Rumen microbiome response to methane inhibition

Background

The rumen is a complex ecosystem that harbours a diverse anaerobic microbiota known as the rumen microbiome, which consists of bacteria, protozoa, fungi and methanogenic archaea, listed in order of relative abundance and diversity.¹ Ruminal bacteria make up the largest component of the microbial biomass, protozoa can account for up to half of this biomass, whereas fungi were initially estimated to be ~8% but may reach 20% in sheep, and the methanogenic archaea comprise ~0.3–4% of the microbial biomass.² These microbial communities (i.e. bacteria, fungi and protozoa) present in the rumen enable the hydrolysis and fermentation of various feed components, including polysaccharides, non-fibre carbohydrates, proteins and lipids consumed by ruminant animals, into substantial amounts of volatile fatty acids (VFAs), carbon dioxide (CO₂), H₂, as well as other end products such as methanol, methylamines and methylated sulfur compounds. However, most of the H₂ produced during fermentation is utilised by methanogenic archaea to reduce CO₂, resulting in methane (CH₄) production. Several H₂-consuming bacteria, including capnophilic bacteria like Prevotella, Fibrobacter, Ruminococcus, Lachnospira and Succinomonas, as well as nitrogen-compound or sulfur-respiring bacteria such as Denitrobacterium detoxificans and Wolinella succinogenes, can compete with methanogens for H₂. Methanogens also form symbiotic relationships with H₂-producing bacteria, protozoa and fungi, which prevent the redirection of H₂ to alternative sinks. These associations facilitate the interspecies transfer of H₂ between H₂ producers and users for CH₄ synthesis, which is fundamental in the normal functioning of an anaerobic ecosystem in an uninhibited rumen.³

Excess electrons in the rumen, particularly H₂, are mainly removed by archaeal methanogens through CH₄ production during the final stages of fermentation, with some contribution from hydrogenotrophic bacteria that produce VFA through nitrate and sulfate reduction.⁴ Therefore, the production of CH₄ prevents the partial pressure of H₂ from increasing to concentrations that could potentially inhibit the effective functioning of hydrogenases involved in electron transfer reactions (i.e. interspecies H₂ transfer), particularly NADH dehydrogenase, which could lead to the accumulation of NADH within microbial cells (i.e. bacteria, protozoa and fungi) and, in turn, a reduction in rumen fermentation and degradation of plant fibre.^3,5 Thus, this paper provides an understanding of the current knowledge on the varied responses of the hydrogenotrophic and fermentative rumen microbiome, with a focus on the potential implications for ruminant productivity from enteric CH₄ inhibition.

Feed additives: methane inhibitors

Rumen microbiota ferment ingested feed into different end products, including VFA, CO₂ and H₂, which are utilised by methanogenic archaea in the rumen to produce CH₄. Enteric CH₄ emissions from ruminant livestock significantly contribute to global anthropogenic GHG emissions and represent a loss of gross energy intake.⁶ Hence, there has been heightened global interest in exploring practical measures to decrease CH₄ emissions from ruminants, driven mainly by the potential benefits of improving production efficiency and reducing contribution to global GHG emissions.⁷ Several strategies have been proposed to mitigate enteric CH₄ emissions, including selective animal breeding, management improvements and enhancing forage quality,⁸ with a particular emphasis on the use of anti-methanogenic feed additives (AMFAs) to manipulate the rumen environment.⁹ Rumen modifiers, such as ionophores, enhance microbial fermentation to improve feed efficiency by promoting the production of propionate and reducing the availability of H₂ for methanogens, which indirectly lowers CH₄ emissions. By contrast, CH₄ inhibitors directly target methanogenesis by blocking key enzymes, resulting in a more immediate and significant reduction in CH₄ production. The red seaweed Asparagopsis spp. (e.g. A. armata and A. taxiformis) and the synthetic compound 3-NOP are two well-known inhibitors that have consistently demonstrated high anti-methanogenic activity and effectiveness in reducing CH₄ emissions.

The effect of CH₄ reduction from Asparagopsis supplementation is due mainly to the bioactive compound bromoform (CHBr₃), found at a concentration up to 100-fold higher than the next most abundant organobromine compound.¹⁰ When fed at minimal effective inclusion levels (0.45–1.58 mg CHBr₃ kg^–1 body weight, BW day^–1), methanogens metabolise CHBr₃ through reductive dehalogenation, which limits the transfer of the active ingredient to animal tissues and food products.¹¹ Romero et al.⁶ reported a 90% degradation within the first 3 h, and that progressive dehalogenation continued to over 99% after 12 h of CHBr₃ inclusion in the rumen. The synthetic compound, 3-NOP (marketed as Bovaer, DSM-Firmenich AG, Kaiseraugst, Switzerland), has been developed that also persistently reduces CH₄ emissions in dairy cows.¹² However, 3-NOP has differential effects on individual methanogenic lineages and thus, the efficacy to inhibit different methanogens is variable among ruminant species,¹³ suggesting structural differences in the subunits composition of the methyl coenzyme M reductase (MCR) enzyme contribute to the variation in tolerance to 3-NOP.¹⁴ Investigations into the metabolism of aliphatic nitro compounds in the rumen have shown that 3-NOP is reduced to 3-nitropropanol, which is then absorbed from the rumen and further metabolised in the liver and other tissues.¹⁵

