REVIEWS

The rumen microbiome: balancing

food security and environmental
impacts
Itzhak Mizrahi 1 ✉, R. John Wallace2 and Sarah Moraïs1

Abstract | Ruminants produce edible products and contribute to food security. They house a
complex rumen microbial community that enables the host to digest their plant feed through
microbial-​mediated fermentation. However, the rumen microbiome is also responsible for the
production of one of the most potent greenhouse gases, methane, and contributes about 18%
of its total anthropogenic emissions. Conventional methods to lower methane production by
ruminants have proved successful, but to a limited and often temporary extent. An increased
understanding of the host–microbiome interactions has led to the development of new
mitigation strategies. In this Review we describe the composition, ecology and metabolism
of the rumen microbiome, and the impact on host physiology and the environment. We also
discuss the most pertinent methane mitigation strategies that emerged to balance food security
and environmental impacts.

1
Department of Life Sciences,
Ben-Gurion University of
the Negev and the National
Institute for Biotechnology
in the Negev, Marcus Family
Campus, Be’er-Sheva, Israel.
2
The Rowett Institute,
University of Aberdeen,
Aberdeen, UK.
✉e-​mail: imizrahi@bgu.ac.il
https://doi.org/10.1038/
s41579-021-00543-6
In recent years, the mammalian gut has been recognized
as a fundamentally important microbial ecosystem1.
Intriguingly, in specific mammalian lineages, complete
obligatory dependence exists between the host and its
associated microorganisms, whereby the microbial
communities perform fundamental processes, such
as digestion of the feed for the host. Among the most
representative and ecologically relevant examples are
ruminants — foregut fermenters that rely critically on
their associated gut microorganisms to digest their plant
feed2. This phenomenon enables ruminants to convert
the complex polysaccharides such as cellulose and
hemicellulose that comprise the major part of the plant
biomass, mostly indigestible to humans, into nutritive
food. This capacity has established ruminants as inte-
gral parts of our societies, culture and even evolution3,
since their domestication in the Neolithic era more than
10,000 years ago. The ability to digest their plant feed is
conferred upon the host animal principally by the func-
tioning of microbial communities residing in the rumen
compartment, situated in their upper digestive tract.
Unlike non-​ruminants, in which microorganisms have a
minor role in the digestion process4, ruminants require
a complex community that codes for a multitude of func-
tions that will enable the animal to exploit and digest
plant fibre5. The function of the rumen microbiome is
tightly linked to host physiology, including to the devel-
opment of the rumen epithelium6,7, possibly involving
the modulation of host gene regulation by short chain
fatty acids (SCFAs)8. Due to the paradigmatic obligatory
dependence of the ruminant host on its microbiome, such
systems are excellent models to understand fundamental
aspects of the ecological and evolutionary relationships
between animal hosts and their microorganisms.
Nevertheless, the rumen microbiome is also respon-
sible for the production of one of the most potent
greenhouse gases, methane. During the last few dec-
ades, worldwide methane emissions from agricultural
livestock have continuously increased (Fig. 1a), with a
significant 1.9-​fold increase from 1961 to 2017. These
emissions are only expected to further increase in the
future, owing to the growing global demand for milk
and beef9. Consequently, methane is one of the six gases
that have required international measures by the Kyoto
Protocol, as it represents 14% of the total greenhouse
gas emitted and has 28 times the global warming poten-
tial of CO2. The mean total methane emissions for the
2010–2015 period were estimated by satellite observa-
tion at 548 Tg year–1 (ref.10). About two-​thirds of these
emissions (356 Tg year–1) are from anthropogenic
sources, of which 100–117 Tg year–1 are from livestock,
emanating either directly by exhalation or from the
biowaste generated by farms, such as manure (Fig. 1a).
More than half of the emitted livestock emissions are
attributed to non-​dairy cattle, with the rest distributed
across other livestock (Fig. 1b). In addition, it seems that
there are no marked differences between grazing and
non-​grazing animals in relation to methane emissions in
areas such as Asia and Africa in which documentation
of these husbandry regimes exists11.

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a Worldwide methane emissions Nevertheless, in view of its relatively short lifespan in

the atmosphere (12 years), methane mitigation strate-
gies seem feasible, and ruminant gut microorganisms
constitute a primary target for such strategies.
Recent years have brought major advances in under-
standing the rumen ecosystem and its interaction
with the host13. Specific community structures, micro­
organisms and their metabolic pathways, together with
Methane specific metabolites that drive community composition
emissions in and ecosystem function, were connected to the host
2017 (Gg) genome and its attributes, such as the host energy har-
2,000 vest efficiency14–22. In this Review, we discuss our current
4,000
6,000 understanding of the composition and ecology of the
8,000 rumen microbiome with regard to methane emissions
10,000 and potential ways to modulate them.
12,000

The rumen microbiome

140,000 Composition of the rumen microbiome. The rumi-
120,000
nal microbiome comprises representatives of bacte-
Methane emissions (Gg)

ria, protozoa, archaea and fungi, with their respective

100,000 abundance and diversity following the same order.
80,000 Unlike the eukaryotic domain, the archaeal and bacte-
rial domains are essential to the viability of the rumi-
60,000
nant host2. The Firmicutes and Bacteroidetes bacterial
40,000 phyla are the most abundant members, at both the
DNA and protein levels23–25, and comprise key fibre
20,000
degraders, which include cellulose degraders, such as
0 Fibrobacter succinogenes, Ruminococcus flavefaciens and
1960 1980 2000 2020 2040 Ruminococcus albus2, and several bacterial species that
Year are highly efficient in hemicellulose degradation, such
b Emissions by animal type as members from the genera Prevotella, Butyrivibrio
Others and Pseudobutyrivibrio26. All of these bacteria belong
Goats to core genera that were found in almost all ruminants
Sheep
across many studies25,27 and were present in 80–100% of
the animals in a recent large cohort survey comprising
1,000 cows28. Therefore, they may have an imperative
Buffaloes role in the metabolism and function of the rumen micro-
Non-dairy cattle
biome (Fig. 2). Members of the Prevotella, Butyrivibrio
and Ruminococcus genera have been particularly studied
as dominant species in the colonization and degradation
of the hemicellulose portion of the fibre, as they com-
Dairy cattle prise a broader fibre-​related enzymatic repertoire that
could enable them to digest the available fibre26,29–32. In
addition, bacteria that were previously shown to utilize
Fig. 1 | Global methane emissions. Methane emission data estimated or calculated the degradation products of cellulolytic bacteria such
from Food and Agriculture Organization of the United Nations (FAOSTAT) enteric as Treponema bryantii33 are also found to be present
fermentation, depicted by region and animal. a | Global annual emissions from 1960 to in all of the 1,000 cows analysed in the cohort study28
2017, and predicted emission trend for 2030 and 2050 (red dots). World map colour- (Fig. 2). Dominant members of the rumen microbiome
coded to indicate low to high methane emissions of data collected in 2017. India, Brazil, also comprise microorganisms that utilize secondary
China, the USA, Argentina and Pakistan exhibit the highest levels of methane emissions. fermentation products of other microorganisms, such
b | Level of contribution to methane emission by animal type. The ‘others’ category on as Selenomonas ruminantium and members of the
the chart encompasses non-​ruminant animals that include (from high to low in order
Succinovibrionaceae family34,35 (Fig. 2).
of importance with regard to methane emission) camels, swine, horses, mules together
with asses, and llamas. Data include enteric methane emissions from both grazing and It is important to note that low-​abundance taxa do
non-​grazing animals11. not translate into less significant members of the micro-
bial community. For example, only very few cellulolytic
species have been isolated from the rumen micro­biome,
Owing to the increase in methane emission, the two and they comprise a very small portion thereof, but
main natural methane sinks (that is, the atmosphere scores off other species that consume the products of
Core microbiome
and the soil) are no longer sufficient for balancing its primary fibre digestion. In this context, the cellulolytic
A group of microorganisms
commonly found within removal10. The accumulation of methane in the atmos- bacterium F. succinogenes occupies only 0.5% of the
the microbiome across phere is equivalent to half of the total global carbon imbal- bacterial diversity36 and occurs in most of the reported
multiple hosts. ance, which greatly contributes to the greenhouse effect12. core microbiome studies and in all of the animals in the

