Dietary, environmental and microbiological aspects ofmethane Production in ruminants

T. A. McAllisterl, E. K. Okine2,3, G. W. Mathison3, and K.-J. Chengl

j Research Centre, Agricutture and Agri-Food Canada, P. O. Bo,x 3000, Lethbridge, Alberta, Canada TIJ 481;2Atbefta Agricutture"iood and RuraiD";t;pr"rt,-obog-t ta st., Edmo-nton, AtQe.rta, Canada T6H 4P2; and

3Department of ngricuittiiit, iooa ana xutitioiitscierces, 310 ng-F?lcentre, IJniversity of Alberta, Edmonton,

Alberta, canada T6G 2p5. Contribution'No. sazesos, ieceived 1 May 1995, accepted 25 February 1996'

McAllister, T. A., okine, E. K., Mathison, G. w. and cheng, K.-J. 1996. Dietary, environmental. and microbiological aspects

of methane production in ruminants. Can. J. Anim. Sci. ft: n1-243. Methani gas is produced in the rumen by methanogenic

bacteria as a metabolic end product. The energy t.i.ur.o by bacteria in the.process-of meihane formation can be used for bacterial

cell formation. Methane formation acts as an electron sink into which the hydiogen from all ruminal microorganisms drains' allowing

a higher yield of adenosine triphosphate. Factors such as the type of carbohydrate in the.diet' level of feed intake, digesta passage

rate, presence of ionophores or lipids in the diet, and ambient temperature influence the emission of methane from ruminants'

Methanobreviborrrr rfp.appear to be the major methanogens in the rumen, but it is likely that phytogenetic analyses will identiff

new species. rne uiochemical reduction of iarbon dioxi-de to methane is well defined, and it has been shown that interspecles

hydrogen transfer between methanogens -o ,urninuitucteria prevents the. accumulation of reduced nucleotides and the inhibition

of feed digestion. The development of ,t ut.gi., to .itlgut. methane.production in ruminants, without causing a negative impact

on ruminant production, continues to be a major challJnge for ruminant nutritionists and microbiologists' Enhancement of the

reduction of carbon dioxide to acetate and direct lenetic"manipulation of methanogens are two interventions that may further

reduce methane losses of ruminants'

Key words: Methane, diet, ruminant, microbiology, methanogen

McAllister, T. A., Okine, E. K., Mathison, G. W. et Cheng, K.-J. 1996. Aspects 1li1-enJlire^s,lnvironnementaux et microbi-

ologiques de la production de m6thane chez les rumini'ntts. Can. J. Anim. Sci. 76: 231-243' Le m6thane est un produit du

m6tabolisme elabor6 Aun, t" *1n.n par les bacteries m6thanogdnes. L'6nergie d6gag6e dlns c-e processus peut 6tre utilis6e pour la

prolif6ration des cellules bact6riennes. r-a m6thano!6ndse agiicomme puits A'eteitrons dans lequel abouti I'hydogdne produit par

tous les micro-organrsmes du rumen, "r6unt

uinri ,in degagiment plus elev6 d'ATP. Le type de glucide utilise dans l'aliment' le

niveau de prise alimentaire, la dur6e du transit gastroinieslinal, la-prdsence d'ionophores ou de lipides dans l'aliment et la tem-

perature ambiante sont autant de facteurs qui infruent sur les 6misslons de methane par les ruminants' Methanobrevibacter sem-

ble 6tre le principal genre de micro-organism. t"lifr"""gere pr6sent dans le *men, bien que les recherches phytog6n6tiques

permettront sans douie d'identifier de nouvelles espdces.ies m6canismes de r6duction biochimique du bioxyde de carbone en

m6thane sont bien connus et on sait que le transfert interspecifique d'hydrogdne entre le-organismes m6thanogdnes et les bact6ries

du rumen empeche l,accurnulation de nucleotides r6duits;t I'inhibitio; de ia digestion. L'elaboration de strat6gies visant d r6duire

la formation du methane chez les ruminants, sans pour autant nuire aux performances des animaux, demeure un probldme de pre-

mier plan pour les nutritionnistes et pour les microbiologistes des.ruminants. La stimulation de la r6duction dl.9Ot en ac6tate et

la manipulation genetique directe des methanogdnes sJnt deux interventions qui pourraient contribuer ir r6duire davantage le

d6gagement de mdthane par les ruminants.

Mots cl6s: Methane, regime alimentaire, ruminant, microbiologiste, m6thanogdne

RUMINANTS AND METHANE PRODUCTION

Amount ProducedIt has been estimated that the world's population of rumi-nants produces 77 000 000 t of methane annually, whichconstitutes about 15% of total afmospheric methane emis-

sions (Crutzen et al. 1986; Lashof and Tirpak 1990; Moss

1993). Others have speculated that domestic animals are

responsible for l210Yo of the total methane production(Anonymous 1990; Crutzen 1995). Ruminants produce

about 97%o of the methane generated by domestic animals.

