SUGARCANE BAGASSE UTILIZATION AS SOLID STATE

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By Inez Areta

14210028

A thesis submitted to the Faculty of LIFE SCIENCES AND TECHNOLOGY

in partial fulfillment of the requirements for the

BACHELOR’S DEGREE in

FOOD TECHNOLOGY

SWISS GERMAN UNIVERSITY EduTown BSD City

Tangerang 15339 Indonesia

August 2014

Revision after the Thesis Defense on 17^th July 2014

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STATEMENT BY THE AUTHOR

I hereby declare that this submission is my own work and to the best of my knowledge, it contains no material previously published or written by another person, nor material which to a substantial extent has been accepted for the award of any other degree or diploma at any educational institution, except where due acknowledgement is made in the thesis.

Inez Areta

_____________________________________________

Student Date

Approved by:

Elisabeth K.Prabawati, STP., MFoodst.

_____________________________________________

Thesis Advisor Date

Irvan S. Kartawiria, S.T, M.Sc

_____________________________________________

Thesis Co-Advisor Date

Irvan S. Kartawiria, S.T, M.Sc

_____________________________________________

Dean Date

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ABSTRACT

SUGARCANE BAGASSE UTILIZATION AS SOLID STATE FERMENTATION SUBSTRATE FOR α-AMYLASE PRODUCTION

By Inez Areta

Elisabeth K.Prabawati, STP., Mfoodst., Advisor Irvan S. Kartawiria, S.T, M.Sc, Co-Advisor

SWISS GERMAN UNIVERSITY

This research investigated the ability of sugarcane bagasse as substrate in SSF to produce α-amylase. The sugarcane bagasse was inoculated with Aspergillus oryzae, Saccharomyces cerevisiae, and the mixed of those pure cultures, in initial moisture content of 50% and 70%. After 72 hour fermentation, α-amylase was extracted and purified partially. Using the DNS method and protein content analysis, it was determined that α-amylase with the highest activity was that of Aspergillus oryzae with 70% of the initial moisture content. The research investigation was continued by combining sugar cane bagasse and rice bran with 5:0, 3:2, and 2:3 ratio to check the influence of substrate type of the enzyme production. The produced α-amylase was analyzed at 24 hour interval. Sugarcane bagasse and rice bran of 5:0 ratio showed the highest enzyme and specific activity with 1.25 mg (ml. Min) ^-1 and 0.47 mg (ml. Min)

-1 respectively at 48 hour fermentation.

Keywords: sugarcane bagasse, α-amylase, solid state fermentation, A. oryzae, activity

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DEDICATION

This works are dedicated for all passionate teachers and lecturers especially, Tabligh Permana, Elisabeth Prabawati, and Irvan Kartawirya

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ACKNOWLEDGEMENTS

The author would like to thank the Almighty God for his guidance, blessing, and grace. Without Him, it will be impossible for this thesis to be finished on time.

The author also acknowledges her advisor, Elisabeth K. Prabawati, STP., MFoodSt., co-advisor, Irvan S. Kartawiria, ST, M.Sc, and Tabligh Permana for their whole support, guidance, and recommendation. Especially to teach me about an integrity, optimistic, and always give the truth. A wish of gratitude is also given to the author’s family for the support during thesis research and writing.

Special thanks are given to author’s best friends, Arief Ramadhi and Angelica Chrissy, for their great support and companionship. Also, thank you to Nikolas Japhar, Harsyadhi Adhiarsa, Michael Reza, Tia Raisha, Ruth Damayanti, and Mascellia Tifanny. Last, thank you for all SGU students and staffs for the support and experience during my 4years study in SGU.

Finally, the author hopes that the result of this thesis research would give contribution to all who read or needed this thesis research. Any comments, criticism, and recommendations are welcomed.

Tangerang, June 13^th, 2014

Inez Areta

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Sugar is the main product of sugarcane plant. The wastes resulted from sugarcane plantation are dried leaves called daduk or klethekan, sugarcane tops, and sogolan. During sugar production, the wastes are sugarcane bagasse, molasses, filter cake and ash. Each sugarcane results 90% bagasse. These wastes have to be recycled, thus lead to zero waste industry (Misran, 2005). ... 15

2.2. Solid State Fermentation ... 17

2.2.1. Substrate Used in SSF ... 18

2.2.2. Biochemical Engineering Aspects in SSF ... 20

2.2.3. Food and Industrial Application ... 22

2.3. α-Amylases ... 24

2.2.4. Physical and Chemical Factors Enzyme Production in SSF ... 24

2.2.5. Purification ... 25

2.2.6. Application ... 26

CHAPTER 3 – RESEARCH METHODS ... 27

3.1. Time and Venue ... 27

3.2. Materials and Equipments... 27

3.2.1. Raw Materials ... 27

3.2.2. Chemical Materials ... 27

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3.3. Deisgn of Experiment ... 28

