Wallace e al. Gene Sel E ol (2017) 49:9
DOI 10.1186/s12711-017-0285-6
REVIEW
Applica ion o me a-omics echniques
ounde s and g eenhouse gas emissions
o igina ing om uminal me abolism
Robe J. Wallace1*, Timo hy J. Snelling1, Ch is ine A. McCa ney1, Ilma Tapio2 and F ancesco S ozzi3
Abs ac
Me hane emissions om uminal e men a ion con ibu e signi ican ly o o al an h opological g eenhouse gas (GHG)
emissions. New me a-omics echnologies a e beginning o e olu ionise ou unde s anding o he umen mic obial
communi y s uc u e, me abolic po en ial and me abolic ac i i y. He e we explo e hese de elopmen s in ela ion o
GHG emissions. Mic obial umen communi y analyses based on small subuni ibosomal RNA sequence analysis a e
no ye p edic i e o me hane emissions om indi idual animals o ea men s. Few me agenomics s udies ha e been
di ec ly ela ed o GHG emissions. In hese s udies, he main genes ha di e ed in abundance be ween high and
low me hane emi e s included a chaeal genes in ol ed in me hanogenesis, wi h o he s ha we e no appa en ly
ela ed o me hane me abolism. Unlike he axonomic analysis up o now, he gene se s om me agenomes may
ha e p edic i e alue. Fu he mo e, me agenomic analysis p edic s me abolic unc ion be e han only a axonomic
desc ip ion, because di e en axa sha e genes wi h he same unc ion. Me a ansc ip omics, he s udy o mRNA
ansc ip abundance, should help o unde s and he dynamic o mic obial ac i i y a he han he gene abundance;
o da e, only one s udy has ela ed he exp ession le els o me hanogenic genes o me hane emissions, whe e gene
abundance ailed o do so. Me ap o eomics desc ibes he p o eins p esen in he ecosys em, and is he e o e a gu-
ably a be e indica ion o mic obial me abolism. Bo h wo-dimensional polyac ylamide gel elec opho esis and
sho gun pep ide sequencing me hods ha e been used o uminal analysis. In ou unpublished s udies, bo h me h-
ods showed an abundance o a chaeal me hanogenic enzymes, bu nei he was able o disc imina e high and low
emi e s. Me abolomics can ake se e al o ms ha appea o ha e p edic i e alue o me hane emissions; uminal
me aboli es, milk a y acid p o iles, aecal long-chain alcohols and u ina y me aboli es ha e all shown p omising
esul s. Rumen mic obial amino acid me abolism lies a he oo o excessi e ni ogen emissions om uminan s, ye
only indi ec in e ences o ni ogen emissions can be d awn om me a-omics s udies published so a . Anno a ion o
me a-omics da a depends on da abases ha a e gene ally weak in umen mic obial en ies. The Hunga e 1000 p ojec
and Global Rumen Census ini ia i es a e he e o e essen ial o imp o e he in e p e a ion o sequence/me abolic
in o ma ion.
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p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license,
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publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed.
Backg ound
Many e ms employ he ‘me a-’ p e ix and ‘-omics’ o
‘-ome’ su ixes. A guably, among all hese, he ou mos
ele an o he umen mic obial communi y and umi-
nal me abolism a e me agenomics, me a ansc ip om-
ics, me ap o eomics and me abolomics. All ou ake
ad an age o echnologies ha ha e only ecen ly become
gene ally a ailable. Me agenomics, he s udy o all he
genes p esen in he ecosys em, and me a ansc ip om-
ics, he s udy o ansc ibed genes, employ high- h ough-
pu DNA-sequencing, which has become inc edibly as
and inexpensi e o e he las decade. Me ap o eomics,
which ca alogues he o al p o ein complemen o he
communi y— he ansla ed genes—now uses high- es-
olu ion mass spec ome y o iden i y pep ides de i ed
om hese p o eins by sho gun hyd olysis. Me abolomics
Open Access
G
ene ics
S
elec ion
E olu ion
*Co espondence: [email p o ec ed]
1 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
Full lis o au ho in o ma ion is a ailable a he end o he a icle
Page 2 o 11
Wallace e al. Gene Sel E ol (2017) 49:9
uses a a ie y o spec oscopic and mass spec ome -
ic me hods and sepa a ion echniques o quan i y he
me aboli es ha a e p esen . Each o he me a-omics
echnologies ells us some hing di e en abou he
mic obial communi y and i s ac i i ies. He e we assess
how hey may help o p o ide e ec i e s a egies o mi i-
ga e he p essing en i onmen al p oblems associa ed
wi h g eenhouse gas (GHG) emissions om uminan
li es ock p oduc ion.
Re iew
Conce ns abou me hane andni ogen emissions
om uminan s
The 2006 publica ion [1] by he Food and Ag icul u e
O ganisa ion (FAO) and he Li es ock, En i onmen
and De elopmen Ini ia i e, ‘Li es ock’s Long Shadow,
En i onmen al Issues and Op ions’, ma ked a wa e -
shed in public and poli ical iews on li es ock and he
en i onmen . The ollowing highly emo i e pa ag aph
in he Execu i e Summa y encapsula es i s message—
“Li es ock’s con ibu ion o en i onmen al p oblems is
on a massi e scale and i s po en ial con ibu ion o hei
solu ion is equally la ge. The impac is so signi ican ha
i needs o be add essed wi h u gency. Majo educ ions
in impac could be achie ed a easonable cos .” Land
deg ada ion, wa e sho age and biodi e si y a e impo -
an , and also he a mosphe e and clima e change. Rumi-
nan s loom la ge in he las conce n, because hey, and
hei exc e a, p oduce la ge amoun s o me hane and
ni ous oxide emi ed o he a mosphe e. The epo con-
cluded ha he li es ock sec o is esponsible o 18% o
o al g eenhouse gas (GHG) emissions and 37% o o al
an h opogenic me hane, which is la gely esponsible o
he o al amoun . While he exac numbe s ha e a ied in
he in e im, and mo e aspec s o he whole sys em ha e
been ac o ed in o he models, i is clea ha uminan
me hane and ni ogen (N) emissions, which o igina e
la gely om umen mic obial ac i i y, mus be add essed
in ou e o s o limi clima e change.
