Ruminal methanogenesis and its alternatives G Fonty, B Morvan

To cite this version:

G Fonty, B Morvan. Ruminal methanogenesis and its alternatives. Annales de zootechnie, INRA/EDP Sciences, 1996, 45 (Suppl1), pp.313-138. ￿hal-00889640￿

HAL Id: hal-00889640 https://hal.archives-ouvertes.fr/hal-00889640 Submitted on 1 Jan 1996

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Ruminal methanogenesis and its alternatives G Fonty, B Morvan

INRA, Laboratoire de Microbiologie, C.R. de Clermont-Ferrand-Theix 63122 Saint-Genès-Champanelle, France

Methanogenesis is the terminal step in carbon nucleotide sequences of archaea and bacteria flow in many anaerobic habitats including indicate early divergence of the two domains marine and freshwater sediments, marshes during evolution. The phylogenetic data based and swamps, rice paddies, geothermal on rRNA analysis and nucleic acid habitats, anaerobic sewage digestion systems hybridization, coupled with physiological, and animal gastrointestinal tracts. Biological immunological and biochemical characteristics methanogenesis plays a major role in the currently support the taxonomic organization of carbon cycle on earth. Methane escaping from methanogenic archaea into three orders anaerobic habitats can serve as a carbon and comprising six families. Nineteen genera energy source for aerobic methanotrophic containing more than 50 species have been bacteria or can escape to the atmosphere, described to date (Boone et al, 1993). where it is a major participant in atmospheric Methanogenic archaea are the dominant chemical reactions and is an important H2 utilizers in the rumen. However despite the greenhouse gas (Zinder, 1993). economic implication of methanogens for The rumen and the large intestine of agriculture, our knowledge of methanogen humans and monogastric animals, do not species in the rumen is still scarce. Our current completely convert organic matter to CH4 and information is based on a few isolates from a C02. They accumulate high concentrations of few animals. Hydrogenotrophic methanogens volatile fatty acids (c.a. 60 mM acetate, 20 mM ,which reduce C02 to CH4, are responsible for propionate and 10 mM butyrate) which are the major part of rumen methanogenesis. available to the animal host as an energy Methanobrevibacter ruminantium (formerly source. The predominant substrates for Methanobacterium ruminantium) was the first methanogens in these ecosystems are H2 and methanogen isolated from the bovine rumen C02. A major difference between intestinal (Smith and Hungate, 1958). It was present in ecosystems and complete bioconversion high numbers and formed CH4 from H2 and systems is the turnover time. Digestive C02- Strains of methanogenic archaea which ecosystems have turnover times of ap- had the morphological and physiological proximately 1 to 2 days, while in complete characteristics of Methanobrevibacter spp. but conversion systems the turnover times are which did not react with rabbit antisera raised weeks to months (Miller, 1991 Thus, the slow- against M. ruminantium were also isolated from growing fatty acids oxiders and acetogenic the bovine rumen (Lovley et al, 1984; Miller et methanogens cannot be sustained in intestinal al, 1986). Methanobrevibacter is considered to ecosystems. be the major methanogenic genus in the rumen (Miller, 1991). Paynter and Hungate (1968) reported the isolation of Methanomicrobium Rumen methanogens mobile from the rumen of a cow, and Methanosarcina species presumably growing Methanogens are a sub-group of the archaea on methylamines and methanol were found in domain (Woese et al, 1987). Bacteria and low numbers (104 ml- ) in a cow rumen methanogenic archaea differ in the (Patterson and Hespell, 1979). Strains composition of their cellular constituents. The ressembling Methanobrevibacter species have characteristic peptidoglycan polymer of the cell also been isolated from the rumen contents of walls of bacteria is absent from archaea. The young lambs (Morvan et al, unpublished data), lipids of methanogens are glycerol ethers but up to now there is no information on the rather than glycerol esters. Ribosomal RNA characteristics of the methanogens of the adult ovine rumen (Miller, 1994). In the rumen, some fermentation of feed. It would appear that the methanogens are associated with protozoa total