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Food webs
The interconnections among
different microbial food chains.
Electron sinks
In this context, microorganisms
within a microbial community
that accept electrons at the
final step of the electron flow.
Hydrogenotrophs
Methanogens that use H2 to
reduce CO2 to methane.
Methylotrophs
Methanogens that use a
methylated compound as the
input metabolite to produce
methane.
Acetoclastic methanogens
Methanogens that use acetate
to produce methane.
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recent cohort study28 (Fig. 2). F. succinogenes is considered
a key cellulolytic species37,38 that fuels the metabolism of
other microbiome members by producing soluble sugars
and succinate from cellulose (Fig. 3).
Another example of important low-​abundance taxa
is the archaeal domain that typically represents a small
proportion of the total rumen microbial richness and
abundance2, and includes methanogens that are the sole
methane producers in this ecosystem. Despite the fact
that environmental conditions such as diet and geo­
graphy were shown to affect the composition of the
methanogenic community18,39,40, methanogens are ubiq-
uitous to all foregut animals and therefore represent part
of the ruminant core microbiome27. This is supported by
the aforementioned recent study28, which showed that
species represented as operational taxonomic units from
the genera Methanobrevibacter and Methanosphaera
occur in 100% and 60% of the animals, respectively,
although they occupy only a small percentage of the
relative abundance (Fig. 2).
Rumen metabolism. In the rumen, metabolic cascades
are carried out by the microbial community in a com-
plex and coordinated manner, whereby successive, cross-​
feeding across food webs exists among different rumen
microorganisms, generating trophic-​like levels41. When
dividing the species comprising the rumen microbiome
into functional groups, it becomes evident that there is a
huge functional redundancy in this ecosystem42. Indeed,
even when considering only the fibrolytic function
located at the top of the metabolic cascades, a multitude
of carbohydrate-​active enzymes are distributed through-
out most of the rumen microbiome26,43. In general, there
is a lack of understanding of the role and importance of
functional redundancy that is apparent at all layers of the
rumen metabolic cascades. This widespread functional
redundancy was exemplified in the recent cohort study
that revealed very high vari­ability among the animals,
with only a small proportion of the core microbiome
existing in 90% of the cohort28. Although these micro­
organisms are likely to be of major importance to the
host, they only represent a fraction of the overall ecosys-
tem richness, which suggests that the rest of the micro-
organisms retain a high degree of redundancy; therefore,
the rumen ecosystem function is carried out by vari-
ous different species that perform similar functions.
Consequently, although species and taxonomic identity
may vary among animals, the functions encoded by the
microorganisms seem to be more stable44. Recently, pro-
teomic data suggested that this functional redundancy
is fostered by microbial niche compartmentalization23.
In broad terms, rumen metabolism can be divided
into three trophic-​like levels of chemical transformations
of plant fibre macromolecules and polymers (Fig. 3a). For
the first level, we focus on the degradation and meta­
bolism of cellulose and hemicellulose, which are the most
abundant and recalcitrant sugar polymers of the plant
cell wall. In this level, the plant cell wall structures are
colonized by microorganisms that utilize enzymes
belonging to different glycosyl hydrolase families to
deconstruct these polymers into soluble forms; these
include shorter polymers and monomers of glucose in
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the case of cellulose, and various pentose sugars in the
case of hemicellulose26. In the second level, these soluble
sugars are imported into the microbial cells by specific
transporters and metabolized via various pathways such
as the Embden–Meyerhoff–Parnas pathway and the
pentose phosphate pathway for the utilization of hexoses
and pentoses, respectively, as well as additional atypi-
cal metabolic pathways suspected to be encoded in the
genomes of the microorganisms in the rumen45,46. These
atypical utilization pathways include an incomplete
Embden–Meyerhoff–Parnas pathway and a pyrophos-
phate (PPi)-​dependent phosphofructokinase pathway.
Due to the anaerobic nature of the metabolism in the
rumen, a great proportion of these sugars are then fer-
mented via various downstream pathways, which results
in the excretion of organic acids and SCFAs, as well as
various metabolites and gases such as carbon dioxide
and hydrogen47–49. Finally, in the last level, some of these
excreted metabolites, such as lactate, succinate, hydro-
gen and carbon dioxide, are further converted into pro-
pionate, butyrate, acetate and methane, among others.
For example, some of the key core microbiome mem-
bers, such as S. ruminantium, Coprococcus catus and
Succiniclasticum ruminis, convert lactate and succinate
into SCFAs (Figs 2 and 3). The high amounts of hydrogen
generated are mainly utilized by methanogens to reduce
carbon dioxide to methane. Hydrogen is also used by
sulfate-​reducing and nitrate-​reducing microorganisms,
and is involved in the production of unsaturated fatty
acids and microbial biomass, but to a lesser extent.
In addition, although homoacetogenic and acetogenic
bacteria are present in the rumen, acetogenesis is thermo-
dynamically less favourable than methanogenesis50, and
reductive acetogenesis mostly occurs in the absence of
methanogens51. There is also evidence for the presence
of methanotrophs (bacteria that oxidize methane into
CO2) in the rumen52, but with limited activity.
The methanogens are essential for generating
electron sinks and represent a crucial driving force for
the entire food chain. All methanogens are members
of the archaeal phylum of Euryarchaeota that com-
prises seven orders, of which Methanobacteriales,
Methanomicrobiales, Methanomassiliicoccales and
Methanosarcinales are commonly found in the rumen
ecosystem. The genus Methanobrevibacter of the
Methanobacteriales order is the most dominant, account-
ing for up to 70% of the rumen archaeal community16.
With respect to their physiology and ecology, three dif-
ferent methanogenic groups can be distinguished, which
alludes to the different ecological roles in the rumen
regarding metabolic precursors of methanogenesis53.
These groups include: hydrogenotrophs, which utilize
hydrogen to reduce CO2, CO or formate to methane54;
methylotrophs, which use methylated compounds such as
methanol or methyl amines; and acetoclastic methanogens,
which use acetate for this process55,56. Members of the
genus Methanosarcina can use all of these precursors,
and thus belong to all of the groups5. Methanogens are
present in the rumen just minutes after birth57. The early
methanogenic community is already active in newborn
animals but differs from the community of mature ani-
mals in terms of substrate utilization58. In fact, whereas
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Previously Prevalent core rumen