Asparagopsis supplementation has been shown to significantly decreased enteric CH₄ emission by up to 67 and 99% in dairy¹⁶ and beef¹⁷ cattle. Similarly, dietary inclusion of 3-NOP resulted in a reduction of CH₄ emission by 20–80% in beef and dairy cattle.^18,19 Overall, the dietary supplementation of said inhibitors has consistently shown significant potential for reducing enteric CH₄ emissions to different extents based on the dosage of the active ingredient, the basal diet and the livestock system.²⁰ However, mitigation of enteric CH₄ emissions in ruminants using feed additive inhibitors leads to inconsistent productivity gains, partly due to the shift in significant fermentative microbes away from acetogenesis and the inability to convert increased ruminal H₂ spared from methanogenesis into absorbable nutrients beneficial to the animals.^16,21

Effects of inhibitors on the rumen microbiome

Supplementation of CH₄ inhibitors in ruminant diets lowers the abundances of rumen methanogen populations, with shifts in bacterial groups and fermentation from acetate to propionate production, using Asparagopsis²² and 3-NOP.²¹ These supplements share a similar mode of action by inactivation of the MCR enzyme to inhibit the final step of methanogenesis.¹¹ However, the inclusion of AMFAs resulted in both positive and negative effects on hydrogenogenic and hydrogenotrophic microbes in the rumen, a consequence of methanogenesis suppression and increased H₂ production.^21,23

Published studies by Krizsan et al.²² and Zhang et al.²³ examined the effects of dietary supplementation of Asparagopsis on the rumen microbiome of dairy cows. Krizsan et al.²² observed that the inclusion of Asparagopsis in a diet composed of high forage resulted in a decrease in dry matter intake (DMI) by 13.8%, a 3% reduction in milk yield, and a five-fold increase in enteric H₂ emissions from dairy cows. Further observations revealed a shift in rumen fermentation from acetate to propionate production, a reduced relative abundance of Prevotella in the bacterial population and a shift from Methanobrevibacter to Methanomethylophilaceae in the archaeal population. Methanomethylophilaceae are methanogens that utilise H₂ as an electron donor and substrates such as methanol and methylamines to synthesise CH₄.²⁴ Zhang et al.²³ evaluated the effects of Asparagopsis supplementation on the rumen microbial communities in lactating dairy cows. The reduction of enteric CH₄ from the supplemented animals was coupled with a decrease in the population of rumen methanogens and a significant reduction in the transcription of methanogenesis pathways. Subsequent findings by Zhang et al.²³ revealed increased H₂ production associated with the upregulation of hydrogenases that mediate hydrogenotrophic metabolism, which may provide a competitive advantage in the conversion of excess H₂ in the rumen. In addition, a trial conducted by Roque et al.¹⁶ that examined Asparagopsis supplementation in a high-forage diet reported a reduction in DMI and milk yield of respectively 38 and 11.6% in lactating dairy cows. The negative effects on production in studies by Krizsan et al.²² and Roque et al.¹⁶ coincided with increased emissions of enteric H₂ from the experimental animals. The findings of Zhang et al.²³ were based on continued analyses to the trial study by Roque et al.¹⁶

Similarly, Ni et al.²¹ analysed rumen microbiota responses to 3-NOP supplementation in dairy calves fed ration composed of low forage (7%) feed. The results showed an increased ruminal H₂ levels, which caused a shift in microbial fermentation away from acetate production towards undesirable compounds such as ethanol, without adverse effects on feed intake or animal performance. Supplementation of 3-NOP in dairy calves by Gaofeng et al.²¹ resulted in a significant reduction in the methanogens population and stimulated reductive acetogens, especially novel uncultivated lineages such as ‘Candidatus Faecousia’. Gruninger et al.²⁵ reported that supplementation of 3-NOP in a high-forage diet reduced the relative abundance of Methanobrevibacter methanogens. This change resulted in a 37-fold increase in H₂ emissions and also increased the relative abundance of Bacteroidota.

Recent studies have provided foundational and new insights into the metagenomic and metatranscriptomic evaluation of the microbiome in rumen fluid from ruminants supplemented with CH₄ inhibitors.^21,23 However, the effects of CH₄ inhibitors on the rumen microbiome, such as shifts in microbial composition, the regulation of methanogenic pathways, and the overall transcriptional dynamics of the rumen microbiome during methanogenesis inhibition, are still not well understood.²³ The lack of information on alternative states of the rumen microbiome under methanogenesis inhibition by AMFAs emphasises a significant gap in the current understanding of rumen microbiology. Inhibition of CH₄ production in the rumen could spare reducing equivalents, such as H₂, which can then be used to produce beneficial nutrients for the host animal²³ and ultimately improve both the feed efficiency and total productivity of ruminants.¹⁶