The umen mic obial communi y andme hane
Mic obio a
The umen is home o a as a ay o mic obes om he
h ee g ea domains o li e. Thei abundance pe g o
diges a anges om 104 o 106 cilia e p o ozoa (al hough
some imes he e a e none), 103 o105 anae obic ungi,
1010 o1011 anae obic bac e ia and 108 o109 a chaea.
The p o ozoa can comp ise up o hal he umen mic o-
bial biomass, he ungi abou 7%, he a chaea 1 o4%
and he bac e ia o m he emainde . In a ecen publi-
ca ion [2], we e iewed he composi ion o he umi-
nal communi y ela ing o me hanogenesis. B ie ly, he
abundance o a chaea has only a weak co ela ion wi h
me hane emissions om indi idual ca le and sheep. The
composi ion o he a chaeal communi y appea s o ha e
a s onge e ec , wi h animals ha ha bou he Me ha-
nob e ibac e ium go schalkii clade ending o be asso-
cia ed wi h g ea e me hane emissions. Al hough cilia e
p o ozoa a e well known o p oduce H2 and ha bou
abundan a chaea, hei numbe s do no ha e a s ong
ela ion o me hane emissions. A me a-analysis o de au-
na ion e ealed me hane emissions o be on a e age 11%
lowe han in auna ed animals [3]. Me hane emissions
a e g ea e om uminan s ha ha e high abundance
o H2-p oducing bac e ia, and lowe when non-H2-p o-
duce s, such as Succino ib ionaceae, a e mo e nume -
ous. Indi idual axa co ela e wi h me hane emissions,
bu no necessa ily in he manne expec ed. Fundamen-
al ques ions ega ding he physiology and me abolism o
indi idual species, bo h cul i a ed and hose no ye cul-
i a ed, need o be add essed in o de o unde s and how
me hane emissions a e a ec ed by he mic obiome.
Me hane
Me hane is a GHG ha is 28 imes mo e po en han
CO2 [4]. A ound 90% o he me hane p oduced by umi-
nan s is de i ed om he umen [5], whe e me hano-
genic a chaea con e he H2 and CO2 p oduced by he
p o ozoa, bac e ia and ungi o me hane [6]. Wo ldwide
esea ch e o s ha e in es iga ed a ious mi iga ion
s a egies, pa icula ly eed addi i es ha migh inhibi
H2 p oduc ion, p o ide an al e na i e H sink o inhibi
he a chaea hemsel es [7–10]. O he s a egies include
chemogenomics and immuniza ion [11–13]. A s a egy
ha could be mos sus ainable, because o i s pe sis ence
and ease o implemen a ion, is gene ic selec ion o low
me hane-emi ing animals [14–16]. I i can be demon-
s a ed ha he di e en olumes o me hane emissions
om di e en animals can be explained by hei di e ing
uminal mic obiomes, and ha he p ope y is pe sis en
and he i able, i should be possible o selec u u e gen-
e a ions o ca le and sheep ha ha e gene ically de e -
mined lowe me hane emissions. Thus a , i has been
demons a ed ha me hane emissions in sheep [14, 15,
17], dai y cows [18] and bee s ee s [19, 20] a e signi i-
can ly he i able. Indeed, he p edic ion o me hane emis-
sions ia milk a y acid composi ion, as desc ibed below,
is he i able [21]. I had been expec ed ha lowe me hane
emissions would imp o e he e iciency o ene gy e en-
ion and he eby inc ease eed e iciency. Howe e , un o -
una ely ha la gely in ui i e p edic ion does no seem o
hold in p ac ice [22, 23], hus weakening he incen i e
o a me s o adop measu es ha would lowe me hane
emissions. Howe e , he e e se is undoub edly ue, i.e.
ha mo e e icien ca le will p oduce less me hane pe
uni p oduc (mea , milk), hus a ocus on eed e iciency
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Wallace e al. Gene Sel E ol (2017) 49:9
may be mo e ui ul, a he han simply me hane o N
emissions alone.
Ni ogen emissions
Ni ous oxide is abou en imes as po en a GHG as
me hane [1]. I is o med by mic obial deni i ica ion in
soil and in anae obic slu ies, bo h o which a e exace -
ba ed by he o e supply o die a y p o ein o ca le. The
quan i y o p o ein lowing om he umen is a majo
ac o ha limi s he p oduc i i y o uminan li es ock
p oduc ion [24, 25]. The p o ein eaching he aboma-
sum consis s o a mix u e o die a y and mic obial p o-
ein and, ollowing diges ion and abso p ion, i p o ides
he amino acids upon which uminan s depend o hei
amino acid equi emen s. Rumen wall issue p o ein
u no e also con ibu es o he p o ein d ain imposed by
uminal mic oo ganisms, because uminal bac e ia end
o in ade and diges uminal epi helial issues [26, 27].
