Optimization of ruminal digestion systems for the production of ...

messengerrushBiotechnology

Feb 22, 2013 (4 years and 6 months ago)

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Lab. of Rumen Microbiology and Biotechnology, GSNU, Korea.


Sung Sill Lee

AL 757

Special Topics in Rumen Microbiology

LEC


10

Rumen

Anaerobic Fungi II (Metabolism)

Digesta

Empty
Sporangium

Release

of
zoospore

Chemotaxis

Diet of

animals

Plant

fragment

Encystment

Germination

Rhizoid

development


Sporangium enlargement

Nuclear division

Differenciation

Attachment

of

zoospore


Life Cycle of
Anaerbic

Fungi in the Rumen

Increasing Content of Fiber in Feed


-

Fungal Population


Increasing Content of Soft Leaf, Young Plant or
Concentrate in Feed

-

Fungal Population


( Soft Leaf, Young Plant
-

Short Turnover Time


Concentrate
-

Low pH)

Adding Nikkomucin in Feed

-

Fungal Population


Adding Nistain in Feed

-

No change in Fungal Population

Chemical Treatment of Rice straw

-

Fungal Population


Population of Anaerobic Fungi in the Rumen

Fungal Population Affected by Host Animal

-

KNC, KNG


>

Holstein Lactating Cow and Sheep

-

Small Animal
(KNG, Sheep)


>

Large Animal
(KNC, Holstein Lactating Cow)

Fungal Population Affected by Fermentation Time

-
Neocallimastix :
Maximum in 15
-

30 min after feeding

-
Caecomyces, Piromyces :
Maximun in 1 hr after feeding


Population of Anaerobic Fungi in the Rumen

Number of
zoospores

Diets

Diets

Number of
zoospores

Effects of Diets


Grass silage
1)


Rye grass (ear emergence stage)
1)


Rice straw + beet pulp + brewer’s
3)


Barley straw
1)


Corn silage + hay
3)


Lucern hay
2)


Lucern hay (day 4)
2)


Maize silage
1)


Corn silage + monensin
2)


Lucern hay + whey(week 4)
2)


Beets (six meals/day)
1)


Barley
1)


40

40

25

20

17
.
3

11
.
1


1


7


2
.
9


0
.
7


1
.
5


0

Effects of Chemical treatment
3)


Untreated
-
rice straw


HaOH treated
-
rice straw


NH3
-
N treated
-
rice straw


11
.
0

43
.
5

65
.
0

Effects of Concentrate:Roughage ratio
3)


Concentrate 100%


Concentrate 85% : Roughage 15%


Concentrate 70% : Roughage 30%


21
.
2

36
.
9

65
.
7

Effects of Animal species
3)


Korean native cattle


Holstein dairy cow


Korean native goat


Sheep


13
.
9

12
.
7

15
.
9

13
.
4

Source :
1)
Grenet
et al
. (1989),
2)
Grenet
et al
. (1989),

3)
Lee
et al
. (1995)


Effects of Feeding System on the Number of Fungal Zoospores

Hydrogenosomes

-
Produce H
2
, CO
2
, Fermentation end
-
products like acetate, ATP

Energy Efficiency

-
2 mol ATP / 1 mol Glucose

Anaerobic Respiration

Fermentation

end
-
products

ATP

SLP

The last electron

acceptor ;
Pyruvate, acetyl
-
CoA,

Lactate, Amino acid, CO, CO2, methanol etc.


Energy Metabolism of Anaerobic Fungi in Rumen


Glucose

PEP

Glycolysis

CO
2

CO
2

Ethanol

Acetate+Formate

Lactate

Acetaldehyde

ADP+PPi

ATP+Pi

NAD

NADH

NAD

NADH

NADH

IDP

ITP

Pyruvate

HYDROGENESOME

Malate

CO
2

Acetate

Succinate

Acetyl

CoA

NAD

NAD(P)

NAD(P)H

PFOR

PFOR

H
2

H
+

H
+

ATP

ADP+Pi

Fdox

Fdred

2 ADP


AMP+ATP

ATP


ADP+Pi

CoASH

Succinate

Succinyl CoA

?

?

?


