The document discusses the production of cellulase enzymes from Aspergillus isolates and its applications. Four Aspergillus isolates were identified as good cellulase producers. One isolate, Aspergillus oryzae AKAL8, produced the highest level of cellulase over time. Crude cellulase was used for denim biostoning and was found to remove more indigo dye than bleach alone. Cellulase was also stable when combined with bleach. Finally, cellulase treatment of banana peel was able to produce cellulosic nanofibers.

1. A Project work
By
Rezwana Khan Nishat
Reg. No. : 2014431035
Session: 2014-2015
B.Sc. (Hons) 4th Year 2nd Semester
Department of Genetic Engineering & Biotechnology
Shahjalal University of Science & Technology, Sylhet
Production of Cellulase from Aspergillus Isolates and It’s
Application for Denim Biostoning and Cellulosic Nanofiber
Preparation from Banana Peel

2. Introduction

3. Naturally occurring mixture of enzymes
Act synergistically or serially
Produced chiefly by fungi, bacteria, and protozoans
Catalyze cellulolysis, the decomposition of cellulose and of some
related polysaccharides
Specific reaction involves hydrolysis of the beta 1,4 glycosidic
bond
Cellulase

4. Classification of Cellulase
 Endo-cellulases
 Exo-cellulases
 Beta-glucosidases
 Oxidative cellulases
 Cellulose phosphorylases
 There are two main types of exo-cellulases
One type working from the reducing end, and
Another type working from the non-reducing end of
cellulose
Classification of Cellulase

5. Economical – produced on large scale within limited space and time
Can be easily extracted and purified
Can grow in a wide range of environmental conditions
Can be genetically manipulated to increase the yield of cellulases
Generally cheaper to produce
Advantages of Using Microbial Sources for
the Production of Cellulase

6. Applications of Cellulase
Textile industry
Paper and pulp industry
Pharmaceutical industry
Detergent industry
Food industry
Fermentation industry
Biofuel Production
Agricultural industry
Waste management

7. Production and partial purification of crude cellulase from Aspergillus
spp
Application of crude cellulase from Aspergillus spp. as denim bio-
stoning agent as well as an additive of commercial bleach
Production of cellulosic nanofiber from banana peel by cellulase
treatment
Objectives of the Present Study

8. Materials and Methods

9. Design of the Experiments
Evaluation of cellulolytic capacity of the fungal isolates
obtained by the previous study
Bio-stoning of
denim
Cellulase production with CMC media
Partial purification of cellulase
Preparation of banana
bran
Preparation of
cellulosic nanofiber
by cellulase
treatment
Compatibility of cellulase
with bleach

10. Results and Discussion

11. Fungal isolates used in the present study were isolated from
municipal solid wastes (Shohag, 2014; Jahed, 2016).
Four best cellulolytic fungal isolates were identified previously based
on morphological, cultural characteristics (Shohag, 2014) and 18S
rDNA sequences (Jahed, 2016).
The stock cultures of the fungal strains were maintained in the
refrigerator at 4°C and subcultures were performed at 15 days
interval.
Obtaining and Maintenance of Aspergillus Isolates

12. Evaluation of Cellulolytic Capacity of the
Fungal Isolates
Aspergillus fumigatus AKAL1 Aspergillus oryzae AKAL4
Aspergillus oryzae AKAL8 Aspergillus flavus AKAL9

13. Production of Cellulase Enzyme from
Fungal Isolates

14. Time Course Study of Aspergillus
Isolates
0.076
0.087
0.083
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
0 24 48 72 96 120 144 168 192 216
EnzymeActivity(U/ml)
Cultivation Period (Hours)
AKAL8
AKAL9
AKAL4

15. Application of Cellulase Enzyme

16. Bio-stoning of Denim Fabric
 Bio-stoning was performed successfully yielding desired look and softening the
denim fabric.
Control Bleach Enzyme Enzyme + Bleach

17. Treatment Process Weight (g) Percentage of weight loss (%)
Control 1.33 0
Bleach 1.22 8.27
Enzyme 1.18 11.28
Enzyme + bleach 1.20 9.77
Bio-stoning of Denim Fabric
 Denim weight was reduced to 1.18g from 1.33g resulting in 11.28% weight loss for
enzymatic treatment

18. Bio-stoning of Denim Fabric
Treatment process OD of released indigo dye
Control 1.9
Bleach 1.7
Enzyme 2.0
Enzyme + bleach 1.8
 Highest amount of indigo dye was removed in case of enzymatic treatment

19. Compatibility of Cellulase Enzyme with
commercial bleach
 Cellulase enzyme stable with 0.3% bleach
0
100
200
300
400
500
600
700
0 0.1 0.2 0.3
Residualcellulaseactivity(U/ml)
Bleach concentration (%)

20. Cellulosic Nanofiber Preparation
from Banana Peel

21. Cellulosic Nanofiber Preparation from Banana
Peel
 Cellulosic nanofiber production from banana peel

22. Cellulosic Nanofiber Preparation from Banana
Peel
Nanofiber suspension after enzymatic treatment (A) and banana bran before
enzymatic treatment (B)
A B

23. Conclusions

24. Conclusions
A significant amount of cellulase was produced from Aspergillus isolates in shake
flask with optimized parameters
The crude enzyme isolated from Aspergillus spp. acted efficiently as an additive of
commercial bleach for bio-stoning of denim fabric yielding desired look and
softening the fabric
Crude cellulase enzyme was proved to be stable with 0.3% of bleach
Crude cellulase enzyme was able to prepare cellulosic nanofiber from banana peel

25. Conclusions