Effects of plant antioxidants and natural vicinal diketones on methane production, studied in vitro with rumen fluid and a polylactate as maintenance substrate

In a simplified model of methane production, lactate based maintenance substrates provided primary metabolites and H2, for methanogenic reduction of CO2, to a rumen fluid inoculum. In batch incubation assays, the polylactate Hydrogen Release Compound eXtended® (HRC-X) as maintenance substrate caused the most protracted methane production when compared to lactate and Hydrogen Release Compound® (HRC), thus offering the advantage of a prolonged period available for bacterial adaptation and interference with methane production. While addition of fumarate as methane controlling agent enhanced methane emission under substrate limiting conditions, it reduced methane production under the influence of excess lactate or HRC-X. The novel methane controlling agents catechin, resveratrol, 2,3-butanedione (diacetyl) and 2,3-pentanedione, and the well-described reference compounds fumarate and 2-bromoethanesulfonate (BES), were tested as additives in terms of their methane reducing capacities. For this purpose, repeatedly fed batch cultures were employed and fed three times with HRC-X plus additive to check if the agents would be effective over a prolonged period of time. To obtain a 20–40% methane reduction in all assays after the first feeding cycle, totals of 1.5 μmol of BES, each 50 μmol of catechin and resveratrol, each 175 μmol of 2,3-butanedione and 2,3-pentanedione, and 1000 μmol of fumarate were added at the beginning, and then again in the two subsequent feedings. Methane and H2 were determined at the end of each feeding cycle, and lactic and short chain fatty acids at the end of the whole experiment. Of the four novel active compounds, catechin reduced methane production with the least side effects in terms of inhibition of substrate degradation or H2 accumulation. An incomplete fermentation with resveratrol indicated that resveratrol does interfere with substrate degradation, which is in agreement with its growth rate reducing effect on bacteria reported in the literature. 2,3-Butanedione, in contrast to resveratrol, did not interfere with substrate degradation, but an elevated H2 concentration indicated that methanogenesis as H2 sink was inhibited, causing a decrease of methane production. 2,3-Pentanedione also diminished methane production, although less efficiently than 2,3-butanedione, with a concomitant accumulation of H2. The different effects observed are worth studying in more detail in view of the need for efficient feed additives in the framework of greenhouse gas emission reduction.