Dietary, environmental and microbiological aspects of ...

Dietary, environmental and microbiological aspects of methane Production in ruminants T. A. McAllisterl, E. K. Okine2,3, G. W. Mathison3, and K.-J. Chengl Can. J. Anim. Sci. Downloaded from pubs.aic.ca by DIRECTORATE OF COLDWATER FISHERIES RES on 09/22/15 For personal use only.

j

Research Centre, Agricutture

481; and Agri-Food Canada, P. O. Bo,x 3000, Lethbridge, Alberta, Canada TIJ

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

Alberta,

environmental. and microbiological aspects McAllister, T. A., okine, E. K., Mathison, G. w. and cheng, K.-J. 1996. Dietary, gas is produced in the rumen by methanogenic Methani n1-243. ft: Sci. Anim. J. Can. in ruminants. of methane production used for bacterial

meihane formation can be bacteria as a metabolic end product. The energy t.i.ur.o by bacteria in the.process-of all ruminal microorganisms drains' allowing from hydiogen the which into sink electron as an acts cell formation. Methane formation level of feed intake, digesta passage in the.diet' carbohydrate a higher yield of adenosine triphosphate. Factors such as the type of of methane from ruminants' emission the influence temperature ambient and diet, in the or lipids rate, presence of ionophores analyses will identiff phytogenetic that likely it is but rumen, the Methanobreviborrrr rfp.appear to be the major methanogens in it has been shown that interspecles and defined, well is methane to dioxi-de of iarbon reduction new species. rne uiochemical inhibition ,urninuitucteria prevents the. accumulation of reduced nucleotides and the hydrogen transfer between methanogens causing a negative impact without in ruminants, methane.production .itlgut. to ut.gi., of ,t development of feed digestion. The the

-o

and microbiologists' Enhancement of on ruminant production, continues to be a major challJnge for ruminant nutritionists are two interventions that may further methanogens of direct and to acetate lenetic"manipulation reduction of carbon dioxide reduce methane losses of ruminants'

Key words: Methane, diet, ruminant, microbiology, methanogen et microbiMcAllister, T. A., Okine, E. K., Mathison, G. W. et Cheng, K.-J. 1996. Aspects 1li1-enJlire^s,lnvironnementaux Le m6thane est un produit du 231-243' 76: Sci. Anim. J. Can. rumini'ntts. les chez production m6thane de ologiques de la dlns c-e processus peut 6tre utilis6e pour la m6tabolisme elabor6 Aun, t" *1n.n par les bacteries m6thanogdnes. L'6nergie d6gag6e dans lequel abouti I'hydogdne produit par puits A'eteitrons agiicomme r-a m6thano!6ndse bact6riennes. cellules prolif6ration des Le type de glucide utilise dans l'aliment' le d'ATP. plus elev6 uinri ,in degagiment tous les micro-organrsmes du rumen, "r6unt ou de lipides dans l'aliment et la temd'ionophores la-prdsence gastroinieslinal, transit du la dur6e prise alimentaire, niveau de par les ruminants' Methanobrevibacter semmethane de 6misslons perature ambiante sont autant de facteurs qui infruent sur les que les recherches phytog6n6tiques bien *men, le pr6sent dans t"lifr"""gere genre micro-organism. de principal le ble 6tre du bioxyde de carbone en biochimique r6duction de m6canismes espdces.ies de nouvelles

permettront sans douie d'identifier entre le-organismes m6thanogdnes et les bact6ries m6thane sont bien connus et on sait que le transfert interspecifique d'hydrogdne L'elaboration de strat6gies visant d r6duire ia digestion. de I'inhibitio; r6duits;t du rumen empeche l,accurnulation de nucleotides des animaux, demeure un probldme de prela formation du methane chez les ruminants, sans pour autant nuire aux performances de la r6duction dl.9Ot en ac6tate et mier plan pour les nutritionnistes et pour les microbiologistes des.ruminants. La stimulation contribuer ir r6duire davantage le qui pourraient la manipulation genetique directe des methanogdnes sJnt deux interventions d6gagement de mdthane par les ruminants.