Phylum Family Genus reported Known core rumen isolates and their morphology microorganisms
Spirochaetes Spirochaetaceae Treponema Treponema bryantii
Fibrobacteres Fibrobacteraceae Fibrobacter Fibrobacter succinogenes
Methanosphaera Methanosphaera stadtmaniae
Euryarchaeota Methanobacteriaceae
Methanobrevibacter Methanobrevibacter ruminantium
Atopobiaceae Olsenella Olsenella umbonata
Actinobacteria Pseudoscardovia Pseudoscardovia suis, pig gut isolate
Bifidobacteriaceae
Bifidobacterium Bifidobacterium ruminale
Succinimonas Succinimonas amylolytica
Ruminobacter Ruminobacter amylophilus
Proteobacteria Succinivibrionaceae Succinivibrionaceae n.d.
Succinivibrio Succinivibrio dextrinosolvens
Succinivibrionaceae n.d.
Prevotellaceae_NK3B31 n.d.
Prevotellaceae_UCG-004
Prevotellaceae_Ga6A1 n.d.
Bacteroidetes Prevotellaceae Prevotellaceae_YAB2003 n.d.
Prevotellaceae_UCG-003 n.d.
Prevotellaceae_UCG-001 n.d.
Prevotella Prevotella ruminicola
Rikenellaceae Rikenellaceae_RC9 n.d.
Veillonellaceae Dialister No gut isolates
Selenomonadaceae Selenomonas Selenomonas ruminantium
Anaerovoracaceae Mogibacterium No gut isolates
CAG-352 n.d.
Ruminococcaceae
Ruminococcus Ruminococcus flavefaciens
Colidextribacter No isolate
Oscillospiraceae UCG-005 n.d.
NK4A214_group n.d.
Lachnospiraceae_XPB1014 n.d.
Oribacterium Oribacterium sp. strain C9
Lachnospira Lachnospira multiparus
Coprococcus Coprococcus sp. Pe15
Lachnospiraceae_UCG-009 n.d.
Firmicutes probable_genus_10 n.d.
Lachnoclostridium Lachnoclostridium clostridioforme
Lachnospiraceae Shuttleworthia No gut isolates
Lachnospiraceae_AC2044_ n.d.
Acetitomaculum Acetitomaculum ruminis
Lachnospiraceae_NK4A136 n.d.
Moryella No gut isolates
Butyrivibrio Butyrivibrio fibrisolvens, B. hungateii, B. proteoclasticus
Pseudobutyrivibrio Pseudobutyrivibrio xylanivorans, P. ruminis
Lachnospiraceae_NK3A20 n.d.
Hungateiclostridiaceae Saccharofermentans No gut isolates
Christensenellaceae Christensenellaceae_R-7 Christensenella minuta, human gut isolate
Acidaminococcaceae Succiniclasticum Succiniclasticum ruminis
Acholeplasmataceae Anaeroplasma Anaeroplasma abactoclasticum
0 20 40 60 80 100
% occupancy in a cow
cohort across Europe
(ESV level)

hydrogenotrophs constitute the majority of methano-
gens in mature animals, newborn calf microbiomes
present a higher proportion of methylotrophs40, sug-
gesting that different metabolic environments present at
different stages of life enrich for metabolically different
methanogenic communities58. Knowledge of such func-
tional dynamics is critical to develop potent community
modulation and intervention strategies.
The food web hierarchy of consumption, from the
macromolecules down to the final electron acceptors,

