Rumen microbiome Ecology and function

R. John Wallace Rowett Institute of Nutrition and Health, University of Aberdeen, UK

Rowett picture

“The Institute aims to conduct research at the forefront of nutrition: to define how nutrition can prevent disease, improve health, and enhance the quality of food

production in agriculture”

The rumen

CA TTLE AND SHEEP

Stomach (Abomasum)

Rumen

Reticulum

Omasum

Jejunum

Ileum

Colon

Caecum

PIG

Stomach

Jejunum

Ileum

Caecum

Colon

Gut anatomy

The three domains of life

Rumen ciliate protozoa

100 µm

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

Fungal picture

Rumen anaerobic fungi

50 µm

Fungal picture

© R.J. Wallace 2004

Fungal life cycle

Rowett picture

1 µm

Rumen bacteria

Proteobacteria M 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 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

High bacterial diversity

Cytophaga-Flexibacter-Bacte [CFB]

Proteobacteria

Low G+C Gram positive

High G+C Gram positive, Fib Spirochaetes, etc

Rumen methanogenic archaea

1 µm

Fermentation

H2 + CO2

CH4

Protozoa, fungi, bacteria Archaea

Methane production in ruminants

109 - 1010 BACTERIA

Up to 106 PROTOZOA

?? ANAEROBIC FUNGI

108 ARCHAEA

per g digesta

FOOD

UNDIGESTED FOOD +

MICROORGANISMS

ACETATE

VFA PROPIONATE

BUTYRATE METHANE

Metabolism in

the rumen

Hot topics in rumen microbiology

• Metagenomics • Methane

Technological advances: 454 sequencing

The latest pyrosequencing platform by 454 Life Sciences (now owned by Roche Diagnostics), can generate 400 million nucleotide data in a 10 hour run with a single machine.

Technological advances: Illumina sequencing

Nature 9 Sept 2010

Science 28 Jan 2011

Metagenomic Discovery of Biomass-Degrading Genes and Genomes from Cow Rumen

Science 28 Jan 2011

Metagenomic Discovery of Biomass-Degrading Genes and Genomes from Cow Rumen

The paucity of enzymes that efficiently deconstruct plant polysaccharides represents a major bottleneck for industrial-scale conversion of cellulosic biomass into biofuels. Cow rumen microbes specialize in degradation of cellulosic plant material, but most members of this complex community resist cultivation. To characterize biomass-degrading genes and genomes, we sequenced and analyzed 268 gigabases of metagenomic DNA from microbes adherent to plant fiber incubated in cow rumen. From these data, we identified 27,755 putative carbohydrate-active genes and expressed 90 candidate proteins, of which 57% were enzymatically active against cellulosic substrates. We also assembled 15 uncultured microbial genomes, which were validated by complementary methods including single-cell genome sequencing. These data sets provide a substantially expanded catalog of genes and genomes participating in the deconstruction of cellulosic biomass.

Fig. 2 (A) Sequence identity of 90 candidate sequences assembled from the switchgrass-associated rumen microbiome and tested for carbohydrate-degrading activity to known

carbohydrate-active enzymes.

M Hess et al. Science 2011;331:463-467

Published by AAAS

Fig. 3 Carbohydrolytic potential of candidate carbohydrate-active enzymes on glycosidic substrates of different complexity.

M Hess et al. Science 2011;331:463-467

Published by AAAS

Science 28 Jan 2011

Metagenomic Discovery of Biomass-Degrading Genes and Genomes from Cow Rumen

Genome Bin Genome Size (Mb) Phylogenetic Order Estimated Complete-ness AFa 2.87 Spirochaetales 92.98% AMa 2.21 Spirochaetales 91.23% AIa 2.53 Clostridiales 90.10% AGa 3.08 Bacteroidales 89.77% AN 2.02 Clostridiales 78.50% AJ 2.24 Bacteroidales 75.96% AC2a 2.07 Bacteroidales 75.96% AWa 2.02 Clostridiales 75.77% AH 2.52 Bacteroidales 75.45% AQ 1.91 Bacteroidales 71.36% AS1a 1.75 Clostridiales 70.99% APb 2.41 Clostridiales 64.85% BOa 1.67 Clostridiales 64.16% ADa 2.99 Myxococcales 62.13% ATa 1.87 Clostridiales 60.41%

