REVIEW Open Access
The uminal mic obiome associa ed wi h
me hane emissions om uminan li es ock
Ilma Tapio
1
, Timo hy J. Snelling
2
, F ancesco S ozzi
3
and R. John Wallace
2*
Abs ac
Me hane emissions om uminan li es ock con ibu e signi ican ly o he la ge en i onmen al oo p in o ag icul u e.
The umen is he p incipal sou ce o me hane, and ce ain ea u es o he mic obiome a e associa ed wi h low/high
me hane pheno ypes. Despi e hei p ima y ole in me hanogenesis, he abundance o a chaea has only a
weak co ela ion wi h me hane emissions om indi idual animals. The composi ion o he a chaeal communi y appea s
o ha e a s onge e ec , wi h animals ha bou ing he Me hanob e ibac e go schalkii clade ending o be associa ed wi h
g ea e me hane emissions. Cilia e p o ozoa p oduce abundan H
2
, he main subs a e o me hanogenesis in he umen,
and hei emo al (de auna ion) esul s in an a e age 11% lowe me hane emissions in i o, bu he esul s
a e no consis en .Di e en p o ozoal gene a seem o esul in g ea e me hane emissions, hough communi y ypes
(A, AB, B and O) did no di e . Wi hin he bac e ia, h ee di e en ‘ umino ypes’ha e been iden i ied, wo o which
p edispose animals o ha e lowe me hane emissions. The wo low-me hane umino ypes a e gene ally cha ac e ized
by less abundan H
2
-p oducing bac e ia. A lowe abundance o P o eobac e ia and di e ences in ce ain Bac e oide es
and anae obic ungi seem o be associa ed wi h high me hane emissions. Rumen anae obic ungi p oduce abundan
H
2
and o ma e, and hei abundance gene ally co esponds o he le el o me hane emissions. Thus, mic obiome analysis
is consis en wi h known pa hways o H
2
p oduc ion and me hanogenesis, bu no ye in a p edic i e manne . The
p oduc ion and u ilisa ion o o ma e by he uminal mic obio a is poo ly unde s ood and may be a sou ce o a iabili y
be ween animals.
Keywo ds: A chaea, Me hane, Mic obiome, Rumen
Backg ound
Me hane is a g eenhouse gas (GHG) wi h a global
wa ming po en ial 28- old ha o ca bon dioxide [1].
Ag icul u e makes a signi ican con ibu ion o o al
GHG p oduc ion, wi h es ima es a ying acco ding o
coun y and calcula ion me hod [2]. None heless, a
global con ibu ion o be ween 7 and 18% o o al an-
h opogenic GHG emissions is gene ally accep ed [2].
Ruminan p oduc ion accoun s o abou 81% o GHG
om he li es ock sec o (calcula ed om H is o e al.
[2]), 90% o which esul s om umen mic obial me h-
anogenesis [3]. Ruminal CH
4
p oduc ion also ep esen s
a loss o ene gy ( om 2 o 12% o g oss ene gy in ake
[4]), which could in p inciple o he wise be a ailable o
animal g ow h o milk p oduc ion. Lowe ing CH
4
emissions he e o e would bene i he en i onmen and
possibly he e iciency o li es ock p oduc ion. Mo e
han 87% o he CH
4
p oduced by sheep has been
es ima ed o be de i ed om he umen [5], whe e a
popula ion o me hanogenic a chaea con e s he H
2
and CO
2
p oduced by a complex communi y o cilia e
p o ozoa, bac e ia and anae obic ungi o CH
4
[6, 7]. A
massi e wo ldwide esea ch e o has in es iga ed
a ious mi iga ion s a egies. Changes in managemen
p ac ices can be simple and e y e ec i e [2], while eed
addi i es ha migh inhibi H
2
p oduc ion, p o ide an
al e na i e me abolic H sink o inhibi he a chaea
hemsel es o e oppo uni ies beyond hose s aigh o -
wa d managemen changes [6–11]. O he oppo uni ies
include chemogenomics and immuniza ion [12–14].
One s a egy ha is o emos in se e al in es iga ions is
gene ic selec ion o he li es ock. I we can demons a e
ha pe sis en ly di e en CH
4
emissions in di e en
animals [14–16] can be explained by hei indi idual
* Co espondence: [email p o ec ed]
2
Rowe Ins i u e o Nu i ion and Heal h, Uni e si y o Abe deen, Fo es e hill,
Abe deen AB16 5BD, UK
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Tapio e al. Jou nal o Animal Science and Bio echnology (2017) 8:7
DOI 10.1186/s40104-017-0141-0
uminal mic obiomes, and ha he cha ac e is ic is
he i able, i should be possible o selec u u e gene a-
ions o uminan s ha ha e in insically lowe CH
4
emissions. All he s a egies po en ially in ol e changing
he uminal mic obiome. The aim o his sho e iew is
o assess ou cu en unde s anding o he ole o di e -
en membe s o he mic obiome in de e mining he
ex en o me hanogenesis in he umen.