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◀ Fig. 2 | Overview of the most prevalent rumen microbiome core members. Phylum,
family and genus-​level assignment of the prevalent exact sequence variants (ESVs)
observed in the Ruminomics survey of 1,000 cows28. Genera of ESVs reported as core in
other large-​scale surveys19,25,27,162–164 are indicated by checkmarks. ESVs with species-level
annotation in the SILVA database are shown in bold whereas ESVs with only genus-
level annotation are assigned with names of known rumen isolates belonging to the
specific genera. ESVs for which defined species annotation was not possible due to
either lack of clear genus-​level annotation or existing isolates are indicated as not
determined (n.d.). ESVs for which isolates can be found in environments other than the
gut are marked ‘no gut isolates’, and ESVs from genera with no existing isolates in any
environment as ‘no isolate’. Cell morphologies of the mentioned isolates are indicated
based on published microscopic images. Bar plot, colour-​coded according to the
family-​level annotation, shows the ESV percentage (%) of appearance across a cohort
of 1,000 cows (termed occupancy).
also gives rise to substantial microbial feedback interac-
tions between the different trophic-​like levels41. These
complex networks yield vast arrays of metabolites pro-
duced by the microorganisms, some of which are uti-
lized by the ruminant host for its energy needs. This
is exemplified by SCFAs, which are absorbed through
the rumen wall and serve up to 70% of the host’s energy
needs2. Furthermore, rumen microorganisms them-
selves are an important protein source for the animal,
being digested, subsequently, in the lower digestive
tract2,59. Hence, the rumen microbial symbionts and
their secreted metabolome (that is, the overall meta­
bolite assortment) form the basis for the welfare of the
animal, affecting global food security as well as methane
production and emission.
Hydrogen-​mediated microbial interactions. Hydrogen
represents a key molecule in the adult rumen ecosys-
tem with regard to methane production and rumen
community dynamics. Hydrogen accumulates during
plant fibre fermentation and is removed by methano-
gens through methanogenesis. Therefore, in this way,
methanogens fulfil their cardinal role as the main elec-
tron sink in the rumen. Moreover, by utilizing hydro-
gen, methanogens potentially affect other microbial
species that are sensitive to hydrogen partial pressure.
A recent study 60 that examined sequenced rumen
microbial genomes and existing rumen metatranscrip-
tomes found that two-​thirds of the genomes encode
and express enzymes that potentially utilize hydrogen,
such as [FeFe] hydrogenases, [NiFe] hydrogenases, [Fe]
hydrogenases and nitrogenases. Indeed, hydrogen pres-
sure has been suggested to influence rumen methane
formation and fermentation balances through microbial
growth kinetics and fermentation thermodynamics61.
A seminal example exists for R. albus and R. flavefaciens.
These major cellulolytic microorganisms are considered
key fibre degraders in the rumen. In vitro, these spe-
cies have been shown to change their metabolism to
increase energy yields in the presence of methanogens
that consume the hydrogen produced and decrease its
partial pressure, thus increasing the free energy asso-
ciated with glucose fermentation and, consequently,
ATP gains and energy yields62–64. Similar examples with
regard to such syntrophic interactions with methano-
gens in the production and consumption of hydrogen
were found with other cellulolytic species, such as the
rumen fungus Neocallimastix frontalis65, the bacterium
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F. succinogenes66, hemicellulolytic bacteria and addi-
tional carbohydrate fermenters67,68. Moreover, hydrogen
utilization by methanogens and the decrease in hydro-
gen partial pressure were suggested to relieve hydrogen-​
mediated product inhibition in biomass hydrolysis,
thereby enabling an increase in anaerobic digestion69.
Indeed, the abundance of methanogens and cellulolytic
microorganisms in rumen samples from various animals
was positively correlated70. Therefore, these two distinct
processes that are situated at the far ends of the rumen
trophic cascade are mutually dependent. This was exem-
plified in a study examining the microbial succession
during the fibre degradation process, showing that an
increased abundance of methanogens was responsible
for the relief in hydrogen partial pressure, which in turn
enabled further fibre degradation and resulted in two
apparent fibre degradation phases30.
These metabolic hydrogen-​t ransfer interactions
potentially require physical proximity between the coun-
terparts. Indeed, methanogenic species are occasionally
found in association with microbial hydrogen producers
in the rumen. An example of such physical proximity
can be observed with the ciliate protozoa, which belong
to the rumen eukaryotic domain. Protozoa are consid-
ered major hydrogen providers and therefore interact
with methanogens71,72. Physical associations have been
observed between several genera, such as Entodinium,
Polyplastron, Epidinium and Ophryoscolex, and the
methanogens73,74. Methanogens were found to colo-
nize protozoa both intracellularly and extracellularly75,
and this interaction results in a mutualistic relation-
ship that is believed to enhance methane formation in
the rumen. Physical interactions were also observed
between methanogens and bacteria. In specific cases,
these interactions were suggested to be mechanically
mediated via an adhesin-​like protein, such as the case
of Methanobrevibacter ruminantium67 where a protein
was identified from its genome that was verified to bind
to protozoa and to the rumen bacterium Butyrivibrio
proteoclasticus76. Such information may shed mecha-
nistic light on the interactions between eukaryotic and
bacterial domains and the methanogens, as it is clear that
they are cardinal in driving methanogenesis.
Rumen microbiome compositional states and functional
outcomes. The energy harvested from the feed and
absorbed by the animal or emitted to the atmosphere in
the form of methane is largely affected by the activity and
composition of its rumen microbiome. Despite the fact
that methanogens are the sole known methane produc-
ers in the rumen, the relationship between methane pro-
duction and methanogen abundance is not clear. Indeed,
in several studies, the abundance of methanogens was
directly correlated to high methane-​emitting ruminants,
whereas in many other studies no direct correlation was
apparent14,77,78. Nevertheless, the composition of the
bacterial79 and eukaryotic communities is suggested to
exert a large influence on the methanogenesis potential
of the rumen microbiome, as methanogens depend on
the activity of other microorganisms. For instance, in a
recent study, the variation in composition of the core
microbiome, mostly comprising non-​methanogenic
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a Trophic level
Polymer
degradation Cellulose
Hemicellulose
I

Soluble sugar
utilization and
fermentation Methanol
Butyrate
CO2 H2
H2 Propionate
II Acetate
Lactate H2
CO2
Succinate

Secondary product
fermentation Succinate Propionate
H2 Butyrate
IIIa Lactate Butyrate
Succinate Propionate
H2 Acetate
Lactate

Methanogenesis
and acetogenesis Butyrate Propionate
Methanol H2 Methane
IIIb CO2 Acetate
CO2 H2 Methane
Methane Propionate

b Utilization Production
rs
se

ga
lo

e
l e

su
lu

Bu nat
l
llu in
He ose

no

ne
at

at

te

M te
el

Pr e
e

e
ce tall

in

in

at

io

ra
ha

ha
ic

bl

at

at

a
rm
cc

cc

CO et

op
m

ty
lu

2
ct

ct
ys

et

et
Known core rumen isolates and their morphology
Ac
So

Su

Su

Fo
La

La
Cr

H
2

Treponema bryantii
Fibrobacter succinogenes
Methanosphaera stadtmaniae
Methanobrevibacter ruminantium
Olsenella umbonata
Bifidobacterium ruminale
Pseudoscardovia suis
Succinimonas amylolytica
Ruminobacter amylophilus
Succinivibrio dextrinosolvens
Prevotella ruminicola
Selenomonas ruminantium
Ruminococcus flavefaciens
Oribacterium sp. strain C9
Lachnospira multiparus
Coprococcus sp. Pe15
Lachnoclostridium clostridioforme
Acetitomaculum ruminis
Butyrivibrio fibrisolvens, B. hungateii, B. proteoclasticus
Pseudobutyrivibrio xylanivorans
Pseudobutyrivibrio ruminis
Succiniclasticum ruminis
Christensenella minuta
Anaeroplasma abactoclasticum

