Abstract
Asparagopsis taxiformis has potent antimethanogenic activity as a feed supplement at 2% of organic matter in in vitro bioassays. This study identified the main bioactive natural products and their effects on fermentation using rumen fluid from Bos indicus steers. Polar through to non-polar extracts (water, methanol, dichloromethane and hexane) were tested. The dichloromethane extract was most active, reducing methane production by 79 %. Bromoform was the most abundant natural product in the biomass of Asparagopsis (1723 µg g−¹ dry weight [DW] biomass), followed by dibromochloromethane (15.8 µg g−¹ DW), bromochloroacetic acid (9.8 µg g−¹ DW) and dibromoacetic acid (0.9 µg g−¹ DW). Bromoform and dibromochloromethane had the highest activity with concentrations ≥1 µM inhibiting methane production. However, only bromoform was present in sufficient quantities in the biomass at 2 % organic matter to elicit this effect. Importantly, the degradability of organic matter and volatile fatty acids were not affected at effective concentrations.
Generated Summary
This research investigates the antimethanogenic activity of the red seaweed *Asparagopsis taxiformis* in in vitro experiments, focusing on identifying its active natural products and quantifying their effects on rumen fermentation. The study employed a series of experiments involving the extraction of compounds from the seaweed using solvents of decreasing polarity, followed by in vitro assays to assess the extracts’ ability to reduce methane production. Gas chromatography-mass spectrometry (GC-MS) was utilized to identify and quantify the major halogenated natural products within the extracts. Further experiments evaluated the antimethanogenic activity of pure natural products, determining the minimum effective concentrations needed to decrease methane production and analyzing their effects on various fermentation parameters. The study also included a positive control using *Asparagopsis taxiformis* biomass and assessed the impact of the identified natural products on key fermentation characteristics.
Key Findings & Statistics
- The positive control (*Asparagopsis taxiformis* 2% OM and Rhodes grass 98% OM) significantly decreased in vitro total gas production (TGP) by 25% (113.6 mL g⁻¹ OM) compared with the Rhodes grass control (100 % OM) (151.4 mL g⁻¹ OM).
- The DCM extract reduced methane production by 79%.
- The positive control of *Asparagopsis taxiformis* 2% OM also had the lowest production of CH₄ (0.7 mL g⁻¹ OM) with a significant reduction of 95% compared to the control (13.5 mL g⁻¹ OM).
- The DCM extract (2.6 mL g⁻¹ OM) also significantly reduced the production of CH₄ with a reduction of 79% compared with the control.
- Bromoform was the most abundant natural product in the biomass of *Asparagopsis* (1723 µg g⁻¹ dry weight [DW] biomass), followed by dibromochloromethane (15.8 µg g⁻¹ DW), bromochloroacetic acid (9.8 µg g⁻¹ DW) and dibromoacetic acid (0.9 µg g⁻¹ DW).
- The DCM extract had the lowest TGP at 143.2 mL g⁻¹ OM (5% lower than control) and the water extract had the highest TGP (165.8 mL g⁻¹ OM).
- The water extract had the highest yield among all solvents with the extract representing 24.9 % of the dry weight (DW) of the biomass. Methanol had a yield of 10.2 % DW, and DCM and hexane had the lowest yields of 1.9 and 0.5% DW, respectively.
- DCM extracted the highest total amount of natural products from the biomass, followed by methanol, hexane and water. The DCM extract had the highest yield of bromoform (Fig. 3c) corresponding to 1723.2 µg g⁻¹ DW of biomass. This is 5.7 times higher than the methanol (301.2 µg g⁻¹ DW) and hexane (297.0 µg g⁻¹ DW) extracts.
- The DCM extract also had a yield of dibromochloromethane (15.8 µg g⁻¹ DW, Fig. 3c) 1.2 times higher than for methanol (13.3 µg g⁻¹ DW) and 5.5 times higher than for hexane (2.9 µg g⁻¹; g⁻¹ DW).
- Bromoform decreased TGP by 23 to 26% at 5 and 10 µM, while dibromochloromethane decreased TGP by 20 to 22%.
- At a concentration of 1 µM, bromoform and dibromochloromethane inhibited the production of CH₄ by an average of 52 and 43%, respectively, compared to the control.
- At the higher concentration of 25 µM, bromoform and dibromochloromethane decreased the production of total VFA by 20 and 30%, respectively, compared with the control.
- In contrast, the production of total VFA increased for bromochloroacetic acid at a concentration of 5 µM and dibromoacetic at concentrations <25 μΜ.
- Bromoform and dibromochloromethane significantly decreased NH3-N by 16 to 17% at a concentration of 1 µM, compared to the control.
Other Important Findings
- DCM extract was most active, reducing methane production by 79 %.
- Bromoform and dibromochloromethane had the highest activity with concentrations ≥1 µM inhibiting methane production.
- Only bromoform was present in sufficient quantities in the biomass at 2 % organic matter to elicit this effect.
- The degradability of organic matter and volatile fatty acids were not affected at effective concentrations.
- The two species, *Asparagopsis taxiformis* and *Asparagopsis armata*, have strong activity against gram-negative and gram-positive bacteria.
- In vitro effects on methanogenesis and fermentation kinetics were evaluated for the four major natural products produced by *Asparagopsis*.
- The effects were much stronger and the production of CH₄ was significantly inhibited to below detection levels by all three compounds at ≥5 µM.
- The production of total VFA for bromoform (minimum of 32.6 mM VFA) and dibromochloromethane (minimum of 33.6 mM VFA) were similar to the control (35.7 mM VFA) for concentrations ≤10 µM.
Limitations Noted in the Document
- The study did not address the long-term effects of bromoform in vivo, and further in vivo studies are required.
- The in vitro experiments might not fully replicate the complex conditions of a rumen environment.
- The study focused on specific natural products; other potentially active compounds in *Asparagopsis* were not explored.
- Variations in the natural product composition of the seaweed, depending on environmental factors or life history stage, were not accounted for.
- The study only included *Bos indicus* steers; the results may not be directly transferable to other breeds or species.
Conclusion
The study successfully identified bromoform as the primary compound responsible for the antimethanogenic activity of *Asparagopsis taxiformis*, confirming its potential as a feed additive in livestock to mitigate methane emissions. Bromoform, along with dibromochloromethane, demonstrated significant inhibition of methane production in vitro. The research highlights the potential of *Asparagopsis taxiformis* biomass at 2% OM in the feed to reduce methane emissions, a finding supported by the effective concentrations of bromoform and dibromochloromethane found in the biomass. The study further supports the mechanism of action of bromoform as a direct suppressor/inhibitor of methanogenesis. The lack of adverse effects on the degradability of organic matter and the production of total volatile fatty acids indicates that fermentation processes were not negatively impacted at effective doses of bromoform and dibromochloromethane. The study suggests a shift in the VFA profile, with a decrease in acetate and an increase in propionate and butyrate, which supports the potential to reduce methane production while maintaining animal productivity. These findings support the consideration of *Asparagopsis* biomass and its natural products in techno-economic analyses, for potential use as feed additives for ruminants. Further long-term in vivo studies are needed to confirm these findings and assess the broader implications of bromoform and other natural products of *Asparagopsis taxiformis*.