World J Microbiol Biotechnol (2009) 25:1557–1566
DOI 10.1007/s11274-009-0041-3
ORIGINAL PAPER
Factors affecting rumen methanogens and methane mitigation
strategies
Sanjay Kumar Æ Anil Kumar Puniya Æ Monica Puniya Æ
Sumit Singh Dagar Æ Sunil Kumar Sirohi Æ
Kishan Singh Æ Gareth Wyn Griffith
Received: 30 July 2008 / Accepted: 2 April 2009 / Published online: 18 April 2009
Ó Springer Science+Business Media B.V. 2009
Abstract The rumen is a highly diverse ecosystem
comprising different microbial groups including methano-
gens that consume a considerable part of the ruminant’s
nutrient energy in methane production. The consequences
of methanogenesis in the rumen may result in the low
productivity and possibly will have a negative impact on
the sustainability of the ruminant’s production. Since
enteric fermentation emission is one of the major sources
of methane and is influenced by a number of environmental
factors, diet being the most significant one, a number of in
vitro and in vivo trials have been conducted with different
feed supplements (halogenated methane analogues, bacte-
riocins, propionate enhancers, acetogens, fats etc.) for
mitigating methane emissions directly or indirectly, yet
extensive research is required before reaching a realistic
solution. Keeping this in view, the present article aimed to
cover comprehensively the different aspects of rumen
methanogenesis such as the phylogeny of methanogens,
their microbial ecology, factors affecting methane emis-
sion, mitigation strategies and need for further study.
Keywords Rumen
Methanogens
Methane
S. Kumar
A. K. Puniya (&)
S. S. Dagar
K. Singh
Dairy Microbiology Division, National Dairy Research Institute,
Karnal 132001, India
e-mail: puniya1234@rediffmail.com
M. Puniya
S. K. Sirohi
Dairy Cattle Nutrition Division, National Dairy Research
Institute, Karnal 132001, India
G. W. Griffith
Institute of Biological, Environmental and Rural Sciences,
University of Wales, Aberystwyth, Ceredigion
SY23 3DD, UK
Introduction
The rumen is characterized by its high microbial popula-
tion density and also by a wider diversity and complexity
of micro-ecological interactions. This bionetwork is com-
prised of different prokaryotes, methanogenic Archaea,
eukaryotes, (protozoa, anaerobic fungi) and bacterio-
phages. Interest in the rumen methanogens has resulted
from the fact that ruminants typically lose 2–15% of their
ingested energy solely as methane (Moss et al. 2000).
Methanogens in the domain of Archaea are character-
ized by their ability to produce methane under highly
anoxic conditions (Guo et al. 2005). Among the different
sources of methane emission viz, wetlands, paddy fields,
energy sectors, ruminants, landfills and biomass burning
(Wright et al. 2004; Mastepanov et al. 2008); methane from
enteric fermentation constitutes the largest source. Miti-
gation of methane may contribute to lowering the green-
house effect that is a topical issue for the scientific
community (Johnson and Johnson 1995; Moss et al. 2000).
However, there is scanty information available concerning
the composition and numbers of methanogens and the
effect of variations in diet on these populations. A number
of species of methanogens have been isolated from rumi-
nants and well characterized by classical microbiological
techniques (Wolin et al. 1997). Conversely, because of the
highly fastidious growth requirements of the rumen meth-
anogens, it is expected, that independent molecular meth-
ods would reveal a greater diversity leading to the
resolution of taxonomic details of these Archaea.
Molecular approaches have already demonstrated the
high diversity of methanogens in anaerobic treatment sys-
tems (Sekiguchi et al. 1998; Casserly and Erijman 2003),
landfills, soil, rice fields, wetlands, peat bogs (Nercessian
et al. 1999; Luton et al. 2002; Galand et al. 2002; Sheppard
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et al. 2005; Sakai et al. 2007) and the rumen ecosystem
(Whitford et al. 2001; Wright et al. 2004), and have
identified several new taxa. Among the factors that influ-
ence ruminant methane production, changes in the rumen
fermentation pattern resulting from altered diets is proba-
bly the most important. Furthermore, various feed supple-
ments have been found to directly or indirectly reduce
methane emissions, including halogenated methane ana-
logues (Ungerfeld et al. 2004), bacteriocins (Lee et al.
