Ru

min

Om

ics

John Wallace

Microbiome – methods and surrogates

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

Fermentation

H2 + CO2

CH4

Protozoa, fungi, bacteria

Archaea

Methane production in ruminants

Ru

min

Om

ics

• Community analysis methods

• Proxies for ruminal digesta

• Metaproteomics

• Milk fatty acids vs. methane

Microbiome – methods and surrogates

Secondary structure

of small

subunit ribosomal

RNA

Community analysis based on ss rRNAsequence analysis

Unclassified

Prevotella

Succiniclasticum

Acidaminococcus

Dialister

Butyrivibrio

Ruminococcus

Treponema

Syntrophococcus

Fibrobacter

Selenomonas

Acetitomaculum

Incertae Sedis

Pseudobutyrivibrio

Atopobium

Mogibacterium

Community analysis based on ss rRNAsequence analysis

Ru

min

Om

icsRuminOmics ring test

• Seven international labs

• DNA from six rumen samples

distributed

• “Just tell us what you think is there”

RuminOmics ring testBacterial phylum summaries

Participating laboratories

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Teagasc AgResearch CNRS CSIRO ARK

Thermoplasmatales

Methanosarcinales

Methanomicrobiales

Methanobacteriales

F B D E F

RuminOmics ring testArchaeal phylum summaries

Participating laboratories

RuminOmics ring testReaction

“bury it”

RuminOmics ring testOngoing investigation on reasons for

differences

• Choice of hypervariable region

• Sequencing platform

• Taxonomic software

• Databases

• What is the correct answer???

Ru

min

Om

ics

Buccal-ruminal-faecal microbiomes

RuminOmics surrogates study

Proxies for rumen sampling – mouth (swab and bolus) and faeces

Proxies for rumen sampling – mouth (swab and bolus) and faeces

Ru

min

Om

ics

Metaproteomics: 2-D gel electrophoresis, different samples

Metaproteomics: 2-D gel electrophoresis, same sample different gels

Metaproteomics: 2-D gel electrophoresis, different samples

Archaealenzymes

Protozoal actins

Bacteroidetes

Firmicutes

Proteobacteria

Archaea

Protists

Fungi

Metazoa

Results of 2-D gel electrophoresis: phylogenetic analysis

• Ultimate 3000 RS LC nano system

• LQT Orbitrap Velos/Q Exactive

• Proteome Discoverer software

Shotgun metaproteomics

UniProt• Taxonomy

• Function• Gene Ontology (GO terms)

Results of shotgun metaproteomics: phylogenetics and ontology

Ru

min

Om

ics

Can milk fatty acids predict methane emissions?

• 1000 cows

• 195 fatty acids in milk

• Four countries

• Seven farms

0.2 0.4 0.6 0.8 1.0 1.2

10

15

20

25

30

35

40

c12-18:1

c12-18:1

CH

4g

.kg

.DM

I

BianchiniCAMBS

FranciosiGandolfiMinkiö

NOTTSRöbäcksdalen

Typical results for a single fatty acid in milk

All FA with significant positive within farm correlations

iso13:0 anteiso13:0 iso14:0 iso15:0 iso16:0

16:0 iso18:0 18:0 20:0 22:0

24:0 c15-24:1 Unid 16 SFA

All FA with significant negative within farm correlations

5:0 7:0 9:0 11:0 13:0 c9-13:1 14:0

c9-14:1 15:0 t8-16:1 t13-16:1 c10-16:1

t9,t13-16:2 + t10-17:1 c9,c12-16:2 c10-17:1

t6-8 18:1 t9-18:1 t10-18:1 t12-18:1 t15-18:1 c11-18:1

c12-18:1 c13-18:1 unid 9 c13-18:1 + unid 9 t16-18:1 + c14-18:1

c15-18:1 c16-18:1 sum trans 18:1 t9,t13-18:2

19:0 t10,t14-18:2 19:0 + t10,t14-18:2 t9,t14-18:2 t9,t12-18:2

c9,t13-18:2 + c10,t14-18:2 c6,c12-18:2 c9,t14-18:2 c9,t12-18:2

t9,c12-18:2 c9,c12-18:2 (+ c9,c15-18:2) c12,c15-18:2 trans 18:2

t12,t14-CLA + t12,c14-CLA t11,t13-CLA t9,t11-CLA + 18:4n-3

t10,c12-CLA + c11,t13-CLA + c15-20:1 t9,c11-CLA sum CLA 18:3(n-6)

