Rumen Lachnospiraceae isolate NK3A20 exhibits metabolic flexibility in response to substrate and coculture with a methanogen

Hydrogen (H 2 ) is the primary electron donor for methane formation in ruminants, but the H 2 -producing organisms involved are largely uncharacterized. This work integrated studies of microbial physiology and genomics to characterize rumen bacterial isolate NK3A20 of the family Lachnospiraceae . Isolate NK3A20 was the first recognized isolate of the NK3A20 group, which is among the ten most abundant bacterial genera in 16S rRNA gene surveys of rumen microbiota. NK3A20 produced acetate, butyrate, H 2 , and formate from glucose. The end product ratios varied when grown with different substrates and at different H 2 partial pressures. NK3A20 produced butyrate as a major product using glucose or under high H 2 partial pressures and switched to mainly acetate in the presence of galacturonic acid (an oxidized sugar) or in coculture with a methanogen. Growth with galacturonic acid was faster at elevated H 2 concentrations, while elevated H 2 slowed growth with glucose. Genome analyses revealed the presence of multiple hydrogenases including a membrane-bound Ech hydrogenase, an electron bifurcating butyryl-CoA dehydrogenase (Bcd-Etf), and an Rnf complex that may be involved in modulating the observed metabolic pathway changes, providing insight into H 2 formation in the rumen. The genus-level NK3A20 group is one of the ten most abundant genera of rumen bacteria. Like most of the rumen bacteria that produce the hydrogen that is converted to methane in the rumen, it is understudied, without any previously characterized isolates. We investigated isolate NK3A20, a cultured member of this genus, and showed that it modulates hydrogen production in response to its growth substrates and the hydrogen concentration in its environment. Low-hydrogen concentrations stimulated hydrogen formation, while high concentrations inhibited its formation and shifted the fermentation to more reduced organic acid products. We found that growth on uronic acids, components of certain plant polymers, resulted in low hydrogen yields compared to glucose, which could aid in the selection of low-methane feeds. A better understanding of the major genera that produce hydrogen in the rumen is part of developing strategies to mitigate biogenic methane emitted by livestock agriculture.