About 75% of this production is from cattle, with the

remainder arising from other ruminant animals, such as

water buffalo, sheep and goats (Crutzen et al. 1986). Given

a world population of approximately 1.3 billion cattle and

calves, each animal is assumed to produce 44 kg of methane

annually. These figures are comparable with our measure-

ments ;f methane-production of approximately 250 L d-l

cow-I, or about 65 kg yr-l (okine et al. 1989). Using our

Abbreviations: ATP, adenosine triphosphate; CHr-S-CoM, methyl-coenzyme M; DM, dry matter; DNA,deoxyribonucleic acid; H4MPT, tetrahydromethanopterin;

HS-CoM, coenzyme M; HS-HTP, 7-mercaptohep-

tanoyltheorine-phosphate; MFR, methanofuran; NADH,nicofinamide-adenine-dinucleotide; RNA, ribonucleic acid;

YFA, volatile fattY acids

231

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Animal typePopulationz

(x l0o)

Methaneproduction

(kg animal I yrl)

Totalmethane

production(t yrr)

232 CANADIAN JOURNAL OF ANIMAL SCIENCE

Table l. Methane production by domestic animals. humans and wildruminants in Canada

1970), which, together with high fermentation rates, mayinhibit methanogenic bacteria and rumen ciliates andincrease propionate production (Demeyer and Henderickx1967;Eadie etal.1970; Van Kessel and Russell 1995).

When intake is increased from maintenance to iwicemaintenance, total production of methane increases, but theamount of energy lost as methane per unit of feed consumeddecreases by 12_30% (Blaxter 196l). A comparison of 42forages indicated that as intake increased from 54.6 to 77.21g DM kg o.75, methane production decreased from 7.0o/o to63% of the gross energy of the diet (G. W. Mathison,unpubl. data). With highly digestible fibre sources like beetpulp, methane losses may fall to 4-5%o of gross energyintake (Kujawa 1994). Moe and Tynell (1979) also showedthat the production of methane was influenced by the natureof the carbohydrate fed, particularly at high feed intakes.Methane production increased when the proportion ofdle1ary roughage increased in steers fed varying proportionsof hay and com either at maintenance or at twice mainte-nance (Blaxter and Wainman 1964). Methane yield from theruminal fermentation of legume forages is generally lowerthan the yield from grass forages (Varga et al. lEgs). Inaddition, the stage of maturity, method of preservaiion,chemical treatment, and physical processing (e.g., chopping,grinding, and pelleting) affect methane production pei unitof forage digested. Methane production in ruminants tendsto rncrease with the maturity of the forage fed (Armstrong1960) and is higher when forage is dried than when it iiensiled (Srurdstol 1981). Energy loss though methane pro-duction is generally higher for coarsely chopped forigesthan for finely ground and (or) pelleted forages (Thomson1972; Hironaka et al. 1996). Chemical treatment of cerealstraws with NaOH or ammonia increases the volume ofmethane produced by wethers but reduces the production ofmethane relative to the intake of digestible organic maffer(Moss et al. 1994). From these studies, it can be concludedthat properties of the forage that decrease the rate of diges-tion or prolong the residency of feed particles within-therumen generally increase the amount of methane producedper unit of forage digested.

Increases in the intake of concentrates (comparison ofeight all-concentrate diets) from 40.0 to 68.a g DM kg-{.zsreduced methane production from 9.2%o to 5.3vo of erossenergy intake, a decline that is even greater tharithatobtained with an increase in forage intake (G. W. Mathison,unpubl. data). High-grain diets (> 90% concenrrare) fed atnear ad libitum intake levels may reduce methane losses to21o/o of gross energy intake (Hutcheson 1994; Johnson andJohnson 1995). Addition of readily fermentable carbohy-drates (e.g., cereal grain) to diets fed at maintenance levelscauses a proliferation in the ciliate population (Bonhomme1990). Ciliates are symbiotic with methanogens (Stummand Zwart 1986; Finlay et al. 7994), and the increase inmethane production when grain is fed at maintenance maybe due to an increase in hydrogen transfer between theslmicroorganisms (Krumholz et al. 1983). Removal of proto-zoa from the rumen reduces the production of methane insheep fed diets high in either starch or fibre (Kreuzer et al.1986). The principal fermentation products of protozoa areacetate and butyrate; therefore, the removal of these

Domestic livestockCattleSheep and goatsPigsHorses

Wild ruminantswHumansTotalTotal for domestic livestock

13.41.0

n.l0.68.3

27.7

65v8.0x1.5r

18.0x

I8.0x0.05r

871 0008 000

l6 650l0 800

t49 400| 370

) 05'7 220906 450

Production by Canadian domestic ruminants aspercentage ofglobal production by livestockv t.lzAdapted from Statistics Canada fl994).YAdapted fiom Okine et al. (1989).xEstimates adapted from Crutzen et al. ( I 9g6) and Adler ( I 994).wlncludes caribou, mountain goat, mountain sheep, muskoxen, wapiti deerand bison. This value is likely the leasr accurare oithose presenred. becauseof inaccuracies in population estimares and a lack olmeasurements ofmethane production from wild ruminants. population estimates fiom paynefi 989).vGlobal methane production by Iivestock (Adler 1994): 79 g00 000 vr l.

estimate, cattle in Canada produce about g71000 t ofmethane annually, or about 96% of the methane producedby livestock (Table l). Wild ruminants also contribute sub_stantially to methane production in Canada. Their produc_tion has been estimated at 149 000 t yr l, but the uc.u.ucyof this estimate is hampered by the inaccuracy of populationestimates and the lack of measurements of methane produc-tion by these animals. Canadian livestock produce about l%of the global methane production attributable to livestock.These estimates do not include the methane that is nroducedwhen manure is anaerobically fermented, as is thl case insome confined-livestock operations. Crutzen et al. 09g6)estimated that the global production of methane from cattleand sheep has been increasing by about 7o/o per year.However, further studies, taking into considerition ttreeffects of such factors as diet, feed intake, digesta passagerates and environmental temperature, are required to refineestimates of methane production from ruminints.

