Carbon-13 stable isotope analysis reveals the existence but insignificance of ruminal methanogenic pathway from acetate in a batch culture system

A series of in vitro batch culture incubations were conducted to investigate the extent of carboxyl group and methyl group in acetate converted to CH4 using a stable isotopic tracer method. Ground lucerne hay was incubated as a substrate with buffered ruminal fluid, and 13C labeled CH313COONa or 13CH3COONa was supplied into the incubation bottles at a dose of 0, 15, 35 or 55 mM. Increasing CH313COONa or 13CH3COONa supplementation linearly increased (P <  0.05) absolute 13CH4 and 13CO2 production (mL/bottle) at all the time points measured. Yet, the conversion rates (CR) of 13CH4 and 13CO2 produced from labeled CH313COONa or 13CH3COONa were very low. For instance, the relative 13CH4 CR (%) was ranged from 0.0028-0.14% with incubation time extended to 48 h, and the relative 13CO2 CR (%) was ranged from 0.054 to 1.3%. Moreover, acetate supplementation linearly increased (P <  0.01) the concentration of butyrate, 16S rRNA gene copies of protozoa, total methanogens, Methanosphaera stadtmanae, Methanobrevibacter smithii and Methanosarcina barkeri. In summary, our study indicates existence of methanogenesis from acetate, but such pathway of methanogenesis makes little contribution to the total CH4 production through in vitro ruminal fermentation.