methane emissions from ruminants (Stumm et al, 1982). Methanogens appear in increases by about 0.5 to 1 % per year. An the rumen of new-born animals very soon after estimation by Crutzen et al (1986) indicated birth since they were found as early as two or that over the last century, CH4 emission by three days of age in the lamb rumen (Fonty et ruminants has increased 4 fold and has almost al, 1987; Morvan et al, 1994). At the end of the doubled within the past 40 years. first week of life their numbers reached 106 Mi- The effects of environmental factors on 1 rumen contents and when the animals were methanogenesis in the rumen are not well two-weeks old the methanogenic population documented because it is difficult to design was close to that observed in adult sheep. experiments that include the effects of pH, toxic substances, composition of the diet etc. However some factors which influence Biochemistry of ruminal methane- methane emission by ruminants have been genesis investigated. The major factors are the followings : C02 is converted to CH4 by sequentially - differences between animal species: The reduced intermediates. The reactions, contributions of different animal species to enzymes and electron carriers involved in this global CH4 emissions are highly variable. Of process have been the subject of numerous the domestic species, cattle, with their large studies during the past decades, and the size and numbers, are the major contributions, results of these investigations on the producing 70°/ of the animal emissions. biochemistry of methanogenesis have been Smaller ruminant species, sheep and goats, intensively reviewed (Vogels et al, 1988; produce approximately 13% of animal Thauer et al, 1993) and will not be detailed in emissions, horses and mules appear to be the this paper. principal non ruminant methane emitters (2%), followed by pigs (1%). Crutzen et al (1986) estimate that wild ruminants globally produce Factors affecting methane production about 5% of total animal methane. Cattle from in the rumen developed countries produce more methane than the cattle from developing countries Owing to the lack of fatty acid oxidizers and (Crutzen et al, 1986; Seiler, 1984). acetotrophic methanogens, the rumen can be - digestibility of feed intake: Approximatively 4 considered as a truncated ecosystem when to 10 per cent of the gross energy ingested by compared to an anaerobic digestor and dairy and feed cattle is lost in the form of considerably less CH4 is produced per unit methane (Vermorel, 1995). The more carbon digested compared to the anaerobic digestible the feed intake, the greater the digestor. However, because of the large proportion of its gross energy that is released amounts of plant material ingested, the rumen in the form of methane. It must be noted, fermentations lead to the emission, by however, that the ingestion of highly digestible belching, of large volume of methane, feedstuffs results in a high level of animal approximately 200-400 liters per day from the performance (Sauvant, 1993). bovine rumen (Miller, 1991; Tyler, 1991) which - feeding level: Blaxter and Clapperton (1965) correspond to a production of 800 to 1600 liters demonstrated that the methane emission of hydrogen. A sheep eructates approximately expressed as the percentage of gross energy 50 liters of CH4 per day. According to Sauvant decreases significantly with an increase in the (1993) there are 65 liters of methane and 152 level of intake, i.e. it is affected by the level of liters of C02 produced in the rumen for each kg production and the rate of passage of the of organic matter fermented. particle in the digestive tract. This means that Ruminants are the second largest for similar diets, ruminants at a high level of anthropogenic methane source in the world production convert a smaller proportion of emitting 80 Tg/year (15% of the total) (Crutzen, ingested feed into methane. 