2002), propionate enhancers, acetogens, immunization,
genetic engineering, phage, fats and probiotics such as
Saccharomyces cerevisiae, Aspergillus oryzae etc. (Boadi
et al. 2004; Moss et al. 2000). All the present strategies
either singly or in combination appear to be quite prom-
ising, but more research is needed so as to validate these
before making any claim.
Phylogeny of methanogens
The methanogens are a large and highly diverse group of
Archaea (Baker 1999) comprising 5 genera and 23 species
isolated in pure culture (Boone et al. 2001). These are
present in diverse natural anaerobic ecosystems (i.e. wet-
lands, marshes, activated sludge, rhizosphere, rumen etc.),
and obtain their energy from the reduction of carbon
dioxide, formate, the methyl moiety of methanol, acetate
and some methyl amines (Jones 1991). In the rumen,
methanogens primarily use hydrogen, carbon dioxide and
formate as a substrate. These microorganisms specifically
possesses three coenzymes i.e., coenzyme F-420, involved
in electron transfer; coenzyme M, involved in transfer of
methyl groups; and a low molecular weight, oxygen labile,
heat stable coenzyme B, involved in the final reaction of
methane production. The free energy change associated
with methanogenesis under environmental conditions pro-
duces 1 mol of ATP while under standard conditions it
Table 1 Cultured methanogenic species from ruminants (Jarvis et al. 2000; Joblin 2005; Rea et al. 2007; Janssen and Kirs 2008)
Host ruminant Gram reaction and morphology
Bovine; ovine Gram variable, filamentous
long rods
Bovine
Bovine, ovine, Gram positive, coccobacilli
cervine
Ovine
Bovine, ovine
Bovine, ovine
Bovine Gram negative, motile curved rods
Bovine, caprine Gram negative, Pseudosarcina
Cervine Methanoculleus olentangyi
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produces 2 mol ATP (Deppenmeier and Muller 2008).
Moreover, there is relevant difference in energy conser-
vation of these archaea with respect to cytochromes.
Methanogens having cytochromes produce energy chemi-
osmotically compared to other methanogens where meth-
anogenesis is energetically coupled to cytoplasmic
enzymes (Thauer et al. 2008). Methanogens and other
Archaea differ from the Bacteria not only in possessing
pseudomurein in their cell walls, but also in having cell
membrane lipids composed of isoprenoids, ether-linked to
glycerol or other sugars (Baker 1999).
Moreover, methanogens are nutritionally fastidious
anaerobes requiring a very low redox potential i.e. below
-300 mV (Stewart and Bryant 1988) and growing most
rapidly in the pH range of 6.0–8.0. Though most species
have hitherto proven resistant to axenic culture (Amann
et al. 1995), the following have been isolated from the
rumen and are listed in Table 1. Of these, Methanobrev-
ibacter ruminantium and Methanosarcina barkeri are
considered of greatest significance, being present at the rate
of over 106 ml-1 in the rumen (Lovley et al. 1984).
Although of limited metabolic diversity, methanogenic
archaea or methanogens possess great phylogenetic and
ecological diversity with G?C contents ranging from
23–61 mol%, bacillary, coccal, and spiral cell morphology
(Brown et al. 1989; Garcia 1990 and Ferry 1993). Cultured
methanogens are grouped into five orders based upon their
phylogeny and phenotypic properties. In addition, uncul-
tured methanogens that may represent new orders are
present in many environments. The ecology of methano-
gens highlights their complex interactions with other
anaerobes and the physical and chemical factors control-
ling their function. Detailed description of the systematics
are reviewed in ‘Bergey’s Manual of Systematic Bacteri-
ology’ (Whitman et al. 2001) and ‘The Prokaryotes’
(Whitman et al. 2006; Garcia et al. 2006; Kendall and
Boone 2006; Liu and Whitman 2008).
