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Studies on the Bio Production of Gibberellic Acid from Fungi

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Studies on the Bio Production of Gibberellic Acid from Fungi Benha University Faculty of Science Botany Department Studies on the Bioproduction of Gibberellic Acid from Fungi A Thesis Submitted for the degree of Doctor Philosophy of Science in Botany (Microbiology) By Doaa Abd El monem Emam Sleem Assistant lecturer-Microbiology Department, National Center for Radiation Research and Technology (NCRRT), Atomic Energy Authority (AEA) Under Supervision of Prof. Dr. Mahmoud M. Hazaa Prof. Dr. seham M. Shash Prof. of Microbiology Prof. of Microbiology and Head of Botany Department, Botany Department, Faculty of Science, Benha University Faculty of Science, Benha University Prof. Dr. Hesham M. Swailam Prof. Dr. Nagi H. Aziz Prof. of Microbiology Late Prof. of Microbiology Microbiology Department, Microbiology Department, NCRRT, AEA. NCRRT, AEA. 2013 ﺻﺪق اﷲ اﻟﻌﻈﻴﻢ Studies on the Bioproduction of Gibberellic Acid from Fungi Submitted By Doaa Abd El monem Emam Sleeem Assistant Lecturer Microbiology Department, NCRRT, AEA This thesis is approved by Supervisor Profession Signature Prof. of Microbiology, and Head of Botany Prof. Dr. Mahmoud A. Hazaa Department, Faculty of Science, Benha University Prof. of Microbiology, Botany Department, Prof. Dr. Seham M. Shash Faculty of Science, Benha University Prof. of Microbiology, Microbiology Prof. Dr. Hesham M. swailam Department , NCRRT, AEA Late Prof. of Microbiology, Microbiology Late Prof. Dr. Nagi H. Aziz Department , NCRRT, AEA Dedication ﺭﺏ ﺍﻭﺯﻋﻨﻰ ﺃﻥ ﺍﺷﻜﺮ ﻌﻤﺘﻚ ﺍﻟﱵ ﺃﻌﻤﺖ ﻋﻠﻰ ﻭﻋﻠﻰ ﻭﺍﻟﺪﻱ ﻭﺍﻥ ﺍﻋﻤﻞ ﺻﺎﳊﺎ ﺗﺮﺿﺎﻩ My work is dedicated to: Allah and I hope to accept it from me and to my father and my mother to whom I owe my life and to my husband who Supported me , my dear Sons Ziad and Mohamed. Also I dedicate this work to my brothers and my sisters. This dissertation has not previously been submitted for any degree at this or at any other university. Doaa Abd El monem Emam Sleem Contents Contents Page 1. Introduction 1 2. Literature Review 4 2.1. Discovery of Gibberellin 4 2.2. Chemistry and Structure of gibberellic acid (GA) 5 2.3. Properties and uses of gibberellic acid 6 2.4. Improvement of the fruit quality by gibberellic acid 7 2.5. Gibberellin Biosynthesis 10 2.6. GA3 formation physiology in fermentative process 15 2.7. Chemical Regulation of GA Biosynthesis 16 2.8. Fermentation techniques 18 2.9. Parameters conditions for Gibberellic acid production 18 2.9.1. Physical conditions 19 2.9.2. Nutritional conditions 23 2.9.3. Inoculum 29 2.9.4. Working volume 29 2.10. Gibberellic acid improvement 30 2.10.1. Effect of gamma irradiation on fungi secondary metabolites 30 2.11. Immobilizaton 35 2.12. Gibberellic acid production from different wastes 39 2.13. Fungal chitosan production 39 2.13.1. Uses of chitosan 42 2.13.2. Influence of gibberellic acid on the growth of Aspergillus 43 niger and chitosan production 3. Material and Methods 45 3.1. Material 45 3.1.1. Samples 45 3.1.2. Standard chemical used 45 3.1.3. Media used 45 3.2. Methods 47 3.2.1. Isolation of gibberellic acid producer fungi on solid medium 47 3.2.2. Isolation of gibberellic acid producer fungi on broth medium 48 3.2.3. Factors affecting gibberellic acid Production by Fusarium 48 moniliforme 3.2.4. Effect of gamma radiation on Fusarium moniliforme activity 53 3.2.4.a. Determination of D10-value 53 3.2.4.b. Effect of gamma irradiation on gibberellic acid production 54 3.2.5. Effect of immobilization on gibberellic acid production by 55 gamma irradiated spores i Contents 3.2.6. Effect of immobilized inoculum age 55 3.2.7. Effect of inoculum density of immobilized cells 56 3.2.8. Time profile of gibberellic acid production by immobilized 56 cells 3.2.9. Recycling batch fermentation using milk permeate as 56 production medium 3.2.10. Toxicological evaluation of the produced gibberellic acid 57 3.2.11. Effect of gibberellic acid on fungal chitosan production 58 3.3. Chemical analysis 59 3.3.1. Estimation of gibberellic acid 59 3.3.2. Extraction of gibberellic acid 60 3.3.3. Isolation and identification of gibberellic acid produced by 60 isolated Fusarium moniliforme 3.3.3.a. HPLC analysis 60 3.3.3.b Infra-red Spectroscopy (FT-IR) 60 3.3.4. Determination of mycelium dry weight (Biomass) 61 3.3.5. Residual sugar 61 3.4. Chemical analysis for chitosan production 61 3.4.1. Fungal chitosan extraction 61 3.4.2. Infrared spectrum 62 3.4.3. Degree of deacetylation 62 3.4.4. Determination of chitosan molecular weight 62 3.5. Definitions 63 4. Results and Discus sion 64 4.1. Isolation of gibberellic acid producer fungi 64 4.2. Screening of isolated fungi for