The umen mic obial communi y
The umen is home o a as a ay o cilia e p o ozoa,
anae obic ungi, anae obic bac e ia and a chaea. The
p o ozoa can comp ise up o hal he umen mic obial
biomass [17, 18], he ungi we e o iginally es ima ed o
be abou 8% o he biomass [19] bu may each 20% in
sheep [20], he a chaea comp ise 0.3–4% [21] and he
bac e ia o m he emainde , ypically he la ges compo-
nen o he mic obial biomass. Ou p esen unde s and-
ing o uminal mic obiology was buil ini ially upon a
ew epoch-changing ad ances made many yea s ago:
G uby & Dela ond’s [22] mic oscopic obse a ions o
p o ozoa; Hunga e’s [23] app ecia ion o he anae obic
na u e o he umen ha led o new, uly anae obic cul-
u e echniques o he bac e ia; O pin’s [24] ealiza ion
ha some lagella e p o ozoa we e in ac zoospo es o
anae obic ungi, un il hen a con adic ion in e ms. The
isola ion and s udy o pu e cul u es was and emains in-
aluable in unde s anding he likely ole o di e en
species o bac e ia, p o ozoa o ungi in he o e all
e men a ion. D awbacks o cul i a ion echniques a e
ha only a e y small numbe o samples can be es ed,
and ha hey su e om bias, whe eby he composi ion
o he g ow h medium, gene ally oo ich, de e mines
which species can g ow [25]. De elopmen o molecula
echniques, based mainly on ssu RNA gene and in e -
genic space sequence ( o he ungi) analyses, opened
new oppo uni ies in umen esea ch. Cloning and
sequencing p o ided communi y analyses ha we e no
p one o he biases imposed by cul i a ion echniques,
al hough di e en bias was in oduced by o he ac o s,
like s o age condi ions [26], he di e en ial e iciency o
DNA ex ac ion om di e en species and ampli ica ion
bias [27–29]. Rela ed echniques o mic obiome analysis
quickly ollowed (DGGE, TGGE, T-RFLP, ARISA).
Quan i a i e PCR and FISH enabled mic obial g oups o
species o be quan i ied [30]. Now, me agenomic se-
quencing enables apid communi y analysis o be ca ied
ou , wi hou he cul i a ion bias o a ia ion associa ed
wi h p ime selec ion o PCR ampli ica ion i egula i ies
[25, 31]. The p oblem o DNA ex ac ion emains, how-
e e , and da abases a e ela i ely weak whe e uminal o -
ganisms a e conce ned [32]. Ne e heless, i we can use
his app oach o de e mine how he unc ional ac i i y
o he umen mic obial communi y in luences me hane
emissions, he knowledge should enable s a egies o
dec ease he en i onmen al impac o li es ock ag icul-
u e. Fu he mo e, i migh be expec ed o imp o e
animal p oduc ion e iciency.
Ruminal communi y analysis ela ing o me hane
emissions
A chaea
The e a e wo main ou es o me hanogenesis in he
umen, bo h ca ied ou by a chaea. The hyd ogeno-
ophic pa hway con e s H
2
and CO
2
p oduced by he
p o ozoa, bac e ia and ungi o CH
4
[3, 6]. I is usually
assumed ha o ma e, which can be used by all he mos
abundan uminal a chaea, is equi alen o H
2
+CO
2
,so
o ma e is included in he hyd ogeno ophic ca ego y
[21, 33]. A second ca ego y o subs a e o me hanogen-
esis is me hyl g oups, such as hose p esen in me hyl-
amines and me hanol [34, 35]. Me hylamines a e de i ed
om glycine be aine ( om bee ) and choline ( om plan
memb anes), while me hanol is de i ed om he
hyd olysis o me hanolic side-g oups in plan polysac-
cha ides. The mos common hyd ogeno ophic a chaea
a e om he genus Me hanob e ibac e , which has been
di ided in o wo subg oups, one known as he SGMT
clade (Mbb. smi hii,Mbb. go schalkii,Mbb. mille ae
and Mbb. haue i), he o he (RO) clade comp ising
p incipally Mbb. uminan ium and Mbb. olleyae [21, 36].
O he signi ican hyd ogeno ophic gene a include
Me hanosphae a, Me hanimic ococcus and Me hanobac-
e ium. The less abundan me hylo ophs (Me hanosa ci-
nales, Me hanosphae a, Me hanomassiliicoccaceae) can
use me hylamines and me hanol, and he e a e a chaea
(Me hanosa cinales) ha p oduce me hane ia he ace i-
clas ic pa hway ( e iewed in Mo ga i e al. [7]). Rumen
me hanogenic a chaeal di e si y is es ic ed o ou
o de s [21] and is highly conse ed ac oss 32 uminan
species collec ed wo ldwide [32].