ENVIRONMENTAL FACTORS INFLUENCINGMETHANE PRODUCTION

Type of Feed and Level of Feed IntakeRuminants offered ad libihrm access to diets rich in starch(Orskov et al. 1968) or infixed with a single dose of a solublecarbohydrate, such as glucose (Demeyer and Van NevelI 975), tend to exhibit increased propionate production, lowermethane production, and a decreased acetate/propionateratio. This shift in rumen fermentation pattern his beenattributed to an increase in the rate of ruminal fermentation,which favours the production of propionate over methane(Demeyer and Van Neve|l975).In addition, feeding solublecarbohydrates or starch typically results in a rumen pHlower than that resulting from feeding forages (Eadie et al.

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microorganisms from the rumen may shift fermentationtoward propionate and decrease the formation of methane(Itabashi et al. 1984; Whitelaw et al. 1984).

Temperature and Digesta Passage RateIt is generally assumed that methane production decreases

with the increase in ruminal passage rates associated withcold adaptation. Graham et al. (1959) recorded a 20%o

decrease in methane production in adult sheep when the

temperature was reduced from 33oC to 8oC' Kennedy and

Miliigan (1978) also reported a 30o/o decrease in methane

production in cold-adapted sheep, while ruminal passage

iate constants of fluid and particulate matter increased by54 and 68o%, respectively. This inverse relationshipbetween methane production and passage rates is consistent

with the 29o/o decrease in methane production observedwhen the fractional passage rate of particulate matter was

increased 63% in steers by placing weights in the rumen(Okine et al. 1989). In addition, a decrease in the

acetate/propionate ratio in cold-acclimated sheep (Kennedy

and Milligan 1978) suggests a shift from methane to propi-

onate production (Fahey and Berger 1988). In contrast to

the trends and relationships reported in the past, VonKeyserlingk and Mathison (1993) observed that methane

production was 25To greater in sheep housed at 4'7"C thanin those housed at 2I"C. This result was related to an 8%o

increase in DM intake, although when expressed as a per-

centage of digestible energy, l4%o more methane was stillproduced in the cold environment (Von Keyserlingk and

Mathison 1993). Thus, cold environments may not alwaysdecrease methane production, especially when the intake ofthe diet is substantially increased. Rogerson (1960) foundthat the effects of temperatures between 20 and 40oC on

methane production were variable. Methane productionwas reduced at a high temperature when feed intake was

high, whereas at intakes below maintenance, temperature

did not affect methane production. The assumption cannot

always be made, therefore, that methane productiondecreases when ruminants are in cold environments.

McALLISTERETAL._METHANEPRoDUcTIoNINRUMINANTS2S3

MICROBIOLOGY OF RUMINAL METHANEPRODUCTION

Taxonomy, DiversitY and EcologyMethanogens are a distinct group of microorganisms, pos-

sessing unique cofactors (e.g., coenzyme M, HS-HTP, F420)

and lipids (e.g., isopranyl glycerol ethers). The cell

envelopes ofthese bacteria can contain pseudomurein, pro-

tein, gfucoprotein or heteropolysaccharides. The 165 rRNAnucleotide sequences ofmethanogens indicate an early evo-

lutionary divergence from the true bacteria. Therefore,

methanogens have been classified in a different domain, the

Archae (formerly Archaebacteria), within the kingdomEuryarchaeota (Balch etal.19791. Noll 1992).

Methanogens are fastidious anaerobes and grow only in

environments with redox potentials below -300 mV(Stewart and Bryant 1988). Sixty-six species of methanogens,

isolated from a variety of anaerobic habitats including sani-

tary landfills, acidic peat bogs, waterlogged soils, salt lakes,

thermal environments, and the intestinal tracts of animals,

have been described to date (Archer and Harris 1986; Mackie

et al. 1992). Only five of these species are isolates from the

rumen (Table 2), and only two, Methanobrevibacter rumi-

nantium (formerly Methanobacterium ruminantium, Smithand Hungate 1958) and Methanosarcina sp, have been found

in the rumen at populations greater than 1 x 100 ml--r (Rowe

et al. 1979; Lovley et al. 1984a; Miller et al' 1986).

Nonetheless, methanogens constitute a fundamental compo-

nent of the rumen microbiota, becoming established veryearly in the life of the ruminant (e.g., within 30 h of birth inlambs) (Morvan et al. 1994). New species from other anaer-

obic habitats are frequently discovered (Jones et al. 1987)'

and it is almost certain that phytogenetic analyses of ruminalmethanogens using 163 rRNA will identify new species.

Hydrogen, formate, acetate, methanol and mono-, di- and

tri-methylamine can all serve as substrates for methanogenesis

(Wolin and Miller 1987). Complete anaerobic bioconver-sion ecosystems (e.g., aquatic sediments, waste digesters)

convert all organic carbon to CO, and methane. Organic

Table 2. Methanogens isolated from the rumenz

Organism

Morphology, cellenvelope composition Energy source Reference

M e th an o b r ev i b a c t er rumi n antium

Methanobrevibacter sp.

M ethanos arcina b arkeri

Methanosarcina mazei

M et h a no b ac t eriu m fo r mic ic um

M ethanomicrob ium mobile

Short rods, requiresCoM, PS

Short rods, synthesizesCoM, PS

Irregular coccilarge clusters, HPS+PR

Cocci, HPS

Long rods and filaments,PS

Short rods, PR

Hr/formate

Hrlformate

Hrimethanolmethylamines/acetate

methanolmethylamines/acetate

H2lformate

Hrlformate

Smith and Hungate (1958)

Lovley et al. (1984a)

Beijer (1952)

Mah (i980)

Oppermann et al. (1957)

Paynter and Hungate (1968)

zAbbreviations: CoM, coenzyme M; PS, pseudomurein; HPS, heteropolysaccharide; PR, protein.