Genus and species Substrate
Methanobacterium formicicum Hydrogen, formate and acetate
Methanobacterium bryantii
Methanobrevibacter ruminantium Hydrogen and formate
Methanobrevibacter smithii
Methanobrevibacter olleyae
Methanobrevibacter millerae
Methanomicrobium mobile Hydrogen, formate and acetate
Methanosarcina barkeri Hydrogen, methanol, methyl
acetate and acetate
Methanoculleus olentangyi Hydrogen, formate and acetate
World J Microbiol Biotechnol (2009) 25:1557–1566
Nolling et al. (1996) reported that Methanopyrus kand-
leri represents a distinct methanogen lineage on the basis of
its methyl-coenzyme M reductase operon. The enzyme
consists of a2b2c2 polypeptide subunit (Harms et al. 1995)
and the genes that encode these subunits are mcrA, mcrB
and mcrG, respectively. These genes are arranged as
mcrBDCGA, within a single transcriptional unit, designated
as MR operon (Klein et al. 1988; Reeve 1992). However,
some methanogens contain a second MR isoenzyme MRII
(Rospert et al. 1990). A phylogenetic tree of the Meth-
anosarcineae based on a 490 bp region of the mcrA locus
from 25 different species was fully congruent with the
phylogeny of methanogens based on 16S rRNA (Nolling
et al. 1996). Goat rumen ciliate-associated bacteria and
methanogenic Archaea were analyzed on the basis of 16S
rRNA gene retrieved from Polyplastron multivesiculatum,
Isotricha intestinalis, and Ophryoscolex purkynjei. Two
Archaea related to Methanobrevibacter smithii were com-
mon in tested ciliate cells (Irbis and Ushida 2004). The
diversity of methanogenic Archaea associated with differ-
ent species of ciliated protozoa in the rumen was analyzed
by amplification of partial fragments of Archaea SSU rRNA
genes from Metadinium medium, Entodinium furca, Oph-
ryoscolex caudatus and Diplodinium dentatum. Sequence
analysis of these fragments revealed that all the new isolates
clustered well with the sequences of known methanogens,
yet there was a difference in the relative distribution of
sequences. Changes in protozoa species due to oil supple-
mentation also led towards increased trend of diversity of
methanogenic Archaea related to Methanosphaera stadtm-
anae, M. smithii and some uncultured groups (Ohene-Adjei
et al. 2008). Luton et al. (2002) used mcrA gene in the
phylogenetic analysis of methanogen population in landfill
as an alternative to 16S rRNA. Denman et al. (2007) found
changes within the mcrA-based clone libraries of ruminal
methanogens in response to bromochloromethane (which
inhibits coenzyme M involved in methanogenesis) and also
revealed that Methanobrevibacter spp. were dominant.
Many novel sequences, clearly of Archaeal origin, have
relatively low identity to the most highly related sequences
present in the Genbank database (Regensbogenova et al.
2004).
Several species of methanogen have also been isolated
from ruminants and some (e.g. M. ruminantium, Methan-
obrevibacter formicicium, Methanomicrobium mobile)
have been consistently found in high numbers (Raskin et al.
1994; Stewart et al. 1997; Wolin et al. 1997). Before the
advent of molecular approaches, cultivation-based tech-
niques allowed isolation and characterization of only lim-
ited number of species in the rumen (Miller et al. 1986).
However, uncultured methanogens can be visualized using
16S rRNA-based FISH probes, which are specific to certain
families of methanogens (Sharp et al. 1998). Tokura et al.