1994). This represents a lost opportunity for - mode of production: According to Sauvant transforming more carbon into useful products, (1993), intensive animal production is not such as meat or milk during the natural associated with increased methanogenesis. In fact, the reverse occurs if methane production cellulose breakdown. The fungal metabolism is evaluated against the units of product shifts towards a greater production of acetate produced. at the expense of lactate and ethanol (Bauchop - nature of the feed: Generally, for equal and Mountfort, 1981; Fonty et al, 1988; Marvin- energy digestibility, low-fibre diets lead to a Sikkema et al, 1990). Fermentation by other lower methane production than high fibre diets. important microbes in the rumen, such as R. Fat supplementation of the feed also reduces flavefaciens, Selemonas ruminantium methanogenesis (Blaxter and Clapperton, influences the partial pressure of H2 and can 1965; Giger-Reverdin et al, 1992). result in interspecies transfer (Wolin and Miller, - composition of the rumen microbial popu- 1988). The effects of methanogens on lation also affects the production of methane by cellulose degradation observed in vitro have ruminants. For example, in the absence of also been demonstrated in vivo in the rumen of protozoa (defaunated animals) the ruminal gnotobiotically-reared lambs (Fonty et al, methane emission is reduced, probably 1994). These shifts in the metabolism of because several species of ciliates produce fermentative or cellulolytic H2-producing hydrogen. Although the physical associa- microorganisms probably play a significant role tions between H2-producing species and in regulating the relative amounts of acetate methanogens are not essential for H2 transfer, and propionate formed in the rumen. close association between the two types of organisms may facilitate interspecies H2 transfer in the rumen (Itabashi et al, 1994; Possible solutions for the reduction of Miller, 1994). Attachment of methanogens to methane emission by ruminants the surface of rumen protozoa has been observed (Stumm et al, 1982; Krumholz et al, Considerable research efforts have been 1983). devoted to the manipulation of rumen fermentations with the aim of reducing methane production and improving ruminant Effects of methanogens on rumen productivity. A low methane-high propionate fermentation. Interspecies electron pattern in the rumen indicates a fermentation transfer with higher efficiency in terms of gross energy retained in non gaseous end-products (0rskov, By using H2 for CH4 production, methanogens 1975). Methanogenesis is a process which can maintain a low partial pressure of H2 (10-4 atm; be easily inhibited by many chemicals 0,01 kPa) in the rumen. The interaction (halogenated methane analogue, sulphite, between H2-producing fermentative bacteria nitrate, unsaturated long-chain fatty acids, and H2-using methanogens is called «inter- ionophore antibiotics etc ...). The effects of species hydrogen transfer» (lanotti et al, 1973) these compounds on rumen fermentation and and has a great influence on fermentation end- animal performance have been intensively products as well as on the ATP yield of reviewed and will not be detailed in this paper fermentative microorganisms. Interspecies H2 (see review of Van Nevel and Demeyer, 1988). transfer eliminates the formation of electron Current knowledge of intestinal fermentations sink products (ethanol, lactate) formed during in wood-feeding termites (Breznak, 1994) and the reoxidation of NADH in monocultures of the in colonic fermentations of non methane- fermenting microorganisms. For example, in producing human subjects (Durand and pure culture Ruminococcus albus produces Bernalier, 1993; Wolin and Miller, 1994) acetate, ethanol, H2 and C02- When co- suggests other strategies for reducing the cultured with a methanogen, its ethanol contribution of ruminants to the global methane production ceases while the production of H2, budget. In these ecosystems H2/CO-Ut’lizing acetate and ATP increases (Wolin and Miller, acetogenic bacteria are frequently the terminal 1988). The anaerobic fungi, which are electron-accepting organisms instead of hydrogen producers, also interact with methanogens. Acetogenic bacteria use the H2 methanogens. In vitro, the association between produced by the fermentative bacteria to fungi and methanogens leads to an increase in reduce C02 to acetate by the Wood-Ljungdahl the fungal biomass (Bernalier et al, 1990) and pathway (2CO2 + 4H2 -j CH3COOH + 2H20) to an increase in the rate and extent of (Ljungdahl, 1986). H2-CO-Utilizing acetogenic bacteria are strains, and usually higher than the ruminal2- H present in the rumen but it is difficult to predict concentration (Boccazzi et