Ruminant Methanogens as Climate Change Target

Reducing atmospheric concentrations of methane, the potent, yet short-lived greenhouse gas, is critical for slowing the rise of global temperatures. Dairy and beef cattle, the world's most numerous ruminants, belch out about 100 teragrams (Tg) of methane (CH4) every year. Globally, enteric methane emissions rival those from the oil and gas industry.

A quest to lower emissions of CH4 from ruminants has led to identification of 3-nitrooxyypropanol (3-NOP), a feed additive that specifically interferes with the final step of methanogenesis. Obtaining a product with an enzyme-specific mode of action is just one of many efforts over the past 75 years, along with development of vaccines and other feed additives, to reduce methanogenesis in the rumen and to understand its influence on animal health and productivity. Development of 3-NOP, now a commercial product, exemplifies the application of microbiological knowledge to mitigation of greenhouse gas emissions from agriculture. The compound, produced by the Dutch company DSM, has been patented and approved for use with dairy cows in Brazil, Chile and the European Union.

Methane is produced by archaea that make up a small proportion (up to 4%) of microbial biomass in the rumen. As the largest of the cow's 4 stomach compartments, the rumen makes up 12-15% of the animal's body mass and houses an anaerobic community comprised of diverse bacteria, protists and fungi. These microbes degrade and ferment lignocellulosic roughage that the animal cannot digest.

Major microbial fermentation products consist of short-chain fatty acids, which are absorbed by the animal, as well as CO2 and H2, which are converted by methanogens to CH4 waste gas. Some undigested material is regurgitated into the buccal cavity, or mouth, where the cud is chewed and swallowed again. Other undigested material passes into the abomasum, where mammalian digestive processes take over before entering the lower intestinal tract.

A typical dairy cow emits about 160 kg of CH4 per year. A minor proportion of CH4 (10-15%) from ruminants is produced in the intestinal tract and exits from their hind ends. The majority of CH4 (> 80%) exits from the mouth during eructation, or belching. The amount of methane produced within the rumen depends on many factors, including feed digestibility, total quantity of carbohydrate fermented, the ratios of fatty acids formed and H2 concentrations.

Most rumen methanogens have hydrogenotrophic metabolisms, meaning they use electrons from H2 to reduce CO2 to CH4, an efficient way to reduce H2 concentrations in the rumen. In a global study of rumen microbiomes from 32 ruminant species, 74% of archaea belonged to just 2 hydrogenotrophic clades representing Methanobrevibacter gottschalkii and Methanobacterium ruminatium. Two other known methanogen groups that produce CH4 from either acetate or methyl-group compounds are far less abundant in the H2-rich rumen habitat.

Methanogenesis can be considered a symbiotic process because it pulls fermentation reactions forward, thereby assisting in continued production of fatty acids for the animal. However, it also represents an energy loss for milk and meat production. Percentages of gross energy intake lost through methane eructation have been estimated at 2-12%, with greater losses associated with forage-rich diets. For decades, improved feed efficiency was the goal for research on lowering ruminant methane through dietary modifications. Greenhouse gas mitigation became a stronger impetus for reducing livestock methane with the first Intergovernmental Panel on Climate Change (IPCC) report in 1992. The IPCC report described the rise in atmospheric CH4 concentrations from 750-1800 parts per billion (ppb) over the previous 100 years. It recognized that global population increase and demand for animal protein would drive greater livestock production, with 1.3 billion cattle estimated to account for 12% of global methane emissions in 1995.

More recently, a targeted, biochemical approach to methanogen inhibition was based on crystal structures of enzymes responsible for methane production (methyl coenzyme M reductase, or MCR) in hydrogenotrophic methanogens like Methanobacter thermoautotrophicum and Methanothermobacter marburgensi. Computational screening identified a group of small molecules, the nitrooxy carboxylic acids, as potential inhibitors that could fit into the MCR active site. Enzyme inactivation was hypothesized to occur when a compound like 3-NOP fit into the MCR active site, inhibiting its ability to carry out a key step in CH4 formation. More specifically, 3-NOP binds to MCR near coenzyme F430, a prosthetic group containing a critical Ni(I) atom. Proximity of the nitrate group of 3-NOP is postulated to cause oxidation of Ni(I) so that it can no longer carry out the final reduction step of CH4 formation.