In ui i ely, a chaea should be he mic obial g oup
mos closely co ela ed wi h me hane emissions. How-
e e , some s udies ha e shown no such co ela ion wi h
hei o e all abundance while in o he s he co ela ion
has been weak. Mo ga i e al. [37], Zhou e al. [38],
Danielsson e al. [39] and Danielsson [40] ound no
co espondence be ween he numbe s o me hanogens
and me hane emissions om dai y cows when measu ed
using me agenomics and qPCR echniques. Ki elmann
e al. [41] and Shi e al. [42] o med a simila conclusion
in sheep. A weak co ela ion be ween a chaeal abun-
dance ela i e o bac e ia was ound in bee s ee s [43]
bu none was ound wi h dai y cows in he RuminOmics
p ojec [h p://www. uminomics.eu/] when exp essed as
he a chaea:bac e ia a io (Fig. 1). Shi e al. [42] also
obse ed ha a chaeal gene exp ession a he han gene
abundance was co ela ed o me hane emissions om
Tapio e al. Jou nal o Animal Science and Bio echnology (2017) 8:7 Page 2 o 11
indi idual sheep. I is easy o see why gene exp ession
migh be a use ul p oxy o me hanogenesis in a s a ic
sys em like soil [44], bu less so in a lowing sys em like
he umen, whe e o physiological easons biomass
mus be di ec ly co ela ed o gene abundance unless
o he p ocesses, such as uncoupled CH
4
p oduc ion
occu [45].
Gi en he high a iabili y o he ela ionship wi h o e -
all a chaeal abundance, i may be ha he composi ion
o he a chaeal communi y a he han jus i s size may
ha e g ea e signi icance wi h ega d o me hane emis-
sions. Zhou e al. [38], Danielsson e al. [39], Shi e al.
[42] and Danielsson [40] all ound a posi i e co ela ion
be ween he ela i e abundance o Me hanob e ibac e
SGMT clade and me hane emissions. Danielsson [40]
in e p e ed his co ela ion in e ms o di e en a ini ies
o H
2
in he wo g oups, wi h he SGMT clade posses-
sing me hyl coenzyme M educ ase isozymes Mc I and
Mc II [12], which enables he a chaea o u ilise H
2
a
highe concen a ions, agains he RO clade ha possess
only Mc I [3, 12]. The dynamics o he o he a chaeal
communi y composi ion and hus he e iciency o H
2
u iliza ion would in u n would be a consequence o
di e ing H
2
p oduc ion by di e en bac e ia [33, 41] and
p esumably also p o ozoal and ungal communi ies.
Fu he mo e, he p opo ion o Me hanosphae a spp. in
o al a chaea was nega i ely associa ed wi h me hane
p oduc ion in sheep [41], al hough no in bee ca le
[46]. Thus, di e ing me hane emissions a e a leas
pa ly due o a ying ela i e abundances wi hin he
communi y o me hanogenic a chaea.
O he obse a ions ega ding he a chaeal communi y,
some imes called he a chaeome, include hose o Pi a
e al. [47], who ound ha a chaeal abundance inc eased
in s ee s su e ing o hy bloa , and Pei e al. [48], who
disco e ed a chaea associa ed wi h he umen epi he-
lium. In he o me case, he CH
4
con en o he gas was
no measu ed, so i is unclea he impac he bloa
would ha e on me hanogenesis. In he la e , he inding
was su p ising because he umen wall is conside ed o
be an ae obic/anae obic in e ace, and he ela i e abun-
dance o O
2
migh be conside ed o supp ess he g ow h
o he ex emely O
2
-sensi i e me hanogens. In ac , one
migh ha e possibly expec ed CH
4
oxidise s o be
p esen , in spi e o hei absence om he deep uminal
diges a [49].
Cilia e p o ozoa
Ruminal cilia es a e in ima ely in ol ed in me hanogen-
esis, pa ly ia hei abundan H
2
p oduc ion [50] and,
aking ad an age o his, hei associa ed me hanogens,
which a e ound bo h as in acy oplasmic commensals
and on he ex e io su ace o he p o ozoa [3, 18, 51–53].
Se e al s udies sugges ed a co ela ion be ween he
abundance o p o ozoa and me hane emissions (colla ed
in [18, 54, 55]), while o he s do no [37, 43]. Guyade e al.
[56] conduc ed a me a-analysis con aining 28 expe imen s
and 91 ea men s. This me a-analysis showed a linea
posi i e ela ionship be ween log
10
p o ozoal numbe s
and me hane emissions exp essed pe uni DMI. An
= 0.96 showed ha he e is indeed a easonably
s ong ela ionship (Fig. 2).
