reduced methane production and yield by between 20-45% as compared to the control diet. As a reminder, saponins are the ones that are deactivated in the gut– so it makes a lot of sense that saponins alone had no effect on methane production.

To assess microbial ecosystems, the scientists took 200 gram samples of goop from different location of each cow’s gut. They washed out the microbes and isolated the RNA and DNA. The RNA was reverse-transcribed into complementary DNA (cDNA), and target genes were then amplified and sent for sequencing. They used a technique called quantitative PCR (qPCR) to determine the relative numbers of these target genes in the cDNA and DNA. Analyzing genes like 16S rRNA for bacteria and mcrAfor methanogens allows scientists to assess relative abundance of the relevant microbes (hydrogen producers and methanogens). Tracking changes in the expression of additional genes necessary for fermentation and methanogenesis allows them to approximate the activity and output of these processes.

To their surprise, the group found that both linseed and nitrate reduce the relative abundance of hydrogen producers called Ruminococcaceae. As expected, they found that nitrate diverts hydrogen to nitrate reducers like Coriobacterialesand Burkholderiales, causing an increase in their relative abundance and activity. Unexpectedly, linseed was also able to divert hydrogen to propionate producers. Previous work had hinted at this result from linseed and nitrate supplementation, but this work showed that linseed alone can cause this effect. Finally, saponins had no effect on microbial numbers or diversity, likely because they are inactivated in the cow gut.

Despite similar changes in methane output, inconsistency was observed between the two studies in the nitrate-supplemented samples. The authors explain this by saying that the effect of nitrate may be more relevant on a functional level than a colony-level remodeling. They assert that assessing the exact mechanisms of methane mitigation should be approached in the metatranscriptome, or the total messenger RNA content of the gut microbiome. This would provide valuable information about the changes in protein production in response to linseed and nitrate. They also suggest that future attempts with saponins should include modifications to the chemical to prevent inactivation in the gut.

Overall, this work attempts to address environmental concerns associated with the meat industry, specifically, to mitigate the methane produced by the animals themselves. Linseed and nitrate supplementation to the ruminant diet could greatly reduce methane emissions from these industries. But questions still remain. For example, how do these additives affect the health of the animals and their ability to absorb nutrients? It also provides valuable insight into the complex webs of microbial metabolisms that occur within all of us.

References:

Popova M, Guyader J, Silberberg M, Seradj AR, Saro C, Bernard A, Gérard C, Martin C, Morgavi DP. 2019. Changes in the rumen microbiota of cows in response to dietary supplementation with nitrate, linseed, and saponin alone or in combination. Appl Environ Microbiol 85:e02657-18. https://doi.org/10.1128/AEM.02657-18.