Post on 24-Apr-2018
John Wallace
Microbial ecology of the rumen
The rumen
The three domains of life
Ruminal microorganisms
50 m
0.5 m
100 m
Ciliate protozoa
106 per g digesta
Anaerobic fungi
103 per g digesta
50 m
Bacteria
1010 per g digesta
Methanogenic archaea
108 per g digesta
2 m
Rumen ciliate protozoa
100 m
Enchelyodon sp.
Amylovorax dehorityi Bitricha tasmaniensis
Amylovorax dogieli Dasytricha ruminantium U57769 Dasytricha ruminantium (France)
Dasytricha ruminantium U27814 Epidinium caudatum
Ophryoscolex purkynjei RS65
Polyplastron multivesiculatum U27815 Polyplastron multivesiculatum (Poland) RS53
Polyplastron multivesiculatum U57767 Polyplastron multivesiculatum (France)
Diplodinium dentatum Ostracodinium dentatum (Poland)
Enoploplastron triloricatum (Slovakia) Eudiplodinium maggii (France) Eudiplodinium maggii (Poland) Eudiplodinium maggii RS61, RS99
Diploplastron affine (Poland) RS59
RS70 RS88, RS33
RS7 RS87
RS24 RS74
RS17 RS100, RS95
RS28 RS2
RS75 RS31
RS57 RS86
RS90 RS94
RS67, RS1 RS58, RS18, RS16, RS3
Entodinium nanellum (Slovakia) RS14 RS89, RS97, RS63, RS13
RS105 Entodinium furca monolobum (Slovakia)
RS26 RS30
RS85 RS77
RS73 RS12 RS4
RS79 Entodinium bursa (Slovakia)
RS71 RS64
Entodinium caudatum
Entodinium caudatum (UK) RS62
RS82, RS32, RS69, RS66, RS22 RS9
Entodinium caudatum (Slovakia) RS92
RS93 Entodinium D?( France)
RS5, RS107 Isotricha intestinalis Isotricha intestinalis (Poland)
Isotricha prostoma (Slovskia) Isotricha prostoma (Poland) Isotricha prostoma (France)
RS19
100
100
73
97 100
100 91 92
100
100 96
94
94
100
77
95
85
69
79
Didinium nasutum
Unexpected protozoal diversity
Wright et al (1997). J Eukar
Microbiol 44, 61-67
Fungal picture
Rumen anaerobic fungi
50 m
Rowett picture
0.5 m
Rumen bacteria
ProteobacteriaM ethanomicrobium mobile (M59142)
0.1
82.7
88.3
Proteobacter ia (OTUs 1-3)
Cytophaga-Flexibacter-Bactero ides Group (OTUs 6-78)
Low G+C Gram Positive Bacteria (OTUs 80-174)
Fibrobacter Group (OTU 175)High G+C Gram Positive Bacteria (OTUs 176 &177)
Chlamydiales -Verrucimicrobia Group (OTUs 1 78 & 179)Spirochaetes (180)
Phylum? (OT U 4)
Phylum? (OTU 5)
Phylum? (OTU 79)
Figure 2
Figure 1: Phylogenetic tree of 16S rDNA rumen library sequence data.
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Bacteroidetes
Proteobacteria
Firmicutes
Fibrobacteraceae
Spirochaetes
Edwards et al. (2004) Anton v
Leeuwen 86, 263-281
Rumen methanogenic archaea
2 m
Phylogenetic dendrogram of total rumen and rumen protozoan-associated archaea and
selected reference sequences.
Janssen P H , and Kirs M Appl. Environ. Microbiol.
2008;74:3619-3625
Methanobrevibacter
usually predominant
genus (60-99%)
Phylogenetic dendrogram of total rumen and rumen protozoan-associated archaea and
selected reference sequences.
Janssen P H , and Kirs M Appl. Environ. Microbiol.
2008;74:3619-3625
‘Rumen Cluster C’
Methanoplasmata
Probably methylotrophic
(e.g. methylamine CH3-
NH2)
Methane production in the rumen
Fermentation
H2 + CO2
CH4
Protozoa, fungi, bacteria
Archaea
Methane production in ruminants
Respiration chambers SRUC, Edinburgh
• 36 Aberdeen Angus, 36 Limousin cross steers, 1-2 years
• Two diets: Forage diet 50:50, forage (grass silage / whole crop silage) : concentrate (barley / barley dark grains ) (n=36) Concentrate diet 8:92, forage (straw) : concentrate (barley / barley dark gains) (n=36)
• Methane emissions measured for 2 days in Green Cow facility; ruminal digesta taken by stomach tube
• Animals slaughtered 1-3 weeks later; ruminal digesta taken again
• Archaea and bacterial 16S rRNA genes measured by qPCR
• Data analysis, GLM procedure of SAS
The Extreme 8 experiment
Selected the 4 highest and 4 lowest methane
emitters for full microbiome/metagenome analysis
Microbiome
The Extreme 8 analysis
Microbiota
Metagenome
What species are
different?