Genome Bin APb 55 Scaffolds 2.41 Mb

Hot topics in rumen microbiology

• Metagenomics – The Hungate 1000 project

Methane, ruminants and the environment

Greenhouse gases: CO2

Methane as a greenhouse gas

CH4 has a global warming potential (“radiative forcing”) 25 times that of CO2 Methane contributes approximately 18% to the overall global warming effect

US Environmental Protection Agency, 2000

Methane as a greenhouse gas

Dlugokencky et al., 2003

t½ of CH4 in atmosphere is 12 years

70% of global methane formation is due to man's activities

Sources of atmospheric methane

US Environmental Protection Agency, 2001

Therefore, 20% of global methane formation is due to ruminants

Sources of atmospheric methane

US Environmental Protection Agency, 2001

And so 20% of the 18% = 3.6% of the total radiative forcing is caused by ruminants

Sources of atmospheric methane

US Environmental Protection Agency, 2001

Ruminants, cars and methane 164 g CO2/km at 19,000 km/year

= 164 × 19000 g CO2/year = 3 × 106 g CO2/year

=

500 L CH4/day = 365 × 500 L/year = 2 × 105 L/year = 2 × 16/22 × 105 g/year = 1.5 × 105 g/year ≅ 25 × 1.5 × 105 g CO2/year ≅ 3 × 106 g CO2/year

Ruminants, cars and methane =

Ruminants, cars and methane =

The New Zealand response

Carbon tax As part of the Climate Change Policy Package, released in 2002, the government will be introducing a carbon tax in New Zealand from April 1, 2007. Hon. Pete Hodgson, Convener of the Ministerial Group on Climate Change, has announced that the carbon tax will be set at $15 per tonne of CO2 and has released a consultation paper on the implementation of the tax. Now New Zealand leads the Global Research Alliance

Methane production in ruminants

95% 5%

Other important topics in rumen microbiology

• Protein metabolism • Fatty acid metabolism • Rumen dysfunction • Feed additives

Protein metabolism in the rumen

Protein

Peptides

Amino acids

Ammonia

Ammonia

Undegraded food protein +

Microbial protein

Food protein

Microbial protein

B C

A

Urea

INEFFICIENCES

Loss of N Microbial protein breakdown Amino acid imbalance

B

C

A

Protein metabolism in the rumen

Proteolytic ruminal microorganisms

Proteinase zymograms from ruminal fluid

Protease in rumen fluid supernatants

• Falconer & Wallace (1998)

Zymogram of proteolytic activity in the rumen of sheep

Properties of rumen proteinases

• Mainly cysteine proteinases • Cell surface-associated • Low activity • Activity increases 3-fold or more in animals

receiving fresh forage

Microbial protein turnover

Fig. 8. Chemical classes of saponins

Steroid

O

O H

O

O

3

22

25 26 27

Steroidal alkaloid

N H

O H

O

O

3

22

2 26

Triterpene O H

3

28

Chemical classes of saponins

R R

R

R = sugars

Multipurpose trees Fuel Shelter Fertilisation Water retention Protein supplement Rumen manipulating agent

0

5

10

15

20

25

30

35

0 1 2 3Incubation time (h)

Deg

rada

tion

of S

. rum

inan

tium

(%)

Influence of Sesbania sesban on the bacteriolytic activity of ruminal protozoa

Control

1 g/l

10 g/l

0

5

10

15

20

25

0 7 14 21 28 35 42

Days of experiment

Num

ber

of p

roto

zoa

(x10

5 /ml)

Influence of Sesbania sesban on protozoal numbers in the sheep rumen

Fig. 8. Chemical classes of saponins

Steroid

O

O H

O

O

3

22

25 26 27

Steroidal alkaloid

N H

O H

O

O

3

22

2 26

Triterpene O H

3

28

Chemical classes of saponins

R R

R

R = sugars

Effect of addition of Enterolobium cyclocarpum N

umbe

r of p

roto

zoa

(105 /m

l)