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234 CANADIAN JOUBNAL OF ANIMAL SCIENCE

Feed components

Wall

Fluminweus albusFib rob rcle r su ei n oge n e sR u m i nrcoau s I I av ef aci e n s

IiSimple Sugars Carbon Skele

Selernmonas ruminantiumTreponema bryantiiMegasphaera elsdenii

Primary andseconoaryfermenters

Biochemistry of MethanogenesisMethanogens convert carbon dioxide to methane throughfour reductive intermediates : formyl, methenyl, methylenyland methyl. These compounds are not present as free inter-mediates, and early investigators hypothesized that the one-carbon unit was attached to a series of carriers duringsequential reduction (Barker 1956).

To date, six coenzymes have been confrrmed as partici-pants in the reduction of carbon dioxide to methane (Fig. 2)(DiMarco et al. 1990). Fixation of carbon dioxide with MFRproduces formyl-MFR, the first stable intermediate ofmethanogenesis (reaction 1) (Leigh et al. 1985). The formylgroup is subsequently transferred to H.MPT (reaction 2),which serves as the carrier for formyl, methenyl, methylenyland methyl intermediates. Reduction of methenyl-ItMpTto methylenyl-HoMPT (reaction 4) and of methylenyl-H4MPT to methyl-HoMPT (reaction 5) is accomplished bythe reduced deazafTavtn, coenzyme Foro (Ferry 1992). priorto reduction to methane, the methyl group of methyl-H4MPT is transferred to coenzyme M (HS-CoM) (reaction6), an essential growth factor for some isolates ofMethanobrevibacter ruminantium (Taylor et al. 1974iLovley et al. 1984a). Coenzyme M is also used as a methylcarier by Methanosarcina barkeri during the metabolism ofacetate and methylamine (Lovley et al. 1984b; Naumannet al. 1984). Methyl-coenzyme M (CH.,-S-CoM) is reducedto methane by methyl-coen-4/me reducta-se, a complex systemof proteins and a number of cofactors (F430, ATP, HS-HTP,FAD) (reaction 7). This reaction completes the cycle and islinked to the activation of carbon dioxide to form formyl-MFR (Romesser and Wolfe 1982).

lEtarcFlI

Rumin&acleramylophifusStrcpteecus bovis

I

V

t Proteinl

Prevotellat ruminicolaButy tiv ib tb I ib ti so lve n s

I

PNHsV

Primaryfermenters

Acetate + Propionate + Butyrate + Hr+ C

Methanobrcvibaclerruminantium MgtnanOggns

Met h a no s arc i n a b ark e r iI

I

V

CH+

Fig. 1. Microbial fermentation in the rumen. Primary digestive microorganisms digest feed to simple monomers which are in tum utilizedby both primary and secondary fermenters. Methanogens prevent the aciumulation of hydrogen by reducing carbon dioxide to methane.

matter within these ecosystems is retained for weeks ormonths, permitting the persistence of the slow-growing(doubling times > 3 d) bacterial populations (e.g., sl,ntrophichydrogen-producing acetogens, acetotrophic methanogens)responsible for the complete conversion of acetate, propi-onate and butyrate to carbon dioxide and methane. In con-trast, the 1-2-d tumover of organic matter in the rumen istoo rapid for complete anaerobic bioconversion ofcarbon tocarbon dioxide and methane (Hobson and Wallace 1982).Substantial concentrations of acetate (60 mM), propionate(20 mM) and butyrate (10 mM) accumulate within the rumenand are absorbed from the digestive tract by the ruminant.

The anaerobic conversion of organic matter to methanein the rumen involves a consortium of rumen mlcroorsan-isms, with the f,rnal step effected by methanogens 1Fig. 1.1.

Primary digestive microorganisms, including bacteria, pro-tozoa and fungi, hydrolyze the proteins, starch and plant-cell-wall polymers, producing amino acids and sugars.These simple products are fermented to VFA, hydrogen andCOrby both primary and secondary digestive microorgan-isms. Methane is then formed by ruminal methanogensusing both hydrogen (80%) and formate (lg%; as iub-strates (Hungate et al. 1970; Whitman et al. 1992).Methanosarcina is an exception in that it produces methanefrom methylamines, methanol or acetate (patterson andHespell 1979; Rowe et al. 1979). This unique biochemicalability enables the slow-growing Methanoiarcina to flour-ish in ruminants fed diets containing ingredients likemolasses that promote the utilization of methylamines,methanol and acetate as substrates for methane production(Rowe et aI. 1979: Vicini et al. 1987).

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M)ALLISTERETAL._METHANEPRoDUcTIoNINRUMINANTS23S

(MFR)

CH3-S-CoM

HaMPT

Methyl-HaMPT H-?-H Tro

_!_tr?H\R

The reduction of CH.-S-CoM and 5,l0-methylenyl-H,MPT are exergonic rea6tions, and both have been impli-

caied as possible sources ofenergy for ATP synthesis (Ferry

1992).IiMethanosarcina spp., ATP synthesis is coupledtomethanogenesis by an electrochemical gradient (Blaut and

Gottschalk 1985; Blaut et al. 1985). The insensitivity ofMethanobrevibacter ruminantium to monensin and lasa-

locid (Chen and Wolin 1979) suggests this organism may

use substrate-level phosphorylation, rather than an electro-

chemical gradient, to generate ATP (Schonheit and

Beimborn 1985; Lancaster 1986).