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◀ Fig. 3 | Overview of rumen microbiome metabolism. a | Trophic-​like levels of the rumen
fermentation cascade and the corresponding isolated microorganisms occupying each
level. At the first trophic-​like level (I), the main polymers of the plant fibre feed (cellulose
and hemicellulose) are degraded into small oligosaccharides by specialist cellulolytic
and hemicellulolytic microorganisms. At the second level (II), the soluble sugars are
fermented by most of the members of the rumen microbiome. The third level (III) can be
divided into two sublevels for utilization of the second-​level fermentation products.
At the first sublevel (IIIa), a small range of secondary fermenters utilize organic acids
to produce short chain fatty acids (SCFAs). At the second sublevel (IIIb), methanogens
and, potentially, acetogens utilize mostly hydrogen and, to a lesser extent, methanol
to produce methane and acetate. The microbial richness at each level is denoted as
the shape area. b | Utilization and production of various metabolites by representative
isolates of core rumen microbiome members, colour-​coded according to their respective
trophic levels in part a.
species, could be used to predict the amounts of meth-
ane emissions from animals with high accuracy17. This
suggests that the metabolic relays between microbiome
members not capable of methanogenesis drive energy
flow towards methane emission.
It seems that the microbial web of interactions in
the rumen microbiome defines the ultimate commu-
nity composition, function and, thus, outcome (that
is, alternative stable community states). Therefore, the
rumen ecosystem can be stabilized at several alternative
microbiome states depending on the microbial interac-
tion types and the strength of the resulting metabolic
feedback41,80. The establishment of alternative stable
community states is connected to methane production
and its emission by the animal41. Indeed, two parallel
studies in geographically distanced locations (Israel and
New Zealand)4,81 identified two distinct community
compositions that were found to correlate to two dif-
ferent rumen ecosystem functions. One state was con-
nected to higher methane emissions and lower host feed
efficiency, whereas the other was linked to higher SCFA
production and higher host feed efficiency. In both
studies, lactate and hydrogen seemed to be bifurcating
connections for functional microbial groups and functional
outcome. The downstream branches of this metabolic
bifurcation are lactate utilization and methanogenesis —
both utilize hydrogen, and therefore are suggested to
compete. The outcome of this competition is suggested
to determine the establishment of the above two com-
munity states. Thus, alternative stable community states
stem from interactions between functional microbial
groups, such as lactate utilizers versus methanogens,
and thus might determine the amount of methane that
is being emitted41. Hence, in this scenario low methane
emissions are characterized by microbial groups that
excrete metabolites that have the potential both to
inhibit methanogens and to function as precursors in
Alternative stable metabolic cascades that compete with methanogenesis.
community states For example, lactate can inhibit methanogenesis through
Assemblages of functional a decrease in pH and, at the same time, is metabolized by
groups that are locked and
pathways that utilize hydrogen, thereby steering rumen
stabilized by metabolic
feedback and result in distinct metabolism away from methanogenesis and towards spe-
composition, function and, cific SCFA production. Within this compositional state,
thus, outcome. species of the genus Sharpea were highlighted as lactate
producers, whereas the Coprococcus and Megasphaera
Functional microbial groups
Groups of organisms that share
genera were designated hydrogen and lactate utilizers
similar functionality within the that produce propionate from these precursors via the
ecosystem. acrylate metabolic pathway14,81,84,87. Hence, a decrease
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in methane production is coupled with an increase in
propionate used as energy by the host, which there-
fore might explain observations connecting reduced
methanogenesis and increased milk production21.
An additional community state that is associated
with low methane emissions is generally characterized
by bacterial groups that directly compete with meth-
anogens for hydrogen. Within these groups, the family
Succinivibrionaceae was highlighted in many studies
and was initially found in high abundance in the micro-
biomes of low methane-​emitting wallabies82 but also in
higher abundance in extremely low methane-​emitting
ruminants14,17,78,83,84. This group was further identi-
fied as one of a limited number of heritable bacteria17.
Thus, the Succinivibrionaceae family could represent
a potential target for a methane mitigation strategy in
microbiome-​directed breeding programmes (see below).
Moreover, another study40 recently recorded a spike in
the abundance of members of the Succinivibrionaceae
family a month after birth, which was replaced by a
spike in methanogen abundance after a dietary shift
at the age of 3 months. These findings suggest that
Succinivibrionaceae and methanogens are mutually
exclusive, and specific conditions would potentially
enable Succinivibrionaceae to outcompete methano-
gens. Indeed, it has been demonstrated that an increase
in hydrogen supply can increase succinate production85
as succinate formation has a lower enthalpy than meth-
ane production86 and is a major metabolic product of the
Succinivibrionaceae family.
These naturally occurring rumen microbiome
compositions that are linked to the production of less
methane and increase feed efficiency without interven-
tion may be the target of future studies for decreasing
methane emission while maintaining food security.
Conversely, several non-​methanogenic microbial groups
have been consistently associated with high meth-
ane emission, among which are taxa from the genera
Ruminococcus, Prevotella and Mogibacterium, as well as
protozoa14,17,78,83,84,88. It remains to be determined how
robust these community state compositions and func-
tions are with regards to dietary changes and whether
they could be manipulated using dietary interventions.
Methane mitigation strategies
Can we mitigate methane emissions from ruminants
and still maintain food security for mankind? More than
50 years have passed since the initial development of miti-
gation strategies to limit the impact of methane produced
by agriculture. Extensive research has been conducted to
find effective strategies to reduce methane emissions from
the agricultural sector. Besides the greenhouse effect,
these emissions also represent a substantial energy loss
and contribute to decreased feed efficiencies21. Studies
suggest a connection between methanogenesis inhibition
and an increase in productivity of the animals89,90. In gen-
eral, the direct inhibition of methanogens affects fermen-
tation patterns of the rumen ecosystem as well as host
physiology, as it changes the metabolic thermodynamics
and directs more energy towards the host in the form of
specific types of SCFAs such as propionate rather than
emission of methane into the atmosphere91. Nevertheless,
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there is considerable variance between studies, and there-
fore it is tempting to speculate that such variation stems
from the potential alternative community states in which
individual microbiomes are locked in, and, consequently,
from their ability to support mitigation treatments and
channel energy from methane production to SCFAs
that the host can use. Given the ecological importance
of ruminal methane emissions, many comprehensive
reviews were published that describe the progress and
problems associated with conventional mitigation
strategies42,92–99. We review and summarize the main
research avenues, including direct and indirect strategies
used in adult animals, as well as strategies at the point
of parturition, and will expand discussions on recently
developed breeding programmes (Fig. 4).
Strategies to reduce methanogenesis. Many strate-
gies to reduce methane emissions directly target the
methanogenic communities or methane itself. These
include inhibitors of the methanogenesis reaction and
treatments that directly affect methanogens or meth-
ane removal by methanotrophic bacteria100. Inhibitory
compounds in the form of structural analogues101–103 or
algae supplementation104–106, which inhibit one or more
enzymatic steps of the methanogenesis pathway, have
been used. However, in general, the effectiveness of these
compounds is often transient107,108, presumably due to
adaptation and resistance mechanisms. It is worth men-
tioning two compounds due to their promising potential:
3-​nitrooxypropanol (3-​NOP)90,102,103,107–109 and the red
macroalgae Asparagopsis taxiformis that contains vari-
ous halogenated methanes103,104. The organic compound
3-​NOP was designed to inhibit the methyl-​CoM reduc-
tase (MCR) complex103, which mediates the final step of
the methanogenesis pathway across all methanogens102.
In most studies, 3-​NOP inhibits methanogenesis in vivo
by up to 60% when given as a food supplement without
compromising animal performance and health90,109.
Supplementing animal feed with A. taxiformis resul­
ted in the substantial reduction of methane production
(95–99% reduction) in an ex vivo rumen microbial com-
munity105,110. Moreover, supplementation of sheep with
A. taxiformis resulted in a consistent and dose-dependent
reduction in methanogenesis, mitigating methane pro-
duction by up to 80% without an effect on the host’s weight
for a period of 72 days111. The inhib­itory effect seems
to be specific, direct and immediate, as the reduction
of methanogenesis precedes the effect on methanogen
abundance with no measurable effect on SCFA produc-
tion as reported in an ex vivo study of the rumen micro­
biome105. Several highly active inhibitory A. taxiformis
compounds such as bromoform have been identified in
studies using ex vivo rumen micro­biomes106. Additional
direct strategies to inhibit methan­ogenesis include meth-
anogenic viruses and vaccines. Although proviruses
have been detected in rumen methanogenic archaeal
genomes67,112, no rumen archaeal viruses have been iso­
lated to date. Nevertheless, a lytic viral enzyme from
M. ruminantium was shown to lyse methanogenic cells
in vitro67, including members from the Methanobrevibacter
and Methanosphaera genera113. Immunization against
ruminal methanogens seems to only have a transient
effect, probably because the vaccine fails to provide
immunity against such a diverse group of methanogens114.

