Responses of methane production and fermentation pathways to the increased dissolved hydrogen concentration generated by eight substrates in in vitro ruminal cultures

Hydrogen (H2) produced in the rumen is a major energy source for the growth of methanogens, and is present in two forms: dissolved H2 (dH2) and H2 gas (gH2). Methanogens use only dH2, and we developed a simple procedure to estimate dH2 in liquid samples. We then used this simple apparatus to measure dH2 in the liquid phase of in vitro ruminal cultures, and to investigate the effects on methane (CH4) and volatile fatty acids (VFA) production of different dH2 concentrations generated by eight substrates of concentrates and forages. Higher dH2 concentrations in the liquid phase did not lead to higher gH2 concentrations in the gas phase, indicating that Henryʹs law might not describe the relationship between the gas and liquid phases for in vitro incubations. The supersaturation factor was larger than unity across eight substrates, indicating that dH2 and gH2 were not in equilibrium, and H2 was supersaturated in the liquid phase. The concentrates generated greater amounts of H2 than the forages, leading to more CH4 per unit of feed. However, the concentrates were degraded to a greater extent, as indicated by the higher VFA concentrations, and less H2 was produced relative to VFA from the concentrates. The dH2 concentration was positively correlated with molar proportion of butyrate (R2 = 0.621) and propionate (R 2= 0.904), and negatively correlated with ratio of acetate to propionate (R2 = 0.853), molar proportion of acetate (R 2= 0.902) and the net H2 production per VFA (R2 = 0.744). The results indicated that higher dH2 concentrations forced the fermentation pathways towards those producing less H2, and more butyrate and propionate, which as electron sinks that spare H2 production. In summary, the dH2 concentration could not be predicted by the gH2 concentration, and the dH2 concentration controlled CH4 formation and the pathways of VFA and H2 production.