Methanogens and Fibre DigestionAlthough methanogens do not produce fibrolytic enz)rynes.

they enhance the energetic efficiency and extent of fibredigestion by other ruminal microorganisms by preventing

the accumulation of reduced nucleotides (e.g., NADH)through interspecies hydrogen transfer (Joblin et al. 1989;

Pavlostatis et al. 1990; Williams et al. 1994). The classic

example of interspecies hydrogen transfer (Table 3) is the

co-culture of Ruminococcus albus ot R. flavefaciens and a

methanogen. When grown on cellulose in pure culture, R.

albus piduces acetate, hydrogen, carbon dioxide, and

ethanol, whereas R. flavefaciens produces acetate, succi-

nate, formate and hydrogen (Miller and Wolin 1973;

Latham and Wolin 1977). In pure cuhure, both bacteria

reoxidize NADH by producing reduced end products: R.

albus reduces acetyl-S-CoA to ethanol, arrd R. Jlavefaciensreduces oxaloacetate to succinate. Neither of these methods

lH,O <4'@/

gt\?:T; Methenyl-HaMPT

T o -'ux?'*-?-To _f>-!X?t F+zoo, r4aorcd

HS-CoM

HHll

MethYlenYl-Ha MPT

Fig. 2. proposed cycle for the reduction of carbon dioxide to methane by methanogens' The numbers refer to the seven steps of the cycle'

(Adapted from Rouviere and Wolfe 1988).

ofelectron disposal is thought to result in the production ofATP. In co-culture with a methanogen' NADH is oxidized

via NADH:ferredoxin oxidoreductase and ferredoxin

hydrogenase, and the resulting electrons are transferred to

.o"trryrn" Foro and used by the methanogen t-o reduce car-

bon dioxide io methane (Glass et al. 1977)' Low concen-

trations of hydrogen in the presence of a methanogen

promote the iransier of electrons to coenzyme Fotq, ut9'only

small amounts of ethanol and succinate are produced'

As a result, acetate production increases, and the ATP yieldof the cellulolytic bacterium and the methanogen is

enhanced (Wolin and Miller 1988). In these co-cultures,

more ATP is available for synthetic reactions, includingproduction of fibrolytic enzymes, and plant-cell-walldigestion is accelerated, in comparison with monocultures

of cellulolytic bacteria.The addition of Methanobrevibacter smithii to cultures of

R. flavefaciens or mixed cultures of R' flavefaciens and

Tr-eponima bryantii dramatically increased -the

rate and

extent of cellulose digestion (Beaudette 199a) Gig' 3)'

Williams et aL (1994) have recently shown similar

improvements in the digestion of xylan byco-cultwes of R'

Jlivefaciens and M. smithii, compared with R. flavefaciensgro*n in monoculture. Epifluoresgslgs rnicroscopy of the

io-culture showed colonies of M. smithii dispersed across

the entirety of the solid cellulose (frlter paper) substrate

and situated adjacent to colonies of R' /lavefaciens, whtch

likely facilitated interspecies hydrogen transfer (Beaudette

r994).

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236 CANADIAN JOURNAL OF ANIMAL SCIENCE

Table 3, Interspecies hydrogen transfer between methanogens and rumen microorganisms2

Subsrrate

lncubation Increase intime digestion(d) (%) Change in fermentation products Reference

Cellulolytic bacteriaRu mi n o c o c c u s .f I av eJ ac i e ns w tthMe t hano brev t baL t er N m i na n r i u m

Ruminococcus albus wrthM e th ano brevib ac ter sm i th r r

Non-cel lulol),tic bacteriaSe leno mo nas ru m i nantium withM. ruminantium

Anaerobic fungiNeoc a I I imas tix Jro nt alis w ithM. smithiiPiromyces communis withM. smithiiNeocallimastix spp. withM. ruminantium or M. smithii

N. frontalis withMe thano b ac terium b ryan ti i orM. smithii

Ne oc a I lim as tix patri ci arum withM. bryanti or M. smithii

P. communis with M. bryantii and,M. smithiiCaecomyces communisx withM. bryantii or M. smithii

N. frontalis with M. snithii

ProtozoaIsotricha spp. withMethanosarcina barkeri

Cellulose

Barley straw

Glucose

Barley straw

Barley straw

Cellulose

Cellulose

Cellulose

Cellulose

Cellulose

Xylan

Glucose

7d

l0 d

JO

t0d

l0 d

8.3 d

14d

t4d

14d

14d

4h

NM

t2.8

5.8

28

4.6

9

l4

r 5.3

6.6

NM

4.6

A, S.', FJ, H2J, CH4T

E<+, H2J, CH4T

AT, FJ, LJ, PJ, CH4T

rJ, lJ, Hr.l., cuot

EJ, LJ, H2J, cH4t

At, EJ, nJ, lI, HrJ, cHot

A1, FJ, FJ, LJ, sJ, H2t, cH4t

At, EJ, rJ, lJ, sJ, H2J, cH4t

Latham and Wolin (1977)

Joblin et al. (1989)

Chen and Wolin (1977)

Joblin et al. (1989)

Joblin et at. (1989)

Bauchop and Mountfort (1981)

Marvin-Sikkema et al. (1990)

Marvin-Sikkema et al. (1990)

Joblin et al. (1990)

Hillman et al. (1988)

A1, EJ, FJ, LJ, SJ, H2J, CH41, Marvin-Sikkema et al. (1990)

At, EJ, FJ, LJ, sJ, H2J, cH41

,q1, EJ, nJ, L.t, H2J, cH4t

- AJ. BJ, Pt. LJ. cH4t

zAbbreviations: NM, not measured; A, acetate; B, butyrate; E, ethanol; F, formate; P, propionate; S, succinate; 1 = increase; J = decrease: and <+ = no changein the amount ofproduct detected in the presence ofmethanosens.xFormerly Sphaeromonas communis, Gold et al. (1988).