OUT IN



IN OUT

Malate

OAt

Acetate

Succinate

Pyruvate


Hydrogenesome of anaerobic rumen fungi

H
2

Organisms

Monocentric Fungi


N. frontalis


N. patriciarum


N. sp.
LM1



P. communis



C. communis


1
.
80

3
.
67


-


3
.
87


4
.
67

CO
2

Formate

Acetate

Lactate

Succinate

Ethanol

Cellulose
fermented
(%)

Fermentation products (mol/10mol of h.u fermented)


3
.
75

3
.
27

3
.
17


3
.
46


6
.
78


6
.
30

6
.
52

7
.
64


7
.
32


8
.
07


6
.
48

7
.
06

6
.
34


7
.
17


9
.
56


6
.
73

5
.
61

8
.
76


4
.
27


1
.
51


0
.
59

0
.
39

1
.
00


0
.
81


0
.
26


4
.
16

3
.
85

1
.
47


4
.
41


5
.
55


89
.
5

85
.
1


-


80
.
2


79
.
1

Polycentric Fungi


O. joyonii


O. intercalaris



A. sp. CP2


A. sp. EB25



-


-



-


-


4
.
55

5
.
27


1
.
91

1
.
77


17
.
30

18
.
24



5
.
58


6
.
21



9
.
10

10
.
53



3
.
82


3
.
53


3
.
80

0
.
67


8
.
78

6
.
69


0
.
12

0
.
43


0
.
11

1
.
44


6
.
91

7
.
41


3
.
81

2
.
20



-


-


-


-


Fermentation end
-
products of cellulose or glucose by rumen fungi

Poly
-

Utilization of saccharides

Bacterial Species

Di/Tri
-

Mono
-

Primary cellulolytics

Secondary cellulolytics

Nonecellulolytics

Fibrobacter succinogenes

Ruminococcus flavefaciens

Ruminococcus albus

Butyrivibrio fibrisolvens

Clostridium longisporum

Clostridium locheadii

Prevotella ruminicola

Ruminobacter amylophilus

Selenomonas ruminantium

Streptococcus bovis

Succinomonas amylolytica

Succinivibrio dextrinosolvens

Cel

Cel, Xyl, Pec

Cel, Xyl

Cel, Xyl, Dex, Pec

Cel

Cel, Dex

Pec, Sta, Dex

Sta

Sta, Dex

Sta

Sta, Dex

Dex, Pec

Cl

Cl

Cl

Ml, Lc, Cl

Cl, Ml, Lc, Sc

Ml, Sc

Lc, Cl, Ml

Ml

Cl, Ml, Lc, Sc

Ml, Lc, Cl, Sc

Ml

Ml, Sc

G

-

G, X, A

G, Ga, M, F, X

G, Ga, F

G

G, Ga, F, X, A, R

-

G, Ga, F, X, A, R

G, Ga, F, X, A

G

G, Ga, M, X, A, F


Utilization patterns of CHO for Anaerobic Fungi

Poly
-

Utilization of saccharides

Species

Di/Tri
-

Mono
-

Holochrichs

Entodinomorphs

Isotricha intestinalis

Isotricha prostoma

Dasytricha ruminantium

Entodinium bursa

Entodinium caudatum

Entodinium simplex

Diplodinium diplodinium

Diplodinium polyplastron

Diplodinium ostracodinium

Diplodinium eremoplastron

Epidinium ecaudatum

Ophryoscolex caudatus

Sta, Pet

Sta, Pet

Sta

Sta, Xyl

Sta

Sta

Cel, Xyl, Sta

Cel, Sta

Cel, Xyl, Sta

Cel, Xyl, Sta

Cel, Xyl, Sta

Cel, Xyl, Sta

Sc

Sc

Cl, Ml

G

G

G

-

Cl, Ml, Sc

-

-

Sc

-

-

Sc, Ml

-

-

G

-

-

G

-

-

-

-


Comparison of carbohydrate utilization patterns of ruminal anaerobic
microbes


Poly
-

Utilization of saccharides

Species

Di/Tri
-

Mono
-

Monocentrics

Polycentrics

Neocallimastix frontalis

Neocallimastix patriciarum

Piromyces communis

Caecomyces communis

Anaeromyce mucronatus

Orpinomyces joyonii

Cel, Xyl, Sta, Pec

Cel, Xyl, Sta, Pec

Cel, Xyl, Sta, Pec

Cel, Xyl, Pec

Xyl, Sta

Cel, Xyl, Sta

Cl, Gn, Ml, Lc, Ra, Sc

Cl, Gn, Ml, Ra, Sc

Cl, Gn, Ml, Lc, Ra, Sc

Cl, Gn, Ml, Lc

F, G, X

G, X

F, G, X

F, G, X

Cl, Gn, Ml, Lc, Sc

Cl, Gn, Ml, Lc, Sc

F, G, X

F, G, X


Comparison of carbohydrate utilization patterns of ruminal anaerobic
microbes


Comparison of rate constant(h
-
1
) for digestion of crystalline
cellulose by various ruminal and non ruminal microbes (Bacteria)

Sub
-

strate

Microbes

Rate

constant

Basis

Clostridium thermocellum

(ATCC27405)