Days of experiment

J J

J

J

J

J J J

J

J J

J J J

J

1 3 5 7 8 9 10 0

5

10

15

20

25 Enterolobium cyclocarpum addition

1 1 12 13 14 16 18 20 21

on protozoal numbers in sheep

Rumen epithelium

Effects of flavomycin in sheep

• Gut tissue turnover >40% of total body protein synthesis; <5% of body weight

• Flavomycin causes 20% decrease in gut protein turnover in sheep

• Flavomycin produced a 20% increase in LWG (MacRae et al. 1999)

Influence of flavomycin on gut tissue turnover in sheep

CONTR +FLAVOMY SE ProbabilRumen 13.9 10.1 1.8 0.075Abomasum 16.6 15.1 3.2 NSDuodenum 48.0 36.7 4.1 0.027Jejunum 42.8 38.3 3.7 NSIleum 36.5 34.7 2.3 NSCaecum 20.2 17.9 2.0 NSLarge 24.5 26.4 5.0 NSLiver 15.5 18.0 3.0 NS

Influence of flavomycin on ruminal bacteria

Gram -ve Species Strain MIC50 (mg/ml) Fusobacterium necrophorum A54, A12 0.25 Fibrobacter succinogenes S85 0.5 Ruminobacter amylophilus WP109 4 Veillonella parvula L59 4 Prevotella albensis M384 8 Megasphaera elsdenii J1 32 Prevotella bryantii B14 32 Anaerovibrio lipolytica 5S >64 Mitsuokella multiacidus 46/5 >64 Prevotella brevis GA33 >64 Prevotella ruminicola 23 >64 Selenomonas ruminantium HD4 >64 Selenomonas ruminantium Z108 >64

Protein metabolism in the rumen

Biphasic Breakdown of Peptides

Dipeptidyl peptidase

Rapidly degraded peptides • Ala or other neutral AA at N-terminus • Neutral or basic peptides

Slowly degraded peptides • Gly or Pro at N-terminus or

(n-1) residue • Acidic AA residues • Blocked N-terminus

NH2-CR-CO-[NH-CO-CR]N-COOH

N-terminal blocking of peptides

NH-CR-CO-[NH-CO-CR]N-COOH CH3-CO-

Acetic anhydride

15N recovery determinedat the ileum

15N-peptides injectedinto jejunumRumen Caecum &

colon

Nutritive value of N-terminally blocked peptides

>97% absorption of acetylated peptides

Proteolytic ruminal microorganisms

Properties of ammonia production

Organism Ammoniaproduction

rate (nmol/mgprotein/min)

Monensinsensitivity

Growth onpeptides

Mixed rumenbacteria

30 Partly +

Megasphaeraelsdenii

19 No -

Selenomonasruminantium

15 No -

Prevotellaruminicola

14 No -

Peptostreptococcusanaerobius

346 Yes +

Clostridiumsticklandii

367 Yes +

Clostridiumaminophilum

318 Yes +

Properties of ammonia production

Organism Ammoniaproduction

rate (nmol/mgprotein/min)

Monensinsensitivity

Growth onpeptides

Mixed rumenbacteria

30 +

Megasphaeraelsdenii

19 No -

Selenomonasruminantium

15 No -

Prevotellaruminicola

14 No -

Peptostreptococcusanaerobius

346 Yes +

Clostridiumsticklandii

367 Yes +

Clostridiumaminophilum

318 Yes +

Hyper-Ammonia-Producing bacteria

• Health implications • Biochemistry of lipases and biohydrogenation • Microbial ecology • Why does biohydrogenation occur? • Plant extracts as modifiers • Conclusions

Biohydrogenation of fatty acids in the rumen

LA

LNA

Saturated fatty acids

BIOHYDROGENATION

unsaturated saturated

LA

LNA C18:2 c9 c12

C18:3 c9 c12 c15

Health implications of biohydrogenation in the rumen

LNA – linolenic acid LA – linoleic acid

Fatty acid composition of feed and ruminal digesta

(% total fatty acids)

