24 May 2016
Ruminant microbiology in cow nutrition
y Intestinal Tract of Ruminant and Its Microbe
y History of Microbiology
y Ecology of Rumen Microbe & Its Function
Ruminant microbiology in cow nutrition
y Analytical Method of Rumen Microbe
y Classification of Rumen Microbe
y Analytical method of eDNA (environmental DNA)
Akio TAKENAKA Ph. D
Deputy Director
Food & Fertilizer Technology Center (FFTC)
y Two Main Function of Rumen Microbe
y Fiber degradation
y Methane production
1
History of Microbiology
ɚ
ɛ
1684
Microorganism was found by Leeuwenhoek’s microscope
1843
1860
First report of rumen ciliate protozoa
Pasteur denied natural occurrence of microorganism
Koch established gelatin medium for colony formation
1882
1950
ɜ
ɝ
1953
Hungate established anaerobic roll tube method
Watson and Crick found double helix formation of DNA
1977
Sanger established the method to analysis DNA sequence
1988
Polymerase chain reaction (PCR) method using heat stable
Taq polymerase was established
1995
First report of complete sequence of bacterial
genome(Haemophilus influenzae)
2003
Complete sequence of human genome was published
A new generation (post genome era) has come.
3
A difference of energy intake and
output is accumulated to a body.
Gastrointestinal system of herbivore
Volume proportion of
gastrointestinal system
Horse
stomach 㻡㻑
intestine
㻥㻡㻑
Cattle
㻡㻑
㻡㻑
㻟㻑
intestine rumen
㻡㻠㻑
㻟㻟㻑
䠎䠑䠌䃛㼙
5
The role of rumen microorganisms
Rumen
Microscopic picture of rumen juice
The global efficiency of rumen microbe
be㸦
㸦per year
ar㸧
3 billion ruminant livestock
㸦increasing 15 million/year㸧
Around 10000MT of cellulosic material
are ingested by domestic ruminants
Meat
Feed
Digested in the rumen
Milk
bacteria
protozoa
archaea
fungi
Rumen microorganisms
The role of rumen mircoorganisms
• Fiber degradation
• Production of proteins
• Production of VFAs
• Breakdown of nutrients
• Methane production
࣭
࣭
࣭
150 MT of carcass
15 MT of milk
7
Feed the people
8
Main rumen bacteria
• Cellulose degrader
• Fibrobacter succinogenes:
• Ruminococcus albus:
• Ruminococcus flavefaciens:
• Hemicellulose, pectin utilizer
• Prevotella ruminicola:
• Butyrivibrio fibrisolvens:
• Starch fermenter
• Ruminobacter amylophilus:
• Streptococcus bovis*:
• Organic acid utilizer
• Megasphaera elsdenii:
• Selenomonas ruminantium:
Morphological classification of rumen ciliate protozoa
G-, rods
G+, cocci
G+, cocci
cilia with whole body
cilia present anterior part
G-, rods
G+, rods
<100䗇
Dasytricha
cilia only at adoral area
Isotricha
G-, rods
G+, rods
Entodinium
G-, cocci
G-, rods
*: Streptococcus bovis is facultative anaerobe, others are
strict anaerobe.
Epidinium
Diplodinium Eudiplodinium
Polyplastron
9
Analytical method of rumen ciliate protozoa
10
Apparent digestibility of dry matter,
energy, crude protein, NDF and ADF.
Rumen 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.
>120䗇
Unf
(n=5)
Dry matter
Energy
Crude protein
NDF
ADF
67.86㼼0.98
66.02㼼0.92
56.56㼼1.24
57.26㼼1.23
54.02㼼0.85
Values are means㼼S.E.
*:p<0.05, **:p<0.01
Roughage:Concentrate=1:1
11
Mono-fau
(n=6)
Poly-fau
(n=6)
70.78㼼1.09
68.63㼼1.24
59.60㼼1.68
58.90㼼1.71
50.38㼼2.65
**
73.15㼼0.81
71.40㼼1.06*
64.20㼼1.84*
**
63.28㼼1.29
**
62.53㼼1.11
Life has already digital protocol
The structure of sugars
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ia䟿
2
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Gß1-4Gß1-4Gß1-4Gß1-4Gß1-4Gß1-4G
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6
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13
1
Many kind of enzymes are needed to degrade lignocellulose
1
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y
Digital words are A, T, G, and C.
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8-Acetylxylan esterase
9-D-Glucuronidase
15
14
Number of genes concerning to β
β-glucanase
-gl
glucanase
e (ce
cellulase
ellulas
se,
se
e,, ce
e
cellobiohydrase
c
ellobiohyd se, xylanase, βglucosidase) and homologues in each family.
Family
Number
1-Cellobiohydrolase
2-Endoglucanase
3-Cellobiase
4-Endoxylanase
5-Xylosidase
6-Arabinofuranosidase
Cellulose
Total Number
From
From
From
Archaea Bacteria Eukaryota
From
Virus
Unclassif From Rumen From Rumen From Rumen
ied
Bacteria
Fungi
Protozoa
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2014
㼀㼔㼑㻌㼚㼡㼙㼎㼑㼞㻌㼛㼒㻌㻳㻴㻌㼒㼍㼙㼕㼘㼥㻌㼓㼑㼚㼑㼟㻌㼑㼤㼕㼟㼠㼕㼚㼓㻌㼕㼚㻌㼠㼔㼑㻌㼣㼔㼛㼘㼑㻌㼓㼑㼚㼛㼙㼑㻌㼟㼑㼝㼡㼑㼚㼏㼑䠄㼜㼍㼞㼠㼕㼍㼘䠅
GH family No.