al, 1991; Mackie their H2-utilizing acetogenic activity in vivo and Bryant, 1994; Morvan and Fonty, since they are capable of utilizing numerous unpublished results). However, the threshold other substrates such as carbohydrates and value varies widely according to the strain and aromatic compounds for their growth (Mackie some acetogenic strains have a H2-threshold and Bryant, 1994; Rieu-Lesme et al, 1994; value comparable to those of methanogenic Morvan, 1995). The main H2-Using aceto- strains (Boccazzi et al, 1991; Morvan and genic species described to date are : Acetito- Fonty, unpublished results). Little is known of maculum ruminis, Eubacterium limosum, the kinetic parameters for H2-CO utilizing Clostridium pfennigii (Mackie and Bryant, ruminal acetogenic bacteria. 1994). Some other isolates which probably In a similar manner to methanogens, in belong to a new species are phylogenetically vitro, H2-using acetogenic bacteria are able to close to Clostridium coccoides, Rumino- interact synergistically with cellulolytic coccus (Peptostreptococcus) productus or H2-producing microorganisms (R. albus, Ruminococcus hansenii and are included in the R. flavefaciens, Neocallimastix frontalis) cluster XIV of Clostridium (Bernalier et al, (Bernalier et al, 1993; Morvan and Fonty, 1995; Rieu-Lesme et al, this issue). unpublished results). In their presence, the rate Acetogenic hydrogenotrophic bacteria are of cellulose breakdown is enhanced and the among the earliest bacterial species able to metabolism of the cellulolytics is shifted colonize the rumen after birth. In newborn towards a greater production of acetate. lambs they are present in quite high numbers Some other ruminal H2-Ut’lizing bacterial one after birth et (105-106 ml-1) day (Morvan al, species such as Wolinella succinogenes, In 1994). the adult ruminant, the acetogenic Selenomonas ruminantium, Anaerovibrio population, estimated by the most probable lipolytica which are not acetogenic, are also number et al, on (MPN) (Dore 1995) (growing members of the ruminal ecosystem (Stewart and is low H2-CO producing acetate) usually and Bryant, 1988). However, their ecological and variable 1995). Prins and (Morvan, role in hydrogen utilization in vivo remains to Lankhorst (1977) measured H2-CO-utilizing be elucidated. Sulfate-reducing bacteria are acetogenic activity by incubating ruminal present in the rumen in low numbers (Morvan contents with H2 and 14C2 0 and found that et al, 1995, unpublished data). They probably only 5% of the label in soluble product was do not make a contribution to incorporated into acetate. In contrast, Leedle significant utilization since the ruminal and Greening (1988) found high numbers of hydrogen concentration of sulfate is low and presumably acetogenic bacteria in the rumen (Durand Kawashima, Moreover, sulfate- of steers. The population was consistently 1980). reduction cannot be an alternative to higher in steers fed a high-grain diet than in because the accumulation of animals fed a high-forage diet (11.3 x 108 methanogenesis sulfurs is toxic for both the animal host and the versus 6.0 x 108 gv rumen contents). Thus, rumen microbes. acetogenesis could be an alternative for H2 utilization in the rumen. The problem for the microbiologist is to understand why acetogenesis outcompetes Conclusion methanogenesis in the human colon and the termite hindgut but does not in the rumen. This Herbivorous animals, especially ruminants requires an evaluation of factors which affect produce far more methane than other animals. the successful competition for hydrogen. Farm cattle represent by far the greatest Thermodynamics, hydrogen thresholds, kinetic methane-producing animal population on the parameters (V!, Km) and mixotrophy are some earth. However if related to the same level of of the factors involved. Acetogenesis is protein produced for man, small ruminants are thermodynamically possible under the in situ in all likelihood just as methanogenous. conditions of the rumen and could theoretically Reduction of methane emission by animal take place (Mackie and Bryant, 1994). populations is an extremely complex problem. Hydrogen thresholds for acetogenic bacteria Individual technical solutions (nature of are usually higher than those for methanogenic the diet, use of feed additives...) can help to reduce methanogenesis. Research