In o de o compensa e o hese ine iciencies, uminan
li es ock p oduce s end o o e supply he animals wi h
ela i ely cheap p o ein sou ces such as soybean meal.
The excess N is exc e ed in u ine and aeces, which hen
p esen a disposal p oblem.
Ni ous oxide emissions a e equi alen o me hane
emissions in Sco land in e ms o GHG om ag icul u e
[28]. Ni ogenous exc e ion om uminan s is he e-
o e ano he a ea ha needs o be add essed. Pa o he
ine iciency s ems om he animal i sel , wi h ine icien
amino acid me abolism, bu he main ine iciency a ises
om he p o eoly ic and bac e ioly ic ac i i ies o umi-
nal mic oo ganisms [24, 25].
Rumen mic obial me agenomics andGHG emissions
The i s applica ion o he me agenome concep o he
umen mic obio a was gene mining, whe eby gene lib a -
ies ha we e sequenced om he o al DNA o uminal
diges a we e sc eened o a ge ac i i ies. This app oach
p o ed success ul in he disco e y and cha ac e isa ion
o many key mic obial enzymes such as glycosyl hyd o-
lases [29–33], polyphenol oxidases [34], and lipases [35,
36]. Du ing anno a ion o whole me agenomes in umen
s udies, i was appa en ha he majo i y o he open
eading ames (ORF) encoded genes ha we e unknown
o no ye included in e e ence da abases. Fu he mo e,
wi h he as majo i y o uminal species ye o be cul i-
a ed in i o [37, 38], he po en ial o me agenome min-
ing in he umen is as .
Pionee ing pape s o explo e he wide po en ial o
me agenomics applied o he umen we e hose by
B ulc e al. [39] and Hess e  al. [40]. B ulc e  al. [39]
we e he i s o epo he esul s o deep sequencing
o he uminal me agenome. They ocussed mainly on
glycosyl hyd olase sequence analysis, in a compa a i e
me agenomics exe cise ha was he i s o i s kind in
he umen. A compa ison o he glycosyl hyd olase and
cellulosome unc ional genes in diges a om h ee s ee s
e ealed ha , in he umen mic obiome, ini ial colo-
niza ion o ib e appea s o be by o ganisms ha pos-
sess enzymes ha a ack he easily a ailable side chains
o complex plan polysaccha ides a he han he mo e
ecalci an main chains, especially cellulose. In an in e -
es ing c oss-species compa ison, B ulc e al. [39] com-
pa ed hei umen da a wi h ha o he e mi e hindgu
mic obiome. Fundamen al di e ences in he glycosyl
hyd olase con en appea ed o be die -dependen , wi h
ca le consuming o ages and legumes compa ed o he
consump ion o wood by e mi es.
Hess e al. [40] we e also d i en la gely by he po en-
ial disco e y o new glycosyl hyd olases ha migh be
o alue in he bio uels indus y, bu hey demons a ed
also he dep h o new in o ma ion ha could be ex ac ed
om me agenomic deep sequencing. Only one cow was
used in his expe imen , ye he weal h o new disco -
e ies was immense. A leas i e ope a ional axonomic
uni s (OTU) we e en iched on he swi chg ass. None o
hese was iden i ied o be a cul i a ed species, indica ing
a majo oppo uni y o isola e he en iched species ha
by implica ion could be in ol ed in swi chg ass deg ada-
ion and he e o e be use ul in he bio uels indus y. Only
12% o he 27,755 ca bohyd a e-ac i e genes ha we e
assembled om he uminal me agenome o swi chg ass-
adhe en mic oo ganisms we e mo e han 75% iden ical
o genes deposi ed in he NCBI non- edundan da abase,
whe eas 43% o he genes had less han 50% iden i y o
any known p o ein. Nine y o he candida e p o eins
we e exp essed in i o, o which 57% we e enzyma ically
ac i e agains cellulosic subs a es. I migh be a gued
ha , since glycosyl hyd olases a e by a he bes cha ac-
e ised enzymes om he uminal ecosys em, e en mo e
no el y would be seen when mining enzymes wi h di -
e en unc ions ha a e impo an o uminal mic oo -
ganisms, such as p o ein o lipid me abolism. The gene
mining so a accomplished has ba ely sc a ched he su -
ace o such a complex enzyma ic ecosys em.
Pe haps he mos ema kable demons a ion o he
Hess e al. [40] analysis was he assembly o 15 bac e ial
genomes om uncul u ed species a comple eness ha
anged om 60 o 93%. The assemblies we e alida ed by
complemen a y me hods including single-cell genome
sequencing. This kind o genome assembly, by analysing
he genes ha a e p esen , can help us o unde s and he
me abolic ole and ecological niche o bac e ia ha ha e
ye o be cul i a ed.