Butyrivivrio fibrisolvens 16/4
Butyrivivrio proteoclasticus B316
Clostridium thermocellum ATCC27405
Fibrobacter succinogenes S85
Prevotella ruminicola 23
Ruminococcus albus 7
Selenomonas ruminantium TAM6421
Homo sapiens
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Many typhoons had occurred in the west Pacific 2013 autumn
䠄
Enteric CH
H4 perspective (2005)
䠅
Indon Thai*
esia*
Philip
pine*
Malay Austr
sia*
alia
NZ
Japan World
238.4
64.8
87.8
26.9
21.1
4.2
127.8
6449
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%
N2O䠖9%
Population, million
CH4䠖18%
CO2䠖72%
Agricultural sector䠖40%
CH4(total), Tg/yr
Natural gas etc䠖30%
From UNFCCC 1994
%
¾
About 20% of GHG emission is methane
¾
About 40% of methane is from Agriculture
¾ A higher contribution rate to methane from
agriculture in the Southeast Asian countries is from
rice paddies and enteric fermentation of livestock.
Rice field
Enteric fermentation
Other agriculture
Non agriculture
* : inventory data of 1994
GHG data from UNFCCC
20
Balance of hydrogen-producing and
hydrogen consuming reactions in the rumen
•
CO2
[2H]
•
•
Butyrate
•
[2H] Fumarate
[2H]
Propionate
Succinate
hydrogen-consuming
reactions
CO2
CO2
Supply propionate
enhancers, malate or
fumarate
Enhance nitrate/nitrite
reduction
Increase sulfate reduction
Supply unsaturated fatty
acids
Enhance reductive acetate
production
Acetate (oxidative acetogenesis)
hydrogen-producing reactions
CH4
21
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㻟㻜
ᑐ↷༊
䝡䞊䝹⢑
⏕⡿䝚䜹
Beer
lees
Rice
bran
12%
12%
㻝㻞䠂ῧຍ༊
㻝㻞䠂ῧ
Vs.Y2Milk
Yield
= 8.19
+ 300/
300/FCM
r = 0.82
50
40
30
20
10
0
b
䠾
㻞㻜
㻲㼑㼑㼐㼕㼚㼓
60
㻞㻚㻜
䠽a
㻡㻜
㻴㼑㼍㼐㼕㼚㼓 㻲㼘㼛㼣㼑㼞㼕㼚㼓
㻝㻞㻜㻜
㻝㻜㻜㻜
㻤㻜㻜
㻢㻜㻜
㻠㻜㻜
㻞㻜㻜
㻜
㻜㻚㻜
㻹㼑㼠㼔㼍㼚㼑㻌㻔㻸㻛㼗㼓㻌㻰㻻㻹㻕
[2H]
Malate
Acryl CoA
•
㻝㻡
Methane production
[2H]
Oxaloacetate
[2H]
Acetyl CoA
㻠㻜
㻞㻜㻑㼞㼑㼐㼡㼏㼠㼕㼛㼚
㻞㻜
litter/
䝯䝍䞁Ⓨ⏕㔞
(liter/kg (FCM)
Lactate
Formate
㻟㻜㻑㼞㼑㼐㼕㼏㼠㼛㼜㼙
㻡㻜
㻹㼑㼠㼔㼍㼚㼑㻌㼜㼞㼛㼐㼡㼏㼠㼕㼛㼚
㻼㼑㼞㻌㻝㼗㼓㻌㼙㼕㼘㼗㻌㼜㼞㼛㼐㼡㼏㼠㼕㼛㼚
Pyruvate
CO2
Increasing methods for hydrogen
consuming reactions
㻔㼘㼕㼠㼠㼑㼞㻛㼗㼓 㻰㻻㻹䠅
[2H]
㻤㻜
㻹㼑㼠㼔㼍㼚㼑㻌㻼㼞㼛㼐㼡㼏㼠㼕㼛㼚
[2H]
㼁㼟㼍㼓㼑㻌㼛㼒㻌㻌㻮㼥㻙㼜㼞㼛㼐㼡㼏㼠㼟
㻴㼕㼓㼔㻌㻲㼍㼠㻌㻲㼑㼑㼐㼕㼚㼓
㻯㼔㼍㼚㼓㼑㻌㼏㼡㼘㼠㼕㼢㼍㼠㼕㼛㼚㻌㼠㼕㼙㼑
CO2
㻹㼑㼠㼔㼍㼚㼑
㻔㼓㻛㼗㼓㻌㻰㻳㻕
Hexose
Cellulose
Hemicellulose
Starch
Technology to reduce the environmental impact
0
5
10
15
20
25
30
35
FCM (kg/day)
(kg/᪥䠅
䠐䠂⬡⫫⿵ṇங㔞
For Animal and
Plant
䞉Breeding
䞉Reproduction
䞉Cultivation
Feeding etc.
Integrated
technology is
needed