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

m6thanogdne

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-

RUMINANTS AND METHANE PRODUCTION

Amount Produced It has been estimated that the world's population of ruminants produces 77 000 000 t of methane annually, which constitutes about 15% of total afmospheric methane emissions (Crutzen et al. 1986; Lashof and Tirpak 1990; Moss

;f methane-production of approximately 250 L d-l cow-I, or about 65 kg yr-l (okine et al. 1989). Using our

ments

Abbreviations: ATP, adenosine triphosphate; CHr-SCoM, methyl-coenzyme M; DM, dry matter; DNA,

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

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

232

CANADIAN JOURNAL OF ANIMAL SCIENCE

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

Total Populationz (x l0o)

Animal type

Can. J. Anim. Sci. Downloaded from pubs.aic.ca by DIRECTORATE OF COLDWATER FISHERIES RES on 09/22/15 For personal use only.

Domestic livestock Cattle Sheep and goats

Methane production (kg animal I

13.4 1.0

n.l

Pigs Horses

0.6 8.3

Wild ruminantsw Humans Total

27.7

methane

production (t yrr)

yrl)

65v 8.0x 1.5r

871 000 8 000 l6 650

l0

18.0x

I8.0x 0.05r

|

)

Total for domestic livestock

800

t49 400 370 05'7 220 906 450

Production by Canadian domestic ruminants as ofglobal production by livestockv zAdapted from Statistics percentage

Canada

t.l

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 deer and bison. This value is likely the leasr accurare oithose presenred. because of inaccuracies in population estimares and a lack olmeasurements of methane production from wild ruminants. population estimates fiom payne

fi 989).

vGlobal methane production by Iivestock (Adler 1994): 79 g00 000

vr

l.

1970), which, together with high fermentation rates, may

inhibit methanogenic bacteria and rumen ciliates

and increase propionate production (Demeyer and Henderickx 1967;Eadie etal.1970; Van Kessel and Russell 1995).

When intake is increased from maintenance to iwice

maintenance, total production of methane increases, but the amount of energy lost as methane per unit of feed consumed decreases by 12_30% (Blaxter 196l). A comparison of 42 forages indicated that as intake increased from 54.6 to 77.21 g DM kg o.75, methane production decreased from 7.0o/o to

63% of the gross energy of the diet (G. W. Mathison,

unpubl. data). With highly digestible fibre sources like beet pulp, methane losses may fall to 4-5%o of gross energy intake (Kujawa 1994). Moe and Tynell (1979) also showed that the production of methane was influenced by the nature of the carbohydrate fed, particularly at high feed intakes.

Methane production increased when the proportion of dle1ary roughage increased in steers fed varying proportions of hay and com either at maintenance or at twice maintenance (Blaxter and Wainman 1964). Methane yield from the ruminal fermentation of legume forages is generally lower than the yield from grass forages (Varga et al. lEgs). In addition, the stage of maturity, method of preservaiion, chemical treatment, and physical processing (e.g., chopping,

grinding, and pelleting) affect methane production pei unit

estimate, cattle

in

Canada produce about g71000 t of methane annually, or about 96% of the methane produced by 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.ucy

of this estimate is hampered by the inaccuracy of population estimates and the lack of measurements of methane production 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 nroduced when manure is anaerobically fermented, as is thl case in some confined-livestock operations. Crutzen et al. 09g6)

estimated that the global production of methane from cattle

and sheep has been increasing by about 7o/o per year. However, further studies, taking into considerition ttre effects of such factors as diet, feed intake, digesta passage rates and environmental temperature, are required to refine estimates of methane production from ruminints.

ENVIRONMENTAL FACTORS INFLUENCING METHANE PRODUCTION

Type of Feed and Level of Feed Intake Ruminants offered ad libihrm access to diets rich in starch (Orskov et al. 1968) or infixed with a single dose of a soluble carbohydrate, such as glucose (Demeyer and Van Nevel I 975), tend to exhibit increased propionate production, lower methane production, and a decreased acetate/propionate

ratio. This shift in rumen fermentation pattern his been

attributed 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 soluble

carbohydrates or starch typically results in a rumen pH lower than that resulting from feeding forages (Eadie et al.

of forage digested. Methane production in ruminants tends

to rncrease with the maturity of the forage fed (Armstrong 1960) and is higher when forage is dried than when it ii

ensiled (Srurdstol 1981). Energy loss though methane production is generally higher for coarsely chopped foriges than for finely ground and (or) pelleted forages (Thomson 1972; Hironaka et al. 1996). Chemical treatment of cereal straws with NaOH or ammonia increases the volume of methane produced by wethers but reduces the production of methane relative to the intake of digestible organic maffer (Moss et al. 1994). From these studies, it can be concluded that properties of the forage that decrease the rate of digestion or prolong the residency of feed particles within-the rumen generally increase the amount of methane produced per unit of forage digested. Increases in the intake of concentrates (comparison of eight all-concentrate diets) from 40.0 to 68.a g DM kg-{.zs reduced methane production from 9.2%o to 5.3vo of eross

energy intake, a decline that

is even greater tharithat

obtained with an increase in forage intake (G. W. Mathison, unpubl. data). High-grain diets (> 90% concenrrare) fed at near ad libitum intake levels may reduce methane losses to 21o/o of gross energy intake (Hutcheson 1994; Johnson and

