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 C-13 labeled (CH3COONa)-C-13 or (CH3COONa)-C-13 was supplied into the incubation bottles at a dose of 0, 15, 35 or 55 mM. Increasing (CH3COONa)-C-13 or (CH3COONa)-C-13 supplementation linearly increased (P < 0.05) absolute (CH4)-C-13 and (CO2)-C-13 production (mL/bottle) at all the time points measured. Yet, the conversion rates (CR) of (CH4)-C-13 and (CO2)-C-13 produced from labeled (CH3COONa)-C-13 or (CH3COONa)-C-13 were very low. For instance, the relative (CH4)-C-13 CR (%) was ranged from 0.0028-0.14% with incubation time extended to 48 h, and the relative (CO2)-C-13 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.