Inhibiting CH₄ production in the rumen, while potentially beneficial for environmental reasons, may also have some negative effects on the animal. Although some research suggests improved animal productivity with CH₄ inhibition, this is not consistently observed and, in some cases, CH₄ inhibition can lead to changes in rumen fermentation, potentially affecting feed digestibility and other metabolic processes. The inhibition of enteric CH₄ production is not an isolated intervention and may have indirect consequences on animal physiology, including inflammation of the rumen epithelium. This has been potentially linked to a dose-dependent negative effect of CHBr₃-containing Asparagopsis supplementation, as reported in some studies.^26,27 However, it remains unclear whether rumenitis arises directly from the additive itself or indirectly due to changes in diet composition following a reduction in DMI. A decline in DMI can impair rumen function, potentially leading to the development of rumenitis, whereas the likelihood of direct toxicity from CHBr₃ at inclusion rates between 0.45 and 1.58 mg CHBr₃ kg^–1 BW appears low when compared with the no-observed-adverse-effect level (NOAEL) of 72 mg CHBr₃ kg^–1 BW reported in mice.²⁸ Therefore, in cases where rumen health impacts such as rumenitis have been reported, these are most likely in response to the direct effects of CH₄ inhibition on DMI and fibre digestion.

Exploring co-supplementation to improve rumen function

Under conditions of high CH₄ inhibition there is currently a paucity of knowledge relating to the mechanisms that regulate the flow of electrons in the rumen, including the processes and microbes involved in H₂ production and consumption.²¹ Mitigation of enteric CH₄ emissions paired with redirection of ruminal H₂ flow toward alternative hydrogenotrophic pathways for the production of absorbable nutrients, such as acetogenesis (as illustrated in Fig. 1), could be one of the most promising strategies to enhance ruminant productivity.²⁹ For example, Romero et al.³⁰ studied the effects of co-supplementation of a CH₄ inhibitor, Asparagopsis, with an electron (H₂) acceptor, phloroglucinol, on the rumen microbiome and fermentation in vitro. Combined treatment of Asparagopsis and phloroglucinol significantly inhibited CH₄ production and lowered the abundance of methanogenic archaea, protozoa and fungi, as well as H₂ accumulation in the supplemented batch compared to the control batch. This co-supplementation also promoted fermentation that resulted in increased acetate production, total VFA and a high acetate-to-propionate ratio (Fig. 1). This combination of supplements shows potential for reducing the negative environmental impact of rumen methanogenesis while maintaining rumen function and enhancing ruminant productivity.

Fig. 1.

A schematic diagram of the effects of dietary co-supplementation of methane inhibitors with electron acceptor on the rumen microbiome and fermentation. (1) Microbial hydrolysis and fermentation of feed by anaerobic bacteria, protozoa and fungi. (2) Substrates for methanogenesis. (3) Methanogenesis by methanogenic archaea using fermentation metabolites. (4) Inhibition of methanogenesis pathways by Asparagopsis bromoform (CHBr₃) or 3-nitrooxypropanol (3-NOP) acting on methyl coenzyme M reductase (Mcr). (5) Decreased methane (CH₄) production. (6) Capture of dihydrogen (H₂) or formate (CH₂O₂) by electron acceptor, phloroglucinol. (7) Degradation of phloroglucinol by ruminal bacteria, particularly strains of Streptococcus and Coprococcus.³¹ (8) Decreased dihydrogen (H₂) concentrations in the rumen fluid. (9) Decreased ruminal dihydrogen (H₂) gas production. (10) Increased ruminal acetate concentrations. (11) Increased carbon dioxide (CO₂) production. (12) Increased carbon dioxide (CO₂) emissions. (13) Increased acetate-to-propionate ratio (A:P). (14) Absorption of volatile fatty acids through the rumen wall, which the animal host utilises as a source of energy, carbon for diverse metabolites and glucose. Abbreviation: acetyl-CoA is acetyl-coenzyme A; Mct is methyl coenzyme M transferase. Created in BioRender (see https://BioRender.com/c505wob).

Conclusions and recommendations

The inhibition of methanogens through dietary AMFAs reduces enteric CH₄ production and alters rumen function, potentially resulting in changes in feed conversion efficiency, DMI and animal productivity. Although there is a growing body of knowledge on the response of the rumen microbiome to CH₄ inhibition, future research needs to explore the role and function of hydrogenotrophic microbes to minimise H₂ accumulation in the rumen and reduce the risk of decreased DMI. Additionally, investigating the potential benefits of co-supplementation with electron acceptors could help optimise rumen function and animal performance. Such advancements are critical for the effective and voluntary adoption of CH₄ inhibitors beyond controlled research and feeding environments, particularly in grazing systems, where day-to-day and animal-to-animal variability in intake and nutrient profile is a key consideration. Without a reliable approach to optimising animal performance under commercial conditions, the adoption of CH₄ inhibitors on-farm may be limited to conservative feeding rates, constraining their potential contribution to CH₄ mitigation.