Development of Multiwell-Plate Methods Using Pure Cultures of Methanogens To Identify New Inhibitors for Suppressing Ruminant Methane Emissions

Abstract

Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H 2 and CO 2 that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen Methanococcus maripaludis strain S2 and the rumen methanogen Methanobrevibacter species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against M. maripaludis as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against M. maripaludis and M. AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions.IMPORTANCE Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions, and new technologies are required to control emissions in the agriculture technology (agritech) sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimized, and validated a highthroughput 96-well microtiter plate assay for growing environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions.KEYWORDS methanogen, greenhouse gas, Methanococcus maripaludis, highthroughput, rumen, Methanobrevibacter M ethane emissions from ruminants are a significant contributor to global greenhouse gas emissions (1). In countries such as New Zealand, with a large pasturebased livestock sector, greenhouse gas emissions from agriculture represent approxi-

Use of Lactic Acid Bacteria to Reduce Methane Production in Ruminants, a Critical Review

“…A similar class of compounds (antimicrobial peptides such as human catelicidin) have also been shown to be strongly inhibitory to a range of methanogens (Bang et al, 2012, 2017). There is no standardized approach to screening methanogen cultures for their susceptibility to bacteriocins, however, the method developed to facilitate screening of small molecule inhibitors (Weimar et al, 2017) should be useful. This employs the rumen methanogen strain AbM4 (a strain of Methanobrevibacter boviskoreani ) which grows without H 2 in the presence of ethanol and methanol (Leahy et al, 2013).…”

Diverse hydrogen production and consumption pathways influence methane production in ruminants

“…Given their major contribution to greenhouse gas emissions, multiple programs are underway to mitigate ruminant methane production [13, 14]. To date, most strategies have focused on direct inhibition of methanogens using chemical compounds or vaccines [15–18]. A promising alternative strategy is to modulate the supply of substrates to methanogens, such as H 2 , for example, through dietary or probiotic interventions [14, 19, 20].…”

Development of a simultaneous bioreactor system for characterization of gas production kinetics of methanogenic archaea at high pressure

“…investigations into required substrates, optimal growth temperature, pH) for a certain biocatalyst are necessary for subsequently optimizing a process in terms of high product yield or biomass formation. The characterization of the biocatalyst by means of screening, optimization, and modification of process parameters is usually performed in microtiter plates, serum bottles, Schott bottles or Erlenmeyer flasks . Serum bottles or Erlenmeyer flasks are vitreous and therefore only withstand slight overpressures (serum bottles p max < 2.0 bar; Schott bottles p max < 1.0 bar).…”