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

Reducing methane (CH 4 ) released from the rumen of cattle is essential for lowering greenhouse gas emissions from livestock. Methanogens, the archaea responsible for producing CH 4 , are the dominant hydrogen (H 2 ) sink in the rumen. Hydrogen is a by-product of normal rumen fermentation, and increased H 2 levels, resulting from inhibiting methanogenesis, may have negative effects on certain rumen fermentation processes. However, increased H 2 levels may also open the possibility for H 2 to be redirected into alternative pathways, harbored by H 2 -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. A culture collection of 182 bacterial isolates was generated. When grown in selective medium with H 2 and CO 2 as substrates, 83 isolates produced acetate and 32 isolates significantly lowered dissolved H 2 (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 CH 4 formation by 66.5% and increased H 2 accumulation by 1400%. However, none of the twelve isolates reduced H 2 or CH 4 in the in vitro system, nor did they alter total gas production, fermentation acid profiles, or feed degradability. Remaining dissolved H 2 measured in pure culture indicated that the isolates required substantially higher dissolved H 2 concentrations than what is theorised to be in the in vitro bottles, where headspace gas was continuously released. Although many newly isolated bacteria were capable of utilizing H 2 under controlled pure-culture conditions, they did not reduce H 2 accumulation in an in vitro rumen system with inhibited methanogenesis under the conditions tested. The predicted low dissolved H 2 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 H 2 sinks when methanogenesis is suppressed.