gibberellic acid production 65 4.3. Isolation and identification of gibberellic acid produced by 67 isolated Fusarium moniliforme 4.4. Optimization of batch culture conditions 69 4.4.1 Influence of specific media 70 4.4.1. Influence of incubation period 71 4.4.2. Effect of incubation temperature 74 4.4.3. Effect of initial pH 76 4.4.4. Effect of agitation speed 78 4.4.5. Effect of carbon sources 81 4.4.6. Effect of fructose concentration 85 4.4.7. Effect of nitrogen source 86 4.4.8. Effect of ammonium sulfate concentrations 90 4.4.9. Effect of different concentrations of rice flour 92 4.4.10. Effect of different concentrations of magnesium sulfate 93 4.4.11. Effect of addition of different concentrations of potassium 95 dihydrogen phosphate: ii Contents 4.4.12. Effect of inoculum type 97 4.4.13. Effect of inoculum age: 99 4.4.14. Effect of inoculum density: 101 4.4.15. Effect of working volume: 103 4.5. Gamma irradiation study: 104 4.5.1. The effect of gamma irradiation on the survival of Fusarium 105 moniliforme 4.5.2. Effect of gamma irradiation on the production of gibberellic 107 acid 4.6. Immobilization study 110 4.6.1. Gibberellic acid production by sponge-immobilized cells of 111 gamma irradiated Fusarium moniliforme 4.6.2. Effect of immobilized inoculum age 113 4.6.3. Effect of immobilized inoculum density 115 4.6.4. Time course of gibberellic acid production by gamma 117 irradiated (0. 5 kGy) immobilized cells of Fusarium moniliforme 4.6.5. Effect of repeated batch fermentation 119 4.7. Toxicological evalution 123 4.8. Chitosan stydy 125 4.8.1. Influence of type of media 125 4.8.2. Influence of addition of gibberellic acid 127 4.8.3. Effect of incubation time 128 4.8.4. Effect of addition of different concentrations of GA in 130 chitosan production Summary 134 Conclusion 137 References 138 Arabic summary 1 iii List of Tables List of Tables Title Page No Table (4-1): Screening of different fungal strains isolated from 66 different sources for their potentiality of gibberellic acid production. Table (4-2): Gibberellic acid production by F. moniliforme (local 70 isolate) grown in various media. Table (4-3): Effect of incubation time on gibberellic acid 72 production by F. moniliforme grown on GPI medium. Table (4-4): Effect of different incubation temperatere (20-40 ºC) 75 on gibberellic acid production by F. moniliforme incubated for 6 days. Table (4-5): Effect of different pH values (3-7) on gibberellic 77 acid production by F. moniliforme incubated for 6 days at 30 ºC. Table (4-6): Gibberellic acid production by F. moniliforme at 80 different aeration regimes (growth conditions: temp. 30 ºC, pH 5 and incubation time 6 days). Table (4-7): Effect of different carbon sources on gibberellic acid 83 production by F. moniliforme (growth condition temp. 30 ºC, pH 5, 6 days, 200 rpm). Table (4-8): Effect of different fructose concentractions (w/v) on 85 gibberellic acid production by F. moniliforme (growth conditions as in Table 4-7). Table (4-9): Effect of different nitrogen sources on gibberellic 88 acid production by F. moniliforme (growth conditions as in Table 4-7 at 6% w/v fructose conc.) Table (4-10): Effect of different ammonium sulfate concentrations 90 on gibberellic acid production by F. moniliforme (growth conditions as in Table 4-9). Table (4-11): Effect of different conc. of rice flour on gibberellic 92 acid production by F. moniliforme (growth conditions as in Table 4-9). Table (4-12): Effect of different conc. of Mg SO4.7H2O on 94 gibberellic acid production by F. moniliforme iv List of Tables (growth conditions as in Table 4-9). Table (4-13): Effect of different conc. of KH2PO4 on gibberellic 96 acid production by F. moniliforme (growth conditions as in Table 4-9). Table (4-14): Effect of type of F. moniliforme inocula on 98 gibberellic acid production (under OMPM). Table (4-15): Effect of inoculum (seed culture) age of F. 100 moniliforme on gibberellic acid production (under OMPM). Table (4-16): Effect of inoculum density of F. moniliforme on 102 gibberellic acid production (under OMPM). Table (4-17): Effect of working volume of production medium on 103 gibberellic acid production by F. moniliforme (under OMPM). Table (4-18): Effect of different doses of gamma radiation on the 106 surviving of F. moniliforme spors. Table (4-19): Effect of different doses of gamma radiation of F. 108 moniliforme for gibberellic acid production (under OMPM). Table (4-20): Gibberellic acid production by immobilized gamma 111 irradiated (0.5 kGy) cells, 24 h age of F. moniliforme (under OMPM). Table (4-21): Effect of inoculum age of gamma irradiated (0.5 114 kGy) immobilized cells of F. moniliforme (0.5g cubes/flask) on gibberellic acid production (under OMPM).
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