11

Akio TAKENAKA Ph. D

Ruminant microbiology in cow nutrition

Deputy DirectorFood & Fertilizer Technology Center (FFTC)

24 May 2016 Ruminant microbiology in cow nutrition

Intestinal Tract of Ruminant and Its MicrobeHistory of MicrobiologyEcology of Rumen Microbe & Its Function

Analytical Method of Rumen MicrobeClassification of Rumen MicrobeAnalytical method of eDNA (environmental DNA)

Two Main Function of Rumen MicrobeFiber degradationMethane production

3

History of Microbiology

1860 Pasteur denied natural occurrence of microorganism1882 Koch established gelatin medium for colony formation

Hungate established anaerobic roll tube methodWatson and Crick found double helix formation of DNA

Polymerase chain reaction (PCR) method using heat stable Taq polymerase was established

1988

1684 Microorganism was found by Leeuwenhoek’s microscope

1953

1977 Sanger established the method to analysis DNA sequence

1995 First report of complete sequence of bacterial genome(Haemophilus influenzae)

2003 Complete sequence of human genome was published

1950

A new generation (post genome era) has come.

1843 First report of rumen ciliate protozoa

A difference of energy intake and output is accumulated to a body.

5

Gastrointestinal system of herbivore

Horse

Cattle

Volume proportion of gastrointestinal system

intestine

stomach

rumenintestine

Microscopic picture of rumen juice

77

The role of rumen microorganisms

Rumen

Feed

bacteria archaea

fungiprotozoa

Rumen microorganisms

The role of rumen mircoorganisms• Fiber degradation• Production of proteins• Production of VFAs• Breakdown of nutrients• Methane production

Meat

Milk

88

The global efficiency of rumen microbebe per yearar

Digested in the rumen

15 MT of milk 150 MT of carcass

Feed the people

3 billion ruminant livestockincreasing 15 million/year

Around 10000MT of cellulosic material are ingested by domestic ruminants

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Main rumen bacteria

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• Cellulose degrader• Fibrobacter succinogenes: G-, rods• Ruminococcus albus: G+, cocci• Ruminococcus flavefaciens: G+, cocci

• Hemicellulose, pectin utilizer• Prevotella ruminicola: G-, rods• Butyrivibrio fibrisolvens: G+, rods

• Starch fermenter• Ruminobacter amylophilus: G-, rods• Streptococcus bovis*: G+, rods

• Organic acid utilizer•Megasphaera elsdenii: G-, cocci• Selenomonas ruminantium: G-, rods

*: Streptococcus bovis is facultative anaerobe, others are strict anaerobe. 10

cilia with whole bodycilia present anterior part

Entodinium

Dasytricha

Isotricha

Epidinium Diplodinium Eudiplodinium Polyplastron

cilia only at adoral area

<100 >120

Morphological classification of rumen ciliate protozoa

1111

Analytical method of rumen ciliate protozoaRumen ciliate protozoa is difficult to culture in vitro.However animals without protozoa is provided by isolation from other animals, because rumen protozoa is infected only by direct touch with other animal.

Faunated: • Normal ruminant has more than one species of rumen protozoa.

Unfaunated: • Ruminants isolated immediately after birth, ruminant without any species of protozoa can be provided.

Defaunated:•Rumen protozoa is removed by any method (wash out, detergent treatment, middle chain fatty acid etc.)

Monofaunated:• Ruminants which have only one species of protozoa.

Apparent digestibility of dry matter, Apparent digestibility of dry matter, energy, crude protein, NDF and ADF.

Unf Mono-fau Poly-fau

*:p<0.05, **:p<0.01

Dry matterEnergyCrude proteinNDFADF

67.86 0.9866.02 0.9256.56 1.2457.26 1.2354.02 0.85

70.78 1.0968.63 1.2459.60 1.6858.90 1.7150.38 2.65

73.15 0.8171.40 1.0664.20 1.8463.28 1.2962.53 1.11

**

****

**

Roughage:Concentrate=1:1

Values are means S.E.

(n=5) (n=6) (n=6)

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Life has universal standard already!yDigital words are A, T, G, and C.

