Investigate different measurements of cellulase screening and the effect of different substrates under solid-state fermentation on conidia production or enzyme activity by Trichoderma

Speaker: Huo, Yi-Hua

Instructor: Chen, Kuo-Lung

June 3, 2013

Abstract

• Because solid-state fermentation is a cost-effective system for sporulation of fungi, the production of cellulases using cheaper substrates is an effective strategy to reduce cost.

• The aim of this study was to investigate different measurements of cellulase screening and the influence of different substrates under solid-state fermentation on conidia production and enzyme activity by different Trichoderma strains.

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• Trail 1, screening strains on agar plates, Congo red test and fermentation in tubes, the strains selected using the fermentation tube assay were cultivated under solid-state fermentation (SSF).

• The SSF substrate was 5 g of a mixture of sugarcane bagasse and wheat bran at a ratio of 1:1. The initial moisture content of the substrate was adjusted to 80% using the nutrient medium. All the SSF cultures were incubated at 30°C for 8 days.

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• The result showed that fermentation in tubes is especially interesting because it requires minimal quantities of nutrients.

• However, no direct correlation was obtained between the results of this procedure and the Congo red test. The existence of a linear correlation between the two methodologies: screening on plates employing Congo red dye, followed by a SSF analysis, therefore, an effective method for selection of cellulase-producing strains of Trichoderma.

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• Trail 2, rice, corn bran, and wheat bran were used as solid substrate to grow Trichoderma harzanium sp., Trichoderma viride sp., Trichoderma koningii sp., and Trichoderma polysporum sp. SSF cultivated at 30°C for 7 days.

• Results showed that wheat bran showed to be the most suitable substrate to produce Trichoderma spores for all strains that were evaluated. High spore counts were obtained for T. harzianum sp. (28.30×108spores/gds) and T. viride sp. (24.10×108 spores/gds). 5

• Trail 3, using apple pomace as SSF substrate, the results show that initial moisture 70%, 32 , ℃inoculum 2×108 spores/flask and adding lactose has the best enzyme activity.

• In conclusion, screening on plates employing Congo red dye, followed by a SSF analysis is an effective method for selection of cellulase-producing strains of Trichoderma and wheat bran is the best substrate of rice and corn bran to produce conidia, on the other hand, apple pomace can be substrate of SSF.

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Outline• Introduction

• Measurements of select the cellulase producing microorganisms

• The influence of different substrates under solid-state fermentation on conidia production by different Trichoderma strains

• Cellulase production by Trichoderma sp. on apple pomace under solid state fermentation

• Conclusion

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Introduction

• Feeding enzymes to poultry is one of the major nutritional advances in the last fifty years.

• Plants contain some compounds that either the animal cannot digest or which hinder its digestive system, often because the animal cannot produce the necessary enzyme to degrade them.

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(Khattak et al., 2006)

Introduction

• Nutritionists can help the animal by identifying these indigestible compounds and feeding a suitable enzyme.

• These enzymes come from microorganisms that are carefully selected for the task and grown under controlled conditions.

• Cellulases are relatively costly enzymes and a significant reduction in cost will be important for their commercial use.

9(Sun et al., 2010)

(Khattak et al., 2006)

• Solid-state fermentation (SSF) is a cost-effective system for sporulation of fungi.

• SSF technology needs less space per unit biomass used as substrate and is less demanding on infrastructure and running costs.

• Production of cellulases using cheaper substrates is an effective strategy to reduce cost.

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(Mekala et al., 2008)

(Cavalcante et al., 2008)

(Sun et al., 2010)

• Microorganisms that generally involved in production of enzymes are; Bacteria (Bacillus subtilis, Bacillus lentus, and Bacillus stearothermophils), Fungus (Triochoderma longibrachiatum, Asperigillus oryzae and Asperigillus niger) and Yeast (S. cerevisiae) .

• Most emphasis has been placed on the use of fungi because of their capability to produce copious amounts of cellulases and hemicellulases which are secreted to the medium for easy extraction and purification.

11(Khattak et al., 2006)

What are Trichoderma?• Trichoderma spp. are fungi that are present in nearly all

soils and other diverse habitats. In soil, they frequently are the most prevalent culturable fungi.

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(http://www.biocontrol.entomology.cornell.edu/pathogens/trichoderma.html)

Fig. 1 T. harzianum

Wheat bran

(http://www.wisegeek.com/what-is-wheat-bran.htm)

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Corn bran

(http://milhao.trustpass.alibaba.com/viewimg/picture.html?picture=http://i01.i.aliimg.com/photo/v0/111271691/Fibramix_Corn_Bran_.jpg)

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Apple pomace

http://www.greenfield.waw.pl/en/food_industry/dried_fruit_pomaces/dried_apple_pomace

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Purpose

• The aim of this study was to investigate different measurements of cellulase screening and the influence of different substrates under solid-state fermentation on conidia production and enzyme activity by different Trichoderma strains.

