The effect of different Trichoderma reesei mutated strains and culture method on the cellulase activity.

1. Effect of different Trichoderma reesei
mutated strains and culture method on the
cellulase activity
Speaker: Huo, Yi-Hua
Instructor: Chen, Kuo-Lung
October 14, 2013

2. Abstract
• Cellulase is an important commercial enzyme, widely used in
food, animal feed, grain alcohol fermentation, starch
processing, malting and brewing industries. Filamentous fungi
Trichoderma reesei are considered to be one of the most
efficient hyper producers of cellulase that is used in industry.
• The methods used to improve fungal enzyme production,
activity or stability, one is mutagenisis and the other one is coculturing.
• The aim of this study was to investigate the influence of
different mutated T. reesei strains and culture method to the
cellulase activity.
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3. • Using T. reesei QM6a, QM9414, RUT C30 and QM9414 MG5
strains, the medium pH was maintained at 3-9. Results showed
that T. reesei QM6a has the best cellulose degradation at pH 3
(69.3%) and its mutants QM9414, RUT C30 and QM9414
MG5 has the best cellulose degradation at pH 6 (64.9、60.3
and 58.0%).
• In specific activity, mutants QM9414 has the best
endoglucanase (5.22mg/min/g protein) at pH 4, and mutants
QM9414MG5 has the best exoglucanase and betaglucosidase(0.87 and 0.80 mg/min/g protein).
3

4. • Using solid state fermentation, co-culture T. reesei LMUC4 and LM-UC4E1 with Aspergillus niger.
• Results showed that coculture LM-UC4E1 with A.niger
has the best dry biomass (0.27 g/g), filter paper activity
(15.5 IU/g) and endoglucanase activity (129 IU/g).
4

5. • Using solid state fermentation, co-culture T. reesei LM-UC4
and LM-UC4E1 with Aspergillus niger. Results showed that
coculture LM-UC4E1 with A.niger has the best dry biomass
(0.27 g/g), filter paper activity (15.5 IU/g) and endoglucanase
activity (129 IU/g).
• Using T. reesei RUT C30 with Aspergillus niger LMA coculture, compare with their monocultures respectively.
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6. • The results of mixed culture experiments exhibited a highly
significant increase in the production of volumetric enzyme
activity (98.4 U/L), filter paper activity (7.1 U/mL),
carboxymethyl cellulase activity (4.7 U/mL), and dry biomass
(21.4 g/ L).
• In conclusion, even if mutants, it can’t able to produce high
levels of the enzymes at the same time while monoculture, the
mutant’s performance can be improved by co-culturing with A.
niger.
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7. Outline
• Introduction
• Compare the Trichoderma reesei wild with its mutant strains
on their cellulase activity
• Compare the culture method of monoculture with mixed
culture on its cellulase activity
• Conclusion
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8. Introduction
• Cellulase is an important commercial enzyme, widely used in
food, animal feed, textile, pulp and paper, grain alcohol
fermentation, starch processing, pharmaceutical, malting and
brewing industries.
• Filamentous fungi Trichoderma reesei are considered to be one
of the most efficient hyper producers of cellulase that is used
in industry.
(Aftab and Patrick, 2008a)
8

9. • The application of different strains and processes which are
selected on the basis of the biomass residues used make
comparisons difficult, if not impossible.
• Nevertheless, the most recent and important improvements in
production/activity of fungal enzymes using different
techniques such as mutagenesis, co-culturing and heterologous
gene expression of cellulases are discussed.
(Dashtban et al., 2009)
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10. Table 1 Some methods which have been used to improve fungal lignocellulolytic
activity or stability
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(Dashtban et al., 2009)

11. Purpose
• The aim of this study was to investigate the influence of
different mutated T. reesei strains and culture method on the
cellulase activity.
11

12. Material and methods
Linear accelerator
Trichoderma reesei QM6a
T. reesei QM9123
T. reesei QM9414
High-voltage electrons
T. reesei QM9414MG5
T. reesei RUT C30
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(Sunil et al., 2011)

13. (Somers et al., 1989)
Fig. 1 The mechanism of cellulase.
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14. Results and discussion
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15. Table 2 Specific activities of cellulytic enzymes and cellulose degradation by T.
reesei wild and its mutant strains at different pHs
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(Sunil et al., 2011)