Fig. 1 A chaea:bac e ia ela i e abundance in ela ion o me hane emissions, p elimina y da a om he 1000-cow RuminOmics p ojec . Dai y cows on
di e en a ms h oughou Eu ope ecei ed g ass o maize silage:concen a e die s o simila nu ien composi ion. Feed in ake was measu ed ei he
di ec ly o calcula ed om aecal long-chain hyd oca bons. Samples o umen con en s we e emo ed by s omach ube and DNA was ex ac ed by
he Yu & Mo ison me hod [110]. Abundances we e calcula ed om qPCR o 16S RNA genes using uni e sal p ime s o a chaea and bac e ia
Tapio e al. Jou nal o Animal Science and Bio echnology (2017) 8:7 Page 3 o 11
De auna ion ( he emo al o he cilia es om he
umen) has he e o e been in es iga ed in ela ion o
me hane p oduc ion. Al hough in some cases he esul s
o de auna ion on CH
4
emissions ha e no been encou -
aging [57–60], Newbold e al. [18] ca ied ou a me a-
analysis o de auna ion s udies and concluded ha CH
4
was dec eased on a e age by 11%. Despi e he lowe CH
4
p oduc ion, he o al a chaeal abundance was no signi i-
can ly dec eased in he Newbold e al. me a-analysis,
sugges ing ha he a chaeal communi y in de auna ed
animals may ha e a lowe CH
4
-emi ing speci ic ac i i y
han ha o he p o ozoa-associa ed communi y.
As wi h he a chaea, he ques ions hen e e o
whe he some indi idual p o ozoal gene a o species,
and hei associa ed a chaea, a e mo e linked wi h me h-
anogenesis han o he s. In gene al, he p o ozoa ha bou
an a chaeal popula ion ha , like he gene al a chaeal
communi y, is domina ed by Me hanob e ibac e spp.
[61–64], al hough di e ences we e obse ed in he
abundance o di e en a chaea ound in he p o ozoa
and in he non-associa ed a chaea [18, 61, 65] ha migh
lead o di e en me hanogenic speci ic ac i i ies in he
wo popula ions. Fu he mo e, a chaeal colonisa ion
abundance may di e be ween di e en p o ozoal spe-
cies [51] and each may be associa ed wi h di e en
p edominan a chaeal gene a/species. Holo ichs in pa -
icula had an a chaeal communi y ha di e ed om
en odiniomo phid p o ozoa [53]. La ge cilia es appea
o be mo e hea ily colonized by me hanogens han
smalle cilia es [53, 66], and also by bac e ia, sugges ing
ha he e is no a selec i e colonisa ion by a chaea [53].
The lowe me abolic ac i i y in e ms o H
2
p oduc ion
o he la ge p o ozoal species pe uni biomass [50, 54,
58] p esumably explains ha smalle p o ozoa, and hei
associa ed a chaea, will be ela i ely mo e ac i e in
me hanogenesis han la ge species. Indeed, in i o
s udies indica ed ha he smalle En odinium spp. we e
mo e associa ed wi h me hane p oduc ion han la ge
species like Polyplas on mul i esicula um [50, 58]. In
i o s udies a e inconsis en , howe e . Re auna ion
expe imen s indica ed ha he abundance o En odinium
spp. [67, 68] o holo ichs [68] co ela ed wi h highe
me hane emissions. A la ge amplicon sequencing s udy
in sheep ne e heless ound no ela ionship be ween he
ela i e abundance o di e en cilia es and me hane
emissions [41]. Fu he mo e, cilia e communi ies all
in o a small numbe o ypes (A, AB, B and O [69])
depending on in e ac ions, p incipally in e -species
p eda ion. Despi e he la ge di e ences in ela i e
abundance o di e en p o ozoa ypes in he di e en
communi y ypes, me hane emissions could no be
co ela ed wi h p o ozoal communi y s uc u e [70]. The
a ying colonisa ion by a chaea depending on he ime
a e eeding [71] is ano he con ounding ac o in ying
o e alua e he ole o p o ozoa in me hanogenesis.
Bac e ia
Ruminal bac e ia o m he mos di e se g oup wi hin
he umen, capable o u ilizing ib e, s a ch, p o ein and
suga s [72]. Among nume ous bac e ial phyla ound in
di e en s udies, Fi micu es, Bac e oide es and P o eo-
bac e ia a e he mos abundan [32]. Fib oly ic bac e ia,
especially celluloly ic Ruminococcus and se e al Eubac-
e ium spp (Fi micu es), a e well s udied H
2
p oduce s.
