The effect of 3-nitrooxypropanol, a potent methane inhibitor, on ruminal microbial gene expression profiles in dairy cows

Abstract

Background: Enteric methane emissions from dairy cows are an environmental problem as well as a gross feed energy loss to the animal. Methane is generated in the rumen by methanogenic archaea from hydrogen (H₂) + carbon dioxide and from H₂ + methanol or methylamines. The methanogenic substrates are provided by non-methanogens during feed fermentation. Methane mitigation approaches have yielded variable results, partially due to an incomplete understanding of the contribution of hydrogenotrophic and methylotrophic archaea to methanogenesis. Research indicates that 3-nitrooxypropanol (3-NOP) reduces enteric methane formation in dairy cows by inhibiting methyl-coenzyme M reductase (MCR), the enzyme responsible for methane formation. The purpose of this study was to utilize metagenomic and metatranscriptomic approaches to investigate the effect of 3-NOP on the rumen microbiome and to determine the fate of H₂ that accumulates less than expected under inhibited methanogenesis.

Results: The inhibitor 3-NOP was more inhibitory on Methanobrevibacter species than methanol-utilizing Methanosphaera and tended to reduce the gene expression of MCR. Under inhibited methanogenesis by 3-NOP, fluctuations in H₂ concentrations were accompanied by changes in the expression of [FeFe] hydrogenases in H₂-producing bacteria to regulate the amount of H₂ production. No previously reported alternative H₂ sinks increased under inhibited methanogenesis except for a significant increase in gene expression of enzymes involved in the butyrate pathway.

Conclusion: By taking a metatranscriptomic approach, this study provides novel insights on the contribution of methylotrophic methanogens to total methanogenesis and regulation of H₂ metabolism under normal and inhibited methanogenesis by 3-NOP in the rumen. Video Abstract.

Keywords: Enteric methane; Hydrogenases; Methane mitigation; Ruminal methanogenesis; Total and metabolically active microbes.

Fig. 1

Rumen archaeal diversity and composition in control and 3-nitrooxypropanol (3-NOP) treated cows at weeks 4, 8, and 12. 16S rRNA archaeal diversity: A species richness, B Shannon diversity, and C comparison of overall community between samples by weighted UniFrac distances in DNA-based and RNA-based 16S rRNA analysis (beta diversity). Archaeal genera: comparison of archaeal composition at genus level for D DNA-based 16S rRNA, E RNA-based 16S rRNA, F metagenomics (metaG), and G metatranscriptomics (metaT). rt-PCR: quantification of selected methanogens at week 8 by real time PCR (rt-PCR) for HMethanosphaera stadtmanae DSM309 (mtaB), IMethanobrevibacter ruminantium M1 (mcrG), JMethanobrevibacter smithii ATCC35061 (mtaB), and K 16S rRNA copy number. PCoA, principal coordinates analysis. NS, no statistical significance in generalized linear mixed model (glmer); *P < 0.05; **P < 0.01; ***P < 0.001. The black circles appearing on the boxplots are the outlier samples

Fig. 2

Comparisons of genes (metagenomics; metaG) and transcripts (metatranscriptomics; metaG) abundance in copies per million (cpm) for enzymes involved in methanogenesis between control and 3-nitrooxypropanol (3-NOP)-treated cows at weeks 4, 8, and 12. A Carbon dioxide (CO₂)/hydrogen (H₂) methanogenesis pathway (KEGG pathway entry MD:M00567), B methanol methanogenesis pathway (KEGG pathway entry MD:M00356), and C methylamine methanogenesis pathway (KEGG pathway entry MD:M00563). NS, no statistical significance in generalized linear mixed model (glmer); *P < 0.05; **P < 0.01; ***P < 0.001. The black circles appearing on the boxplots are the outlier samples

Fig. 3

Effect of 3-nitrooxypropanol (3-NOP) on rumen bacteria via changes in dissolved hydrogen (H₂) concentrations. A Effect of 3-NOP on dissolved H₂ in lactating dairy cows; the bacterial genera appearing in the oval shapes are rapid (orange) and slow (blue) fermenters that were increased in 3-NOP treated cows at week 8 and week 12, respectively (see panel B). B Fold change (log 10) between control and 3-NOP-treated cows at weeks 4, 8, and 12 in the relative abundance of selected bacterial genera in DNA-based 16S rRNA analysis. The selection of bacterial genera was based on significant differences (glmer test) between treatment groups (P < 0.05). C Fold change (log 10) between control and 3-NOP treated cows at weeks 4, 8, and 12 in hydrogenase content in metagenomes (metaG) and metatranscriptomes (metaT). Hydrogenase content is shown based on hydrogenase subgroup. These are divided into fermentative hydrogenases (H₂-producing; group A1, A2, B [FeFe]-hydrogenases), bifurcating hydrogenases (bidirectional; group A3, A4 [FeFe]-hydrogenases), respiratory hydrogenases (H₂-uptake; group 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 1i, 1j [NiFe]-hydrogenases), respiratory hydrogenases (H₂-evolving; group 4b, 4d [NiFe]-hydrogenases), alternative and sensory hydrogenases (H₂-uptake; 2a, 2b [NiFe]-hydrogenases), cofactor-coupled bidirectional hydrogenases (3b, 3d, [NiFe]-hydrogenases), methanogenic hydrogenases (H₂-uptake; group 1k, 3a, 3c, 4h, 4i [NiFe]-hydrogenases, [Fe]-hydrogenases), energy-converting hydrogenases (bidirectional; group 4a, 4c, 4e, 4f, 4g [NiFe]-hydrogenases), and sensory hydrogenases (group C [FeFe]-hydrogenases). Positive and negative log 10-fold change is the increased and decreased relative abundance, respectively, in 3-NOP compared with controls cows.

Fig. 4

Associations between rumen bacteria and fermentation profiles. Correlation between bacteria (DNA-based [top] and RNA-based 16S rRNA sequencing analysis [bottom]) and fermentation profiles. Different colors of the bacterial genera show the corresponding phylum. dH₂, dissolved hydrogen; VFA, volatile fatty acids; mol%, molar proportion

Fig. 5

Comparisons of metagenomic (metaG) and metatranscriptomic (metaG) abundance for enzymes involved in the butyrate pathway (A) and propanoate pathway (B) between control and 3-nitrooxypropanol (3-NOP) treated cows at weeks 4, 8, and 12. cpm, copies per million; NS, no statistical significance in generalized linear mixed model (glmer); *P < 0.05; **P < 0.01; ***P < 0.001. The black circles appearing on the boxplots are the outlier samples