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(1999) on the basis of phylogenetic analysis of ciliate-
associated methanogens found that M. smithii was domi-
nant over M. ruminantium. Whitford et al. (2001) analyzed
41 methanogen-related rDNA sequences from the bovine
rumen. The most abundant clones in this library were from
M. ruminantium and this was found to be the most sig-
nificant species of the rumen methanogens followed by
M. stadtmanae, not previously described in the rumen, but
there was also a cluster of unidentified methanogens con-
taining six highly similar clones with a sequence similarity
of 99.0–99.8%, suggesting some novel groups of metha-
nogens. The molecular diversity of the rumen methanogens
from sheep was also shown on the basis of 16S rRNA,
whensheep were fed on different diets. On the basis of
sequence similarity, 65 phylotypes were found to be of
order Methanobacteriales and greater diversity was found
to be present in pasture-grazed sheep as compared to the
sheep fed with oaten or Lucerne hay (Wright et al. 2004).
Uncultivable methanogenic Archaea were also identified
using 16s rRNA gene libraries prepared from pooled rumen
liquor from merino sheep. These Archaea showed 72–75%
similarity to Thermoplasma acidophilum and Thermo-
plasma volcanium (Wright et al. 2006). Molecular analysis
of 16S rRNA of complex microbial communities degrading
long chain fatty acids in methanogenic bioreactors identi-
fied two major groups of methanogens, closely related to
Methanobacterium (Diana et al. 2007). In a study con-
ducted on reindeer rumen, methanogens from the families
Methanobacteriaceae and Methanosarcinaceae were high-
lighted on the basis of 16s rRNA sequence similarity
(Sundset et al. 2008). Hook et al. (2009) studied the effect
of monensin on the rumen methanogens and concluded that
long-term monensin supplementation could not signifi-
cantly alter the quantity or diversity of methanogens in the
rumens of lactating dairy cattle.
Microbial ecology of the rumen methanogens
The rumen consists of complex, anaerobic microbial popu-
lations including 1010–1011 bacteria, 108–109 methanogens,
106 ciliate protozoa and 106 fungi/ml. The establishment and
maintenance of the stable population of methanogens is
affected by the type of diet, level and frequency of feeding.
The rumen methanogens utilize reducing equivalents that are
mostly produced by fermentative hydrogen-producing bac-
teria, anaerobic fungi and ciliate protozoa. The symbiotic
association of hydrophobic methanogens with hydrogen
producers is usually realized by attachment or by floc for-
mation (Conrad et al. 1985; Thiele et al. 1988; Lange et al.
2005). Among these ciliates, protozoa are the only ones for
which such interaction can be microscopically demonstrated
(Vogels et al. 1980). The symbiotic relation between
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methanogens and ciliates may generate up to 37% of rumen
methane emission (Finlay et al. 1994).
In the rumen, hydrogen is produced during plant cell-
wall degradation as an intermediate compound by cellu-
lolytic bacteria, (i.e. Ruminococcus albus, Ruminococcus
flavifaciens) and anaerobic fungi (Stewart and Bryant
1988). Hydrogen never accumulates, as it is rapidly utilized
by methanogens in ruminants (Wolin and Miller 1988) to
produce methane and generate ATP (Ferry and Kastead
2007; Albers et al. 2007). Indeed, even traces of hydrogen
inhibit sugar oxidation in the rumen (McAllister and
Newbold 2008).
Factors affecting methane production
The major factors that affect methane production in the
ruminants are pH, volatile fatty acids, diet, feeding strat-
egy, animal species and environmental stresses. The opti-
mum pH for methane production is 7.0–7.2, but the gas
production can occur in the pH range of 6.6–7.6. However,
beyond this range, the activity of fiber degraders reduces
(Arglyle and Baldwin 1988; Dijkstra et al. 1992). Diet has
an important impact not only on methanogen numbers but
also on methane production, as both the quantity and
quality of feed can alter the rumen fermentation pattern.