On he o he hand, he p ominen celluloly ic genus,
Fib obac e , does no p oduce H
2
, while Bac e oide es
a e ne H
2
u ilize s [72]. Mic obiome analysis has iden i-
ied h ee di e en ‘ umino ypes’ ha seemed o be
associa ed wi h a ia ions in me hane p oduc ion by sheep
[41]. The low-CH
4
p oduc ion umino ype Q was cha ac-
e ised by high ela i e abundances o he p opiona e-
p oducing Quinella o alis.Low-CH
4
umino ype S had
Fig. 2 Rela ionship be ween me hane emission and umen p o ozoa concen a ion in a me a-analysis o 28 di e en expe imen s. The black dashed
line ep esen s he a e age wi hin-expe imen ela ionship. Rep oduced om [56] wi h pe mission
Tapio e al. Jou nal o Animal Science and Bio echnology (2017) 8:7 Page 4 o 11
highe abundances o lac a e- and succina e-p oducing
Fib obac e spp., Kandle ia i ulina,Olsenella spp., P e o-
ella b yan ii,andSha pea azabuensis. The high-CH
4
p oduc ion umino ype H had highe ela i e abundances
o species belonging o Ruminococcus, o he Ruminococ-
caceae, Lachnospi aceae, Ca abac e iaceae, Cop ococcus,
o he Clos idiales, P e o ella, o he Bac e oidales, and
Alphap o eobac e ia. The o e all in e p e a ion would be
ha me hane emissions depend on he abundance o he
H
2
-p oducing bac e ia p esen ; a co olla y o his is he
obse a ion ha chemical inhibi ion o me hanogenesis in
goa s led o inc eases in he abundance o H
2
-consuming
P e o ella and Selenomonas spp.[73]. P o eobac e ia we e
4- old less abundan (2.7 s. 11.2% o bac e ia) in high
emi ing bee ca le [46] and a simila inding was made in
dai y cows [40]. The dominan amily among P o eobac-
e ia was Succini ib ionaceae. This inding seems o
pa allel he high numbe s o Succini ib ionaceae in he
Tamma wallaby [74], which, like he uminan , is a
he bi o ous o egu e men e . I p oduces only abou
one- i h o he me hane pe uni o eed in ake o umi-
nan s, which is a ibu ed o he la ge communi y o
Succino ib ionaceae. An in iguing addi ional obse a ion
common o hese s udies [40, 41] was ha wi hin di e en
P e o ella OTUs, some we e co ela ed wi h a high CH
4
pheno ype, while o he s we e associa ed wi h low emis-
sions. The di e en OTUs seem o clus e oge he
(Fig. 3), sugges ing unc ional e sa ili y wi hin he
P e o ella genus. Fu he in es iga ion o he pheno-
ypes o hese dominan uminal bac e ia is needed,
which may well p o ide clues o u u e exploi a ion,
pa icula ly as some P e o ella a e epo ed o p oduce
o ma e [72].
In a dai y ca le s udy [75] wi h wo CH
4
-mi iga ing
eed addi i es, g apema c and a combina ion o lipids
and annins, i was ound ha he mic obiome di e ed
om he con ol die in a simila way. Faecalibac e ium
p ausni zii was o e - ep esen ed in he low- CH
4
die s,
and o he mic obiome ma ke s ha could be p edic i e
o low-CH
4
pheno ypes we e iden i ied. F. p ausni zii is
a bac e ial species ha is abundan in he human colon
[76] bu is seldom men ioned in he con ex o he
umen. I may p o e a use ul ma ke , bu i is no
ob ious how i s p ope ies could be mechanis ically
connec ed o he low-CH
4
pheno ype.
Anae obic ungi
The anae obic ungi, like he p o ozoa, p oduce abun-
dan amoun s o H
2
, along wi h CO
2
, o ma e and
ace a e as me abolic end p oduc s [77]. Six ungal gene a
ha e been de ec ed in he umen bu ecen molecula
esea ch sugges s exis ence o se e al new axa [78], wi h
unc ions s ill o be unde s ood. Me hanogens a e ound
in close associa ion wi h ungal hyphae [79]. Al hough
he e is eason o suppose ha ungal abundance migh
be ela ed o me hane emissions, epo s a e ew.
Ki elmann e al. [41] no ed no di e ence in ungal com-
muni y s uc u e in ela ion o me hane emissions om
sheep. In he RuminOmics p ojec , howe e , p elimina y
esul s sugges ha wo ungal species, Caecomyces
communis and Neocallimas ix on alis, a e nega i ely
ela ed o me hanogenesis ( = -0.50 and -0.45, P< 0.001;
R.J. Wallace e al., unpublished]. The me a-analysis o
Newbold e al. [18] no ed ha one o la ges e ec s o
de auna ion, which leads o lowe CH
4
p oduc ion, was
a dec ease in ungal abundance. Whe he his dec ease
is a majo o di ec cause o lowe CH
4
p oduc ion in
de auna ed animals is unclea .