Johnson 1995). Addition of readily fermentable carbohydrates (e.g., cereal grain) to diets fed at maintenance levels causes a proliferation in the ciliate population (Bonhomme 1990). Ciliates are symbiotic with methanogens (Stumm and Zwart 1986; Finlay et al. 7994), and the increase in methane production when grain is fed at maintenance may be due to an increase in hydrogen transfer between thesl microorganisms (Krumholz et al. 1983). Removal of protozoa from the rumen reduces the production of methane in sheep fed diets high in either starch or fibre (Kreuzer et al. 1986). The principal fermentation products of protozoa are

acetate and butyrate; therefore, the removal

of

these

McALLISTERETAL._METHANEPRoDUcTIoNINRUMINANTS2S3


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

Temperature and Digesta Passage Rate It is generally assumed that methane production decreases with the increase in ruminal passage rates associated with cold 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 by 54 and 68o%, respectively. This inverse relationship between methane production and passage rates is consistent with the 29o/o decrease in methane production observed when the fractional passage rate of particulate matter was increased 63% in steers by placing weights in the rumen

In addition, a decrease in the ratio in cold-acclimated sheep (Kennedy acetate/propionate and Milligan 1978) suggests a shift from methane to propionate production (Fahey and Berger 1988). In contrast to the trends and relationships reported in the past, Von Keyserlingk and Mathison (1993) observed that methane production was 25To greater in sheep housed at 4'7"C than in those housed at 2I"C. This result was related to an 8%o (Okine et

al.

1989).

increase in DM intake, although when expressed as a percentage of digestible energy, l4%o more methane was still produced in the cold environment (Von Keyserlingk and

Mathison 1993). Thus, cold environments may not always decrease methane production, especially when the intake

of

the diet is substantially increased. Rogerson (1960) found that the effects of temperatures between 20 and 40oC on methane production were variable. Methane production was 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 production decreases when ruminants are in cold environments.

MICROBIOLOGY OF RUMINAL METHANE PRODUCTION

Taxonomy, DiversitY and Ecology Methanogens are a distinct group of microorganisms, possessing unique cofactors (e.g., coenzyme M, HS-HTP, F420) and lipids (e.g., isopranyl glycerol ethers). The cell

envelopes ofthese bacteria can contain pseudomurein, protein, gfucoprotein or heteropolysaccharides. The 165 rRNA nucleotide 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 kingdom

Euryarchaeota (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 sanitary 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 ruminantium (formerly Methanobacterium ruminantium, Smith and 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 very early in the life of the ruminant (e.g., within 30 h of birth in lambs) (Morvan et al. 1994). New species from other anaerobic habitats are frequently discovered (Jones et al. 1987)' and it is almost certain that phytogenetic analyses of ruminal methanogens 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 bioconversion ecosystems (e.g., aquatic sediments, waste digesters) convert all organic carbon to CO, and methane. Organic

Table 2. Methanogens isolated from the rumenz Morphology, cell envelope composition

Organism M e th an

o b

r ev i b a c t er rumi n antium

Short rods, requires

CoM,

CoM,

Reference

Smith and Hungate (1958)

Hrlformate

Lovley et al. (1984a)

PS

Short rods, synthesizes

Methanobrevibacter sp.