3.4. Research Procedure ... 28

3.4.1. Preliminary Research ... 29

3.5. α-Amylase Production ... 30

3.5.1. Substrate Preparation ... 30

3.5.3. Solid State Fermentation ... 31

3.5.4. Microbial Analysis ... 31

3.5.5. Enzyme Extraction and Partial Purification ... 32

3.5.6. α-Amylase Activity Analysis ... 32

3.5.7. Protein Content Analysis ... 33

3.5.8. Specific Activity Analysis ... 33

3.5.9. Statistical Analysis ... 33

CHAPTER 4 – RESULT AND DISCUSSION ... 34

4.1. Substrate Preparation ... 34

4.2. Preliminary Research: Determination of Optimum Culture Type and Initial Moisture Content ... 35

4.2.1. Total Fungi Number ... 35

4.2.2. Protein Content, Enzyme, and Specific Activity ... 38

4.2.3. Relation of the Effect of Preliminary Research on Microbial Number, Protein Content, Enzyme and Specific Activity ... 42

4.3. Main Research ... 44

CHAPTER 5 – CONCLUSION AND RECOMMENDATION ... 51

5.1. Conclusion ... 51

5.2. Recommendation ... 51

REFERENCES ... 52

APPENDICES ... 65

Abbreviation ... 65

Appendix 1: 50% Moisture Content and Additional Water in Pretreatment ... 65

Appendix 2: 70% Moisture Content and Additional Water in Pretreatment ... 66

Appendix 3: Microbial Number in Pretreatment ... 66

Appendix 4: Glucose Standard Curve ... 66

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Appendix 6: Glucose Mass in Preliminary Research ... 68

Appendix 7: Enzyme Activity Calculation ... 68

Appendix 8: Protein Content in Pretreatment ... 70

Appendix 9: Specific Activity Calculation ... 70

Appendix 10: Moisture Content and Additional Water in Treatment for 5:0 ... 72

Appendix 14: Glucose Mass in Treatment for 5:0 ... 74

Appendix 17: Enzyme Activity of Ratio 5:0 ... 76

Appendix 20: Spesific Activity of Main Research ... 79

Appendix 21: Statistical Analysis of Microbial Number in Preliminary Research ... 79

Appendix 22: Statistical Analysis of Specific Activity in Main Research ... 82

CURRICULUM VITAE ... 84

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Figure 1. The Saccharum officinarum L. plant ... 15

Figure 2. Zoom in on how a potato starch tuber is build up ... Error! Bookmark not defined. Figure 3. Flowchart for α-amylase production using sugarcane bagasse as SSF substrate ... 29

Figure 4. Grounded dried sugarcane bagasse ... 35

Figure 5. Total Fungi Number ... 36

Figure 6. Log Base Graphic of Microorganism Living Phase ... 38

Figure 7. Comparison between glucose mass, enzyme Activity, and specific Activity of preliminary research ... 44

Figure 8. Fermented Substrate at 72 h Fermentation ... 46

Figure 9. Comparison of enzyme activity at 72 hours of various substrate ratios ... 47

Figure 10. Specific activity for 72 hour fermentation of various substrate ratios ... 49

Figure 11. The extracted of various substrate ratio during 72 h incubation ... 50

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LIST OF TABLES

Table 1. Chemical composition comparison of agro-industrial products ...Error!

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Table 2. Advantages and disadvantages of SSF over SmF ... 18 Table 3. Uses of amylases in various sectors of industry ... 26 Table 4. Summary of 2-Way ANOVA of Microbial Growth Affected by Moisture Content and Culture Types during 72 h Fermentation... 37 Table 5. Summary of 2-way ANOVA of microbial growth affected by culture type with 50% moisture content during 72 hour fermentation ... 37 Table 6. Summary of 2-way ANOVA of microbial growth affected by cultyre ype with 70% moisture content during 72 hour fermentation ... 37 Table 7. Summary of 2-way ANOVA of enzyme activity at various culture types and moisture content after 72 hour fermentation ... 40 Table 8. Summary of Specific Activity on Pretreatment at 72 h Fermentation ... 41 Table 9. Summary of 2-Way ANOVA of Specific Activity on Pretreatment at 72 h Fermentation ... 42 Table 10. Glucose concentration after extraction and starch addtion ... 42 Table 11. Summary of 2-way ANOVA of enzyme activity for 72 hour fermentation at various substrate ratio ... 47 Table 12. Combination of sugarcane bagasse and rice bran as SSF substrate ...Error!

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SUGARCANE BAGASSE UTILIZATION AS SOLID STATE

ABSTRACT

DEDICATION

TABLE OF CONTENTS