Gene al conside a ions on a ia ions in me hanogenesis
and he mic obiome
Con ibu ion o non-hyd ogeno ophic me hanogenesis
The main subs a es o me hanogenesis in he umen
a e known o be H
2
+CO
2
, o ma e and compounds
con aining me hyl g oups like he me hylamines and
me hanol [21]. In he e iews al eady men ioned he e,
o ma e and H
2
+CO
2
a e usually conside ed o be
equi alen as subs a es o me hanogenesis and o ma e
is no ea ed sepa a ely. Fo ma e eeds di ec ly in o he
me hanogenesis pa hway a he e y beginning ia o -
ma e dehyd ogenase [80]. Hunga e e al. [81] es ima ed
ha 18% o me hane was o med ia o ma e a he han
H
2
+CO
2
. Ye he e a e some impo an aspec s o
o ma e me abolism abou which ou unde s anding is
incomple e. The ela ionship be ween bac e ial abun-
dances om mic obiome es ima es, abo e, was discussed
in ela ion o whe he bac e ia o m H
2
, as in o he ana-
lyses [33, 41, 43, 46], wi h li le indica ion abou o ma e
p oduce s. The e is a la ge unce ain y abou bac e ial
o ma e p oduc ion, e lec ed in he summa y ables o
S ewa e al. [72]. Al hough many species p oduce some
o ma e, p ecise amoun s a e no known and he e o e
he impo ance o his p oduc ion is di icul o es ima e.
Pe haps he Hunga e 1000 collec ion (www. mgne wo k.o g/
hunga e1000.h ml) could be used as a esou ce o make such
measu emen s. A p esen , he Hunga e 1000 p ojec has i s
emphasis on s eng hening gene ic da abases [3], bu much
pheno ypic in o ma ion is being collec ed alongside he main
h us o he p ojec . Assessing bac e ial o ma e p oduc ion
is u he complica ed by he knowledge ha co-cul u e
expe imen s demons a e ha he me abolism o some
bac e ia and ungi g own in he p esence o me hanogens
can be pulled in he di ec ion o H
2
o o ma e p oduc ion
[82–85], so i is e y di icul o be su e wha he ole o
di e en species migh be in he mixed umen communi y.
And pe haps mos c ucially, me hanogenesis is no
he sole a e o o ma e in he umen. Hunga e e al.
[81] no ed o ma e u ilisa ion in he absence o
Tapio e al. Jou nal o Animal Science and Bio echnology (2017) 8:7 Page 5 o 11

Fig. 3 Neighbo Joining ee o P e o ella-like OTUs ha had a nega i e (blue do s) o posi i e ( ed do s) ela ion o me hane (exp essed in e ms o
g me hane/kg DMI) in he 1,000-cow RuminOmics p ojec . Mul iple alignmen was done using MUSCLE [111]. The Neighbo Joining ee was cons uc ed
using p-dis ance and pai wise-dele ion pa ame e s. The ee was esampled1,000 imesandboo s ap aluesa eindica ed.The linea ized ee was
compu ed using MEGA 5.1 [112] by using mos abundan Bac e oidales OTUs o c ea e an “ou g oup”
Tapio e al. Jou nal o Animal Science and Bio echnology (2017) 8:7 Page 6 o 11
me hanogenesis, p esumably by bac e ia. Species like
Wolinella succinogenes use o ma e as an ene gy
sou ce [72]. So, al hough i is usually s a ed ha u-
minal a chaea u ilise ei he H
2
+CO
2
o o ma e [3],
i is unclea whe he hey a e indeed equi alen o
di e en a chaea. Fo example, in co-cul u es be ween
umen anae obic ungi and h ee me hanogens, all he
me hanogens used H
2
bu o ma e was only u ilised
simul aneously by M. smi hii [86]. The di e en ial exp es-
sion o o ma e dehyd ogenase was one o he la ges
di e ences be ween high- and low-emi ing sheep [42].
The o ma e dehyd ogenase o M. uminan ium M1 was
induced by co-cul u e wi h he o ma e-p oducing Bu y i-
ib io p o eoclas icus [12]. Thus he e a e se e al easons
o conclude ha hinking abou o ma e as a subs a e in
he con ex o mic obiomes di e ing in hei me hano-
genic ac i i y migh p o e ui ul. Fu he mo e, despi e
he emphasis on H
2
p oduced by cilia e p o ozoa, he
quan i y o o ma e p oduced seems o be many imes
g ea e han H
2
[65].
The me hylamines and me hanol a e me hyl dono s
o me hanogenesis by me hylo ophic a chaea, as
desc ibed abo e. Thei con ibu ion o me hanogenesis
will depend o some ex en on he concen a ion o
me hylamines in he die [34, 35]. Bu how e icien is
he p ocess? A e me hylamines con e ed quan i a i ely
o CH
4
, and a e me hylamine, dime hylamine and
ime hylamine equi alen in ha espec ? I is possible
ha a ia ion in CH
4
emissions be ween indi idual
animals on some die s may be due o di e en e icien-
cies whe eby me hylamines a e eleased om eed ma e-
ials and con e ed o CH
4
.