c7-19:1 c10-19:1 c11-19:1 c10-19:1 + c11-19:1 sum cis 19:1

t13-20:1 c13-20:1 20:2(n-6) + unid 14 20:3(n-6) Unid 1 Unid 3 Unid 6 Unid 24 Trans fatty acids PUFA c9-20:1/(c9-20:1+20:0)

All FA with significant positive within farm correlations

iso13:0 anteiso13:0 iso14:0 iso15:0 iso16:0

16:0 iso18:0 18:0 20:0 22:0

24:0 c15-24:1 Unid 16 SFA

All FA with significant negative within farm correlations

5:0 7:0 9:0 11:0 13:0 c9-13:1 14:0

c9-14:1 15:0 t8-16:1 t13-16:1 c10-16:1

t9,t13-16:2 + t10-17:1 c9,c12-16:2 c10-17:1

t6-8 18:1 t9-18:1 t10-18:1 t12-18:1 t15-18:1 c11-18:1

c12-18:1 c13-18:1 unid 9 c13-18:1 + unid 9 t16-18:1 + c14-18:1

c15-18:1 c16-18:1 sum trans 18:1 t9,t13-18:2

19:0 t10,t14-18:2 19:0 + t10,t14-18:2 t9,t14-18:2 t9,t12-18:2

c9,t13-18:2 + c10,t14-18:2 c6,c12-18:2 c9,t14-18:2 c9,t12-18:2

t9,c12-18:2 c9,c12-18:2 (+ c9,c15-18:2) c12,c15-18:2 trans 18:2

t12,t14-CLA + t12,c14-CLA t11,t13-CLA t9,t11-CLA + 18:4n-3

t10,c12-CLA + c11,t13-CLA + c15-20:1 t9,c11-CLA sum CLA 18:3(n-6)

c7-19:1 c10-19:1 c11-19:1 c10-19:1 + c11-19:1 sum cis 19:1

t13-20:1 c13-20:1 20:2(n-6) + unid 14 20:3(n-6) Unid 1 Unid 3 Unid 6 Unid 24 Trans fatty acids PUFA c9-20:1/(c9-20:1+20:0)

Methane α Fatty acid saturation• Reflects H2 availability for

both

All FA with significant positive within farm correlations

iso13:0 anteiso13:0 iso14:0 iso15:0 iso16:0

16:0 iso18:0 18:0 20:0 22:0

24:0 c15-24:1 Unid 16 SFA

All FA with significant negative within farm correlations

5:0 7:0 9:0 11:0 13:0 c9-13:1 14:0

c9-14:1 15:0 t8-16:1 t13-16:1 c10-16:1

t9,t13-16:2 + t10-17:1 c9,c12-16:2 c10-17:1

t6-8 18:1 t9-18:1 t10-18:1 t12-18:1 t15-18:1 c11-18:1

c12-18:1 c13-18:1 unid 9 c13-18:1 + unid 9 t16-18:1 + c14-18:1

c15-18:1 c16-18:1 sum trans 18:1 t9,t13-18:2

19:0 t10,t14-18:2 19:0 + t10,t14-18:2 t9,t14-18:2 t9,t12-18:2

c9,t13-18:2 + c10,t14-18:2 c6,c12-18:2 c9,t14-18:2 c9,t12-18:2

t9,c12-18:2 c9,c12-18:2 (+ c9,c15-18:2) c12,c15-18:2 trans 18:2

t12,t14-CLA + t12,c14-CLA t11,t13-CLA t9,t11-CLA + 18:4n-3

t10,c12-CLA + c11,t13-CLA + c15-20:1 t9,c11-CLA sum CLA 18:3(n-6)

