Background Reducing methane (CH4) released from the rumen of cattle is essential for lowering greenhouse gas emissions from livestock. Methanogens, the archaea responsible for producing CH4, are the dominant hydrogen (H2) sink in the rumen. Hydrogen is a by-product of normal rumen fermentation, and increased H2 levels, resulting from inhibiting methanogenesis, may have negative effects on certain rumen fermentation processes. However, increased H2 levels may also open the possibility for H2 to be redirected into alternative pathways, harbored by H2-utilizing (hydrogenotrophic) bacteria that would normally not be competitive with methanogens. This study aimed to isolate hydrogen-utilizing bacteria, characterize their hydrogen metabolism in pure culture, and evaluate whether selected strains could reduce hydrogen accumulation in an in vitro rumen system where methanogenesis was chemically inhibited. Results A culture collection of 182 bacterial isolates was generated. When grown in selective medium with H2 and CO2 as substrates, 83 isolates produced acetate and 32 isolates significantly lowered dissolved H2 (by 7.7 to 84.6%.) compared with controls. Twelve isolates with the highest hydrogen-utilizing potential were tested in an in vitro rumen batch system (rumen fluid amended with corn silage), where methanogenesis was inhibited with iodoform. Iodoform reduced CH4 formation by 66.5% and increased H2 accumulation by 1400%. However, none of the twelve isolates reduced H2 or CH4 in the in vitro system, nor did they alter total gas production, fermentation acid profiles, or feed degradability. Remaining dissolved H2 measured in pure culture indicated that the isolates required substantially higher dissolved H2 concentrations than what is theorised to be in the in vitro bottles, where headspace gas was continuously released. Conclusions Although many newly isolated bacteria were capable of utilizing H2 under controlled pure-culture conditions, they did not reduce H2 accumulation in an in vitro rumen system with inhibited methanogenesis under the conditions tested. The predicted low dissolved H2 concentrations in the system likely prevented the isolates from growing autotrophically. These findings indicate that alternative hydrogen-utilizing bacteria may require different in vitro conditions or in vivo environments to effectively act as H2 sinks when methanogenesis is suppressed.

Challenges in redirecting rumen hydrogen flow in vitro: insights from new hydrogenotrophic isolates

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2026年7月9日

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Samantha Joan Noel,
Ole Højberg,
Mirka Thorsteinsson

摘要

Background

Reducing methane (CH₄) released from the rumen of cattle is essential for lowering greenhouse gas emissions from livestock. Methanogens, the archaea responsible for producing CH₄, are the dominant hydrogen (H₂) sink in the rumen. Hydrogen is a by-product of normal rumen fermentation, and increased H₂ levels, resulting from inhibiting methanogenesis, may have negative effects on certain rumen fermentation processes. However, increased H₂ levels may also open the possibility for H₂ to be redirected into alternative pathways, harbored by H₂-utilizing (hydrogenotrophic) bacteria that would normally not be competitive with methanogens. This study aimed to isolate hydrogen-utilizing bacteria, characterize their hydrogen metabolism in pure culture, and evaluate whether selected strains could reduce hydrogen accumulation in an in vitro rumen system where methanogenesis was chemically inhibited.

Results

A culture collection of 182 bacterial isolates was generated. When grown in selective medium with H₂ and CO₂ as substrates, 83 isolates produced acetate and 32 isolates significantly lowered dissolved H₂ (by 7.7 to 84.6%.) compared with controls. Twelve isolates with the highest hydrogen-utilizing potential were tested in an in vitro rumen batch system (rumen fluid amended with corn silage), where methanogenesis was inhibited with iodoform. Iodoform reduced CH₄ formation by 66.5% and increased H₂ accumulation by 1400%. However, none of the twelve isolates reduced H₂ or CH₄ in the in vitro system, nor did they alter total gas production, fermentation acid profiles, or feed degradability. Remaining dissolved H₂ measured in pure culture indicated that the isolates required substantially higher dissolved H₂ concentrations than what is theorised to be in the in vitro bottles, where headspace gas was continuously released.

Conclusions

Although many newly isolated bacteria were capable of utilizing H₂ under controlled pure-culture conditions, they did not reduce H₂ accumulation in an in vitro rumen system with inhibited methanogenesis under the conditions tested. The predicted low dissolved H₂ concentrations in the system likely prevented the isolates from growing autotrophically. These findings indicate that alternative hydrogen-utilizing bacteria may require different in vitro conditions or in vivo environments to effectively act as H₂ sinks when methanogenesis is suppressed.