Ruminococcus albus 8

Ruminococcus flavefaciens

(FD
-
1)

Fibrobacter succinogenes

(S85)

Cellulomonas uda

(ATCC21399)

Cellulomonas flavigena

(JC3)

AV


AV

SC


SC


AV


AV

Weight loss, chemostat


Weight loss, chemostat

Weight loss, chemostat


Weight loss, chemostat


Heat production, batch


Weight loss, batch

Lynd et al. (1986)


Pavlostathis et al. (1986)

Shi & Weimer (1992)


Weimer (1992)


Dermoun & Belaich

(1985)

Chesson (1988)

Reference

0.16


0.05

0.08


0.07


0.027


0.006

Comparison of rate constant(h
-
1
) for digestion of crystalline
cellulose by various ruminal and non ruminal microbes (Fungi)

Sub
-

strate

Microbes

Rate

constant

Basis

Neocallimastix frontalis

(RE1)

Orpinomyces joyonii SG4

White
-
rot fungi (5 species)

Brown
-
rot fungi (8 species)

FP


FP

CT

CT

Weight loss, batch


Weight loss, batch

Weight loss, soil block

Weight loss, soil block

Lee et al. (1997a)


Lee et al. (1997b)

Highley (1988)

Highley (1988)

Reference

0.040


0.037

<0.004

<0.004

Interaction of Rumen Fungi
with Methanogenic Bacteria

Incubation with

Methanogenic Bacteria


Stabilization


of Incubation


Fungal Population



Hydrolysis Rate



Cellulolysis Rate


Cellulolytic Activity


Electron Sink Products


(Like Lactate, CH
4
)

Acetate, CO
2
, H
2

etc.

Interaction of Rumen Fungi with
Non
-
methanogeic Bacteria

Interaction by Competition, Symbiosis and Synergy

Incubation with Hydrogentropic Bacteria

Stabilization of Incubation, Fungal population,

Cellulolysis rate

Incubation with Cellulolytic Bacteria

Inhibit or no influence on Cellulolysis & Xylolysis rate of
fungi

Incubation with Saccharolytic Bacteria

Xylolysis rate of fungi

Incubation with Lactate Utilization Bacteria

Cellulolysis & Xylolysis rate of fungi

Interaction of Rumen Fungi
with Protozoa

Predation of Zoospore and Fungal Mycelium

Increase the turnover rate of anaerobic fungi in the rumen

Secreting Chitinase

Inhibit the growth rate & enzymen activities of rumen fungi

Interaction of Rumen fungi

with Protozoa are not defined

Exactly up to now

Excretion form of

rumen fungal enzymes

Cellulase

Mostly
extracellular

excretion type

Protease

Amylase

Over 80% is
extracellular

excretion type

Comparison of cellulase activities (U/ml


h
-
1
)of the
supernatant of medium containing rice straw as a carbon
source incubated with various microorganisms

24

Incubation time (h)

Microbial strains

48

96

Rumen mixed microorganism

Ruminal Fungi

Commercial microoganisms

Piromyces fhizinflata

Orpinomyces joyonii

Neocallimastix frontalis

Aspergillus oryzae

Saccharomyces cerevisiae

3.272

3.128

4.633

Ruminal Bacteria

Fibrobacter succinogenes

Ruminococcus albus

Ruminococcus flavefaciens

4.034

4.000

0.170

0.305

0.302

3.996

3.990

4.067

6.656

3.910

3.899

0.791

0.487

0.842

4.113

4.692

4.278

7.435

3.902

3.944

1.401

0.857

1.067

4.283

Bermuda grass leaf

Filter paper

Digestibility(%)

Neocallimastix

Piromyces

R. flavefacience

Rice straw

B. succinogenes

68

30

23

15

97

96

23

3

59

48

18

4


Digestion of roughage by anaerobic fungi and bacteria

Filter paper

Rice Straw

A. oryzae

S. cerevisiae

KNGF
-
2

Alfalfa

KNGF
-
6

40,300

30,374

5,045

6,247

40,300

53,258

9,729

4,943

40,300

56,834

Enzyme activities (U/ml
.
h
-
1
)


Xylanase activities of anaerobic ruminal fungi


Enzymes activity


Anaerobic fungus

Control B

Control A

Commercial Enz.