Things a e now mo ing apidly in ela ing me agenom-
ics o me hane emissions. Denman and McSweeney [41]
and McAllis e e al. [7] published ex ensi e e iews less
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Wallace e al. Gene Sel E ol (2017) 49:9
han wo yea s ago, o which he eade is e e ed. Since
hen, se e al undamen al esea ch pape s ha e been
published using me agenomics o unde s and GHG emis-
sions. The me hanogenic a chaeal communi y and i s
gene complemen we e cha ac e ized by me agenomics
analysis in he bu alo umen [42]. Genes encoding all he
key s eps o me hanogenesis we e ound. Mo eo e , and a
po en ially signi ican inding was he disco e y o genes
in ol ed in he ace ogenesis pa hway, a possible al e na-
i e o me hanogenesis in he umen. Howe e , in goa s,
he con ibu ion o educ i e ace ogenesis in edi ec ing
H2 away om me hanogenesis was minimal, e en when
me hanogenesis was inhibi ed by b omochlo ome hane
[43]. Ins ead, genes in ol ed in p opiona e o ma ion ia
he andomizing pa hway, and numbe s o co espond-
ing bac e ia among P e o ella and Selenomonas spp.,
inc eased in he p esence o b omochlo ome hane, while
he genes in ol ed in me hanogenesis dec eased.
Ano he example s udy in bee demons a ed signi i-
can di e ences (P<0.05) in he abundance o 21 o he
mos nume ous (>0.1%) genes when he umen mic obial
me agenomes om high and low me hane-emi ing bee
s ee s we e compa ed [44]. Eigh o he nine mos sig-
ni ican ly di e ing genes we e associa ed wi h me hane
me abolism, bu he o he s we e no . Indeed, hei link
wi h me hanogenesis was no ob ious. The abundance
o he 21 genes in o al explained 88% o he a ia ion in
me hane p oduc ion, hus possibly o ming he basis o
gene ic selec ion o animals wi h a low-me hane geno-
ype. The same expe imen s showed ha si e-p ogeny
g oups di e ed in hei me hane emissions. Fu he anal-
ysis [20] demons a ed ha he abundance o 49 genes
explained 86% o he a ia ion in eed e iciency. Once
again, he easons ha unde lie hese co ela ions we e
no ob ious, al hough i was no ed ha hos -mic obio a
c oss alk gene exp ession (TSTA3 and FucI) we e signi i-
can ly associa ed wi h eed e iciency. These esul s sug-
ges , as p oposed by Taxis e al. [45], ha u u e s udies
o he whole animal-gu mic obiome ne wo ks hold high
p omise o unde s anding he ‘supe o ganism’ [8].
A signi ican ecen pape on uminal me agenom-
ics explo ed eed e iciency in dai y cows [46]. Me hane
emissions we e also measu ed ex i o, while me agen-
omes we e s udied om deep sequencing [46]. Species
di e si y was lowe in he mo e e icien animals, as was
gene di e si y (Fig. 1). Mo eo e , me hane emissions
we e also signi ican ly lowe in he e icien animals,
as was ound p e iously in ca le [47]. Ruminal diges a
con ained mo e p opiona e, bu y a e and iso ale a e in
e icien animals. Mos s iking o all, me abolic pa h-
way analysis showed ha genes o he non- andomizing
ac yla e pa hway o p opiona e p oduc ion we e much
mo e p e alen in he e icien ca le. The ac yla e pa h-
way is ound p incipally in he dis inc i e, la ge G am-
nega i e coccus, Megasphae a elsdenii, which has been
iden i ied wi h a s abilising e ec on uminal e men a-
ion because o i s apid con e sion o lac a e o p opi-
ona e and bu y a e [48, 49]; M. elsdenii also p oduces
iso ale a e and ale a e as end-p oduc s o i s amino
acid- e men ing abili y [50, 51]. RNA gene amplicon
analysis showed ha M. elsdenii abundance was much
g ea e in e icien animals, co esponding o he ac yla e
gene abundance. In he s udy by Wallace e al. [44] in
bee ca le, al hough no epo ed in he pape i sel , he
abundance o M. elsdenii was 13- old highe in he low-
me hane s ee s, hus en i ely consis en wi h he esul s
o Shaba e al. [46]. M. elsdenii has been ialled wi h
some success as a p obio ic o uminan s on he g ounds
Fig. 1 Communi y pa ame e s o e icien and ine icien cows’ mic obiomes ( om Shaba e al. [46]). a, b Mic obiome ichness wi h coun s calcu-
la ed and exp essed as simple ichness: a Species (based on 16S RNA amplicon sequencing) and b genes (based on me agenomics sequencing).
Ke nel densi y o he e icien and ine icien his og ams emphasizes he di e en dis ibu ion o coun s in each mic obiome g oup. P alues o he
di e ence in ichness be ween e icien and ine icien cows a e shown
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Wallace e al. Gene Sel E ol (2017) 49:9
o i s pH-s abilizing p ope ies [52, 53]. Thus, hanks o
hese s udies, a pic u e is eme ging whe eby i can be
seen ha di e ences in he abundance o H2-p oducing
bac e ia, non-H2-p oducing bac e ia and H2 u ilise s,
oge he wi h he abundance o pH-s abilizing bac e ia,
a ec he quan i y o me hane ha a uminan animal
p oduces and i s eed e iciency.
