Optimization of ruminal digestion systems for the production of ruminant animals

1
AL 757 Special Topics in Rumen Microbiology
LEC 10
Rumen Anaerobic Fungi II (Metabolism)
Lab. of Rumen Microbiology and Biotechnology,
GSNU, Korea.
Sung Sill Lee
2
Life Cycle of Anaerbic Fungi in the Rumen
Chemotaxis
Attachment of zoospore
Release of zoospore
Plant fragment
Digesta
Diet of animals
Empty Sporangium
Encystment
Differenciation
Germination
Rhizoid development
Sporangium enlargement Nuclear division
3
Population of Anaerobic 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
4
Population of Anaerobic Fungi in the Rumen
Fungal Population Affected by Host Animal - KNC,
KNG gt Holstein Lactating Cow and Sheep -
Small Animal (KNG, Sheep) gt 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
5
Effects of Feeding System on the Number of
Fungal Zoospores
Number of zoospores
Diets
Diets
Number of zoospores
Effects of Diets Grass silage1) Rye grass
(ear emergence stage) 1) Rice straw beet pulp
brewers 3) Barley straw1) Corn silage
hay3) Lucern hay2) Lucern hay (day 4)2)
Maize silage1) Corn silage monensin2)
Lucern hay whey(week 4) 2) Beets (six
meals/day) 1) Barley1)
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 ConcentrateRoughage ratio3)
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)
6
Energy Metabolism of Anaerobic Fungi in
Rumen
Hydrogenosomes -Produce H2, CO2, Fermentation
end-products like acetate, ATP
Energy Efficiency -2 mol ATP / 1 mol Glucose
Anaerobic Respiration
SLP
Fermentation end-products
ATP
The last electron acceptor Pyruvate, acetyl-CoA,
Lactate, Amino acid, CO, CO2, methanol etc.
7
Hydrogenesome of anaerobic rumen fungi
Glucose
Glycolysis
AcetateFormate
Ethanol
Lactate
PEP
ADPPPi
CO2
NAD
ATPPi
NAD
IDP
CO2
NADH
ITP
NADH
Acetaldehyde
OAt
Pyruvate
NADH
HYDROGENESOME
NAD
CO2
Acetyl CoA
Malate
Acetate
?
?
Acetate
Pyruvate
Malate
NAD(P)
NAD(P)H
PFOR
?
Succinate
PFOR
Succinate
Succinyl CoA
Succinate
OUT IN
Fdox
Fdred
H2
H
CoASH
ATP
ADPPi
IN OUT
ATP
ADPPi
2 ADP
AMPATP
H
8
Fermentation end-products of cellulose or
glucose by rumen fungi
Cellulose fermented ()
Fermentation products (mol/10mol of h.u fermented)
Organisms
H2
CO2
Formate
Acetate
Lactate
Succinate
Ethanol
Monocentric Fungi N. frontalis N.
patriciarum N. sp. LM1 P. communis C.
communis
1.80 3.67 - 3.87 4.67
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
- - - -
9
Utilization patterns of CHO for Anaerobic
Fungi
Utilization of saccharides
Bacterial Species
Poly-
Di/Tri-
Mono-
Primary cellulolytics
Fibrobacter succinogenes Ruminococcus
flavefaciens Ruminococcus albus
Cel Cel, Xyl, Pec Cel, Xyl
Cl Cl Cl
G - G, X, A
Secondary cellulolytics
Butyrivibrio fibrisolvens Clostridium
longisporum Clostridium locheadii
Cel, Xyl, Dex, Pec Cel Cel, Dex
Ml, Lc, Cl Cl, Ml, Lc, Sc Ml, Sc
G, Ga, M, F, X G, Ga, F G
Nonecellulolytics
Prevotella ruminicola Ruminobacter
amylophilus Selenomonas ruminantium Streptococcus
bovis Succinomonas amylolytica Succinivibrio
dextrinosolvens
Pec, Sta, Dex Sta Sta, Dex Sta Sta, Dex Dex, Pec
Lc, Cl, Ml Ml Cl, Ml, Lc, Sc Ml, Lc, Cl,
Sc Ml Ml, Sc
G, Ga, F, X, A, R - G, Ga, F, X, A, R G, Ga, F,
X, A G G, Ga, M, X, A, F
10
Comparison of carbohydrate utilization patterns
of ruminal anaerobic microbes
Utilization of saccharides
Species
Poly-
Di/Tri-
Mono-
Holochrichs
Isotricha intestinalis Isotricha
prostoma Dasytricha ruminantium
Sta, Pet Sta, Pet Sta
Sc Sc Cl, Ml
G G G
Entodinomorphs
Entodinium bursa Entodinium caudatum Entodinium
simplex Diplodinium diplodinium Diplodinium
polyplastron Diplodinium ostracodinium Diplodinium