c7-19:1 c10-19:1 c11-19:1 c10-19:1 + c11-19:1 sum cis 19:1

t13-20:1 c13-20:1 20:2(n-6) + unid 14 20:3(n-6) Unid 1 Unid 3 Unid 6 Unid 24 Trans fatty acids PUFA c9-20:1/(c9-20:1+20:0)

Methane α (1/Odd number FAs) • Reflects fatty acid synthesis

from propionate• Propionate ↓ as CH4↑

All FA with significant positive within farm correlations

iso13:0 anteiso13:0 iso14:0 iso15:0 iso16:0

16:0 iso18:0 18:0 20:0 22:0

24:0 c15-24:1 Unid 16 SFA

All FA with significant negative within farm correlations

5:0 7:0 9:0 11:0 13:0 c9-13:1 14:0

c9-14:1 15:0 t8-16:1 t13-16:1 c10-16:1

t9,t13-16:2 + t10-17:1 c9,c12-16:2 c10-17:1

t6-8 18:1 t9-18:1 t10-18:1 t12-18:1 t15-18:1 c11-18:1

c12-18:1 c13-18:1 unid 9 c13-18:1 + unid 9 t16-18:1 + c14-18:1

c15-18:1 c16-18:1 sum trans 18:1 t9,t13-18:2

19:0 t10,t14-18:2 19:0 + t10,t14-18:2 t9,t14-18:2 t9,t12-18:2

c9,t13-18:2 + c10,t14-18:2 c6,c12-18:2 c9,t14-18:2 c9,t12-18:2

t9,c12-18:2 c9,c12-18:2 (+ c9,c15-18:2) c12,c15-18:2 trans 18:2

t12,t14-CLA + t12,c14-CLA t11,t13-CLA t9,t11-CLA + 18:4n-3

t10,c12-CLA + c11,t13-CLA + c15-20:1 t9,c11-CLA sum CLA 18:3(n-6)

c7-19:1 c10-19:1 c11-19:1 c10-19:1 + c11-19:1 sum cis 19:1

t13-20:1 c13-20:1 20:2(n-6) + unid 14 20:3(n-6) Unid 1 Unid 3 Unid 6 Unid 24 Trans fatty acids PUFA c9-20:1/(c9-20:1+20:0)

Methane α Branched FAs • Reflects fatty acid synthesis

from short-chain branched VFA

Predominant substrate (from Hobson and Stewart, 1997), fermentation end products (from Russell and Rychlik, 2001) and OBCFA profile (g/100 g fatty acids; original references in Vlaeminck et al., 2006a) of

some major bacteria involved in rumen carbohydrate fermentation. Predominant OBCFA is underlined. Indication of main substrate is given by superscript letter.