Adult (direct) Adult (indirect) Birth Prior to birth

• Direct inhibitors of
methanogenesis or
methanogenic archeae
(A. taxiformis and 3-NOP)
• Anti-methanogen
vaccines
• Chemical composition of the • Defaunation • Breeding towards
fibre • Modulation of low methane-
• Concentrate level in ration rumen microbiome emitting animals
• Lipids and plant extracts development from • Breeding towards
• Alternative metabolic birth (historical modulation of
pathways and hydrogen sinks contingency effects) specific microbiome
(for example, favouring composition
propionate)
• Antimicrobials (monensin)
• Supplementation of
alternative electron sinks
(sulfate and nitrate)

Fig. 4 | Methane mitigation strategies. Microbiome-​guided examples of methane mitigation strategies and interventions
that can be applied at different stages of animal development. In mature animals, antimicrobial feed additives that directly
affect methanogens, such as Asparagopsis taxiformis and 3-​nitrooxypropanol (3-​NOP), represent promising direct strategies.
Indirect approaches in mature animals include feed supplementation with alternative chemical electron sinks (for example,
nitrate and sulfate) that are of risk to the animal, antimicrobials (for example, monensin) that are commonly used, and
microbiome modulation for alternative metabolic electron sinks (for example, propionate-​producing pathways) that are
still in research and development. Among the strategies to intervene and mitigate methane at birth, defaunation was proven
to be effective but likely inapplicable at the farm scale; historical contingency effects are still in research and hold great
promise. Prior to birth, breeding programmes are already deployed but show negative effect on animal productivity and
microbiome-​guided breeding is still in research.

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Box 1 | Antimicrobial resistance in the rumen microbiome

As the use of antimicrobials is still a current mitigation practice in many
countries, and could also be reconsidered as a major strategy if an
efficient antibiotic would come to light in the future, we discuss the
current global effect of such treatments on our environment and human
life. We additionally elucidate general mechanisms of gene transfer
studied in this system that could augment antimicrobial resistance (AMR)
gene spread.
In livestock agriculture, substantial amounts of antibiotics are used to
treat, prevent and control disease165–167. Growth promoters are also used
to improve feed efficiency, which are sometimes administered orally
as feed additives. A major concern for human health is the threat that,
upon antibiotic supplementation, AMR determinants found in microbial
genomes will be selected, enriched and transferred across the food
chain168. Compared with other livestock sectors, the quantities of
antibiotics used for cattle are much lower than those used for chicken
and pigs167,169. Nevertheless, with a density of 1 × 1010–1 × 1013 bacterial
cells per gram of rumen contents2, microbial richness of hundreds of
species and large phylogenetic diversity, the rumen ecosystem could be
a fertile ground for horizontal gene transfer (HGT)170.
A large part of the potential for gene mobility is carried by the plasmi-
dome171. The rumen plasmidome was shown to be highly mosaic in nature,
even at high phylogenetic levels, notably the phylum level, thus supporting
the high frequency of gene-​shuffling events that occur by HGT via plas-
mids, with an estimated 7% of these genes being associated with viru-
lence171. An analysis of a ruminal microbial genome database43 revealed
that, across the main phyla, most genomes harboured resistance to at least
one antibiotic class. The most abundant AMR genes were related to resis­
tance to β-​lactams, glycopeptides, tetracycline and aminoglycosides172.
The tetracycline resistance genes were suggested to spread via plasmid
conjugative mechanisms into non-​phylogenetically related bacteria,
thus raising concern for further dissemination into the environment.
The mobile genetic pool and HGT are tightly linked to environmental
changes, such as those imposed by husbandry regimes16,173. It was
reported that AMR genes varied in abundance in a diet-​specific
manner174. Similarly, age — a known driver of rumen microbiome
composition — also affects the composition of the resistome175.
Together, these studies suggest that AMR genes that naturally occur
in rumen microbial genomes are changing in abundance, as a result
of fluctuations in microbiome composition, regardless of antibiotic
treatments. Nevertheless, the impact of antibiotics on the resistome
remains a debate, as several studies have reported the conservation
of AMR genes in the rumen microbiome, independent of the examined
antibiotic treatments176,177, whereas others have reported an increase
in specific AMR genes following antibiotic treatment177,178.
The resistome in ruminant waste was analysed in wastewater, manure
and soil from beef and dairy farms in an attempt to quantify the potential
environmental spread of AMR genes via agricultural waste and to
determine how or if they could affect human health175. Differences in
AMR genes were noted in each sample group, suggesting that specific
waste management regimes should be applied for each type of waste.
Furthermore, it was reported that the increase in AMR genes in soils
fertilized with manure was only temporary in countries with limited
antibiotic usage policies179. This could infer that differences in microbial
compositions, ecology and phylogenetics in these two environments
of manure and soils render HGT between their microbial inhabitants180.
Nevertheless, across seven different European countries, strong
correlations were reported between specific antibiotics used in livestock
and AMR in Escherichia coli isolates from treated livestock181. Policies to
decrease antimicrobials use in livestock agriculture were established and
have already taken effect, but, nevertheless, caution should be used when
administrating antibiotics to livestock owing to the possible threat of
AMR emergence and spread.