Shorland et al. (1955) Nature 175:1129-1130

Fatty acid Clover pasture Ruminal digesta

C16:0 8.9 16.9

C16:1 7.9 1.8

C18:0 2.8 48.5

C18:1 9.5 19.4

C18:2 8.1 2.9

C18:3 58.9 3.3

CLA Stimulates Immune Response

Helps Prevent Heart Disease

Helps Prevent Cancer

Health implications of biohydrogenation in the rumen

cis-9, cis-12 linoleic acid

cis-9, trans-11 linoleic acid

trans-10, cis-12 linoleic acid

Conjugated linoleic acids (CLA)

CLA in foods

CLA and cancer

Incidence of carcinogen-induced mammary tumours in mice (%)

01020304050607080

0 5 10 15CLA in diet (g/kg)

CLA and atherosclerosis

Severity of cholesterol-induced aortic lesions in rabbits (on a scale 0-4)

Kritchevsky (2000)

0

0.5

1

1.5

2

2.5

0 1 5CLA in diet (g/kg)

CLA and body composition

Influence of dietary CLA (5 g/kg for 32 d) on body fat composition in mice (%)

Park et al. (1997)

02468

101214161820

Protein Fat

ControlCLA

CLA and immune modulation

• Enhanced immune function: – Lymphocyte proliferation in pigs (Chew et al., 1997a)

• CLA suppress inflammatory bowel disease • Protection from metabolic effects of infection:

– Chicks fed CLA resisted growth suppression by LPS injection (Chew et al., 1997b)

CLA in foods

To provide 10 g of CLA/day requires 3.6 kg cheese

How can we increase our intake of CLA?

Aims

• Aims To increase the CLA

content of meat and milk To increase PUFAs in

meat and milk To increase MUFAs in

meat and milk

Lipase

Bacterial lipases in the rumen

• Lipases hydrolysing triacylglycerol (TAG) : Anaerovibrio lipolytica

• Lipases hydrolysing phospho- and galactolipids : Butyrivibrio spp.

• Lipolysis essential for fatty acid biohydrogenation to occur

Rumen Animal tissues

C C C C C

C C C C C

2H

vaccenic acid

C C C C C

9 12 C C C C C

C C C C C

2H

C C C C C

2H

linoleic acid

vaccenic acid (VA)

stearic acid

cis cis

cis cis trans trans

trans trans

cis-9, trans-11 CLA

cis-9, trans-11 CLA

CLA is an intermediate in the biohydrogenation of linoleic acid

Ruminal microorganisms

50 µm

0.5 µm

100 µm

Ciliate protozoa 106 per g digesta

Anaerobic fungi 103 per g digesta

50 µm

Bacteria 1010 per g digesta

Methanogenic archaea 108 per g digesta

0.5 µm

Role of protozoa in ruminal fatty acid metabolism

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 2 5

Time after feeding (h)

cis9

, tra

ns11

C18

:2 (u

g/m

g pr

otei

n)

ProtozoaBacteria

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

0 2 5

Time after feeding (h)

tran

s11

C18

:1 (u

g/m

g pr

otei

n)ProtozoaBacteria

From Devillard et al. (2006) Br. J. Nutr. 96, 697-704

0

2

4

6

8

10

12

14

Isotric

ha pr

ostom

a

Entodin

ium na

nnelum

Entodin

ium fu

rca m

onolo

bum

Anoplo

dinium

dentic

ulatum

Entodin

ium ca

udatu

m

Epidinium

ecau

datum ca

udatu

m

Ophryo

scole

x caud

atus

Diplop

lastro

n affin

e

Fatty

aci

ds (g

/100

g to

tal f

atty

aci

ds)

C18:1 t11Total CLA

CLA VA

C C C C C

9 12 C C C C C

C C C C C

2H

C C C C C

2H

linoleic acid

conjugated linoleic acid

vaccenic acid

stearic acid

-10

0

10

20

30

40

50

60

70

0 5 10 15 20 25

Time (h)