The Hunga e 1000 p ojec andGlobal Rumen Census
Thus a , combined unde s anding o unc ion and phy-
logene ic iden i y in me agenomics da a has been lim-
i ed by he ela i ely ew comple ed umen bac e ial
genomes and in u n by he numbe o anno a ed genes
and p o ein sequences o uminal species. This issue is
being add essed by he Hunga e 1000 p ojec (www.hun-
ga e1000.o g.nz). The p ojec i le e e s o he pionee ing
wo k in cul u ing s ic ly anae obic uminal bac e ia ca -
ied ou by Robe E. Hunga e [54]. The aim o he p o-
jec is o p oduce a e e ence se o 1000 umen mic obial
genome sequences om cul i a ed umen bac e ia and
me hanogenic a chaea, oge he wi h ep esen a i e
cul u es o umen anae obic ungi and cilia e p o o-
zoa. The p ojec is unded by he New Zealand Go e n-
men in suppo o he Li es ock Resea ch G oup o he
Global Resea ch Alliance on Ag icul u al G eenhouse
Gases. The sequencing e o ob ained suppo om he
US Depa men o Ene gy Join Genome Ins i u e Com-
muni y Sequencing P og am, and he o e all p ojec is
a global collabo a ion be ween membe s o he Rumen
Mic obial Genomics Ne wo k, es ablished o accele -
a e knowledge de elopmen and mi iga ion solu ions in
he umen mic obial genomics esea ch a ea. The e e -
ence genome in o ma ion ga he ed will be used o acili-
a e genome-enabled esea ch aimed a unde s anding
umen unc ion in o de o ind a balance be ween ood
p oduc ion and GHG emissions, and o suppo in e na-
ional e o s o de elop me hane mi iga ion and umen
adap a ion echnologies. Once he Hunga e 1000 p ojec
is comple ed, genes disco e ed om deep me agenome
sequencing will be able o be pinned wi h much g ea e
ce ain y o known species.
The mos ex ensi e explo a ion o he uminal mic o-
biome ha was ecen ly published was he Global Rumen
Census, an in e na ional e o ha analysed he mic o-
bial communi y in 742 samples om 32 animal species
om 35 coun ies [55]. The esul s e ealed a common
co e mic obiome in all samples, and p omp ed he con-
clusion ha signi ican new axonomic g oups we e
unlikely o be disco e ed. The au ho s also commen ed
on likely unc ional edundancy, wi h di e en axa
pe o ming essen ially he same unc ion using ela ed
genes, a opic ha has also been e iewed ecen ly [56].
Indeed u u e unde s anding o umen unc ion, and
i s ela ionship wi h he hos genome, is likely o be
expanded mos signi ican ly by explo ing and linking
gene ne wo ks [45]. An impo an o e all conclusion
o he Global Rumen Census was ha die , a he han
gene ics o geog aphical loca ion, had he g ea es in lu-
ence on he uminal mic obiome.
Me a ansc ip omic analysis
Simila o me agenomics, me a ansc ip omics was used
i s as a ool o gene mining by Qi e al. [57], again wi h
he p incipal objec i e o iden i ying no el lignocel-
luloly ic and glycosyl hyd olase genes in he muskoxen
umen, wi h he in e es ing hypo hesis ha new genes,
pa icula ly om he euka yo ic communi y, migh be
ound. The in es iga ion was highly success ul, achie ing
an 8.7× highe a e o o al ca bohyd a e ac i e enzyme
disco e y han ha ound in p e ious me agenomics
analyses. The me a ansc ip omic app oach o e s he
unique possibili y o es ic he analysis only o an-
sc ibed genes, hus emo ing he o en e y high noise
o non- ansc ibed po ions o he genome, which a e by
con as always p esen in me agenomic expe imen s.
Shi e  al. [58] in es iga ed me hane p oduc ion in a
coho o New Zealand sheep using me agenomics and
me a ansc ip omic echniques ha aimed a unde -
s anding mic obiological di e ences be ween animals
ha p oduced low and high amoun s o me hane. The
pape illus a ed he powe o deep sequencing in unde -
s anding he mic obial communi y and i s ac i i y. Fou
ams wi h a high-me hane pheno ype, iden i ied om a
pool o 22 animals, we e compa ed wi h ou ams wi h a
low-me hane pheno ype and ou ams wi h an in e me-
dia e pheno ype. The di e ence in me hane p oduc ion
be ween he high and low pheno ypes was abou 1.7-
old, simila o he bee ca le s udy [44] discussed abo e.
Mic obial communi y s uc u es we e compa ed by
ex ac ing RNA gene sequence in o ma ion om deep
sequencing and also by qPCR o RNA and mc A/mc T
genes. No di e ences we e de ec ed in he di e en
mic obial g oups. Fu he de ailed analysis o he a chaeal
communi y ound highe abundances o Me hanob e i-
bac e go schalkii in high p oduce s, an obse a ion ha
has been epo ed in o he s udies [2]. Me hanogenic
gene abundances also did no di e be ween he animal
g oups. I was only he me a ansc ip ome ha di e ed,
whe e he abundance o mRNA sequences was com-
pa ed. Th ee o he en mos inc eased ansc ip s in he
high p oduce s coded o enzymes in he me hanogenesis
pa hway. The idea ha he ansc ip ome is mo e espon-
si e as a measu emen o me hane emissions has gained
cu ency. This a gumen was challenged [44] because
ATP p oduc ion in me hanogens is en i ely dependen on
me hane o ma ion and he g ow h yield, mola g ow h

Page 6 o 11
Wallace e al. Gene Sel E ol (2017) 49:9
yield (g biomass/mol ATP u ilised) (YATP), is p opo -
ional o ATP p oduc ion. Howe e , mola g ow h yield
[g biomass/mol CH4 p oduced (Yme hane)] a ies signi i-
can ly acco ding o g ow h condi ions, wi h excess H2
appa en ly leading o uncoupling [59] analogous o ha
obse ed in bac e ia whe e g ow h is limi ed by a nu i-
en o he han a suga as ene gy sou ce [60]. Gi en he
ex ensi e na u e o elec on- anspo -linked me abo-
lism in me hanogens [61], uminal a chaea may well use
simila mechanisms o main ain cellula me aboli es du -
ing pe iods o s ess, and hei abundance may he e o e
no be p opo ional o he quan i y o me hane o med.