is of methane relevance to atmosphere chemistry. necessary to establish a set of re- Proc Soc Nutr Physiol 3, 175 commendations on this subject. The microbial Crutzen PJ, Aselmann I, Seiler W (1986) Methane potential for a C02-acetate fermentation production by domestic animals, wild ruminants, other herbivorous fauna and humans. Tellus 33B, already exists in the rumen and biotechnology 271-284 could be applied to replace the methanogenic Gouet population of the rumen with the population Dore J, Morvan B, Rieu-Lesme F, Goderel I, which carries out the C02-acetate Ph, Pochart P (1995) Most pobable number enumeration of bacteria fermentation. More research is required to H2-utilizing acetogenic from the digestive tract of animals and man. understand the and of ecology physiology FEMS Microbiol Lett 130, 7-122 acetogenic bacteria and to devise practical Durand M, Kawashima R (1980) Influence of minerals methods for effecting a shift to the in rumen microbial digestion. In: Digestive in order hydrogenotrophic acetate production, physiology and metabolism in ruminants to increase the efficiency of ruminant (Ruckebush Y, Thivend P, eds) MTP Press production and decrease their contribution to Limited, Lancaster, 375-408 the global methane budget. Durand M, Bernalier A (1993) Reductive acetogenesis in animal and human gut. In: Physiological and Clinical Aspects of Short Chain Fatty Acid Literature cited Metabolism (Cummings JH, Rombeau JL, Sakaha T, eds) Cambridge University Press, Bauchop T, Mountfort DD (1981) Cellulose Cambridge, 107-1177 a in fermentation by rumen anaerobic fungus Fonty G, Gouet Ph, Jouany JP, Senaud J (1987) of rumen both the absence and presence Establishment of the microflora and anaerobic Environ Microbiol 42, 1101- methanogens. Appl fungi in the rumen of lambs. J Gen Microbiol 133, 1110 1835-1843 Bernalier A, G, Gouet Ph Cellulose Fonty (1990) Fonty G, Gouet Ph, Sant6 V (1988) Influence d’une degradation by two anaerobic fungi in bacterie methanogene sur I’activitb et le monoculture or in coculture with rumen bacteria. m6tabolisme de deux espèces de champignons Anim Feed Sci Tech 32, 131-136 cellulolytiques du rumen. Rbsultats pr6liminaires. Bernalier A, Fonty G, Gouet Ph (1993) Effect of Reprod Nutr Dev 28, 133-134 Eubacterium limosum, a ruminal hydrogenotropic Fonty G, Williams AG, Bonnemoy F, Morvan B, Gouet bacterium, on the degradation and fermentation Ph Effects of methanogens on microbial of cellulose by three species of rumen anaerobic (1994) activities in the rumen of fungi. Reprod Nutr Dev 33, 577-584 gnotobiotically-reared lambs. Proc Soc Nutr Physiol3, 178 Bernalier A, Rieu-Lesme F, Willems A, Fonty G, Sauvant Collins MD (1995) Diversity of H2/C0-utilizing Giger-Reverdin S, Vermorel M, D, (1992) acetogenic bacteria from the digestive tract of Facteurs de variations de la production de methane au cours de la des aliments animals and man. In: Proceeding of the digestion chez les ruminants. Ann Zootech International Symposium on Exploration of composes 41, Microbial diversity, Groslar, June 12-15, 37-38 Germany, lanotti EL, Kafkewitz D, Wolin MJ, Bryant MP (1973) Blaxter KL, Clapperton JL (1965) Prediction of the Glucose fermentation products of Ruminococcus amount of methane produced by ruminants. Brit J albus grown in continous culture with Vibrio Nutr 19, 511-522 succinogenes , changes caused by interspecies transfer of J Bacteriol 114, 1231-1240 Boccazzi P, Patterson JA, Wilsey BJ, Schaeffer DM H2. (1991) Hydrogen threshold values for one ltabashi H, Wahia Y, Takenaka A, Oda S, lshibashi T methanogenic and form acetogenic isolates from (1994) Effects of a controlled fauna on the rumen. In: Rumen fonction conference, methanogenesis, fibre digestion and rate of Chicago, USA, 37 growth in calves. Proc Nutr Soc Physiol3, 179 Boone DR, Whitman WB, Rouvibre P (1993) Diversity Krumholz LR, Forsberg CW, Veira DM (1983) and taxonomy of methanogens. In: Association of methanogenic bacteria with rumen Methanogenesis (Ferry JG, ed) Chapman of Hall, protozoa. Can J Microbiol29, 676-680 New York, London, 35-80 Leedle JAZ, Greening RC (1988) Postprandial Breznak JA (1994) Acetogenesis from carbon in changes in methanogenic and acetogenic termite guts. In: Acetogenesis (Drake HL, ed) bacteria in the rumen of steers fed high- or low- Chapman and Hall, New-York, 