One o he mo e su p ising indings in he Mbb.
uminan ium M1 genome was he p esence o h ee genes
encoding alcohol dehyd ogenase [12]. I has been demon-
s a ed ha e hanol can be used as a C sou ce, bu no as
sole C sou ce [3]. Thus, he a ailabili y o e hanol om
bac e ial e men a ion may in luence he dependence o
a chaea on me hanogenesis o ATP p oduc ion, and
he e o e a ec he quan i y o CH
4
p oduced.
In luence o die and mi iga ion measu es
An impo an p inciple unde lying his e iew is ha
some mic obiomes lead o di e en CH
4
emissions when
o he ac o s emain cons an . Thus, key membe s o he
mic obiome leading o high o low emissions should be
able o be iden i ied. In he RuminOmics p ojec , all dai y
cows ecei ed die s ha we e as nu i ionally simila as
was possible gi en he di e en loca ions. Only by keeping
as many o he ac o s as possible unchanged will i be
possible o dissec he ole o di e en membe s o he
mic obial communi y in de e mining low- and high-
emi ing indi iduals. I should be no ed he e ha we ha e
chosen o exp ess CH
4
p oduc ion in e ms o DMI, o he
simple eason ha i makes i easie o iden i y a low-CH
4
mic obiome a he han a mic obiome ha o ms less CH
4
only because he hos animal ea s less.
The esul s o mic obiome analysis so a we e
expec ed in some espec s, in he sense ha die s high
in s a ch con en a e known o lead o lowe me hane
emissions, because s a ch u ilising bac e ia end o
p oduce less H
2
han o he s, o example [33, 72]. In a
simila way, he changed e men a ion s oichiome y
linked wi h me hane emissions is a e y long es ablished
obse a ion [87, 88]. New ques ions ha e been
highligh ed ega ding di e en species associa ed wi h
high and low CH
4
emissions unde simila condi ions.
Unexpec ed co ela ions ha e been ound. Bu many
ques ions emain. I is also wo h no ing ha widely
di e en axa may ha e simila me abolic ac i i ies [89],
so he e a e se e al di e en mic obio a ha could lead
o simila me abolic p ope ies.
Mi iga ion measu es ha e been desc ibed comp ehen-
si ely elsewhe e [2, 3, 6–10]. Pe haps he mos p omising
o hese is 3-ni ooxyp opanol, a molecule ob ained
a ionally by i s s uc u al simila i y o me hyl-CoM
[90–92]. As ye we do no know he ull implica ions o
3-ni ooxyp opanol, bu encou agemen can be ob ained
ha he conce n ha H
2
accumula ion migh inhibi
o e all e men a ion does no seem o be such a p oblem
as was sugges ed by some in i o expe imen s [33, 93]. I
is also wo h no ing ha a 50% educ ion in he g ow h
a e o me hanogens would be su icien o cause hei
washou om he umen [3, 33]. Comple e inhibi ion o
g ow h is he e o e no necessa y.
Me hane and eed e iciency
CH
4
p oduc ion and eed e iciency a e linked, in he
sense ha a low eed e iciency, exp essed as esidual eed
in ake (RFI), is accompanied by lowe CH
4
p oduc ion
[94–96]. The e e se does no apply, howe e , as has been
ound in dai y cows in he RuminOmics p ojec . The
indings ha he abundance o ce ain P e o ella changes
acco ding o eed e iciency in bee ca le [97, 98] and
many o he axa change in abundance [98] u he empha-
sises ou need o unde s and he ole o P e o ella and i s
di e en bio ypes on uminal e men a ion and me hano-
genesis. Shaba e al. [99] disco e ed ha Megasphae a
elsdenii was mo e abundan in low-e iciency cows, as
we e genes o he ac yla e pa hway, used by M. elsdenii in
p opiona e o ma ion. The explana ion o lowe e iciency
was ha M. elsdenii in oduced a ype o u ile cycle in he
p oduc ion and subsequen u ilisa ion o lac a e, an
ene ge ically ine icien p ocess.
The in luence o he hos animal
Many esea che s belie e, and some s udies a e begin-
ning o show, ha he hos animal exe s a con olling
Tapio e al. Jou nal o Animal Science and Bio echnology (2017) 8:7 Page 7 o 11
e ec on i s own gu mic obio a [100–102]. The mech-
anism could concei ably be a a molecula le el, pe haps
ia complex in e ac ions wi h ecep o s in he umen
wall [103, 104] o an ibodies in sali a [3, 105, 106]. Mo e
likely, howe e , is ha he physical s uc u e and dynam-
ics o gu diges a a e di e en in di e en animals.
Goopy e al [15] ound ha lowe me hanogenesis in
sheep was he i able and accompanied by he animals’
ha ing smalle umen olumes and he e o e al e ed
luxes o nu ien s h ough he ac . This would ha e he
e ec ha less eed would be e men ed in he umen,
leading o lowe me hanogenesis. Va ia ions in sali a
p oduc ion could lead o a simila esul [107]. Bo h
would likely in luence he uminal mic obiome. The e o e,
cau ion should be exe cised in in e p e ing mic obiome
analyses – he changed mic obiome may be associa ed
wi h, bu no cause, a dec ease in me hanogenesis.