R. albusa

Fermentation productsd

A

Anteiso C13:0

–

Anteiso C15:0

9.4

Anteiso C17:0

1.3

Iso C13:0

–

Iso C15:0

–

Iso C17:0

0.7

Iso C14:0

20.6

Iso C16:0

11.0

C13:0

–

C15:0

10.3

C17:0

1.4

C17:1

–

B. fibrisolvensa A, B, F 6.4 16.2 8.6 6.8 10.4 5.7 10.8 11.1 2.9 7.8 4.3 3.5

R. flavefaciensa

S. amylolyticab

A, S

A, P

– 2.3 2.9 – 35.7 5.2 2.5 7.3 0.1 3.2 0.5 –

N6 – – – – 52.6 10.8 1.6 5.3 1.6 5.0 – –

B24 – – – – 0.1 0.3 – 0.6 1.4 3.3 1.3 0.6

Prevotellab , c A, S 1.2 36.7 4.2 3.0 14.7 2.3 3.3 3.0 1.2 12.1 2.1 –

L. multiparusb , c A, L, F – 4.0 2.6 – 1.1 1.1 1.2 1.8 0.3 2.9 0.8 0.1

S. dextrinosolvensc A, S 0.8 3.6 1.0 – 0.1 – 0.6 1.5 0.5 4.0 0.7 –

R. amylophilusb A, S, F – 1.1 – – – – – – 0.5 1.1 0.3 0.1

F. succinogenesa A, S 3.9 7.7 1.2 – 0.1 0.2 3.6 3.4 9.0 30.2 2.1 –

S. bovisb L – 0.9 – – – – 0.4 0.2 0.6 1.7 1.2 0.2

M. elsdeniic

E. ruminantiumb

A, P, B

B, L, F

– 2.8 – 0.1 0.2 0.2 1.5 0.5 1.5 6.0 4.5 3.0

B1C23 – – – – 17.7 1.4 – – 5.4 49.0 1.5 –

GA195 – 30.1 1.7 – 0.4 0.2 6.1 3.7 0.4 6.5 0.4 –

S. ruminantiumb A, P, L – 0.1 – – 0.2 – 0.3 0.1 1.3 6.0 2.9 2.6

a Bacteria fermenting cellulose and hemicellulose.

b Bacteria fermenting starch.

c Bacteria fermenting sugar and pectin.

d A: acetate; S: succinate; B: butyrate; F formate; P: propionate; L: lactate.

Different ruminal bacteria contain different minor fatty acids

Fievez et al. (2012)

All FA with significant positive within farm correlations

iso13:0 anteiso13:0 iso14:0 iso15:0 iso16:0

16:0 iso18:0 18:0 20:0 22:0

24:0 c15-24:1 Unid 16 SFA

All FA with significant negative within farm correlations

5:0 7:0 9:0 11:0 13:0 c9-13:1 14:0

c9-14:1 15:0 t8-16:1 t13-16:1 c10-16:1

t9,t13-16:2 + t10-17:1 c9,c12-16:2 c10-17:1

t6-8 18:1 t9-18:1 t10-18:1 t12-18:1 t15-18:1 c11-18:1

c12-18:1 c13-18:1 unid 9 c13-18:1 + unid 9 t16-18:1 + c14-18:1

c15-18:1 c16-18:1 sum trans 18:1 t9,t13-18:2

19:0 t10,t14-18:2 19:0 + t10,t14-18:2 t9,t14-18:2 t9,t12-18:2

c9,t13-18:2 + c10,t14-18:2 c6,c12-18:2 c9,t14-18:2 c9,t12-18:2

t9,c12-18:2 c9,c12-18:2 (+ c9,c15-18:2) c12,c15-18:2 trans 18:2

t12,t14-CLA + t12,c14-CLA t11,t13-CLA t9,t11-CLA + 18:4n-3

t10,c12-CLA + c11,t13-CLA + c15-20:1 t9,c11-CLA sum CLA 18:3(n-6)

c7-19:1 c10-19:1 c11-19:1 c10-19:1 + c11-19:1 sum cis 19:1

t13-20:1 c13-20:1 20:2(n-6) + unid 14 20:3(n-6) Unid 1 Unid 3 Unid 6 Unid 24 Trans fatty acids PUFA c9-20:1/(c9-20:1+20:0)

Ruminococcus albus - H2 producer Ruminococcus flavefaciens - H2

producer

Fibrobacter succinogenes – non-H2 producer

Ru

min

Om

ics

Conclusions

• (Unspoken) problems of

community analysis are being

resolved

• Oral samples are useful proxies

for ruminal community analysis

• Metaproteomics interesting for

microbiome, but practical

usefulness is limited

• Milk fatty acids vs. methane

makes sense, but predictive

value between farms is limited