eremoplastron Epidinium ecaudatum Ophryoscolex
caudatus
Sta, Xyl Sta Sta Cel, Xyl, Sta Cel, Sta Cel, Xyl,
Sta Cel, Xyl, Sta Cel, Xyl, Sta Cel, Xyl, Sta
- Cl, Ml, Sc - - Sc - - Sc, Ml -
- G - - G - - - -
11
Comparison of carbohydrate utilization patterns
of ruminal anaerobic microbes
Utilization of saccharides
Species
Poly-
Di/Tri-
Mono-
Monocentrics
Neocallimastix frontalis Neocallimastix
patriciarum Piromyces communis Caecomyces communis
Cel, Xyl, Sta, Pec Cel, Xyl, Sta, Pec Cel, Xyl,
Sta, Pec Cel, Xyl, Pec
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
Polycentrics
Anaeromyce mucronatus Orpinomyces joyonii
Xyl, Sta Cel, Xyl, Sta
Cl, Gn, Ml, Lc, Sc Cl, Gn, Ml, Lc, Sc
F, G, X F, G, X
12
Comparison of rate constant(h-1) for digestion of
crystalline cellulose by various ruminal and non
ruminal microbes (Bacteria)
Microbes
Basis
Reference
Sub- strate
Rate constant
Clostridium thermocellum (ATCC27405) Ruminococcus
albus 8 Ruminococcus flavefaciens (FD-1) Fibrobact
er 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)
0.16 0.05 0.08 0.07 0.027 0.006
13
Comparison of rate constant(h-1) for digestion of
crystalline cellulose by various ruminal and non
ruminal microbes (Fungi)
Microbes
Basis
Reference
Sub- strate
Rate constant
FP FP CT CT
0.040 0.037 lt0.004 lt0.004
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)
Neocallimastix frontalis (RE1) Orpinomyces
joyonii SG4 White-rot fungi (5 species) Brown-rot
fungi (8 species)
14
Interaction of Rumen Fungi with Methanogenic
Bacteria
Stabilization of Incubation Fungal
Population Hydrolysis Rate Cellulolysis
Rate Cellulolytic Activity Electron Sink
Products (Like Lactate, CH4)
Incubation with Methanogenic Bacteria
Acetate, CO2, H2 etc.
15
Interaction of Rumen Fungi with Non-methanogeic
Bacteria
Interaction by Competition, Symbiosis and Synergy
Incubation with Hydrogentropic Bacteria Stabilizat
ion 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
16
Interaction of Rumen Fungi with Protozoa
Predation of Zoospore and Fungal
Mycelium Increase the turnover rate of anaerobic
fungi in the rumen
Interaction of Rumen fungi with Protozoa are not
defined Exactly up to now
Secreting Chitinase Inhibit the growth rate
enzymen activities of rumen fungi
17
Excretion form ofrumen fungal enzymes
Cellulase
Mostly extracellular excretion type
Protease Amylase
Over 80 is extracellular excretion type
18
Comparison of cellulase activities (U/ml? h-1)of
the supernatant of medium containing rice straw
as a carbon source incubated with various
microorganisms
Incubation time (h)
Microbial strains
24
48
96
Rumen mixed microorganism
4.113
4.283
3.996
Ruminal Bacteria
Fibrobacter succinogenes Ruminococcus
albus Ruminococcus flavefaciens
0.170 0.305 0.302
0.791 0.487 0.842
1.401 0.857 1.067
Ruminal Fungi
3.990 4.067 6.656
4.692 4.278 7.435
Piromyces fhizinflata Orpinomyces
joyonii Neocallimastix frontalis
3.272 3.128 4.633
Commercial microoganisms
3.910 3.899
3.902 3.944
Aspergillus oryzae Saccharomyces cerevisiae
4.034 4.000
19
Digestion of roughage by anaerobic fungi
and bacteria
Neocallimastix
R. flavefacience
97
96
B. succinogenes
Piromyces
Digestibility()
68
59
48
30
23
23
18
15
4
3
Bermuda grass leaf
Filter paper
Rice straw
20
Xylanase activities of anaerobic ruminal
fungi
56,834
53,258
40,300
40,300
40,300
A. oryzae
30,374
9,729
S. cerevisiae
Enzyme activities (U/ml.h-1)
6,247
5,027
KNGF-2
5,045
4,943
4,464
KNGF-6
Filter paper
Rice Straw
Alfalfa
21
Enzymes activity
F-1
F-2
F-3
F-4
F-5
Cont
Control A
Anaerobic fungus
Control B
Commercial Enz.