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Material and methods

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1. Trichoderma Isolates

2. Screening Step on Agar Plates Containing Avicel

3. Screening Step Using the Congo Red Test

4. Screening Step Using Fermentation in Tubes

5. Solid-State Fermentation (SSF)

6. Enzyme Assay

(Florencio et al., 2012)

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78 Trichoderma strains

potato dextrose agar (PDA), 30℃

• NaNO3 (3.0 g·L-1)• K2HPO4 (1.0 g·L-1)• MgSO4 (0.5 g·L-1)• KCl (0.5 g·L-1) • FeSO4·7H2O (0.01 mg·L-1)• agar (20.0 g·L-1)• Avicel (5.0 g·L-1)• pH 5.0

(Florencio et al., 2012)

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49 Trichoderma strains

• NaNO3 (3.0 g·L-1)• K2HPO4 (1.0 g·L-1)• MgSO4 (0.5 g·L-1)• KCl (0.5 g·L-1) • FeSO4·7H2O (0.01 mg·L-1)• agar (20.0 g·L-1)• carboxymethylcellulose (CMC) (5.0 g·L-1)

(Florencio et al., 2012)

300C, 4 days

enzymatic index (EI) higher than 1.50

Figure 2 Observation of the clear zone around a colony of T. reesei Rut C30 using Congo red dye. 20

(Florencio et al., 2012)

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• NaNO3 (3.0 g·L-1)• K2HPO4 (1.0 g·L-1)• MgSO4 (0.5 g·L-1)• KCl (0.5 g·L-1) • FeSO4·7H2O (0.01 mg·L-1)• Filter paper 1×10 cm (90mg)

(Florencio et al., 2012)

300C, 4 weeks

analysis

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(Florencio et al., 2012)

• mixture of sugarcane bagasse : wheat bran = 1 : 1 (5 g)• initial moisture 80%

30 , 8 days℃

Extraction and assay

adding 100 mL of distilled water to each flask agitating at 120 rpm for 40 min at 30°C

Endoglucanase (EGase)

Results and discussion

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• A total of 49 test strains (62.8% of all strains evaluated), as well as the reference strain (T. reesei RUT C30), were able to hydrolyze this cellulosic substrate and exhibited obvious growth.

• The 29 strains that showed no potential for Avicel degradation were discarded at this stage.

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(Florencio et al., 2012)

Table 1 Enzymatic indices of filamentous fungi belonging to the genus Trichoderma.

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(Florencio et al., 2012)

Figure 3 Production of EGase in tubes: (-○-) T. harzianum CEN 139; (-□-) T. harzianum CEN 155; (- -) T. sp104 NH; (-×-) T. reesei Rut C30; (-*-) T. asperelum ◇CEN 201; (-̇ -) T. harzianum CEN 241; (- -) T. harzianum CEN 248; (-■-) T. harzianum ◆CEN 238; (-+-) T. sp CEN 156; (-▲-) T. koningii CEN 142; (- -) T. sp CEN 159; (- →-) △T. sp CEN 90; (- ← -) T. sp CEN 97.

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(Florencio et al., 2012)

Rut C30

CEN 90

CEN 139

104 NH

Figure 4 EGase production (UI·mL−1) versus enzymatic index of strains selected for fermentation in tubes.

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(Florencio et al., 2012)

Figure 5 Production of EGase using solid-state fermentation of selected Trichoderma strains and comparison with T. reesei Rut C30.

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(Florencio et al., 2012)

Figure 6 EGase production (UI·g−1) versus enzymatic index of strains selected for solid-state fermentation.

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(Florencio et al., 2012)

( R2 = 0.977 )

Brief 1

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• Although fermentation in tubes is especially interesting because it requires minimal quantities of nutrients, no direct correlation was obtained between the results of this procedure and the Congo red test.

• The existence of a linear correlation between the two methodologies: screening on plates employing Congo red dye, followed by a SSF analysis.

• An effective method for selection of cellulase-producing strains of Trichoderma.

Trial 2

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Purpose

• Comprises the elevation of the use of by-products of the cereal industry (corn bran and wheat bran) as raw materials to produce Trichoderma sp. conidia without the need of the addition of nutrients (C and N sources) to increase spore concentration.