There is a different profile of volatile fatty acids and
methane production with different carbohydrates fermented
(Murphy et al. 1982; Arglyle and Baldwin 1988; Friggens
et al. 1998). Simple carbohydrates produce more methane
(0.45 moles/mole of hexose) as compared to complex
carbohydrates (0.3 moles/mole of hexose). Forage: con-
centrate ratio also influence the acetate: propionate ratio,
and methane emission decreases drastically from 6–12%
(forage-based diet) to 2–3% when diet with concentrate
([90%) predominates (Johnson and Johnson 1995). Feed-
ing of a high concentrate: low roughage diet produce less
methane as compared to low concentrate: high roughage
diet (Whitelaw et al. 1984). Singh and Singh (1997)
reported a decrease in the numbers of methanogens and
cellulolytic bacteria when concentrate: roughage diet
(75:25) was given to the cattle.
Das and Singh (1999) analyzed the effect of different
proportions of berseem (Trifolium alexandrium) with
wheat straw on in vitro methane production and found that
methane decreased from 51 to 42% when wheat straw was
partially replaced with berseem. Singh and Mohini (1999),
also observed that in vitro methane production decreased
by replacing the wheat straw with berseem and legume
fodder at different levels. A similar trend of reduced
methane emission was also reported in a prediction model
by Benchaar et al. (2001) with legumes in the diet of
ruminants. This lower methane emission can be attributed
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to lower portion of carbohydrates, faster passage rate,
hence, shifting the fermentation pattern towards higher
propionate production (Johnson and Johnson 1995).
Aside from diet, a higher intake and passage rate of
digesta can also effect methane production in the rumen.
The residence time in the rumen (i.e. the opportunity for
microbial action) decreases with increased passage rate,
feed intake and the digestion occurs in small intestine
rather than in the rumen, which in turn reduces the extent
and rate of ruminal dietary fermentation. Methane pro-
duction is also influenced by host species; with methane
emissions from crossbred cattle (Holstein Friesian 9 Har-
yana) being higher than those from exotic breeds (Lal et al.
1987). Similarly methane emission have been reported in
Indian cattle (Srivastava and Garg 2002) and Buffalo
calves (Mohini and Singh 2001) suggesting that the vari-
ations among the animal species also show variations in
methane emission.
Mitigation of methane emission from ruminants
If methane emissions continue to rise in direct proportion
to livestock number a 60% increase in global methane
production is predicted by 2030, (FAO 2003). However,
changes in feeding regime could remodel the present sce-
nario of methane emission from livestock and thus mitigate
some of this increase. According to the US-EPA (2006)
pooled methane emission from enteric fermentation and
manure management attributable to domesticated livestock
are predicted to increase by 21% from 2005–2020. Possible
changes in animal production practices for reducing
methane emission are discussed below.
Chemical inhibitors
Methanogenesis can be directly inhibited by halogenated
methane analogues (i.e., 2- bromoethanesulphonate,
3-bromopropanesulphonate), lumazine, propionic acid and
ethyl 2- butynoate (Van Nevel and Demeyer 1995). Chloral
hydrate (which is converted to chloroform in the rumen)
inhibits methane production in vivo (Mathers and Miller
1982) but it can also lead to liver damage and death in
sheep after prolonged feeding (Lanigan et al. 1978). In
contrast, amichloral appeared to be safer and was found to
increase live weight gain in sheep.
Denman et al. (2007) reported that bromochloromethane
could decrease the number of methanogenic Archaea by
34% as assayed by qPCR and thus reduce methane pro-
duction. But the anti-methanogenic activity of bromochlo-
romethane has been reported to be transient (Sawyer et al.
1974). However, a combination of bromochloromethane
with a-cyclodextrin was found to be more stable, and was
World J Microbiol Biotechnol (2009) 25:1557–1566
shown to suppress methane emission in sheep and cattle
over a prolonged period (McCrabb et al. 1997). In recent
report bromochloromethane inhibited methane emission
during batch fermentation and the effect also persisted in
continuous fermentation (Goel et al. 2009).