Interspecies hydrogen transfer is not restricted to cellu-lolytic bacteria. Interactions with methanogens have beendocumented for non-cellulol1.tic bacteria (e.g., Selenomonasruminantium; Chen and Wolin 1977), as well as protozoa(Hillman et al. 1988; Finlay et al. 1994) and tungi (Joblinet al. 1989; Marvin-Sikkema et al. 1990) (Table 3).Methanogens have been shown to associate closely with thehyphae of fungi (Bauchop and Mountfort 1981), to adhere tothe pellicles of protozoa (Vogels et al. 1980; Stumm et al.1982), and to live in an endosymbiotic relationship withsome ciliates (Finlay etal. 1994). Rumen fungi and protozoaboth produce hydrogen in membrane-bound organellescalled hydrogenosomes (Yarlett et al. 1984; Yarlett et al.1986). Presumably, the physical proximity of methanogensto these organelles facilitates hydrogen transfer (Krumholzet al. 1983; Wolin and Miller 1988).

As with cellulolytic bacteria, co-culnring of several generaof rumen fungi with a variety of methanogens decreased theformation of reduced fermentation products (e.g., ethanol,

8EoE(EL

UEo(t,o=oO

80

60

40

20

0

-20

_-4z-r -..u

.--{tr-'.'..D

-j -O

R.f.R.f. + Ib.R.f. + M.s.

r- - -r R.f. + T.b. + M.s.

0 20 40 50 80 100

Time (hours)

Fig. 3. Percentage of cellulose degraded in cultures ofRuminococcus Jlavefaciens, (R.f)., co-cultures of R. flavefaciensand Treponema bryantii, (R.f. + T. b.), R. flavefaciens andMethanobrevibacter smithii (R.f. + M.s.) and tri-cultures of allthree bacteria (R.f. + T.b. + M.s.). (From Beaudette 1994).

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succinate and lactate) and enhanced (5-28%) the digestion

of cell-wall components (Marvin-Sikkema et al' 1990;

Joblin et al. 1990) (taUte 2). Stewart and Richardson (1989)

showed that co-culture with a methanogen diminished the

inhibitory effects of ionophores on ruminal firngi, and they

oostulated that the enhanced ATP synthesis in fungi-methanogen cultures offset the disruptive effects ofionophores on the proton motive force of the fungi'

GENETICS OF THE METHANOGENSMethanogens share genetic characteristics with both eukary-

otes and eubacteria. They possess a circular chtomosome,

ransins in size from 1.7 x 106 to 3.0 x 106 base pairs, com-

p-Ea irittr 4.1 x 106 base pairs for Escherichia coli (Brown

"t al. teal;. The structure of the genome is similar to that of

eubacteria, with genes arranged in operons, but complete

operator sequences have not been identified (Jones et al'

t'ggZ). Introns have been discovered in the tRNA genes and

rRNA genes of some members of the Archaea but not in

methanogens (Brown et al' 1989). As is characteristic of all

eubacterii, all RNA in methanogens is synthesized by a single

DNA-dependent RNA polymerase. However, the structure

of the methanogens' RNA polymerase is considerably more

complex than that of the eubacteria and, in many ways'

t"setttbl"t the RNA poll'rnerases of eukaryotes (Zillig et al'

1988; Hausner et al. 1991). Transcription is initiated

through a TATA box-like sequence' which is not unlike that

found in eukaryotic promoters. As in eubacteria, transcrip-

tion appears to be terminated through an inverted repeat

r"q.r"ni" that forms a stem-loop structure (Muller et al'198S). However, in some methanogens, transcription of the

rRNA operon is terminated by an oligo-thymidine sequence

similar to that found in eukaryotic rRNA and IRNA genes

(Mackie et al. 1992). The enzymological characteristics ofthe DNA polymerases and DNA gyrases (enzymes respon-

sible for DNA replication) are not consistent among

methanogens. For example, DNA polymerase ofMethanococcus vannielii is inhibited by aphidicolin, as is

the type A enzyme of eukaryotes (Zabel et al. 1987), but the

DNA-polymerase of thermophilic Methanob acterium ther-

moautotrophicum is insensitive to this inhibitor and resem-

bles the DNA polymerase I of E coli (Klimczak et al. 1986)'

Although most genetic information on methanogens has

been derived from non-ruminal species' some promisingresearch on ruminal isolates has been done. Several genes

from some of the less common ruminal methanogens have

been cloned into E coli, including the formate dehydroge-

nase gene from Methanobacterium formicicum (Shuber et al'

1986), an ATPase gene from Methanosarcina barkeri(Inatomi et al. 1989), and the heat-shock gene fromMethanosarcina mazei (Macario et al' 1991). Drug-resistant(e. g., 8-azaguanine, unisomycin) spontaneous mutant strains

of Methanosarcina barkeri and Methanobacterium formici-cumhave also been isolated (Hummel and Bock 1985; Knoxand Harris 1988). and these traits could serve as powerfulselective markers for the engineering of these strains.

