Understanding the molecular-level responses of cattle rumen microbiota to kaolin clay as a feed additive in vitro

Abstract Enteric methane emissions from ruminants, primarily produced by methanogenic archaea during rumen fermentation, contribute substantially to greenhouse gas emissions and represent a loss of dietary energy. Clay mineral-based feed additives have been explored as potential methane mitigation strategies; however, their mechanisms remain poorly understood. This study investigated the mechanistic role of halloysite–kaolinite mixed mineral (kaolin) supplementation in modulating rumen fermentation and microbial functional potential, including bacteria, protozoa, and archaea, using an in vitro system. Approximately 5% clay was incorporated into 2 g of basal diet and incubated under anaerobic conditions using ANKOM technology. Samples were collected at defined intervals up to 48 h, and integrated 16S and 18S rRNA sequencing, and PICRUSt2 functional prediction analyses. Clay supplementation reduced methane by ~21%, from 35.43 ± 3.50 to 27.85 ± 2.31 mL CH 4 /g dry matter. Although bacterial and archaeal community composition shifted in response to supplementation, the dominant bacterial taxa ( Firmicutes and Bacteroidota ), and protozoal populations were largely preserved. Predicted methanogenesis pathway abundance was significantly reduced by ~47% ( p < 0.01), alongside methane reduction. Predicted functional analysis further revealed potential alteration in enzyme-mediated hydrogen utilisation ( p < 0.001). This might indicate a redistribution of metabolic hydrogen among competing pathways without accumulation. Microscopic observations further showed spatial associations between microbial cells and clay surfaces. These findings suggest that kaolin reduces methane production through changes in predicted functional potential related to methanogenesis and hydrogen utilisation, alongside shifts in bacterial and archaeal community composition, while fermentation-related parameters remained largely stable. Key points • Kaolin clay in feed suppresses rumen methanogenesis without disrupting fermentation or fibre digestion. • Clay may redirect hydrogen away from methane production, offering a practical methane-mitigation strategy. • Direct microbial attachment to clay coincided with altered predicted methanogenesis potential, while dominant taxa were largely preserved.