A bromine analogue of coenzyme M (2-bro-
moethanesulphonate), involved in methyl group transfer
during methanogenesis, was found to be a potent methane
inhibitor (Martin and Macey 1985; Wolfe 1982). However,
when tested in vivo, the inhibition in methanogenesis was
temporary, inducing the adaptation of the methanogens
(Van Nevel and Demeyer 1995). Oral administration of
nitroethane and 2-nitropropanol resulted in methane
reduction and nitroethane was found to be the better of the
two inhibitors (Anderson et al. 2006).
Quaternary ammonium compounds inhibit methano-
genesis at concentration of 25 mg/l or above but at pro-
longed periods of incubation the effect become transient
(Tezel et al. 2006). Anthraquinone has also been shown to
inhibit methanogenesis in vitro and in lambs by inhibiting
the methyl-coenzymeM reductase (Garcia-Lopez et al.
1996; Kung et al. 1998). Lovastatin and Mevastatin,
inhibitors of the enzyme hydroxymethylglutaryl-SCoA
reductase also directly reduced methane emission up to
50% by inhibiting the growth of Methanobrevibacter
without affecting the feed utilization efficiency (Miller and
Wolin 2001). Lila et al. (2004) found a significant decrease
in methane production both in vitro and in vivo without
affecting other rumen micro-flora using cyclodextrin dial-
lyl maleate in the diet.
Ionophores
Monensin, an ionophoric antibiotic has been shown to
depress methane production by mixed rumen microflora in
vitro (Van Nevel and Demeyer 1992). The ionophore affects
methanogens indirectly by inhibiting protozoa and cellulo-
lytic bacteria (Hino et al. 1993), which in turn changes the
dominant product of sugar fermentation from acetate to
propionate (Chen and Wolin 1979; Newbold et al. 1995).
However, the protozoan population can adapt to ionophores
present in low or high concentrate diets. Even rotation of
ionophores could not prevent protozoa adaptation to iono-
phores (Guan et al. 2006). Van Nevel and Demeyer (1995)
claimed that monensin lowered methane production to the
extent of 25% in the short term but did not persist in the
longer term (Johnson et al. 1991). In contrast, altered pat-
terns of volatile fatty acid production were found to persist in
monensin-treated animals during long term trials (Rogers
et al. 1997). The effect of salinomycin on methane produc-
tion also appeared to be lasting (Wakita et al. 1986). How-
ever, there is a risk that polyether ionophores may get
absorbed from the rumen and reach meat or milk. Russell
1561
and Rychlik (2001) indicated that ionophore resistance
counteracts the improvement in feed efficiency.
Propionate enhancers
Inclusion of dicarboxylic organic acids in the diet (e.g.,
malic acids and fumaric acid) results in shifting the rumen
fermentation towards propionic acid. Hence, less methane
production (Martin 1998; Lopez et al. 1999a) was reported.
Addition of sodium fumarate in vitro decreased methane
production and also increased dry matter digestibility
(Lopez et al. 1999b). Asanuma et al. (1999) found that
fumarate also reduced methane production in vitro and
suggested it to be an economical feed additive. Similarly,
malate which is converted via fumarate in the rumen was
found to stimulate propionate production and to inhibit
methanogenesis in vitro (Martin and Streeter 1995).
However, in vitro experiments by Carro et al. (1999)
reported that although malate did decrease methane pro-
duction per unit of dry matter digested but enhanced fiber
digestion resulted in a net increase in methane production.
The proportion of higher acids (i.e. fumaric acid)
decreases with increased concentration of acetic and pro-
pionic acids (Isobe and Shibata 1993). Itabashi et al. (2000)
reported that the molar concentration of propionic acid
increased with 16% decrease in methane when Holstein
steers were fed with fumaric acid supplemented with sal-
inomycin. However, a 23% reduction in methane emission
was reported when fumaric acid was supplemented along
with sorghum silage. Ungerfeld et al. (2007) suggested that
fumaric acid at low concentration would be more effective
by being converted into propionic acid and hence, reducing
methane production.