Cont

F
-
1

F
-
2

F
-
3

F
-
4

F
-
5

Improved productivity

DFM

Enzymes

Feeds

Additives

Silage

Inoculants



D

I

E

T





Industrial Application of Rumen anaerobic Fungi in Diets

Rumen Nutritionist


Enzymologist

Microbiologist

Gene
-

Designer


Major Research Areas for Rumen Anaerobic Microbes

RUMEN


ANAEROBIC

FUNGI

Bioremediator

Botanist

Glucose

Aspartic acid

Glutamic acid

Oxaloacetate

Citrate

Oxoglutarate

Acetyl CoA

Shikimate

Mevalonate

Terpenoid
s

(Wortmannin)

Polyketides

patulin

griseofulvin

ochratoxin A

aflatoxins

Polyacetylenes

(brefeldin A)

Secondary

Metabolites

Secondary

Metabolites

Glucose

Pyruvate

Pyruvate

Glycosides

Kojic acid


muscarine

Fatty acids

Amino acids

HMP
1)

Cycle

HMP

Cycle

Aromatic

amino acids

Triose
phosphate

Aromatic
secondary

metabolites

(gliotoxin)


Peptides

Penicillin

Monensin

Neomysin

Verginiamycin

Lasalosid

Bambemycin

Novomycin

Tylosine

Subtilin

Enzyme Characteristics of
Rumen Anaerobic Fungi

Factors influencing the
activities of fungal enzymes

Species of ruminal fungi

Types of growth substrates

Incubation time

Host animals

pH and temperature

Enzyme activities of
various rumen fungi

CMCase, b
-
glucosidase, Xylanase activities:

Polycentric fungi

>
Monocentric fungi

CMCase, Xylanase activities (On straw media):

Caecomyces
>

Piromyces, Neocallimastix

Enzyme activities of various
rumen fungi

Enzyme activities of
O. joyonii, N. patriciarum
and

P. communis

on cellobiose substrate

Proteolytic activity:

Amylolytic activity:

O. joynii
>

P.communis
>

N. patriciarum

O. joynii
>
P.communis
>

N. patriciarum

Enzyme characteristics as
influenced by substrates

Catabolite

End
-
product

Repress the enzyme activities of rumen fungi

monosaccharides

or
disaccharides

polysaccharides
(cellulose, xylan)

Enzyme activities as influenced
by incubation time & Host Animal



Increase in proportion to the incubation time


(Akin et al.,1990; Lee et al.,1995)

Reach maximum activity on 5 days of 7 days


(Ho et al., 1996; Boreman et al., 1989)

KNG, KNC

>
Holstein lactating cow, sheep


Small animal (KNG, sheep)



>
Large animal (KNC, Holstein cow)

Control of Enzyme Synthesis


Catabolic Repression

:


Phenomena that inhibit the enzyme production to use 2nd


substrate when easier metabolizable substrates or metabolites


exist

Cotrolled by cAMP system

Osmosis,

Control of O
2
:CO
2
Ratio

Structual Transformation of Enzyme

Isoenzyme, Chemotaxis,

etc.

Glucose & Cellobiose


Concentration of
intracellular cAMP


Inhibit cellulolytic enzymes

Control Enzyme

Synthesis

Effect of Small
-

& Medium
-
sized Protozoa on
Cellulose Digestion by
Neocallimastix frontalis

Uninoculate

Protozoa

Substrate weight loss (%)


Small Protozoa
1)


Medium Protozoa
2)


3

1

1)

Small
-
sized(<30

m) protozoa,

2)

Medium
-
sized(30
-
70

m) protozoa.

Fungi

Fungi+Protozoa



7


1


74

69


88


2

H
2
-
gas production (
ml
)


Small Protozoa


Medium Protozoa


-

-


tr


-



1
.
5


1
.
1



-


-

CH
4
-
gas production (
ml
)


Small Protozoa


Medium Protozoa


-

-



0
.
1


0
.
13



-


-



4
.
1


0
.
23

(Fonty and Joblin, 1991)

Percentage of Cellulose & Xylan degraded
by Rumen Fungi in absence or in presence of
H
2
-
utilizing Rumen Methanogenic Bacteria

Fungi alone

Organisms

Fungi+Methanogenes

N. frontalis

P. communis

C. communis

O. joyonii
3)

68
.
5

87
.
6


9
.
7

77
.
1

1)

From filter paper, after 8days of incubation, Fonty and Joblin, (1989).

2)

From xylan, after 7days of incubation, Joblin et al. (1990).

3)

From cellulose, after 8days of incubation, Hodrová et al. (1995).

Fungi alone

Fungi+Methanogenes

80
.
1

90
.
3

19
.
1

87
.
2

81
.
9

83
.
3

52
.
3


-

88
.
5

86
.
6

80
.
5


-

1)
Cellulose degraded by

2)

Xylan degraded by

New Useful Products

Enzymes

Antibiotics

Aromatic compounds

GENE

Feed additives

Medicine

Penicillium notatum

2unit/mL(0.6ug)

P. Chrysogenum

100unit/mL(30ug)

Anaerobic fungi

50,000unit/mL


Commercialization of new useful products