Metabolic-methane mitigation by combination of probiotic Escherichia coli strain Nissle 1917 and biochar in rumen fluid in vitro fermentation of dairy cow

In response to climate change, there have been various trials to reduce methane emissions from ruminant animals in the livestock industry, and one of the typical candidates is probiotics used as feed additives. We employed Escherichia coli Nissle 1917 (EcN) as a promising probiotic for the reduction of methane emissions and investigated the molecular mechanism of methane-reducing effects using CRISPR/Cas9. The anaerobic culture of EcN with biochar (BC) could significantly increase acetate and hydrogen production. Additionally, RNA-seq analysis revealed an upregulation in the expression of acetate synthesis genes, namely, pta, poxB, and ackA. Subsequently, treatment of rumen fluid with EcN significantly decreased methane and increased acetate and propionate production, as observed through the analysis of short-chain fatty acids, and these effects could accelerate with additional supplementation of BC. The observation of changes in the microbial composition of rumen fluid following treatment with EcN and BC was made through rumen metagenomic analysis and RT-qPCR. The results revealed an increase in acetogen and propionate producing bacteria abundance and a decrease in methanogen abundance. Based on these findings, the CRISPR/Cas9 system was employed to elucidate the molecular mechanism of the methane-reducing capability of EcN. Gene deletion was performed targeting the genes poxB, ackA, and pta as key factors in acetate pathway in E. coli. The knockout of poxB, ackA, and pta could lead to the elimination of the methane-reduction of EcN as well as acetate-producing abilities. Collectively, the methane reduction ability of EcN in rumen fluid is associated with its acetate production capability, and the addition of BC significantly enhances methane mitigation capability of EcN.