Effect of the macroalgae Asparagopsis taxiformis on methane production and rumen microbiome assemblage

Abstract

Background: Recent studies using batch-fermentation suggest that the red macroalgae Asparagopsis taxiformis has the potential to reduce methane (CH₄) production from beef cattle by up to ~ 99% when added to Rhodes grass hay; a common feed in the Australian beef industry. These experiments have shown significant reductions in CH₄ without compromising other fermentation parameters (i.e. volatile fatty acid production) with A. taxiformis organic matter (OM) inclusion rates of up to 5%. In the study presented here, A. taxiformis was evaluated for its ability to reduce methane production from dairy cattle fed a mixed ration widely utilized in California, the largest milk producing state in the US.

Results: Fermentation in a semi-continuous in-vitro rumen system suggests that A. taxiformis can reduce methane production from enteric fermentation in dairy cattle by 95% when added at a 5% OM inclusion rate without any obvious negative impacts on volatile fatty acid production. High-throughput 16S ribosomal RNA (rRNA) gene amplicon sequencing showed that seaweed amendment effects rumen microbiome consistent with the Anna Karenina hypothesis, with increased β-diversity, over time scales of approximately 3 days. The relative abundance of methanogens in the fermentation vessels amended with A. taxiformis decreased significantly compared to control vessels, but this reduction in methanogen abundance was only significant when averaged over the course of the experiment. Alternatively, significant reductions of CH₄ in the A. taxiformis amended vessels was measured in the early stages of the experiment. This suggests that A. taxiformis has an immediate effect on the metabolic functionality of rumen methanogens whereas its impact on microbiome assemblage, specifically methanogen abundance, is delayed.

Conclusions: The methane reducing effect of A. taxiformis during rumen fermentation makes this macroalgae a promising candidate as a biotic methane mitigation strategy for dairy cattle. But its effect in-vivo (i.e. in dairy cattle) remains to be investigated in animal trials. Furthermore, to obtain a holistic understanding of the biochemistry responsible for the significant reduction of methane, gene expression profiles of the rumen microbiome and the host animal are warranted.

Keywords: 16S rRNA community profiling; Asparagopsis taxiformis; Feed supplementation; Greenhouse gas mitigation; In-vitro rumen fermentation; Macroalgae; Rumen microbiome.

Fig. 1

In-vitro rumen system set-up. Extraction: Rumen fluid and rumen solids were collected from 2 dairy cows. Mixing: Rumen fluid was homogeneously mixed and rumen solids were homogeneously mixed. After mixing, rumen fluid was separated into two Erlenmeyer flasks, where treatment was then assigned. 24 Hour Equilibration: The control flask received 30 g of mixed rumen solids and 30 g of SBR and the treatment flask received 30 g of mixed rumen solids, 30 g of SBR, and 1.5 g of A. taxiformis. After each flask received their treatment, the 24 h equilibration period began. After the equilibration period, each flask was then divided into 3 vessels, then fed their respective treatments (control = 10 g SBR/vessel, treatment = 10 g SBR/vessel & .2 g A. taxiformis)

Fig. 2

Total gas, CH₄, and CO₂ production during in-vitro fermentation. Production of total gas, CH₄ and CO₂ [ml/(g OM)] from vessels without (n = 3) and with (n = 3) A. taxiformis as additive at 4, 12, and 24 h over the course of the experiment. a Total gas production; b CH₄ production; c CO₂ production. Measurement were performed in triplicates. “**” indicates significant difference (p value ≤0.05), “*” indicates trend toward significance (0.05 > p value ≤0.1)

Fig. 3

Volatile fatty acid production during in-vitro fermentation. Volatile fatty acid concentrations [ppm] of fermentation fluid of vessels without (n = 3) and with (n = 3) A. taxiformis as additive, determined 4, 12, and 24 h after feeding over 4 days. a Acetic acid; b Propionic acid; c Isobutyric acid; d Butyric acid; e Isovaleric acid f Valeric acid; g Propionate/Acetate Ratio. Measurement were performed in triplicates

Fig. 4

Effects of seaweed amendments on composition of in-vitro rumen microbiome. a Genus-level β-diversity between pairs of vessels throughout the duration of the experiment. b β-diversity across multiple taxonomic groups measured between pairs of samples versus sampling time for each of the 6 vessels. 95% bootstrap confidence intervals are shown. Regression slopes identified as significant (p < 0.001) by a permutation test are indicated with an asterisk. c Genus-level β-diversity within individual vessels across different sampling times

Fig. 5

Relative abundance of phyla during in-vitro fermentation. Fermentations were performed in three in-vitro vessels (n = 3). Incubation times annotated with “C” represent control conditions

Fig. 6

Relative abundance of Euryarchaeota during in-vitro fermentation. Fermentations were performed in three in-vitro vessels (n = 3). Error bars indicate standard error of the mean