Effects of hydrogen in headspace and bicarbonate in media on rumen fermentation, methane production and methanogenic population using in vitro gas production techniques

• H2 addition increased CH4 production efficiency from bicarbonate in media.
• NaHCO3 addition decreased CH4 production efficiency from bicarbonate in media.
• H2 and NaHCO3 additions influenced in vitro rumen fermentation and methanogens.

An in vitro batch culture study was conducted to investigate (1) 13C-labeled methane (CH4) production efficiency (PE) which was produced from the reduction of 13C-labeled sodium bicarbonate (NaH13CO3) in media; and (2) the effects of NaHCO3 and hydrogen (H2) additions on ruminal fermentation, CH4 production and methanogenic populations in a 4 × 3 factorial design with lucerne hays as substrates. Factors were NaH13CO3 concentration in media (0, 11.9, 23.8 or 47.6 μmol/ml) and H2 volume in headspace (0, 36 or 60 ml). The 13CH4 PE produced from NaH13CO3 was linearly increased (P < 0.01) with H2 addition, but quadratically decreased (P < 0.01) in response to NaH13CO3 addition. Moreover, addition of H2 quadratically increased (P < 0.01) total CH4 production, apparent dry matter degradability (ADMD), total volatile fatty acids (VFA), molar proportion of acetate and populations of Methanosarcina barkeri (M. barkeri), Methanosphaera stadtmanae (M. stadtmanae), Methanobrevibacter ruminantium M1 (M. ruminantium) and Methanobrevibacter smithii (M. smithii). Quadratic effects (P < 0.041) were observed for total CH4 production, populations of M. barkeri, M. stadtmanae, M. ruminantium and M. smithii in response to NaHCO3 addition. Additionally, the addition of NaHCO3 linearly increased (P < 0.01) the total VFA. The results of methanogenic populations during incubation showed that the copy number of 16S rRNA gene for M. barkeri was increased (P = 0.023) by incubation time with the highest value being at 24 h, but no differences were observed for the M. stadtamanae, M. ruminantium and M. smithii gene copy numbers among the incubation time points. These results demonstrate that both CO2 and H2 concentrations affect methanogenesis in the rumen. Furthermore, the present study suggests that headspace hydrogen and media bicarbonate play roles in regulating feed fermentation, CH4 production and methanogenic populations in vitro.