Fu he me a ansc ip omic s udies, linked possibly o
me abolomics analysis, migh be use ul in in es iga ing
his poin .
Me ap o eomic analysis
The p o eome di e s om he p e ious -omes in ha ,
while he o he s p edic wha genes a e p esen and how
hey a e ansc ibed, he p o eome e lec s he end-p od-
uc , he p o eins ha a e ac ually exp essed. The e has
no been a conce ed e o o cha ac e ise he p o eomes
o di e en pu e cul u es o uminal mic oo ganisms.
Indeed, i appea s ha he echnology and in e es ha e
jumped ha pa icula s ep o s udy he me ap o eome,
i.e. he en i e complemen o p o eins ha is exp essed
by he uminal mic obiome. Me ap o eomic analysis
aims a cha ac e ising he en i e p o ein con en o an
en i onmen al sample a a gi en poin in ime [62]. A
i s , i may seem imp obable ha such a complex com-
muni y, comp ising hund eds o species each wi h hou-
sands o genes, would p esen a p o eome ha would
be su icien ly disc imina ed o enable he iden i ica ion
o indi idual p o eins. None heless, ea lie examples o
me ap o eomic analyses om he human gu [63] and
soil [64] ha e shown ha i is in ac echnically easible.
Two main echnical app oaches a e a ailable in p o -
eomics. The i s is he long es ablished wo-dimensional
SDS polyac ylamide gel elec opho esis (2D SDS-PAGE)
echnology ha was o igina ed by O’Fa ell [65]. Sepa-
a ion o he o al p o ein is accomplished by isoelec ic
poin in he i s dimension and molecula size in he
second. The p o eome is isualised using a s ain, e eal-
ing indi idual spo s ha can be iden i ied by mass spec-
oscopic analysis ollowing ypsinisa ion o spo s cu
om he gel. P o ein iden i ica ion depends hea ily on
sea ches o e e ence da abases ha con ain ela i ely
ew umen mic obial p o eomes. A ecen de elopmen
in p o eome echnology uses s a e-o - he-a mass spec-
ome e s ha a e capable o analysing complex mix-
u es o pep ides de i ed by pa ial hyd olysis o o al
p o ein mix u es. Raw da a a e gene a ed as a massi e
se o mass spec a which a e con e ed in o a long lis
o sho pep ide sequences ( he me apep idome). These
a e assembled in o p o eins by mapping o a e e ence
da abase in a simila way o sho gun DNA sequencing,
hence he name sho gun me ap o eomics. Many belie e
ha he sho gun me hod, wi h he much la ge olume
o da a gene a ed, will supplan he gel-based me hod.
The e a e s ill a numbe o echnical issues ha need
o be add essed be o e sho gun me ap o eomics can
be used o compa a i e analysis. The i s is a eliable
me hod o quan i a e da a. This has been ca ied ou p e-
iously using spec um coun ing bu can also be achie ed
by labelling samples wi h s able iso opes. Mo eo e , he e
is a lack o bioin o ma ics analysis suppo and, simila
o 2D SDS-PAGE, he iden i ica ion o p o eins elies on
mapping da a o amino acid sequence da abases in which
he g ea majo i y o uminal species a e no ep esen ed.
The umen ecosys em sha es some cha ac e is ics
wi h mic obial communi ies in he en i onmen and
human gu ha ha e p e iously been cha ac e ised using
me ap o eomics, such as mic obial di e si y and ela i e
abundance o mic oo ganisms in some s udies [64, 66–
68] and he abundance o nu ien s in o he s [63], bu i
p o ides a unique challenge in he combina ion o hese
p ope ies. The me ap o eome will p o ide a di e en
insigh o he unc ion o he umen mic obial commu-
ni y compa ed o he nucleic acid me a-omes, a guably
one ha migh p o e mo e use ul as pa o he campaign
o lowe me hane emissions and o be e unde s and he
ole o key enzymes in ol ed in eed u ilisa ion e iciency
in uminan s.
The RuminOmics p ojec (www. uminomics.eu) in es-
iga ed SDS-PAGE me hods o gene a ing me ap o -
eomic in o ma ion om uminal diges a [69]. Resul s
we e a iable acco ding o he sample. In some gels, dis-
inc spo s we e obse ed, while in o he s in e e ence by
humic subs ances ha a e de i ed om he plan ma e i-
als consumed by he animal, esul ed in no dis inc p o-
ein spo pa e n. In he gels whe e spo s we e esol ed,
andem mass spec um analysis indica ed ha s uc u al
p o eins om p o ozoa we e mos abundan , an expec ed
esul conside ing he high p opo ion o hei biomass in
he umen. A su p ising disco e y was he s ong eso-
lu ion o key enzymes associa ed wi h me hanogenesis
om he a chaea ha o m a ela i ely small p opo ion
o he umen mic obial communi y. In a compa ison o
he 2D PAGE me ap o eomes o high- and low-me hane
emi ing dai y cows, no signi ican di e ence was e iden
al hough his was possibly due o he lack o p ecision
using his echnique.