What genes are
different?
Secondary structure
of small
subunit ribosomal
RNA
The Extreme 8 microbiota analysis
Variation in methane production: microbiota analysis
Methane
0
5
10
15
20
25
30
LOW HIGH
Met
han
e (g
/kg
DM
I)
P = 0.004
0
0,5
1
1,5
2
2,5
3
3,5
4
LOW HIGH
Ab
un
da
nce
(%
)
Archaea
0
2
4
6
8
10
12
LOW HIGH
Ab
un
dan
ce (
%)
Succinovibrionaceae
P = 0.014P = 0.002
Methane emission status of beef cattleFour highest and four lowest from 72 beef cattle
Herbivore
Foregut fermenter
Produces 20% of methane per DMI
compared to cattle
Herbivore
Foregut fermenter
Has large numbers of
Succinovibrionaceae
Microbiome
The Extreme 8 analysis
Microbiota
Metagenome
What species are
different?
What genes are
different?
Variation in methane production: the geneticist’s slant
KEGG ID/Diet Description bEstimate cVIP
K06001 Tryptophan synthase beta chain [EC:4.2.1.20] 0.13310 1.233
Diet conc Concentrate based diet -0.14695 1.204
Diet mixed Mixed forage-concentrate diet 0.14695 1.204
K02118 V/A-type H+-transporting ATPase subunit B [EC:3.6.3.14] 0.07984 1.151
K02117 V/A-type H+-transporting ATPase subunit A [EC:3.6.3.14] 0.08564 1.133
K00638 Chloramphenicol O-acetyltransferase [EC:2.3.1.28] 0.06182 1.082
K00200 Formylmethanofuran dehydrogenase subunit A [EC:1.2.99.5] 0.06042 1.070
K03531 Fell division protein FtsZ 0.07526 1.065
K00201 Formylmethanofuran dehydrogenase subunit B [EC:1.2.99.5] 0.04859 1.049
K00399 Methyl-coenzyme M reductase alpha subunit [EC:2.8.4.1] 0.03843 1.021
K00123 Formate dehydrogenase, alpha subunit [EC:1.2.1.2] 0.03417 1.013
K03388 Heterodisulfide reductase subunit A [EC:1.8.98.1] 0.02734 0.997
K14126 F420-non-reducing hydrogenase subunit A [EC:1.12.99.-] 0.00384 0.933
K01079 Phosphoserine phosphatase [EC:3.1.3.3] 0.03424 0.932
K00401 Methyl-coenzyme M reductase beta subunit [EC:2.8.4.1] 0.00484 0.909
K00527 Ribonucleoside-triphosphate reductase [EC:1.17.4.2] 0.03449 0.898
K02337 DNA polymerase III subunit alpha [EC:2.7.7.7] -0.05338 0.886
K02837 Peptide chain release factor RF-3 0.00883 0.852
K01893 Asparaginyl-tRNA synthetase [EC:6.1.1.22] -0.06673 0.812
K00925 Acetate kinase [EC:2.7.2.1] -0.00903 0.812
K00656 Formate C-acetyltransferase [EC:2.3.1.54] -0.00834 0.787
K02948 Small subunit ribosomal protein S11 -0.00400 0.734
Rainer Roehe
SRUC
“The genes identified in Table 3 to be important
explained the variation in methane emissions by 88%.”
Ru
min
Om
ics
Tools, resources, legacy
• Proxies Buccal-ruminal-faecal microbiomes
Proxies for rumen sampling – mouth (swab and bolus) and faeces
Proxies for rumen sampling – post-mortem samples
y = 1.012xR² = 0.35
0
2
4
6
8
10
12
14
16
18
0 5 10 15
Ch
am
ber
sam
ple
s A
:B r
ati
o
(%)
Post-mortem A:B ratio (%)
Conclusions on methane microbiome research
• Low-methane phenotype has low
archaea and high Succinovibrionaceae
abundance
• Differences in abundance of 22
microbial genes explained the variation
in methane emissions by 88%
• Post-mortem sampling of digesta is a
useful proxy
• Oral samples also a useful proxy
RuminOmics Kevin Shingfield, Ilma Tapio, Johanna Vilkki, Pierre Taberlet, Aurelie Bonin
Methane John Rooke, Rainer Roehe, Mick Watson, Tim Snelling, Graham Horgan
Funders European CommissionBBSRC (UK Research Council)RESAS (Scottish Government)
Acknowledgements