Ross e al. [108] ound good co ela ions be ween CH
4
emissions and he b oad cha ac e is ics o he mic o-
biome. Now, me agenomics has shown ha he
abundance o ce ain g oups o mic obial genes can be
highly p edic i e o CH
4
emissions [46, 109] and eed
e iciency [99]. Fo example, 20 mic obial genes ex-
plained 81% o a ia ion in CH
4
emissions om bee
ca le, while 49 genes explained 86% o a ia ion in RFI
[109]. Fu he mo e, he animal’s gene ic backg ound was
a ac o in de e mining hese gene abundances [109].
This is he ea ly phase o wha is su e o be a e ile a ea
in which animal-mic obiome-emissions can be delin-
ea ed by me agenomics p o iling, and animal b eeding
based on hese gene abundances may lead o animals
wi h lowe CH
4
emissions.
Conclusions
Recen la ge scale p ojec s such as he Global Rumen
Census, he Hunga e 1000 and RuminOmics, om
which some p elimina y esul s a e p esen ed he e, ha e
p o ided new dep h o insigh in o he composi ion and
unc ion o he umen mic obial communi y. By e eal-
ing he some o he ela ionships be ween he mic o-
biome and he animal pheno ype, hey ha e shown how
unde s anding he ole o he umen mic obio a can
help in he e o s o educe he en i onmen al impac
o li es ock ag icul u e, in pa icula wi h he amelio -
a ion o g eenhouse gas emissions. The a chaea ha e
been he main a ge o esea ch, being di ec ly
associa ed wi h me hane p oduc ion in he umen. How-
e e , o he majo mic obial g oups such as he cilia e
p o ozoa, he anae obic ungi, Succino ib ionaceae and
P e o ella, among o he s, ha e shown o be associa ed
wi h bo h high and low me hane p oduc ion. The esul s
illus a e ha he e a e basic pheno ypic cha ac e is ics,
such as o ma e me abolism, ha a e insu icien ly
unde s ood. When placed in he con ex o he many as
ye uncul i a ed mic obial species o he umen, i
becomes clea ha he powe ul ool o molecula analysis
mus be accompanied by cul u al and me abolic/pheno-
ypic analysis i we a e o uly unde s and he ela ion
be ween he uminal mic obiome and me hanogenesis.
Abb e ia ions
ARISA: Au oma ed ibosomal in e genic space analysis; DGGE: Densi y
g adien gel elec opho esis; FISH: Fluo escence in i o hyb idiza ion;
qPCR: Quan i a i e polyme ase chain eac ion; TGGE: Tempe a u e g adien
gel elec opho esis; T-RFLP: Res ic ion agmen leng h polymo phism
Acknowledgemen s
The au ho s g a e ully acknowledge he wo k o , and discussions wi h, o he
membe s o he RuminOmics conso ium and hank hem o hei
pe mission o use p elimina y da a o illus a e his a icle.
Funding
The Rowe Ins i u e is unded by he Ru al and En i onmen Science and
Analy ical Se ices Di ision (RESAS) o he Sco ish Go e nmen . This s udy
was inancially suppo ed by Ruminomics (p ojec no. 289319 o EC 7 h
F amewo k P og amme: Food, Ag icul u e, Fishe ies and Bio echnology).
A ailabili y o da a and ma e ials
The au ho s a e bound by he Collabo a ion Ag eemen eached by he
RuminOmics conso ium, in which all da a gene a ed in he p ojec become
eely a ailable in Janua y 2018, bu canno be eleased wi hou consen o
he conso ium be o e hen. The o iginal da a unde pinning Fig. 1 and Fig. 3
a e subjec o hese condi ions. Indi idual applica ions o elease he da a
be o e 2018 should be di ec ed o he co esponding au ho .
Au ho s’con ibu ions
The au ho s w o e he manusc ip oge he , RJW ha ing ini ia ed he p ojec .
All au ho s ead and app o ed he inal manusc ip .
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
Consen o publica ion
All au ho s ha e gi en hei consen o submission o his a icle. The
submission has also been app o ed by he publica ions commi ee o he
RuminOmics conso ium.
E hics app o al
Animal expe imen a ion was conduc ed using p o ocols complying wi h
na ional legisla ion and app o ed by local e hics commi ees.
Au ho de ails
1
G een Technology, Na u al Resou ces Ins i u e Finland, Jokioinen, Finland.
2
Rowe Ins i u e o Nu i ion and Heal h, Uni e si y o Abe deen, Fo es e hill,
Abe deen AB16 5BD, UK.
3
PTP, Via Eins ein - Loc. Cascina Codazza, 26900
Lodi, I aly.
Recei ed: 31 July 2016 Accep ed: 3 Janua y 2017
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