303-330 forage diets once daily. Appl Environ Microbiol Crutzen JP (1994) The role of ruminants as a source 54, 502-506 Ljungdahl LG (1986) The autotrophic pathway of Seiler W (1984) Contribution of biological processes acetate synthesis in acetogenic bacteria. Ann to the global budget of CH4 in the atmophere. In: Rev Microbiol 40, 415-450 Current Perspectives in Microbial Ecology (Klugg Lovley DR, Greening RC, Ferry JG (1984) Rapidly MJ, Reddy CA, eds) American Society for DC, 468-477 growing rumen methanogenic organisms that Microbiology, Washington synthetizes coenzyme M and has a high affinity Smith PH, Hungate RE (1958) Isolation and for formate. Appl Environ Microbiol48, 81-87 characterisation of Methanobacterium rumi- Mackie RI, Bryant MP (1994) Acetogenesis and the nantium n. sp.. J Bacterio175, 713-7188 rumen: syntropic relationship. In: Acetogenesis Stewart CS, Bryant MP (1988) The rumen bacteria. (Drake HL, ed) Chapman and Hall, New York, In: The Rumen Microbial Ecosystem (Hobson 331-364 PN, ed) Elsevier Applied Science, London, New Marvin-Sikkema FD, Richardson AJ, Stewart CS, York, 21-75 Gottschal JC, Prins RA (1990) Influence of Stumm CK, Cijzen HJ, Vogels GD (1982) Association hydrogen-consuming bacteria on cellulose of methanogenic bacteria with ovine rumen degradation by anaerobic fungi. Appl Environ ciliate. BrJNutr47, 95-99 Microbiol 56, 3793-3797 Thauer RK, Hedderich R, Fisher R (1993) Reactions Miller TL, Wolin MJ, Hongxue Z, Bryant MP (1986) and enzymes involved in methanogenesis from Characteristics of methanogens isolated from CO2 and H2. In: Methanogenesis (Ferry JG, ed) bovine rumen. Appl Environ Microbiol 51, 201- Chapman and Hall, London, 209-252. 202 Tyler SG (1991) The global methane budget. In: Miller TL (1991) Biogenic sources of methane. In: Microbial Production and Consumption of Microbial Production and Consumption of Greenhouses Gases, Methane, Nitrogen Oxides Greenhouse Gases, Methane, Nitrogen Oxides and Halomethanes (Rogers JE, Whitman WB, and Halomethanes (Rogers JE, Whitman WB, eds) American Society for Microbiology, American for eds) Society Microbiology, Washington DC, 7-38 Washington DC, 175-187 Van Nevel CJ, Demeyer Di (1988) Manipulation of Miller TL of methane and (1994) Ecology production rumen fermentation. In: The Rumen Microbial sinks in the rumen. Proc Soc Nutr hydrogen PN, Elsevier Applied 176 Ecosystem (Hobson ed) Physiol3, Science, London, New York, 387-443 Morvan B des (1995) Ecologie et physiologie Vermorel M Emissions annuelles de methane des (1995) microorganismes hydrog6notrophes les bovins en France. Etude de d’origine digestive par 6cosyst6mes digestifs. particuli6re Variations selon le d’animal et le niveau de ruminal. These de Doctorat, type 1’6cosystbme Prod Anim Université Claude Bernard, Lyon I, production. 8(4), 265-272 GD, JT, Van der Drift C Morvan B, Dore J, Rieu-Lesme F, Foucat L, Fonty G, Vogels Keltjens (1988) of methane In: Gouet Ph (1994) Establisment of hydrogen- Biochemistry production. Biology of Anaerobic (Zehnder AJB, ed) utilizing bacteria in the rumen of the newborn Microorganisms 707-770 lamb. FEMS Microbiol Lett 117, 249-255 Wiley, New-York, Woese CR, Kandler 0, Wheelis JL Towards a 0rskov ER (1975) Manipulation of rumen (1990) fermentation for maximum food utilization. World natural system of organisms, proposal for the Rev Nutr Diet 22, 153-182 domains Archaea, Bacteria and Eucarya. Proc Nat Acad Sci USA 87, 4576-4579 Patterson JA, Hespell RB (1979) Trimethylamine and Wolin Miller TL Microbe-Microbe methylamine as growth substrates for rumen MJ, (1988) bacteria and Methanosarcina barkeri. Curr interactions. In: The Rumen Microbial Ecosystem Microbiol3, 79-83 (Hobson PN, ed) Elsevier Applied Science, London, New-York, 343-359 Paynter JB, Hungate RE (1968) Characterisation of Methanobacterium mobilis, sp. nov., isolated Wolin MJ, Miller TL (1994) Acetogenesis from C02 in from the bovine rumen. J Bacteriol 95, 1943- the human colonic ecosystem. In: Acetogenesis 1951 (Drake HL, ed) Chapman and Hall, New York, London, 365-385 Sauvant D (1993) La production de methane dans la biosphere, le r6le des animaux d’61evage. Zinder SH (1993) Physiological ecology of Courrier de la cellule Environnement de I’INRA, methanogens. In: Methanogenesis (Ferry JG, ed) 18, 65-70 Chapman of Hall, New York, London, 128-206