How many rumen methanogens are there?

Abstract

Animal Production in Australia 1998 Vol. 22 HOW MANY RUMEN METHANOGENS ARE THERE? S. K. BAKER A B AB , N.L. CHAMBERLAIN A, A. KICIC B, L. KLEIN A and K.A. MUNYARD B CSIRO Division of Animal Production, Floreat Park, WA 6014 The University of Western Australia, Nedlands, WA 6907 For nearly 70 years in ruminant nutrition the loss as methane of gross energy intake by ruminants has been measured, but surprisingly little is known about the populations of methane-producing microorganisms (methanogens) in the rumen. Numbers of methanogens in the cattle rumen are ca. 108/mL rumen contents, and differ little with time after feeding or with the roughage content of the diet (Leedle and Greening 1982). Methanogens that have been isolated from the rumen and from faeces of ruminants belong to the genera Methanobrevibacter, Methanomicrobium and Methanosarcina (Miller et al. 1986, Wolin and Miller 1997). Methanobrevibacter spp. and Methanomicrobium spp. are present in high numbers in the rumen (ca. 108/mL), but the sarcina are present in low numbers (ca. 103/mL) except in animals fed unusual diets where rumen dilution rates are very low. Interestingly, few of the type species are rumen isolates (Sowers and Schreier 1995). Here we report observations of the size and composition of the populations of methanogens in sheep and cattle from several studies. An agar medium (Miller and Wolin 1982) was used for enumeration and isolation of methanogens. Methanogens were identified by their autofluorescence at 420nm and production of methane in culture. Methane was determined by gas chromatography with flame ionisation or thermal conductivity detectors. Serum antibodies raised in rabbits were used as probes to indicate possible phylogenetic relatedness of isolates, or of cells in rumen contents, observed using immunofluorescence microscopy (see Conway de Macario et al. (1982) for more detail). In rumen inocula from 12 sheep fed oaten hay, lupin grain and a mineral mix (88:10:2) the size of the methanogen population, estimated from colony counts in the agar medium, varied between sheep from less than 105 up to 108/mL rumen inoculum. In the culture dilution series, the final dilutions at which methane in the head-space was detectable also varied, from dilutions of 105 to more than 109/mL rumen inoculum. From two cattle fed either hay or hay silage, methanogens were isolated from dilutions of 105 to 108/mL rumen contents. The isolates were cocci, coccobacilli or rods and when they were tested against antisera raised to various methanogens they bound most strongly antibodies to either Methanobrevibacter ruminantium M1 or Methanobacterium formicicum MF. There was little or no cross-reactivity with antibodies to Methanobrevibacter smithii B181 or Methanobrevibacter arboriphilicus DC. From a sheep fed silage the morphology of methanogens that were isolated (in the laboratory of M.J. Wolin, New York State Department of Health) resembled Methanobrevibacter spp. Antisera (in the laboratory of A. J. L. Macario, New York State Department of Health) raised against Mb. ruminantium M1, Mb. smithii PS, ALI, Mb. arboriphilicus DH1, AZ, DC, Methanobacterium br yantii M.o.H.G, M. formicicum MF, Methanomicrobium mobile BP, Methanosarcina mazei S6 and Methanosarcina barkeri MS were used to probe methanogens in rumen fluid samples. Coccobacilli that morphologically resembled Methanobrevibacter spp. and strongly bound antibodies to Mb. smithii PS were most numerous followed by cocobacilli that strongly bound antibodies to Mb. arboriphilicus DH1 and DC. Low numbers of large cocci strongly bound antibodies to Ms. mazei S6 and Ms. barkeri MS. Since antibody probes revealed a greater number and diversity of methanogens in the rumen population than did culture techniques, ecological studies of rumen methanogens using culture techniques also should employ complementary techniques such as the use of antibody or oligonucleotide probes. CONWAY DE MACARIO, E., WOLIN, M.J., and MACARIO, A.J.L. (1982). J. Bacteriol. 149, 316-9. LEEDLE, J.A.Z. and GREENING, R.C. (1988) Appl. Environ. Microbiol. 54, 502-6. MILLER, T.L., WOLIN, M.J. and KUSEL, E.A. (1986). System. Appl. Microbiol. 8, 234-8. MILLER, T.L. and WOLIN, M.J. (1982). Arch. Microbiol. 131, 14-8. SOWERS, K.R. and SCHREIER, H.J. (1995). (Eds) Archae. A Laboratory Manual. Methanogens. (Cold Spring Harbour Press: New York). WOLIN, M.J. and MILLER, T.L. (1997). In The Rumen Microbial Ecosystem, 2nd ed. (Eds P.N. Hobson and C.S. Stewart) p. 467. (Chapman and Hall: London). 350