Application of propionate-producing bacterial consortium in ruminal methanogenesis inhibited environment with bromoethanesulfonate as a methanogen direct inhibitor

Abstract

Methane production in ruminants is primarily due to the conversion of metabolic hydrogen (H₂), produced during anaerobic microbial fermentation, into methane by ruminal methanogens. While this process plays a crucial role in efficiently disposes of H₂, it also contributes to environmental pollution and eliminating methane production in the rumen has proven to be challenging. This study investigates the use of probiotics, specifically propionate-producing bacteria, to redirect accumulated H₂ in a methane-mitigated environment. For this objective, we supplemented experimental groups with Lactiplantibacillus plantarum and Megasphaera elsdenii for the reinforced acrylate pathway (RA) and Selenomonas ruminantium and Acidipropionibacterium thoenii for the reinforced succinate pathway (RS), as well as a consortium of all four strains (CB), with the total microbial concentration at 1.0 × 10¹⁰ cells/mL. To create a methane-mitigated environment, 2-bromoethanesulfonate (BES) was added to all experimental groups at a dose of 15 mg/0.5 g of feed. BES reduced methane production by 85% in vitro, and the addition of propionate-producing bacteria with BES further decreased methane emission by up to 94% compared with the control (CON) group. Although BES did not affect the alpha diversity of the ruminal bacteriome, it reduced total volatile fatty acid production and altered beta diversity of ruminal bacteriota, indicating microbial metabolic adaptations to H₂ accumulation. Despite using different bacterial strains targeting divergent metabolic pathways (RA and RS), a decrease in the dominance of the [Eubacterium] ruminantium group suggesting that both approaches may have a similar modulatory effect. An increase in the relative abundance of Succiniclasticum in the CB group suggests that propionate metabolism is enhanced by the addition of a propionate-producing bacterial consortium. These findings recommend using a consortium of propionate-producing bacteria to manage H₂ accumulation by altering the rumen bacteriome, thus mitigating the negative effects of methane reduction strategies.

Keywords: alternative hydrogen sink; methane emission; methanogen direct inhibition; probiotics; propionate production; ruminant.

Figure 1

VFA characteristics after 48 h of the in vitro fermentation experiment. BES was added to the RA, RS, and CB groups at the same concentration as that (15 mg/0.5 g) in the BES group. The RA group comprised Lactiplantibacillus plantarum and Megasphaera elsdenii; RS group, Selenomonas ruminantium and Acidipropionibacterium thoenii; and CB group, all four bacterial strains. CON, control group; BES, 2-bromoethanesulfonate group; RA, reinforced acrylate pathway group; RS, reinforced succinate pathway group; CB, propionate-producing bacterial consortium group; VFA, volatile fatty acid; A:P ratio, acetate-to-propionate ratio. ^A–D: significant differences (p ≤ 0.05) between treatment groups.

Figure 2

NMDS plot based on Bray-Curtis distance matrix. BES (15 mg/0.5 g of feed) was added to all the groups except the control group. Three-dimensional NMDS plot including the control group (A), two-dimensional NMDS plot comparing the BES-supplemented groups (B), Q-values resulting from pairwise multiple tests adjusted using the Benjamini–Hochberg method (C). NMDS, non-metric multidimensional scaling; BES, 2-bromoethanesulfonate group; RA, reinforced acrylate pathway group; RS, reinforced succinate pathway group; CB, propionate-producing bacterial consortium group; VFA, volatile fatty acid; A:P ratio, acetate-to-propionate ratio.

Figure 3

Analysis of multivariate associations in the ruminal bacteriome at the genus level between the CON and BES groups. Coefficient scores of ruminal bacteriome data showing significant differences (Q ≤ 0.05) between the CON and BES groups at genus level. BES, 2-bromoethanesulfonate group; UC, unclassified; UCG, uncultured genus.

Figure 4

Relative abundance of major taxa at the genus level in the BES-supplementation groups. Relative abundance results of microbial taxa indicating significant differences (Q ≤ 0.05) between the BES, RA, RS, and CB groups at genus level. Relative abundance of major genus-level taxa with 100% occurrence in all groups among the BES-supplementation groups. RA, reinforced acrylate pathway group; RS, reinforced succinate pathway group; CB, propionate-producing bacterial consortium group; UCG, uncultured genus.

Figure 5

Heatmap of Pearson correlation coefficients between VFA characteristics, methane production (mL), methane yield (mL/g dDM), and three supplemented bacterial strains showing significant changes, as well as relevant bacterial genera. Significant differences are observed between the BES group and the groups supplemented with propionate-producing bacterial strains. The three supplemented bacterial strains were analyzed for correlation based on copy number, while the other relevant bacterial genera were analyzed based on relative abundance. UCG, uncultured genus; VFA, volatile fatty acid; A:P ratio, acetate-to-propionate ratio; CH₄ (mL/g dDM), quantity of methane production per degraded gram of dry matter; *p ≤ 0.05, **p < 0.01, ***p < 0.001.