Stimulation of acetogens
Reductive acetogenesis by acetogenic bacteria during
hindgut fermentation (Demeyer and De Graeve 1991;
Fonty et al. 2007a) could act as an alternative hydrogen
sink and be exploited to reduce rumen methanogenesis
(Moss et al. 2000). Lopez et al. (1999a) found that aceto-
gens suppressed methane production when added to the
rumen fluid in vitro but that they did not persist in the
rumen. However, they concluded that it might be possible
to achieve the same metabolic activity using acetogens
provided as a daily-fed feed additive. Similarly, Joblin
(1999) reported that artificially increasing the concentra-
tion of acetogens through exogenous inoculation could be
useful for competing against methanogens as they have
many-fold greater hydrogen threshold concentration than
methanogens. The strategy was further validated by in vivo
trials on gnotobiotic lambs that were inoculated with a
functional microflora lacking methanogens. On the basis of
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feed intake, volatile fatty acids, population density and
hydrogen utilization pattern it was suggested that reductive
acetogenesis can sustain a functional rumen in absence of
methanogens (Fonty et al. 2007b).
Methane oxidizers
Methane accumulation is the difference between methane
production and its oxidation. Methane-oxidizing bacteria
have been isolated from the rumen (Moss et al. 2000) and
other environments (Stock and McCleskey 1964) and have
been identified based on molecular phylogeny (Heyer et al.
2002). In vitro studies revealed that the extent of methane
oxidation in the rumen is very limited i.e. 0.3–8% (Valdes
et al. 1996; Boadi et al. 2004). However, increase in the
activity or number of these groups of bacteria may be of
much significance in mitigating the methane and hence,
reducing the green house gases in the environment through
the rumen intestinal manipulations. However, the impli-
cation seems to be quiet impractical, as methane oxidizers
need oxygen for their growth.
Defaunation
Vogels et al. (1980) observed methanogenic bacteria on the
exterior surface of the rumen ciliate protozoa and some
methanogens have also been shown as endosymbionts of
these protists (Finlay et al. 1994). Newbold et al. (1995)
reported that methanogens associated with ciliate protozoa
were responsible for 9–25% of the methanogenesis in the
rumen fluid. Ohene-Adjei et al. (2007) studied the estab-
lishment and association patterns of protozoa with metha-
nogenic archaea in the ovine rumen and suggested that the
association is linked to the evolution of commensals and is
symbiotic. In a similar observation, the defaunation of the
rumen leads to decreased methane production (Ushida et al.
1997) but because of the toxicity of defaunating agents to
the host animal, this is not a practical approach (Williams
and Coleman 1992). In a study on comparison of individual
species of protozoa on methane emission, it was concluded
that elimination of Entodinium caudatum could reduce
methane emission from the rumen with no adverse effect on
feed degradation (Ranilla et al. 2007). In a contrasting
report, the effects of the absence of protozoa in lambs on
methane emission from birth or from weaning lamb were
studied, indicating that there is no main effect of rumen
protozoa on methane emission (Hegarty et al. 2008). Plant
secondary metabolites i.e., saponin-containing plants are
reported to suppress or inhibit protozoa and certain bacteria
in the rumen (Cheeke 2000). Saponins are glycosides which
interact with the cholesterol present in the membrane of
protozoa and causes cell lysis (Hess et al. 2003). Guo et al.
(2008) suggested that supplementation of saponins
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indirectly inhibit methane production with no adverse effect
on rumen function. In a contrasting report, Goel et al.
(2008) reported that there is no obligatory relationship
between defaunation by saponins and methanogens, as not
all saponins could be promising in methane inhibition.