16. Brief 1
• Results showed that T. reesei QM6a has the best cellulose
degradation at pH 3 (69.3%) and its mutants QM9414,
Rut C30 and QM9414MG5 has the best cellulose
degradation at pH 6 (64.9、60.3 and 58.0%).
• In specific activity, mutants QM9414 has the best
endoglucanase (5.22mg/min/g protein) at pH 4, and
mutants QM9414MG5 has the best exoglucanase and
beta-glucosidase(0.87 and 0.80 mg/min/g protein).
• Even if mutants, it can not able to produce high levels of
the enzymes at the same time.
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17. Material and methods
T. reesei LM-UC4
T. reesei LM-UC4E1
Aspergillus niger ATCC 10864
7 days
1.5-2 days
1.5-3ays
C:N
= 10:1
(Gutierrez-Correa et al., 1999)
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18. □ LM-UC4
■ LM-UC4+ A. niger
△ LM-UC4E1
▲ LM-UC4E1+ A. niger
○ A. niger
Fig. 2 Growth kinetics of T.
reesei LM-UC4 (□,■ ) or T.
reesei LM-UC4E1 (△,▲)
with A. niger (○) in single
(open symbols) and mixed
(closed symbols) culture
SSF on bagasse
supplemented with
ammonium sulfate and
urea (A) or soymeal (B).
(Gutierrez-Correa et al., 1999)
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19. □ LM-UC4
■ LM-UC4+ A. niger
△ LM-UC4E1
▲ LM-UC4E1+ A. niger
○ A. niger
Fig. 3 Time course
profiles of enzyme
production by single and
mixed culture SSF on
bagasse supplemented
with ammonium sulfate
and urea (A) or soymeal
(B).
(Gutierrez-Correa et al., 1999)
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20. □ LM-UC4
■ LM-UC4+ A. niger
△ LM-UC4E1
▲ LM-UC4E1+ A. niger
○ A. niger
Fig. 4 Time course
profiles of enzyme
production by single
and mixed culture SSF
on bagasse
supplemented with
ammonium sulfate and
urea (A) or soymeal (B).
(Gutierrez-Correa et al., 1999)
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21. □ LM-UC4
■ LM-UC4+ A. niger
△ LM-UC4E1
▲ LM-UC4E1+ A. niger
○ A. niger
Fig. 5 Time course
profiles of enzyme
production by single
and mixed culture SSF
on bagasse
supplemented with
ammonium sulfate and
urea (A) or soymeal (B).
(Gutierrez-Correa et al., 1999)
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22. Table 3 Maximum growth and enzyme activities in mixed culture
solid substrate fermentation on sugar cane bagasse
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(Gutierrez-Correa et al., 1999)

23. Table 4 Comparison of maximum growth and enzyme activities in mixed
culture solid substrate fermentation on sugar cane bagasse
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(Gutierrez-Correa et al., 1999)

24. Brief 2
• Mixed culturing is advantageous in nutrient-limited conditions,
where symbiotic associations may overcome such limitations.
• In practical terms, it means that cheaper media may be used for
enzyme production by mixed cultures than by single cultures,
without sacrificing enzyme yields.
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25. Material and methods
T. reesei RUT C30
Aspergillus niger LMA
T. reesei RUT C30 + Aspergillus niger LMA
Stirred tank
bioreactor
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(Gutierrez-Correa et al., 1999)

26. Table 5 The extent of cellulolytic enzyme production in T. reesei RUT-C30
strain grown in different culture media: cellulose–yeast extract (A); corn
steep–glucose (B); cellulose–yeast extract–peptone (C); and cellulose–yeast
nitrogen base–CMC (D) with lactose fed at regular intervals in a 7 L New
Brunswick stirred tank bioreactor maintained at 30 ◦C, an agitation of 250
rpm, and culture pH of 4.8
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(Aftab and Patrick, 2008a)

27. Table 6 Comparison of growth and cellulase enzyme productivity from mono-culture (A.
niger) and mixed culture (T. reesei and A. niger) strains grown in a Cellulose–Yeast extract
culturemedium with lactose fed at regular intervals in a 3 L New Brunswick stirred tank
bioreactor maintained at 30 ◦C, an agitation of 250 rpm, and culture pH of 4.8
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(Aftab and Patrick, 2008b)

28. Table 7 Comparison of growth and cellulase enzyme productivity from monoculture (T. reesei RUT-C30 and A. niger) and mixed culture (T. reesei and A.
niger) strains grown in a Cellulose–Yeast extract culturemedium with lactose
fed at regular intervals in a 3 L New Brunswick stirred tank bioreactor
maintained at 30 ◦C, an agitation of 250 rpm, and culture pH of 4.8
Mono culture
T. reesei RUT-C30
A. niger
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(Aftab and Patrick, 2008a; Aftab and Patrick, 2008b )

29. Brief 3
• Mixed culture can increase in the production of volumetric
enzyme activity, filter paper activity, carboxymethyl cellulase
activity and dry biomass than monoculture.
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30. Conclusion
• Even if mutants, it can not able to produce high levels of
the enzymes at the same time while monoculture, the
mutant’s performance can be improved by co-culturing
with A. niger.
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31. Thank you for your attention!
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32. Questions & Answers
Oct. 21, 2013

33. Question 1 What is heterologous expression of
cellulases? (東隴)
• Answer
Heterologous expression is a powerful technique to improve
production yield of enzymes, as well as activity. In order to make
a robust lignocellulolytic fungal strain, many different fungal
cellulases with higher and/or specific activity based on the need
for a functional cellulase system in the organism have been
cloned and expressed.
For example, thermostable β-glucosidase (cel3a) from
thermophilic fungus T. emersonii was expressed in T. reesei
RUT-C30 using a strong T. reesei cbh1 promoter. The expressed
enzyme has been shown to be highly thermostable (optimum
temperature at 71.5 °C) with high specific activity.
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34. Question 2 Why the unit is mg/min/g protein?
(連老師)
• Answer
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35. Question 3 How much is strain? (耀庭)
• Answer
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36. 36

37. Question 4 What is the principle of Linear
Accelerator? (群翔)
• Answer
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