Effects of several inhibitors on pure cultures of ruminal methanogens* E.M. Ungerfeld 1 , S.R. Rust 1 , D.R. Boone 2 and Y. Liu 2 1 Department of Animal Science, Michigan State University, East Lansing, MI, and 2 Department of Biology, Portland State University, Portland, OR, USA 2003/1174: received 21 December 2003, revised 14 April 2004 and accepted 14 April 2004 ABSTRACT E.M. UNGERFELD, S.R. RUST, D.R. BOONE AND Y. LIU. 2004. Aims: To examine the effects of five inhibitors of methanogenesis, 2-bromoethanesulphonate (BES), 3-bromopropanesulphonate (BPS), lumazine, propynoic acid and ethyl 2-butynoate, on CH 4 production of the ruminal methanogens Methanobrevibacter ruminantium, Methanosarcina mazei and Methanomicrobium mobile. Methods and Results: Methanogens were grown in MS medium including 25% (v/v) clarified ruminal fluid. Methane production was measured after 4 and 6 days of incubation. Methanobrevibacter ruminantium was the most sensitive species to BES, propynoic acid and ethyl 2-butynoate. Methanosarcina mazei was the least sensitive species to those chemical additives, and Mm. mobile was intermediate. BPS failed to inhibit any of the methanogens. All three species were almost completely inhibited by 50- and 100%-lumazine saturated media, but the inhibition was somewhat lower with a 25%-lumazine saturated media. Conclusions: There were important differences among species of methanogens regarding their sensitivity to the different inhibitors. In general, Ms. mazei was the most resistant to inhibitors, Mb. ruminantium the least resistant, and Mm. mobile was intermediate. Significance and Impact of the Study: Differences among methanogens regarding their resistance to chemical inhibitors should be considered when designing strategies of inhibition of ruminal methanogenesis, as selection of resistant species may result. Keywords: chemicals, inhibitors, methanogens, resistance, rumen. INTRODUCTION Methane formation in the rumen implies an energy loss for ruminants, and also contributes to global warming. Therefore, there is an interest in decreasing CH 4 production in the rumen (Moss 1993). Chemical additives with different modes of action have been used to decrease ruminal methanogenesis. Some compounds have been shown to considerably decrease CH 4 production of mixed ruminal cultures (Van Nevel and Demeyer 1996; Nagaraja et al. 1997). However, it should be stressed that inhibiting methanogenesis alone does not improve the efficiency of ruminal energetic transformations if reducing equivalents are not relocated into sinks that are nutritionally useful to the host animal. Inefficient relocation of electrons spared from methanogenesis often results in decreased fermentation and accumulation of H 2 and other compounds that are not energy substrates for ruminants (Van Nevel and Demeyer 1996; Le Van et al. 1998). Although the main interest when evaluating potential inhibitors of methanogenesis is their effects on the mixed ruminal microbial community, it is important to know how individual species of methanogens are affected. If some species are resistant to an inhibitor, its long-term use in vivo could result in their selection, and CH 4 formation could return to the levels observed before the inhibitor was fed. For example, 2-bromoethanesulphonate (BES) is a very *The work was carried out at the Department of Biology, Portland State University, Portland, OR 97207, USA. Correspondence to: Emilio M. Ungerfeld, 4119 Department of Animal and Avian Sciences, College Park, MD 20742-2311, USA (e-mail: emu@umd.edu). ª 2004 The Society for Applied Microbiology Journal of Applied Microbiology 2004, 97, 520–526 doi:10.1111/j.1365-2672.2004.02330.x