Horizontal gene transfer
(HGT). The lateral mobilization
of genetic material between
distinct microorganisms.
Rumen plasmidome
The collective plasmid
population in the rumen.
Resistome
The collection of antibiotic
resistance genes in a given
environment.
Newly sequenced genomes of isolated methanogens
and recently assembled archaeal genomes115 could help
to identify and develop broad-​spectrum molecules and
treatments that impair methane formation67.
Indirect mitigation strategies do not directly target
methanogens or methanogenesis, but influence methane
formation by modifying rumen metabolic cascades. Such
strategies would involve modulations of the trophic net-
works to decrease hydrogen availability for methane pro-
duction and to create less thermodynamically favourable
conditions for the establishment of methanogens86.
In this context, homoacetogens are a group of bacte-
ria that can reduce CO2 to acetate using hydrogen, and
could therefore theoretically compete with methanogens
(Fig. 3). Studies suggest that acetogenic microorganisms
could be used to replace methanogens in the rumen
microbiome51,116 to mitigate methane production, but
such attempts have essentially failed and only transient
effects were observed117,118. This is probably due to the
fact that hydrogenotrophic methanogenesis is thermo-
dynamically more favourable than the reduction of CO2
to acetate64.
By contrast, a more viable strategy is the use of
feed additives, such as nitrate, nitroethane and sulfate,
that are thermodynamically more favourable electron
acceptors for hydrogen than CO2 and can be reduced
by several prevalent microbial inhabitants of the rumen
ecosystem119–122. A drawback of such feeding additives is
that as the inhibitory effect follows stoichiometric rules,
large dietary inclusions of these compounds are likely
to be uneconomic. In addition, the use of nitrate and
sulfate compounds in large quantities would lead to the
formation of products poisonous to the microbiome or
the animal97,123. Furthermore, nitrate, sulfate and other
sulfur-​based compounds are known to decrease feed
intake97,123.
The above-​mentioned strategies relate to alternative
hydrogen sinks, which could compete with the methano­
genic sink, but additional fermentation pathways,
upstream in the redox potential, such as propionate
and butyrate fermentation, use reducing equivalents or
directly utilize hydrogen and thus could stoichiometri-
cally lower hydrogen availability to methanogenesis86,124.
Indeed, supplementation with intermediates of carbo­
hydrate fermentation, such as acrylate, fumarate or
malate, results in lower methane emission, but the
levels vary considerably between experiments117,118,125.
Alternatively, selective antimicrobials that alter the com-
position of the rumen microbiome to produce less hydro-
gen (that is, less substrate for methane formation) were
proposed. These include unsaturated fatty acids, mixtures
of lipids, essential oil compounds (see below)98,126–128 and
ionophores such as monensin129. Although ionophore
antibiotics are not currently used in human or animal
therapeutics130, their use in animal production is con-
troversial and some were even banned for use in mem-
ber countries of the European Union131 (Box 1). Hence,
other compounds such as the bacterial peptide nisin132
that, similar to monensin, increase the permeability of
cell membranes of microorganisms were suggested as