Feeding trials of 3-NOP to sheep, lactating cows and beef cattle in feedlots have resulted in an average 30% reduction of CH4 emissions, confirming that 3-NOP works in vivo. In the laboratory, however, inhibition by 3-NOP on growth of different methanogens in pure cultures varies widely. Very low (micromolar) concentrations prevent growth of hydrogenotrophic methanogens like M. ruminantium, while 100 times higher concentrations are needed to inhibit other hydrogenotrophs like Methanomicrobiium mobile and Methanosarcina barkeri. Phylogenetic and physiological diversity among methanogens may make it difficult to bypass the symbiotic relationship established between ruminants and archaeal consumers of H2 in the rumen.

Next-generation sequencing of extracted microbial DNA and RNA from rumens has become a boon for more comprehensive understanding of the effects of 3-NOP on the microbiome. Concomitant with reduced CH4 emissions, for example, supplementation of 60 mg 3-NOP per kg of feed dry matter in dairy cows over 4-12 weeks resulted in a decline of dominant Methanobrevibacter spp. and increases in unclassified Methanobacteriaceae and Methanosphaera spp. compared to controls. Because non-methanogen members of rumen microbiomes were not significantly affected, alterations in methanogen composition by 3-NOP appeared to explain the increases in propionic acid (a short chain fatty acid) and H2 that were measured during the study.

Notably, as methanogens inhibited by 3-NOP die, H2 concentrations may accumulate and interfere with carbon and energy flow before other methanogens can take over. This may necessitate the addition of other electron acceptors to assist efficient fermentation. Whether 3-NOP will be used more extensively will depend on its acceptance, cost and research confirming that animal health and productivity are not affected in the long term.

Most recently, feeding ruminants with the red seaweed Asparagopsis taxiformis to reduce methane emissions has attracted attention in the industry. In vitro studies using these algae have shown methane inhibition of up to 99% without adverse health effects. The proposed mode of action is introduction of a bioactive brominated compound (bromoform) contained within the seaweed biomass, which is a halogenated methane analogue that inhibit methanogenesis. For the specific purpose of climate mitigation, considerable venture capital is being directed toward production of adequate supplies of red seaweed in locations that would lower transportation costs.

In addition to many dietary supplements tested for methanogen suppression, anti-methanogenic vaccines have been developed based on the recognized dominance of relatively few hydrogenotrophic species. To that end, vaccines have been derived from either whole cells or cell components of mixed cultures of methanogens. However, in vivo studies measuring methane emissions, mainly in sheep, following inoculation with mixed-culture vaccines have reported little or no reductions, possibly reflecting greater breadth of methanogen diversity. To address this challenge, antibody cross-reactivity using immunocapture beads can be tested in vitro with diverse methanogens in rumen fluids prior to in vivo trial. Still, with a lack of vaccine success, dietary modifications remain the most common approach to reducing CH4.

Consistent patterns in the magnitude of methane emissions are observed among species and breeds of domestic ruminants. Some breeds of cattle emit consistently lower levels of methane than others, with beef cattle generally emitting less methane than dairy cows. Evidence that host genetics affect microbiome composition and energy flow through the rumen is leading to efforts that integrate animal genetics and microbiome analyses. Buccal sampling of saliva and mouth contents are being evaluated for biomarkers that reflect animal traits for faster and more frequent data collection.

In some countries, the rise of confined feedlot systems for raising and finishing cattle has resulted in increased feeding of concentrates containing more digestible carbohydrates and more protein than high-roughage forages. Methane emissions from cattle fed with concentrate are lower than from cattle fed high-roughage diets. Genetic selection of cattle can also result in lowered methane emissions, with high-efficiency feeders producing less methane than low-efficiency feeders.

However, concentrate feeding, use of selected breeds and feed additives are not as readily implemented in developing nations due to less availability of resources and research infrastructure. Still, options are available for agriculturalists in developing countries to lower methane emission from ruminants. Raising ruminants not only provides milk and meat, but also provides crucial ecosystem service of converting low-quality cellulosic materials to high-quality protein on land that cannot otherwise be cultivated. And, moving forward, it will be critical to ensure equitable global access to multiple agricultural innovations that reduce methane emissions as we look to protect human, animal and environmental health worldwide.

Marry Ann Bruns, Ph.D., the author of this article, will be convening a session about Microbial Technologies to Mitigate Methane Emissions," at ASM Microbe 2023 on June 16, 2023 from 1:45-3:45 p.m. Interested in learning onre about reducing or preventing methanogenesis?