There are no reports ofthe cloning of genes or the isolation

of mutant strains of the predominant ruminal methanogen,

Methanobrevibacter ruminanfium. However, functional vec-

tors and gene transfer systems, critical in any attempt to

develop rJcombinant microorganisms, have- been developed

for non-ruminal strains of methanogens' Numerous cryptic

nlasmids have been isolated from marine and thermophilic

methanogens, and phage-like particles have been shown to

infect Mbthanobrevib act er smithii and Met hanob ac t erium

thermoautotrophicum Sagle 1989). An insertion element

(ISMI) isolated ftom Methanobrevibacter smithii is of

iarticular interest to geneticists of rumen microbiology'iince Methanobrevibacter smithii shares a number of bio-

chemical properties with Methanobrevibacter ruminantium

(Stewart indRichardson 1989)' The insertion element is

i183 bure pairs long, and approximately 10 copies are pre-

sent in eu"h geno-" (Hamilton and Reeve 1985)' Their

unique biochemistry provides methanogens resistance to

many of the antibiotiCs (e.g., ampicillin, streptomycin and

tetracycline) commonly used as selectable markers in shrdies

with iecombinant eubacteria. Methanogens are sensitive to

bacitracin and pseudomonic acid, however, so introduction

into ISM1 of genes that confer resistance to these antibiotics

would produci a transposon potentially useful for the genetic

manipulation of Methanobr evib acter ruminanti um'

leihaps the most completely characterized plasmid from

a methanogen is pME200l from Methanobacterium ther-

moautotrophicum slrain Marburg (Bokranz et al' 1990)'

This nlasmid has been used to build constructs that confer

resistance to specific inhibitors and that replicate in eubac-

teria and yeist (Meile and Reeve 1985)' Recently,

Methanocolcus voltae and MethanococcLts maripaludis

were transformed using an integration vector containing the

Streptomyces alboniger pac gene, ,which codes for a

puromy"itt acetyl transferase and confers puromycin resis-

iance (Sandbeck and Leigh l99l)' However,. methanogens

transformed with this construct have not established a self-

replicating plasmid (Reeve 1992). By incubating recipient

celts with donor DNA, Methanobacterium thermoau-

totrophicum, Methanococcus maripaludis and Methano co ccus

vottie (Jordan et al. 1989; Sandbeck and Leigh 1991;

Bertani and Baresi 1987) have been induced to incorporate

heterologous genes into their chromosomes. Additionally,transduciion of chromosomal DNA between auxotrophicmutants and wild-type mutants of Methanobqcterium ther-

moautotrophicumhisbeen accomplished using phage ryMI '

However, the specifrcity of phage 'qMl to Methanobacterium

thermoautotrophicum strain Marburg may limit its use in

the genetic manipulation of other methanogens (Leisinger

and Meile 1990).

CHEMICAL AND BIOTECHNOLOGICALSUPPRESSION OF RUMINAL METHANOGENESISThe substantial loss of ruminant feed energy as methane has

prompted research on chemical methods of reducing rumi-

nal methane production (Prins et al. 1972)' Several com-

pounds, including chlorinated methane analogues (Bauchop

io6z;, pyro-eltiilc diimiae (Linn et al. 1982)' tricloroethyladipit€ (Clapperton 1977) and bromoethanesulfonic acid

(Gunsalus et al. 1978), are toxic to methanogens' However,

ruminal microbial populations in vivo have been found to

adapt to (Clapperton 1977) or degrade (Martin and Macy

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238 CANADIAN JOURNAL OF ANIMAL SCIENCE

1985) many ofthese compounds, and favourable effects onanimal performance have rarely been observed (Davies et al.1982; Demeyer et al. 1986).

Several methanogens are sensitive in the short term toionophores (Hilpert et al. 1981) but adapt to them after pro-longed exposure (Chen and Wolin 1979; Rumpler et al.1986; Saa et al. 1993). Ionophores inhibit merhanogens invitro, initially by suppressing the production of hydrogenfrom formate (Dellinger and Ferry 1984). However, asreduction of carbon dioxide remains a viable metabolic routefor the formation of methane (Van Nevel and Demeyer 1977),it is doubtful that this phenomenon signifrcantly affectsmethane production in the rumen. Ionophores are notablyinhibitory to bacteria that produce hydrogen and formate andless so to bacteria that produce succinate and propionate(Chen and Wolin 1979). Thus, ionophore-mediated suppres-sion of methane production by ruminants appears to resultmore from the inhibition of growth of microorganisms pro-viding subshates for methanogens, rather than a direct toxiceffect on the methanogens themselves (Stewart andRichardson 1989). This shift in fermentation products, com-bined with a ionophore-mediated improvement in feed effi-ciency, may reduce methane production by as much as 25o/o(Johnson and Johnson 1995).

Alternate electron acceptors (e.g., sutphate, nitrate, unsat-urated fatly acids) direct electron flow toward microorgan-isms other than methanogens (Czerkawski et al. 1966;Marty and Demeyer 1973; Allison and Macfarlane i988),hence, their addition to the rumen seems a logical means ofreducing ruminal methane production. This afproach is Ilr-ther suppofied.by the fact that reduction of nitrate (AGo'=

-163 KJ mol l) and sulphate (A6o'= -152 KJ mol l-1 arethermodl,namically favoured over carbon dioxide reduction(AG''=-130 KJ mol-l), which provides nitrate- and sulphate-reducing species a competitive advantage over methanogensfor hydrogen (Oremland 1988). Sulphate-, nitrate- andnitrite-reducing bacteria occur naturally in the rumen(Coleman 1960; Hungate 1966; Howard and Hungate 1976;Cheng et al. 1988), and experiments with sheep and cattleindicate that these bacteria adapt as the level of their respec-tive electron acceptors in the ruminant diet is increised(Alaboudi and Jones 1985; Gould et al. 1993). However,dietary intake ofnitrate often exceeds the reductive capacityof ruminal microorganisms, and excess nitrite is thenabsorbed through the rumen wall into the bloodstream.Nitrite is a potent vasoconstricter and oxidizes hemoglobinto methemoglobin, which does not transport oxygen (Deeband Sloan 1975), and nitrate and sulphate depress feedintake (Farra and Satter 1971; Nakamura et al. 19g1;Kandylis 1984). Thus, these compounds, even as altemateelectron acceptors, cannot be recommended for use asinhibitors of methane production.