The i s analysis o he uminal me ap o eome using
sho gun pep ide me hodology was published in 2015
[70]. Rema kably, axonomic in o ma ion assigned o
he p edic ed p o eins enabled a communi y analysis o
Page 7 o 11
Wallace e al. Gene Sel E ol (2017) 49:9
be ca ied ou , in which he ela i e abundance o di e -
en bac e ial and a chaeal amilies and euka yo e phyla
we e calcula ed. The composi ion o he mic obial com-
muni y was di e en om hose mos commonly seen
in he umen, bu no compa a i e DNA-based analysis
was p esen ed. I would be e y in e es ing o examine
he co espondence o he mic obiome deduced om
he me agenome o ha p edic ed om he co espond-
ing me ap o eome. Ano he ea u e o he analysis was
he high abundance o plan -de i ed pep ides de ec ed
by he sho gun me hod (Fig.2) compa ed o none being
de ec ed by he 2-D me hod [69]. The plan -de i ed
pep ides would be he p o eoly ic p oduc s o mic obial
diges ion o plan p o ein in he eed. Me hanogenesis-
associa ed p o eins we e only men ioned in passing,
bu p esumably he me ap o eome may be as use ul
in p edic ing me abolic pa hways as i was in desc ib-
ing pa hways o s a ch me abolism [70]. Once again, no
di e ences in he mic obial communi y based on he
me ap o eome be ween high- and low-me hane emi e s
we e e iden in dai y cows in he RuminOmics p ojec
(Fig.2).
Me abolomic analysis
Me abolomics p o ides a de ailed a ay o in o ma ion
abou uminal me abolic ac i i y, which is complemen-
a y o he DNA- and p o ein-based me hods desc ibed
abo e. The eade is di ec ed o he g ound-b eaking
pape by Saleem e  al. [71] on his opic. He e, ou
me abolomic analyses will be discussed ha ela e spe-
ci ically o me hanogenesis.
Shaba e al. [46] analysed he uminal me abolome in
cows ha a ied in eed e iciency, i.e. he con e sion o
eed o p oduc . Highe concen a ions o sho -chain
a y acids we e obse ed in mo e e icien cows, accom-
panied by lowe me hane emissions. O he me aboli es
we e no signi ican ly di e en , excep o pu escine,
which was p esen a highe concen a ions in e i-
cien cows. Whe he his e lec s a key aspec o umi-
nal me abolism ha a ec s e iciency is unclea . A la ge
numbe o me aboli es was de ec ed by Zhao e al. [72],
ela ed o bo h ni ogen and p o ein me abolism. The
me abolome was highly dependen on he die a y com-
posi ion, and no a emp was made o co ela e me abo-
li es wi h emissions, bu signi ican di e ences we e seen
in amino acid me aboli es and in me hylamines ha a e
subs a es o me hylo ophic me hanogenesis, sugges -
ing possible u u e alue in hese measu emen s o s ud-
ies o uminan GHG emissions.
The aecal me abolome associa ed wi h me hane
includes he dis inc i e memb ane lipids o he a chaea,
namely dialkyl glyce ol die he s (DGDG) and glyce ol
dialkyl glyce ol e ae he (GDGT). The mos common
o ms o DGDG and GDGT a e a chaeol and calda -
chaeol, espec i ely (Fig.3). A chaeol has ecei ed he
mos a en ion, and has had i s ela ionship wi h me h-
ane p oduc ion analysed ac oss a ange o die s in s udies
on bee and dai y ca le [73–75]. These s udies concluded
ha he e is conside able be ween-animal a ia ion in
he ela ionship, al hough he ela ionship is signi ican
when compa ing he ea men means. Be ween-animal
a ia ion could be a ibu ed o di e ences in he loca-
ion and kine ics o me hanogens in he uminan diges-
i e ac , and a lack o ela ionship be ween a chaeol
measu emen s in he umen and he aeces [76]. Ano he
po en ial cause o he a ia ion could be he o e sigh o
he p esence o calda chaeol in he me hanogen mem-
b ane. In compa ison o a chaeol, which o ms a bilaye ,
calda chaeol o ms a monolaye and is less pe meable o
p o ons. McCa ney e al. [77] ound ha he p opo ion
o calda chaeol in he aeces inc eased ma kedly when
he animal was ed a die high in s a ch, and hus pe haps
p o ec ing he me hanogens om he esul an d op in
uminal pH. Fu he mo e, concen a ions o calda chaeol
and o al e he lipids we e ound o be mo e p opo ional
o measu ed me hane p oduc ion han a chaeol con-
cen a ions. In summa y, a chaeol is po en ially a use ul
al e na i e ma ke o de e mining me hanogen abun-
dance, howe e , as a me hane p oxy, mo e wo k is needed
o u he in es iga e bo h a chaeol and calda chaeol.