Fatty acid addition
Fatty acid supplementation of cattle diet increases the
energy density and milk yield, besides modifying the fatty
acid profile of milk fat (Ashes et al. 1997). However, fatty
acids, especially those of medium chain length (C8–C16)
(e.g., coconut oil, canola oil, kernel oil etc.) have been
found to decrease methane production (Machmuller and
Kreuzer 1999; Dohme et al. 2000) and in study a 22%
reduction was achieved by addition of sunflower oil to the
diet (McGinn et al. 2004). Fatty acids in the diet provide
an alternative hydrogen sink and also inhibit protozoa
(Johnson and Johnson 1995). However, Beauchemin and
McGinn (2006) found that whilst use of canola oil as a
feed additive did reduce methane emission, animal per-
formance was reduced, due to lower feed intake and
decreased fiber digestibility. In an another report inclusion
of refined soya bean oil reduced methane emission in
comparison to whole soya bean with no effect on dry
matter intake with low forage diet (Jordan et al. 2006).
Odongo et al. (2007) supplemented the diet of Holstein
dairy cows with myristic acid and reported a decrease in
methane production to the extent of 36% with no signif-
icant decrease in the conjugated linoleic acid content of
milk. However, the effect of some of these oils is pH- and
diet-dependent, and their use may be beneficial only under
specific conditions (Calsamiglia et al. 2007). By employ-
ing Archaea-specific PCR and Denaturing Gradient Gel
Electrophoresis it was studied that dietary fat transiently
stimulates Methanosphaera stadmanae but Methanobrev-
ibacter sp strain AbM4 (Yu et al. 2008).
Bacteriocins
Bacteriocins, bactericidal peptides from bacteria can be
used directly for the suppression of methanogens (Klieve
and Hegarty 1999), although the exact mechanism is still
not clear. Nisin, a well known bacteriocin produced by
Lactococcus lactis was used as an alternative to monensin
by Callaway et al. (1997) who reported increased propio-
nate production in vitro along with 36% reduction in
methane emission. In contrast, feeding trials of cattle
revealed that addition of nisin to the diet had no effect on
the acetate: propionate ratio (Mantovani and Russell 2001;
Russell and Mantovani 2002) and the authors suggested the
nisin was either degraded or that the target methanogens
quickly developed resistance.
World J Microbiol Biotechnol (2009) 25:1557–1566
Lee et al. (2002) studied the effect of bovicin HC5 from
Streptococcus bovis and observed that it inhibited as much
as 50% methane emission in vitro. Kalmakoff et al. (1996)
surveyed 50 strains of Butyrivibrio fibrisolvens from that
50% showed bacteriocin-like activity. Endogenous bacte-
riocins have been identified in Butyrivibrio spp. from a
variety of rumen samples (Kalmakoff et al. 1996; Teather
and Froster 1998), to broaden the exogenous bacteriocins.
Thus bacteriocins may play a promising role in reduction
of methane from ruminants, if produced by the bacteria that
inherently reside in the rumen but it also needs to be
established that these compounds are actually secreted in
vivo.
Recombinant technology
A derivative of wild type E. coli W3110 with elevated
nirBD expression was constructed by replacing the
endogenous promoter with the constitutive tac promoter
using recombinant DNA technology. The methane pro-
duction was markedly decreased by the addition of nitrate
and nitrite, as nitrite reductase activity was approximately
twice in recombinant E. coli nirPtac, while compared to the
wild type. Hence, anaerobic cultures of E. coli W3110 and
E. coli nirPtac may decrease methane production in vivo
(Sar et al. 2005) if they could be administered and estab-
lished in the rumen.
Need for further study
Feeding strategies need to be developed that could mini-
mize the energy loss from ruminants leading to increased
productivity either by reducing the number or the activities
of methanogens. Although methane production can be
inhibited with the present strategies, due to variety of
adaptive mechanisms all the strategies have been found to
be effective only for a short period. Therefore, further
research is required to study the effect of diets as well as
inhibitors on the rumen fermentation pattern with special
reference to methane production and change in methano-
gens population, if any.
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