µg

fatt

y ac

ids/

mg

prot

ein

CLA and VA formation from LA

VA, bacteria VA, total CLA, total

CLA, bacteria

VA, protozoa CLA, protozoa

Role of protozoa in ruminal fatty acid metabolism

1 2 3 4 5 6 8 7 9 10 11

Role of protozoa: incubations with [14C]stearic acid

C18:0

C18:1

Protozoa Bacteria

Protozoal fatty acid metabolism

C C C C C

C C C C C

2H

trans-11-vaccenic acid

C C C C C

9 12 C C C C C

C C C C C

2H

C C C C C

2H

linoleic acid

trans-11-vaccenic acid

stearic acid

cis cis

cis cis trans trans

trans trans

cis-9, trans-11 CLA

cis-9, trans-11 CLA

• Contain high concentrations of PUFA, including CLA and VA

• Carry out neither biohydrogenation nor desaturation

• Ingested bacteria carry out biohydrogenation. Other observations at least partly due to ingestion of chloroplasts

Microbial ecology: role of protozoa

Role of anaerobic fungi in fatty acid metabolism

Anaerobic fungi produce cis-9,trans-11-18:2 from LA

Nam & Garnsworthy (2007). J. Appl. Microbiol. 3, 55-56

OUR DATA Neocallimastix frontalis produced 25 µg/ml CLA from 50 µg/ml LA in 96 h

Maia et al. (2007) Ant. v. Leeuw. 91, 303-314

Microbial ecology: role of bacteria

>350 bacterial species

PrevotellaJK668JK669G222JK205SU6C-proteoH17c SANCDO 2435JK724X2D62UC142Bu43NCDO 2432NCDO 2434NCDO 222210295JK611NCDO 2398JK612Mz9Mz3JK614JK609JK615WV1ATCC19171C211NCDO 2221JK662OB156LP1265JK618SH1JK663O110JK23/210296H17cNCDO 2223NCDO 2397Mz5B835NCDO 2249JL5JK626JK10/1JK86AR11SR8510316Mz7JK729S2/1010317D6/1JW11DSM9787Mz6Mz4JK170SH13Mz8pC-XS6A46pC-XS7NCDO 2399pC-XS2JK633JK730

100

74

95

78

99

97

79

9283

100

9480

77

99

98

82

92

89

Butyrivibrio proteoclasticus

Butyrivibrio hungatei

Butyrivibrio fibrisolvens and Pseudobutyrivibrio spp.

CLA, VA formed

from LA

Produce C18:0

Microbial ecology: role of bacteria

Seven dairy trials: two in

Reading, five in MTT, Finland

Intake, milk production Rumen VFA, NH3, pH

Milk fatty acid composition

qPCR of bacterial population

LIPGENE

Microbial ecology, LIPGENE project results

Seven dairy trials: two in

Reading, five in MTT, Finland

Intake, milk production Rumen VFA, NH3, pH

Milk fatty acid composition

qPCR of bacterial population

LIP GE

Microbial ecology, LIPGENE project results

AtB

Bfi

Bhu

Cpr

Sbo

SA But

Pac

Ori

IFor

age

DM

ICon

c D

MIA

dded

Oil

ITot

al O

ilID

MIO

MIN IC

PIS

tarc

hIA

shIW

SCIN

DF

IAD

FIiN

DF

IpN

DF

IME

I12:

0I1

4:0

IC15

:0I1

6:0

I18:

0I1

8:2

n-6

I18:

3(n-

3)I1

8:4(

n-3)

I20:

0IC

20:1

c5IC

20:1

c8I2

2:0

I22:

5(n-

6)I2

2:5(

n-3)

I22:

6(n-

3)I2

4:0

Isat

urat

edIM

UFA

IPU

FAIT

otal

Rumen microbiology and dietary intake

C4:0C6:0C8:0C10:0C12:0C14:0C14:1_c9C15:0C16:0C16:1_totalC16:1_c9C17:0C18:0t_4t_5t_6_8t_9t_10t_11t_12t_13c_9t_15c_11c_12c_13t_16c15c16trans_18:1_totaC18:2C6t_8tt_6_c_8c_t_6_8C7t_9tC7t_9cC8t_10tC8t_10cTotal_CLAC18:3n_6C18:3n_3C18:3_totalC20:0C20:1C20:2Total_saturatesTotal_MUFATotal_PUFATotal_trans

Milk

com

p

Heatmap showing correlations between (vertically) components of milk composition and (horizontally) rumen microbiology and dietary intake