Effects of Methanogenic Inhibitors on Methane Production and Abundances of Methanogens and Cellulolytic Bacteria in In Vitro Ruminal Cultures

Abstract

The objective of this study was to systematically evaluate and compare the effects of select antimethanogen compounds on methane production, feed digestion and fermentation, and populations of ruminal bacteria and methanogens using in vitro cultures. Seven compounds, including 2-bromoethanesulphonate (BES), propynoic acid (PA), nitroethane (NE), ethyl trans-2-butenoate (ETB), 2-nitroethanol (2NEOH), sodium nitrate (SN), and ethyl-2-butynote (EB), were tested at a final concentration of 12 mM. Ground alfalfa hay was included as the only substrate to simulate daily forage intake. Compared to no-inhibitor controls, PA, 2NEOH, and SN greatly reduced the production of methane (70 to 99%), volatile fatty acids (VFAs; 46 to 66%), acetate (30 to 60%), and propionate (79 to 82%), with 2NEOH reducing the most. EB reduced methane production by 23% without a significant effect on total VFAs, acetate, or propionate. BES significantly reduced the propionate concentration but not the production of methane, total VFAs, or acetate. ETB or NE had no significant effect on any of the above-mentioned measurements. Specific quantitative-PCR (qPCR) assays showed that none of the inhibitors significantly affected total bacterial populations but that they did reduce the Fibrobacter succinogenes population. SN reduced the Ruminococcus albus population, while PA and 2NEOH increased the populations of both R. albus and Ruminococcus flavefaciens. Archaeon-specific PCR-denaturing gradient gel electrophoresis (DGGE) showed that all the inhibitors affected the methanogen population structure, while archaeon-specific qPCR revealed a significant decrease in methanogen population in all treatments. These results showed that EB, ETB, NE, and BES can effectively reduce the total population of methanogens but that they reduce methane production to a lesser extent. The results may guide future in vivo studies to develop effective mitigation of methane emission from ruminants.Methane (CH 4 ) emissions from ruminants can result in a significant loss of feed efficiency: up to a 12% loss of gross energy intake for forage-fed cattle and 4% for concentrate-fed cattle (14). Because methane is 25 times more potent than carbon dioxide as a greenhouse gas (11), methane emitted from ruminants amounted to 141 teragrams of CO 2 equivalents (Tg CO 2 eq), accounting for 25% of total methane emissions from anthropogenic activities in the United States in 2008 (26). To mitigate the negative impact on climate change and to improve feed efficiency, numerous strategies for reducing methane emission from ruminant livestock have been tested. Plant extracts (7, 9), vaccines (28), ionophores (27), and dietary strategies (21) have been evaluated for their efficacy in reducing ruminal methane emission. However, only monensin has been used in animal-feeding operations, and it typically achieves only transient reductions in methane production (12). More importantly, the monensin-driven reduction in methane reduction is largely attributable to decreased feed digestibility (4,19...

Effects of nitrate adaptation by rumen inocula donors and substrate fiber proportion on in vitro nitrate disappearance, methanogenesis, and rumen fermentation acid

“…From this trial, nitrate-N could be used as an electron acceptor in place of CO 2 . However, Zhou et al (2011) showed that sodium nitrate also significantly decreased methanogen population using qPCR.…”

Design, implementation and interpretation of in vitro batch culture experiments to assess enteric methane mitigation in ruminants—a review

“…This prompted the hypothesis that headspace gas composition, CO 2 in particular, which is in exchange with H 2 CO 3 /HCO 3 − in the medium, depending on concentration, acid-base balance and gas pressure, may affect fermentation characteristics and gas production in ruminal in vitro cultures. A range of initial headspace composition of in vitro cultures has been reported, including 100% CO 2 (Anderson et al, 2003;Weimer et al, 2005), 100% N 2 (Hoover et al, 1976) and a mixture of gases typical of an anaerobic chamber (85% N 2 , 10% H 2 , and 5% CO 2 ; Zhou et al, 2011;Patra et al, 2012). Patra and Yu (2013) investigated the effects of three different headspace gases (N 2 + CO 2 + H 2 in the ratio of 90:5:5, 100% CO 2 , and 100% N 2 ) and the interaction with type of substrate (alfalfa hay or alfalfa hay and concentrate) and media bicarbonate concentration on gas and CH 4 production.…”

Targeting methanogenesis with a nitrooxypropanol bullet

“…However, monensin is relatively nonspecific and impairs beneficial processes, like acetic acid formation by acetogenic bacteria (7,8). Other antimethanogenesis compounds that have been developed include 2,4-bis (trichloromethyl)benzo [1,3] dioxins (9), an inhibitor of methanopterin biosynthesis (10), and nitrogen oxides (nitrate, nitrite, NO, and N 2 O) (11)(12)(13)(14). Whereas the former two compounds have complex structures, the third is simple.…”