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Material and methods

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Rice

(Cavalcante et al., 2008)

Corn bran Wheat bran

4 Trichoderma species

T. harzianum T. viride T. konigii T. polysporum

Immersion time (min) Water amount (ml)

Table 2 Moisture and water activity of the rice and corn bran used as solid substrates

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(Cavalcante et al., 2008)

Table 3 Moisture and water activity of wheat bran used as solid substrates

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(Cavalcante et al., 2008)

Table 4 Spores produced using rice, corn bran, and wheat bran as solid substrates

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(Cavalcante et al., 2008)

Brief 2

• Results showed that wheat bran showed to be the most suitable substrate to produce Trichoderma spores for all strains that were evaluated.

• High spore counts were obtained for T. harzianum sp. (28.30×108 spores/gds) and T. viride sp. (24.10×108 spores/gds).

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Trial 3

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Material and methods

Trichoderma sp. GIM 3.0010

Apple pomace

oven at 80°C, crushedAverage size of 300 - 500 μm

moisture temperature inoculum size supplementation

Figure 7 Time course of cellulase production by Trichodermasp. on apple pomace.

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(Sun et al., 2010)

Table 5 Effect of initial moisture level of the medium oncellulase production.

41(Sun et al., 2010)

Table 6 Effect of incubation temperature on cellulaseproduction.

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(Sun et al., 2010)

Table 7 Effect of particle size on cellulase production.

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(Sun et al., 2010)

Table 8 Effect of supplementation of apple pomace withdifferent carbon sources.

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(Sun et al., 2010)

Brief 3

• Apple pomace could be a good substrate for cellulase synthesis.

• Optimum initial moisture level, incubation temperature and inoculum size were 70%, 32°C and 2×108 spores/flask, respectively.

• The supplement of lactose favored the enzyme formation markedly.

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Conclusion

• Screening on plates employing Congo red dye, followed by a SSF analysis is an effective method for selection of cellulase-producing strains of Trichoderma and wheat bran is the best substrate of rice and corn bran to produce conidia, on the other hand, apple pomace can be substrate of SSF.

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Thank you for your attention!

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Questions & AnswersJune 10, 2013

Question 1 How to define water activity? (俊緯 )

• Answer• Water activity is a measure of how efficiently the water

present can take part in a chemical (physical) reaction. If half the water is so tightly bound to a protein molecule that it could not take part in a hydrolysis reaction the overall water activity would be reduced. Water activity (aw) is defined as

• aw=p/po

• where p and po are the partial pressures of water above the food and a pure solution under identical conditions respectively.

• 30℃ 49

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Question 2 How to assay extracellular enzyme? (純宜 )

• Answer• The enzymatic activity of EGase in the extracts was

quantified and the results were expressed as activity units per gram of dry substrate.

• The cellulolytic activity of endoglucanase was determined by the CMC method.

• Samples of the enzyme complexes (0.5mL) were incubated together with 0.5mL of the CMC substrate (4%) at 50◦C for 10 min.

• After incubation, 1.0mL of DNS was added to the tubes for quantification of the reducing sugars.

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Question 3 What is endo-glucanase? what is the different between endo and exo-β-1,4-gulcanase? (連老師、吳老師、林老師 )

• Answer• β-1,4-glucan glucanhydrolase

• Random hydrolyze β-1,4-glucosidic linkages.

• β-1,4-D-gulcan cellobiohydrolase

• From reducing ends or Non-reducing ends.

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Question 4 What is the different between Avicel and CMC? (琳蘋 )

• Answer

• Avicel →Exo-cellobiohydrolase

• CMC → Endo-glucosidase

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Enzyme Substrates Productsendo-β-1,4-gulcanase, E.C. 3.2.1.4.

1.Carboxymethyl-cellulose2.Hydroxyethyl-cellulose3.Amorphous cellulose

Regucing sugar and/or loss in viscosity

exo-β-1,4-gulcanase, E.C. 3.2.1.91

1.Crystalline cellulose (Cotton)2.Avicel3.Cellodextrins4.Heterobiosides

1.Cellobiose2.Cellobiose3.Cellobiose4.4-Nitrophenol/glucose

β-glucosidase, E.C. 3.2.1.21

1. Cellobiose2.Cellodextrins

1.Glucose2.Glucose/ Cellodextrins

Cellulase

1.Filter Paper 2.Avicel3.Hydrocellulose4.Dyed cellulose5.Tritiated cellulose6.Thread

1.Reducing sugar/loss in weight/reduction on absorbance2.Dye release3.Scintillation counts4.Breaking strength

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Question 5 How many substrate in the trial? (琳蘋 )

• Answer

• immersing 40 g of each substrate

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• 28.6%• 33.3%• 60%• 66.7%• 71.4%

Question 6 What kind of rice in the trial? (逢展 )

• Answer

• All substrates were purchased from the municipal market (Mercado São Sebastião, Fortaleza, CE, Brazil).

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