Life is multimedia1

ia

Life has already digital protocol

Multimedia has universal standard protocol: digital words

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The structure of sugars

Glucose

Maltose

Sucrose

Cellobiose

Starch Cellulose

long chain will be

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Many kind of enzymes are needed to degrade lignocellulose

mGu:4-O-methylgluculonic acid

Xß1-4Xß1-4Xß1-4Xß1-4Xß1-4Xß1-4Xß1-4X3

Gß1-4Gß1-4Gß1-4Gß1-4Gß1-4Gß1-4Gß1-4GGß1-4Gß1-4Gß1-4Gß1-4Gß1-4Gß1-4G

1

Gß1-4G

Af

Fer-Fer-O-Fer

Xß1-4X2G5

2X

Xß1-4Xß1-4Xß1-4Xß1-4Xß1-4Xß1-4Xß1-4X

mGu

5

5Af

3Ac3

3

Af5Fer

Lignin

Lignin

6

21

mGu

FerLignin

1-Cellobiohydrolase2-Endoglucanase3-Cellobiase4-Endoxylanase5-Xylosidase6-Arabinofuranosidase7-Feruloyl esterase8-Acetylxylan esterase9- -GlucuronidaseAc:Acetic acid

Af:ArabinoseFer:Ferulic acidG:Glucose

X:Xylose

12

3Af

1

3

46

7

6

2

4

8 91

1

Number of genes concerning to ββ-β-glucanasee (e ((((((((((((((((((((((((cellulasese, e, cellobiohydrasese, xylanase, ββ-concerning to βββ lucanaseglg ee (( ellulasce((((((((( see, ellobiohydcecglucosidase) and homologues in each family.

FamilyNumber Total Number From

ArchaeaFrom

BacteriaFrom

EukaryotaFromVirus

Unclassified

From RumenBacteria

From RumenFungi

From RumenProtozoa

GH family No.Butyrivivrio fibrisolvens 16/4

Butyrivivrio proteoclasticus B316Clostridium thermocellum ATCC27405

Fibrobacter succinogenes S85Prevotella ruminicola 23

Ruminococcus albus 7Selenomonas ruminantium TAM6421

Homo sapiens

Many typhoons had occurred in the west Pacific 2013 autumn

2014

CO2 72%

CH4 18%

N2O 9%

Agricultural sector 40%

Natural gas etc 30%

%From UNFCCC 1994

About 20% of GHG emission is methaneAbout 40% of methane is from AgricultureA higher contribution rate to methane from

agriculture in the Southeast Asian countries is from rice paddies and enteric fermentation of livestock.

Rice fieldEnteric fermentation Other agricultureNon agriculture 202020

Enteric CHH44 perspective (2005)

GHG data from UNFCCC

Indonesia*

Thai* Philippine*

Malaysia*

Australia

NZ Japan World

Population, million 238.4 64.8 87.8 26.9 21.1 4.2 127.8 6449

CH4(total), Tg/yr 6.4 3.2 1.5 2.2 4.3 1.2 1.1 92

CH4(enteric)/CH4(agric) 23% 22% 27% 15% 87% 97% 46% 59%

CH4(agric)/CH4(total) 51% 91% 66% 15% 60% 91% 64% 51%

CH4(total)/GHG(total) 15% 23% 31% 32% 20% 35% 1.9% 18%

GHG(agric)/GHG(total) 9.4% 8.0% 33% 4.8% 16% 48% 2.2%

* : inventory data of 1994

2121

Balance of hydrogen-producing and hydrogen consuming reactions in the rumen

Hexose

[2H]

Oxaloacetate

Malate

Pyruvate

Formate

Acetyl CoA

Propionate Succinate

Fumarate

Lactate

Butyrate

Acetate (oxidative acetogenesis)

Acryl CoA

CH4

[2H]

[2H]

CO2

CO2

CO2

CO2

CelluloseHemicelluloseStarch

[2H]

[2H][2H]

[2H]

CO2

[2H]

hydrogen-producing reactions hydrogen-consuming reactions

• Supply propionate enhancers, malate or fumarate

• Enhance nitrate/nitrite reduction

• Increase sulfate reduction• Supply unsaturated fatty

acids• Enhance reductive acetate

production

Increasing methods for hydrogen consuming reactions

0102030405060

0 5 10 15 20 25 30 35FCM (kg/day)

(lite

r/kg

FCM

)

Y2 = 8.19 + 300/FCMr = 0.82

(kg/

(litt

er/

0102030405060

0 5 10 15 20 25 30 35FCM (kg/day)

Met

hane

pro

duct

ion

(lite

r/kg

FCM

)

Y2 = 8.19 + 300/r = 0.82

Technology to reduce the environmental impact

For Animal and Plant

BreedingReproductionCultivationFeeding etc.

Integrated technology is needed

Beer lees12%

Rice bran12%

ab

Vs. Milk Yield