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Biohydrogenation
The microbial transformation
of unsaturated fatty acid
to saturated fatty acid.
Residual feed intake
A parameter that describes
feed efficiency, measuring the
difference between the actual
feed intake and the predicted
intake, based on an animal’s
body weight, weight gain and
milk composition, and is a
proxy for the animal’s ability
to extract energy from its feed.
562 | September 2021 | volume 19
alternatives. Although monensin and nisin differed in
their effect on rumen fermentation and microbial com-
position in an ex vivo rumen microbiome study, nisin
was shown to have a similar functional effect of decreas-
ing methanogenesis and increasing propionate produc-
tion but, unlike monensin, did not negatively affect fibre
digestibility133.
As mentioned above, lipids and plant extracts (tan-
nins, saponins and essential oils) were proposed as
possible antimicrobial strategies because they might
also reduce hydrogen bioavailability via microbial
biohydrogenation98. However, the potential of biohydro-
genation to function as a hydrogen sink is too small, as
only 1–2% of the hydrogen produced during micro-
bial fermentation is consumed by biohydrogenation134.
Hence, the mode of action is not entirely clear but
several notions have been put forward, including
membrane disruption, alterations in chemotaxis and
substrate acquisition, lowering accessibility of die-
tary particles by coating them and decreasing cation
availability due to salt formation and toxicity due to
obstruction of nutrients135. Meta-​analyses support a
consistent reduction in methane production upon
lipid supplementation126,127, which is correlated with
the supplemented amounts98. Another meta-​analysis of
23 in vivo studies also demonstrated an improvement in
feed efficiency, fat and protein-​corrected milk following
supplementation with Agolin oil during periods longer
than 1 month128.
Dietary polysaccharides influence methanogenesis in
various ways. For example, an increase of starch content
in the feed decreases methanogen activity, possibly as an
increase in lactate production due to starch fermenta-
tion decreases the pH and inhibits methanogenesis, and
therefore propionate formation is promoted due to the
increase availability of reducing equivalents14,136–138.
In addition, the plant particle retention time in the rumen
is also thought to have a major role in methanogenesis —
the longer the retention time, the higher the amount
of methane produced11. A thermodynamic model of
rumen fermentation implied that not only the quantity
of hydrogen produced is connected to methane emission
but also the rate of digestion. Higher rates and shorter
retention times led to negative feedback with insufficient
time for methanogens to establish and utilize the hydro-
gen, which could be rerouted to propionate production61.
Thus, a major recommended methane mitigation prac-
tice is increasing forage digestibility to reduce the reten-
tion time139 by techniques such as feed grinding140,141 or
introduction of exo­genous fibrolytic enzymes as feed
supplements. It was also established that smaller rumen
volumes decrease the feed retention time and, sub­
sequently, methane production, although such a strategy
might negatively affect the energy harvest efficiency141.
On the basis of those findings, it was suggested that
physical and genetic characteristics of the animal can
influence methane production141,142.
Breeding and genetics to mitigate methanogenesis.
Methane emission is a heritable trait with measured
heritability estimates of 0.3–0.45 g day–1 of dry matter
intake143–146. Breeding programmes have been initiated
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to select for lower methane-​emitting animals, targeting
smaller rumen sizes and, therefore, low particle retention
times142, but it is noteworthy that this breeding strategy
also lowers feed efficiency146. To launch breeding pro-
grammes, specific host genetic markers linked to lower
methane emission but that do not affect productivity
need to be identified. As mentioned above, particular
microbiome compositions have been connected to lower
methane emission and higher feed efficiency (approx-
imated by the residual feed intake). Now, an increased
understanding of the relationships among host genet-
ics, rumen microbiome composition and methane emis-
sion is central to develop microbiome-​guided breeding
strategies.
In this context, microbiome composition, host traits
and genetics have been associated in several recent
studies17,19,28,147. The identification of heritable micro­
organisms is based on statistical connection and does
not provide any mechanistic insights into the under-
lying mechanisms of host–microorganism interac-
tions; never­theless, one could hypothesize that the host
immune system and other physiological factors such as
the buffer’s strength of host saliva that influences rumen
pH, directly affect the abundance of specific micro­
organisms in the rumen. Indeed, changes in gene regu­
lation of rumen epithelial cells and their morphology
correlate with methanogenesis, SCFA production and
rumen microbiome composition, thus connecting host
physiological factors to rumen microbiome composition
and function6,8,148.
A recent study of 1,000 cows28 from different farms
throughout Europe revealed 39 microorganisms that
are heritable and central in the microbiome inter­action
networks. Seven of those microorganisms could be
strongly associated with the methane production trait.
These include one member from the Succinivibrionaceae
genus, four from the Prevotella genus, one member from
the Bacteroidales order and one from the Shuttleworthia
genus along with protozoal species of the subclass
Trichostomatia. It would be extremely interesting to pursue
the biological nature of these heritability associations to
host genetics and to methane emissions. The fact that
these heritable microorganisms are more interconnected
than others in the interaction networks suggests that they
might have a pleiotropic effect on microbiome compo-
sition with a potential for lowering methane emission if
they could be manipulated via breeding programmes.
Furthermore, as the host genome and rumen micro-
biome were shown to be also independent in their
contribution to methane emissions, future strategies
should possibly target both host genetics via breeding
and rumen microbial composition via complementary
mitigation methods147.
Strategies to mitigate methanogenesis during microbial
succession. Another suggested strategy to manipulate the
rumen microbiome towards lower methane emissions
is to intervene at the first stages of the succession and
assembly process of the rumen microbiome (early-​life
intervention)149–151. For example, targeting the proto-
zoal population, which is colonized by methanogens,
has been extensively investigated152–155, and defaunation
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(that is, the removal of protozoa) decreased methano-
genesis by 13–35%88,153,156. Nevertheless, in most of the
studies, long-​term effects have not been considered
and only short-​term effects were documented157. Only
recently, a major high-​resolution study spanning 3 years
of the cows’ life revealed that the initial species pool after
birth has a long-​lasting effect on the assembly process
and adult composition of the rumen microbiome40.
In the latter study, the authors investigated the effects of
caesarean section and vaginal delivery on the assembly
and development of the rumen microbiome. Comparing
rumen microbiome assembly of these cohorts, which
had uniform dietary regimes, rearing conditions and
very similar genetic backgrounds, the authors found
that historical contingency effects, such as access to the
maternal species pool, shape rumen microbial commu-
nity development, with long-​lasting consequences on
temporal dynamics and taxon composition at the adult
stage40. This was suggested to reflect the outcome of
niche modification by microbial groups in each of the
delivery modes, which was deemed a strong determi-
nant in defining microbiome composition and assem-
bly trajectories15,128,158. Furthermore, a change in dietary
regime (from a high-​starch diet to a fibre-​rich diet) during
development of the rumen microbiome was followed
by an increase in Methanobacteriaceae and a decline
in the hydrogen-​utilizing family Succinivibrionaceae.
Therefore, the dietary regime in early life might be
another potential way to select for low methane emission
during animal development40.
Conclusions
In this Review, we summarized 60 years of research
into rumen microbial ecology and host microbiome
interactions, while focusing on the factors that affect
methan­ogenesis, as well as the main characteristics of
the methanogenic community, how they are intercon-
nected with other members of the rumen microbial
community and how these connections are intertwined
with developing methods for methane mitigation.
A large part of our existing knowledge for the function
of rumen microbial groups has mainly been gained
from isolated microorganisms. To further increase our
understanding of the function of the different microbial
groups in the rumen, we need to increase the repertoire
of known rumen isolates, as there still exists a large
discrepancy between the typical rumen composition
gained from culture-​independent techniques and the
reported cultured microorganisms of rumen origin159,160.
A better understanding of isolated rumen microbiome
members is crucial to unravel the metabolic interactions
among the resident microorganisms, and to enable the
rational design of the microbiome. Specifically, catego-
rizing bacteria using similar input and output metabo-
lites into functional groups will guide future microbial
assemblies41. In addition, the development of methods
to investigate the methanogenic population associated
with protozoa could provide useful insights161. Moreover,
future breeding strategies would benefit from fundamen-
tal research of isolated heritable microorganisms that
show strong associations with the methane production
trait and the mechanism underlying these associations.
These microorganisms should then be targeted during
rational microbiome engineering for intervention in the
rumen microbiome.
Many mitigation strategies suffer from drawbacks
such as microbial adaptation or modification of ani-
mal productivity. The main drawbacks, as we see them,
stem from a lack of mechanistic knowledge of the rumen
microbiome assembly, development and function and of
the host–microbiome interactions. An increased under-
standing of those aspects would enable predictions of
the effects of different treatments on microbiome devel-
opment and on mature community states. Such know­
ledge could be gained from longitudinal studies with high
resolution that are still very scarce.
The development of new technologies currently
available for microbial ecology, such as advanced
microscopy, including cryo-​e lectron microscopy,
deep sequencing, broad-​scale metabolomics of thou-
sands of different metabolites, proteomics and stable
isotope probing using nanoscale secondary ion mass
spectrometry technologies, integrated with classical
culturing techniques and accumulated knowledge of
the rumen microbiome creates the opportunity to fully
understand the rumen ecosystem and modulate it to
decrease methane emissions while maintaining food
security.
Finally, for these findings and technologies to be
adapted by policy-​makers and the agricultural sector,
research should focus on combining the various afore-
mentioned approaches, while keeping in mind that
mitigation strategies should only interfere to a minimal
extent with the protein necessity of humanity.
Published online 12 May 2021

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Acknowledgements
The authors acknowledge the collaborative nature of the work
on the rumen microbiome by thanking the global scientific
community involved in rumen microbiome research and the
deciphering of its mysteries. The authors also thank E. Jami
(Agricultural Research Organization, Volcani Center, Israel)
and E. A. Bayer (The Weizmann Institute of Science, Israel) for
critical reading of the manuscript. Research in the authors’
laboratory was supported by grants from the European
Research Council (No. 640384) to I.M. and from the Israel
Science Foundation (ISF No. 1947/19) to I.M. and S.M.
Author contributions
I.M. and S.M. researched data for the article, substantially
contributed to discussion of content and wrote the article.
I.M., R.J.W. and S.M. conceptualized, reviewed and edited
the manuscript before submission.
Competing interests
The authors declare no competing interests.
Peer review information
Nature Reviews Microbiology thanks S. Huws, T. McAllister
and the other, anonymous, reviewer(s) for their contribution
to the peer review of this work.
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Related links
Food and Agriculture Organization of the United Nations
(FAOsTAT) enteric fermentation: http://www.fao.org/
faostat/en/#data/GE
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