22
Industrial Application of Rumen anaerobic
Fungi in Diets
Improved productivity
Silage Inoculants
DFM
D I E T
Feeds Additives
Enzymes
23
Major Research Areas for Rumen Anaerobic
Microbes
Rumen Nutritionist
Enzymologist
RUMEN ANAEROBIC FUNGI
Botanist
Bioremediator
Microbiologist
Gene - Designer
24
HMP Cycle
Secondary Metabolites
Secondary Metabolites
Glucose
Triose phosphate
Glycosides Kojic acid muscarine
Aromatic secondary metabolites (gliotoxin)
Aromatic amino acids
Shikimate
Terpenoids (Wortmannin)
Pyruvate
Mevalonate
Peptides Penicillin Monensin Neomysin Verginiamyc
in Lasalosid Bambemycin Novomycin Tylosine Subtili
n
Amino acids
Polyketides patulin griseofulvin ochratoxin
A aflatoxins
Acetyl CoA
Aspartic acid
Oxaloacetate
Citrate
Polyacetylenes (brefeldin A)
Fatty acids
Glutamic acid
Oxoglutarate
25
Enzyme Characteristics of Rumen Anaerobic Fungi
Factors influencing the activities of fungal
enzymes
Species of ruminal fungi
Types of growth substrates
Incubation time
pH and temperature
Host animals
26
Enzyme activities of various rumen fungi
CMCase, b-glucosidase, Xylanase activities
Polycentric fungi gt Monocentric fungi
CMCase, Xylanase activities (On straw media)
Caecomyces gt Piromyces, Neocallimastix
27
Enzyme activities of various rumen fungi
Enzyme activities of O. joyonii, N. patriciarum
and P. communis on cellobiose substrate
Proteolytic activity
O. joynii gt P.communis gt N. patriciarum
Amylolytic activity
O. joynii gt P.communis gt N. patriciarum
28
Enzyme characteristics as influenced by substrates
Catabolite End-product
Repress the enzyme activities of rumen fungi
monosaccharides or disaccharides
polysaccharides(cellulose, xylan)
29
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 gt Holstein lactating cow, sheep Small
animal (KNG, sheep) gt Large animal (KNC,
Holstein cow)
30
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
Glucose Cellobiose ? Concentration of
intracellular cAMP ? Inhibit cellulolytic enzymes
Osmosis, Control of O2CO2Ratio Structual
Transformation of Enzyme Isoenzyme,
Chemotaxis, etc.
Control Enzyme Synthesis
31
Effect of Small- Medium-sized Protozoa on
Cellulose Digestion by Neocallimastix frontalis
Uninoculate
Protozoa
Fungi
FungiProtozoa
Substrate weight loss () Small Protozoa1)
Medium Protozoa2)
3 1
7 1
74 69
88 2
H2-gas production (ml) Small Protozoa
Medium Protozoa
- -
tr -
1.5 1.1
- -
CH4 -gas production (ml) Small Protozoa
Medium Protozoa
- -
0.1 0.13
- -
4.1 0.23
(Fonty and Joblin, 1991)
1) Small-sized(lt30?m) protozoa, 2)
Medium-sized(30-70?m) protozoa.
32
Percentage of Cellulose Xylan degraded by Rumen
Fungi in absence or in presence of H2-utilizing
Rumen Methanogenic Bacteria
1)Cellulose degraded by
2) Xylan degraded by
Organisms
Fungi alone
FungiMethanogenes
Fungi alone
FungiMethanogenes
N. frontalis P. communis C. communis O. joyonii 3)
68.5 87.6 9.7 77.1
80.1 90.3 19.1 87.2
81.9 83.3 52.3 -
88.5 86.6 80.5 -
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).
33
Commercialization of new useful products
Enzymes
Antibiotics
Aromatic compounds
New Useful Products
Penicillium notatum 2unit/mL(0.6ug)
GENE
Feed additives
P. Chrysogenum 100unit/mL(30ug)
Medicine
Anaerobic fungi 50,000unit/mL