Free fatly acids, particularly long chain, unsaturated fatfyacids, have been shown to inhibit methanogenesis andincrease the production of propionate both in vitro and invivo (Czerkawski et al. 1966: Van Nevel and Demeyer1981; Van der Honing et al. 1983). The fatty-acid-induceddecline in methane production was originally attributed topartitioning of available hydrogen between hydrogenation

of unsaturated fatly acids and reduction of carbon dioxide(Lennarz 1966). In the artificial rumen system, however,coconut oil (which contains high concentrations of Cr+,0 )inhibited methane production from concentrate and foragediets to a greater extent than did oils (e.g., canola oil and codliver oil) with high concentrations of unsaturated fatty acids(Dong et al. 1994). Long-chain fatty acids are directly toxicto methanogens, protozoa and gram-positive cellulolyticbacteria (Henderson 1973;Maczulak et al. 1981; Broudiscouet al. I 990), which accounts for the depressed fibre digestionand reduced ruminal acetate and butyrate production associ-ated with diets containing high concentrations of faffy acids(Van Nevel 199 1). Gram-negative, propionate-producingbacteria, however, are not significantly inhibited by fattyacids (Van Nevel and Demeyer 1988), thus the reduction inmethane production observed when fatty acids are includedin the diets of ruminants results primarily from a shifttoward the production of propionate.

The inhibitory effects of fatty acids on fibre digestionhave been partially overcome by feeding fatty acids as insol-uble calcium soaps (Elmeddah et al. 1991). Calcium salts ofstearic, palmitic or oleic acid did not affect the amount orrelative proportions of VFA produced in the rumen(Chalupa et al. 1986). The calcium salt of the unsaturatedfatty acid, linoleic, was found to undergo biohydrogenationin the rumen without altering fibre digestion (Fotouhi andJenkins 1992). The extent to which calcium salts of unsatu-rated fatty acids could serye as alternative hydrogen acceptorsand reduce ruminal methane production without adverselyaffecting digestion remains to be determined.

Although formation of methane is the predominantmethod of carbon dioxide reduction in the rumen. acetogenscapable ofreducing carbon dioxide to acetate have been iso-lated from rumen contents (Greening and Leedle 1989).Acetogens appear to be present in the mmen in numberssimilar to those of methanogens (Leedle and Greening1988) and are capable ofinterspecies hydrogen transfer withR. albus in co-culture (Miller 1995). However, formation ofacetate from carbon dioxide in rumen contents was notdetected in studies with laC-carbon dioxide (Prins andLankhorst 1977). Pinder and Patterson (1995) isolated anacetogen that was also capable of growth on glucose, raisingthe possibility that substrates other than hydrogen are uti-lized by acetogens competing with ruminal methanogens.The development of methods that shift fermentation from acarbon dioxide-methane fermentation to a carbon dioxide-acetate fermentation could reduce methane oroduction whileenhancing the production of an energetically valuable VFA.

The information available on gene regulation inmethanogens is inadequate. In the short term, more progress inbiotechnological research will likely be made in direct study ofthe ruminal methanogens than in alteration of methane pro-duction. The size and species composition of ruminalmethanogenic populations could be estimated with geneprobes, srmilar to those developed for the detection and poten-tial quantification of other ruminal bacteria, e.9., Synergi.stesjonesii (McSweeney et al. 1993) arLd Streptococcus bovis(Odelson et al. 1993). Genes encoding the subunits of CH.-S-CoM are highly conserved among methanogens lWeil 6t

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al. 1989) and are a logical choice for the development ofoligonucleotide probeJ for such a project. Application ofsigial amplificaiion methods (e.g., the polymerase chain

reiction) to species-specific probes developed for identifi-

cation of metfiuttog"ttic species will allow quantification ofthese organis-t uttd determination of the species proftle oftheir unique ruminal subpopulation. Many of the recombi-

nant DNA techniques necessary for characterization of gene

expression and regulation in methanogens are presently

being developed. As these systems are defined, new meth-

ods iimed at regulating the expression of specific genes in

methanogens may provide additional means of altering

ruminal methane Production.

CONCLUSIONSPresently, methane production is an inescapable conse-

quence of the fermentation of carbohydrates in the rumen'

The generation of methane in the rumen can be decreased by

prot*tittg a shift in fermentation toward propionate produc-

iion, butiannot be eliminated without adverse effects on

ruminant production. Production of methane by livestock is

a global ph.no-"non, to which the contribution made by

ro*ittuttti in Canada is practically insignificant. It is appar-

ent that a substantial reduction in methane emissions by

ruminants would require a global effort, but many of the

approaches currently promoted to curtail methane production

(e4., high-energy diets, ionophores, accelerated market fin-

istrlng strategiei) are impractical for developing countries,

where low-quality forages are the norm and mminalts are

used primarily for draught. Any effective strategy, whether itis chemical or biotechnological, must include the provision

of alternative electron acceptors within the rumen, or the

efficiency of fermentation will be compromised. Ultimately'the decision to pursue a substantial reduction in methane

emissions by ruminants could be an ethical one, in which the

value of ruminant conversion of cellulosic feeds unsuitable

for human consumption to high-quality protein sources (i'e',

meat and milk) muit be weighed against the contribution ofthe resultant methane emissions to global warming'

ACKNOWLEDGEMENTSThis review was undertaken as a result of financial support

from the Greenhouse Gases Initiative and the Alberta

Agricultural Research Institute. The authors thank K. Jakober

and.J. Kastelic for their insightfril comments on the maluscript'

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