Energy source

Hr/formate

PS

M ethanos arcina b arkeri

Irregular cocci large clusters, HPS+PR

Hrimethanol methylamines/acetate

Beijer (1952)

Methanosarcina mazei

Cocci, HPS

methanol methylamines/acetate

Mah (i980)

Long rods and filaments,

H2lformate

Oppermann et al. (1957)

Hrlformate

Paynter and Hungate (1968)

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

ic um

PS

M ethanomicrob ium mobile

Short rods, PR

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

234

CANADIAN JOUBNAL OF ANIMAL SCIENCE

Feed components Wall

lEtarcFl

t Proteinl

I

Rumin&acler

Fluminweus albus

amylophifus

Fib rob rcle r su

Strcpteecus bovis

R u m inrcoau s I Iav efaci e n s

I

i

V

Prevotellat ruminicola Buty tiv ib tb I ib ti so lve n s

V

Simple Sugars

Primary fermenters

PNHs

I

I


ei n oge n e s

Carbon Skele

Selernmonas ruminantium

Primary and seconoary fermenters

Treponema bryantii

Megasphaera elsdenii

Acetate + Propionate + Butyrate + Hr+ C Methanobrcvibacler

ruminantium

Met h a no s arc i n a

b

MgtnanOggns

ark e r i

I I

V

CH+

Fig. 1. Microbial fermentation in the rumen. Primary digestive microorganisms digest feed to simple monomers which are in tum utilized by 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 or months, permitting the persistence of the slow-growing (doubling times > 3 d) bacterial populations (e.g., sl,ntrophic hydrogen-producing acetogens, acetotrophic methanogens) responsible for the complete conversion of acetate, propionate and butyrate to carbon dioxide and methane. In contrast, the 1-2-d tumover of organic matter in the rumen is too rapid for complete anaerobic bioconversion ofcarbon to carbon dioxide and methane (Hobson and Wallace 1982). Substantial concentrations of acetate (60 mM), propionate (20 mM) and butyrate (10 mM) accumulate within the rumen and are absorbed from the digestive tract by the ruminant. The anaerobic conversion of organic matter to methane in the rumen involves a consortium of rumen mlcroorsanisms, with the f,rnal step effected by methanogens 1Fig. 1.1. Primary digestive microorganisms, including bacteria, protozoa and fungi, hydrolyze the proteins, starch and plant-

cell-wall polymers, producing amino acids and sugars. These simple products are fermented to VFA, hydrogen and COrby both primary and secondary digestive microorganisms. Methane is then formed by ruminal methanogens using both hydrogen (80%) and formate (lg%; as iubstrates (Hungate et al. 1970; Whitman et al. 1992).

Methanosarcina is an exception in that it produces methane from methylamines, methanol or acetate (patterson and Hespell 1979; Rowe et al. 1979). This unique biochemical ability enables the slow-growing Methanoiarcina to flour-

ish in ruminants fed diets containing ingredients like

molasses that promote the utilization of methylamines, methanol and acetate as substrates for methane production (Rowe et aI. 1979: Vicini et al. 1987).

Biochemistry of Methanogenesis Methanogens convert carbon dioxide to methane through four reductive intermediates : formyl, methenyl, methylenyl and methyl. These compounds are not present as free intermediates, and early investigators hypothesized that the onecarbon unit was attached to a series of carriers during sequential reduction (Barker 1956). To date, six coenzymes have been confrrmed as participants in the reduction of carbon dioxide to methane (Fig. 2) (DiMarco et al. 1990). Fixation of carbon dioxide with MFR

produces formyl-MFR, the first stable intermediate of methanogenesis (reaction 1) (Leigh et al. 1985). The formyl group is subsequently transferred to H.MPT (reaction 2), which serves as the carrier for formyl, methenyl, methylenyl and methyl intermediates. Reduction of methenyl-ItMpT to methylenyl-HoMPT (reaction 4) and of methylenylH4MPT to methyl-HoMPT (reaction 5) is accomplished by the reduced deazafTavtn, coenzyme Foro (Ferry 1992). prior to reduction to methane, the methyl group of methylH4MPT is transferred to coenzyme M (HS-CoM) (reaction

6), an essential growth factor for some isolates of Methanobrevibacter ruminantium (Taylor et al. 1974i Lovley et al. 1984a). Coenzyme M is also used as a methyl carier by Methanosarcina barkeri during the metabolism of acetate and methylamine (Lovley et al. 1984b; Naumann et al. 1984). Methyl-coenzyme M (CH.,-S-CoM) is reduced to methane by methyl-coen-4/me reducta-se, a complex system of proteins and a number of cofactors (F430, ATP, HS-HTP, FAD) (reaction 7). This reaction completes the cycle and is linked to the activation of carbon dioxide to form formylMFR (Romesser and Wolfe 1982).

M)ALLISTERETAL._METHANEPRoDUcTIoNINRUMINANTS23S


(MFR)

CH3-S-CoM HaMPT

HS-CoM

l

HH ll

Methyl-HaMPT H-?-H

H,O Tro

gt \

_!_tr?H

R \