The u ina y me abolome has p o ided much use-
ul in o ma ion on N e en ion and luxes in he animal
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Low Emie s High Emie s
O he
Fib obac e succinogenes
(s ain ATCC 19169 / S85)
Acnobac e ia (high G+C
G am-posi e bac e ia)
P o eobac e ia
Fi micu es
Bac e oide es
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Low Emie s High Emie s
O he (No assigned o
phylum)
Vi idiplan ae
Opis hokon a
Al eola a
Fig. 2 Me ap o eomics—bac e ial (uppe panel) and euka yo ic
(lowe panel) p o eins om sho gun pep ide sequencing (Snelling
and Wallace [69])
Page 8 o 11
Wallace e al. Gene Sel E ol (2017) 49:9
o many yea s [78]. The pu ines p o ide a use ul p oxy
measu e o mic obial p o ein low om he umen [79,
80] and u ea i sel is o cou se he mos impo an me ab-
oli e associa ed wi h he e iciency o N e en ion. How-
e e , a ecen s udy in p o ozoa-deple ed lambs e ealed
ha he u ina y me abolomes o auna ed and he p o-
ozoa-deple ed animals we e almos comple ely pola -
ised in e ms o p o ein-de i ed me aboli es ollowing
disc iminan analysis [81]. In spi e o he complexi y o
he da a, he clea sepa a ion o he me abolome acco d-
ing o he di e en ea men s gi es an indica ion o he
alue o u he in es iga ion in o he u ina y me abo-
lome. Co ela ion wi h he composi ion o he mic o-
bio a also sugges s he possibili y o using he u ina y
me abolome o p edic umen mic obial me abolism. Fo
ins ance, me aboli es o yp ophan we e linked no only
o he abundance o p o ozoa bu also o bac e ial axa
mos ly dis an ly ela ed o known species. In iguingly,
he u ina y me abolome s udy also e ealed a possible
link o me hanogenesis. Me hane emissions we e no
measu ed, bu a nega i e ela ionship was ound be ween
u ina y ime hylamine-N-oxide and he uminal abun-
dance o he me hylo ophic me hanogenesis o de
Me hanomassiliicoccales.
The a y acid composi ion o milk, some imes called
he milk lipidome, can also be use ul in p edic ing umi-
nal me abolism, including me hanogenesis, in dai y cows.
A me a-analysis [82] concluded ha milk a y acid con-
cen a ions o C10:0, C12:0, C14:0-iso, C14:0, cis-9 C14:1,
C15:0, and C16:0 we e posi i ely ela ed o me hane yield
pe uni o milk, while C4:0, C18:0, ans-10+11 C18:1,
cis-9 C18:1, cis-11 C18:1, and cis-9,12 C18:2 in milk a
we e nega i ely ela ed. Ma hema ical analysis enabled
p edic ion equa ions o be o mula ed ha had mode -
a e po en ial o p edic ing me hane yield pe uni o
eed and a sligh ly lowe po en ial o p edic ing me h-
ane yield pe uni o milk. Subsequen expe imen s sug-
ges ed ha mid-in a ed spec oscopy was a use ul ool
in p edic ing me hane emissions om milk a y acid
composi ion [83]. In spi e o hese obse ed co ela ions,
in he RuminOmics p ojec , he p edic i e alue o indi-
idual a y acid concen a ions in mo e han 200 a y
acids measu ed was weak o me hane emissions. The
link be ween milk a y acids and me hane is he uminal
mic obio a. Key species di e in hei a y acid compo-
si ion [84], hus di e en mino a y acids de i ed om
hese species appea in milk depending on he abundance
o di e en membe s o he mic obial communi y. Since
he communi y o H2-p oducing bac e ia, o example,
has an in luence on me hanogenesis, he quan i ies o
hei a y acids in milk can indica e hei abundance in
he umen and he e o e indi ec ly hei e ec on me ha-
nogenesis. In he RuminOmics p ojec , he p edic i e
alue o indi idual a y acid concen a ions o me hane
emissions was weak. Mul iple co ela ions we e ound,
howe e , ew o hem had been obse ed p e iously.
Conclusions
Cu en -omics echnologies can p o ide de ailed in o -
ma ion abou he animal genome, he uminal me age-
nome and hei espec i e unc ional ac i i ies om he
me a ansc ip ome and me ap o eome. Compa a i e
analysis using hese echnologies allows us o cha ac-
e ise he in e ac ion be ween he animal and i s umen
mic obio a. A he p esen ime, i is mainly he powe
and po en ial o me agenomics, me a ansc ip omics and
Fig. 3 S uc u e o he co e memb ane lipids o he a chaea including glyce ol dialkyl glyce ol die he (DAGE) and glyce ol dialkyl glyce ol e a-
e he (GDGT). PHG pola head g oup. Rep oduced om [77] wi h pe mission
Page 9 o 11
Wallace e al. Gene Sel E ol (2017) 49:9
me ap o eomics ha a e being in es iga ed, wi h ewe
s udies in es iga ing hei applica ion o p oblems asso-
cia ed wi h animal p oduc ion. Fu he mo e, in eg a -
ing he esul s o a ious me a-omics analyses emains a
challenge. Imp o ing he da a p esen in public da abases
o include p og essi ely mo e in o ma ion on umen
mic obial species is a p io i y. Indeed, esea ch g oups
a ound he wo ld a e joining o ces o mee hese chal-
lenges. A much la ge knowledge base o umen mic o-
bial genomics will allow hese me hods o become mo e
obus o he de ec ion o ele an species as well as
o a co ec iden i ica ion and quan i ica ion o mic o-
bial genes and p o eins di ec ly ela ed o umen me a-
bolic pa hways, which could ha e an impo an ole in
he imp o emen o li es ock p oduc ions and b eeding
p og ammes. Some p og ess has been made wi h me h-
ane emissions. Howe e , an a guably mo e accep able
s a egy, pa icula ly o he li es ock p oduce , would be
o ocus on he e iciency o eed u ilisa ion a he han
me hane i sel . The equally impo an issue o N emis-
sions has ecei ed oo li le a en ion.
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 .
Au ho de ails
1 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. 2 G een Technology, Na u al Resou ces Ins i u e Fin-
land, Jokioinen, Finland. 3 PTP, Via Eins ein - Loc. Cascina Codazza, 26900 Lodi,
I aly.
Acknowledgemen s
The Rowe Ins i u e o Nu i ion and Heal h 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).
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
Recei ed: 9 July 2016 Accep ed: 6 Janua y 2017
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