OPTIMIZATION OF
α
- AMYLASE PRODUCTION FROM Bacillus subtilis NRRL B3411USING CASSAVA MEAL
RAMIL ILAO CUEVAS
SUBMITTED TO THE FACULTY OF THE COLLEGE OF ENGINEERING AND AGRO-INDUSTRIAL TECHNOLOGY UNIVERSITY OF THE PHILIPPINES LOS BAÑOS IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
BACHELOR OF SCIENCE IN CHEMICAL ENGINEERING
ABSTRACT
Cuevas, Ramil Ilao. University of the Philippines Los Baños. Optimization of α-Amylase Production From Bacillus subtilis NRRL B3411 Using Cassava Meal.
Adviser: Dr. Jovita L. Movillon Co-Adviser: Dr. Pham Binh Chay
Optimization of α-amylase production has been studied from Bacillus subtilis NRRL B3411 using cassava meal in a batch fermentor. The fermentations were carried out using 1 liter stirred tank fermentor. The process parameters were nitrogen sources, pH of the media, and temperature of
cultivation. Various nitrogen sources were used such as yeast extract, peptone, beef extract, malt extract, (NH4)2SO4, ammonium chloride and ammonium acetate. The optimum pH was also determined by doing batch fermentation at different pH values of 6.0,7.0, 8.0,8.5 and 9.0. The optimum process
temperature was also determined by doing batch study at from30ºC up to 50ºC with increment of 5ºC. Samples were taken every 6 hrs and analyzed for α- amylase activity and biomass concentration.
The optimum pH, temperature, nitrogen source and incubation period obtained are 7.0, 50ºC, yeast extract, and 42 hrs. The results were subjected to statistical analysis to determine correlation with results and the parameters varied. Based on the analysis of variance, a strong dependence of
α-amylase activity with process variables(i.e. pH of the medium, temperature, incubation time and nitrogen source) was established.
TABLE OF CONTENTS
CHAPTER PAGE
Abstract 2
Table of Contents 6
List of Tables 8
List of Figures 1 Introduction 1.1 Significance of the Study 9
1.2 Statement of the Problem 12
1.3 Objectives of the Study 13
1.4 Scope and Limitations of the Study 14
1.5 Date and Place of the Study 14
2 Review of Literature 15
1.6 α- Amylase Producing Microorganism 15
1.7 Medium for α-Amylase Fermentation 17
1.8 Effect of Nitrogen Sources 19
1.9 Effects of pH and Temperature 21
1.10 Yield 22
1.11 Yield of Enzyme 22
1.12 Downstream Processing of α-Amylase Produced by Bacillus subtilis 24
2.6.1. Determination of Sugar 25
2.6.2. Determination of α-Amylase Activity 25
3 Methodology 27
3.1. Stock Culture 27
3.1.1. Microorganism 27
3.1.2. Agar Media 28
3.2. Seed Medium 28
3.3. Fermentation Medium 29
3.4. Fermentation Process 29
3.5. Analytical Methods 33
3.5.1. Bacillus subtilis Growth 33
3.5.2. Partial Purification of the Enzyme 34
3.5.3. Determination of Residual Biomass Concentration 34
Concentration and α-Amylase Activity
4.3.1 Effect of Incubation Time on α-Amylase Activity 42
4.4 Statistical analysis of Results and Analysis of Variance 47
5 Summary and Conclusion 54
6 Recommendations 55
References 57
Appendices 61
List of Tables
TABLES PAGE
Table 1 Composition of Culture Agar 28
Table 2 Composition of the Seed Medium 29
Table 3 Composition of the Fermentation Medium 30
Table 4 α-Amylase Activity as Affected by the Different Sources of Nitrogen at pH 6.98 and 50°C 37
Table 5 Effect of Nitrogen Sources on the Biomass- Cell Production of Bacillus subtilis NRRL B3411 at 50°C and pH 6.98 39
Table 6 Effect of Different pH Values on α- Amylase Activity at 30°C Using Cassava Meal as Carbon Source and Yeast Extract as Nitrogen Source. 40
Table 7 Effect of Incubation Time at pH 7.0 and 50°C 42
Table 8 Effect of Incubation Time at pH 7.0 and 45°C 43
Table 9 Effect of Incubation Time at pH 7.0 and 40°C 43
Table 10 Effect of Incubation Time at pH 7.0 and 35°C 44
Table 11 Effect of Incubation Time at pH 7.0 and 30°C 45
Table 12 Variables Entered Removed for Statistical Analysis 47
Table 13 Model Summary 48
Table 14 Analysis of Variance 49
