Methane production from in vitro rumen incubations with Lotus pedunculatus and Medicago sativa, and effects of extractable condensed tannin fractions on methanogenesis

Abstract

Inhibitory effects of condensed tannins (CT) on methane, hydrogen, total gas and volatile fatty acid (VFA) formation were explored for the forage legumes Lotus pedunculatus (also known as L. uliginosus; CT 0.10 of DM) and Medicago sativa (lucerne; CT < 0.001 of DM) in the presence and absence of polyethylene glycol (PEG; a tannin binding reagent) and 2-bromo-ethylsulfonic acid (BES; a methanogen inhibitor). Treatment × forage interactions were explored using a factorial design for in vitro batch incubations with rumen fluid and minced forages over a 12 h period. The accumulated methane volume (ml/0.5 g DM) at 12 h for M. sativa (12.5 ml) was higher than L. pedunculatus (8.8 ml; P < 0.05). PEG increased methane production for L. pedunculatus (17%, P < 0.001) but not for M. sativa. BES inhibited methane (>80%) and increased accumulated hydrogen for all treatments. For L. pedunculatus, addition of PEG in the presence of BES increased hydrogen accumulation (70%, P < 0.001) compared to L. pedunculatus and relative production (49%, P < 0.01), with no effect of PEG in combination with BES for M. sativa. The VFA (i.e., C2–C5) concentrations were determined at 12 h for the batch incubations of rumen fluid with forage. Addition of PEG increased total VFA production ( P < 0.01) for L. pedunculatus (16.9%) and M. sativa (1.8%). Addition of PEG increased C2–C5 concentrations for L. pedunculatus (5.9, 30, 22, 53%, respectively) with M. sativa having an increase only for C2 (13%). Branched chain VFA (bC4 and bC5) increased with PEG addition for L. pedunculatus (67 and 59%, respectively), but not for M. sativa. Inhibitory effects of extractable condensed tannin fractions from L. pedunculatus on the common rumen methanogens Methanobrevibacter ruminantium, strains YLM-1 and DSM1093, were evaluated. Oligomeric CT fractions were inactive against both strains in broth culture, as determined by methane production measurements. A polymeric fraction completely inhibited methane production. Inhibitory effects in broth culture for strain YLM1 were bacteriostatic, while strain DSM1093 did not recover growth, as indicated by methane production, even upon prolonged incubation. In a plate assay, the zone of inhibition with the polymeric fraction remained after a further week of incubation. This is the first demonstration of inhibition of growth of a rumen methanogen by a polymeric CT fraction. Results indicate that CT action on methanogenesis can be attributed to indirect effects via reduced hydrogen production (and presumably reduced forage digestibility) and via direct inhibitory effects on methanogens.