Studies on the Bio Production of Gibberellic Acid from Fungi

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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

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). Table (4-22): Effect of inoculum density of gamma irradiated 116 (0.50 kGy) immobilized cells of F. moniliforme on gibberellic acid production (under OMPM). Table (4-23): Time course of gibberellic acid production by 118 gamma irradiated (0.5 kGy) immobilized cells of F. moniliforme (under OMPM). Table (4-24): Gibberellic acid production from milk permeate in 121 repeated batch process1 by 0.50 kGy gamma

irradiated free2 or immobilized cells3 of F. moniliforme under optimized fermentation conditions4.

v List of Tables

Table (4-25): Effect of gibberellic acid produced by F. 124 moniliforme on chicken embryos. Table (4-26): Effect of different media on mycelial growth and 126 chitosan production growth conditions (72 h, 120 rpm, 30 °C). Table (4-27): Influence of gibberellic acid on growth of 127 Aspergillus niger and chitosan production. (MSM medium, 5% molasses, 3 mg GA, 120 rpm, 72 h). Table (4-28): Effect of incubation time on chitosan production 129 (MSM medium, 5% molasses, 3 mg GA, 120 rpm). Table (4-29): Effect of addition of different concentrations of GA 131 in chitosan production: (MSM medium, 5% molasses, 48 h, 120 rpm).

vi List of Figures

List of Figures

Title Page No Fig (2.1): Bakanae disease of rice. Rice plants infected with F. 4 moniliforme are yellow and elongated relative to shorter and greener healthy plants.

Fig (2-2): ent-Gibberellane skeleton and structures of C20- and 6 C19-GAs. Four rings, A-D, and a carbon numbering system are shown for ent-gibberellane. Fig (2-3): The gibberellin biosynthesis pathways in plants. 12 Fig (2-4): The gibberellin biosynthesis pathways in fungi. 14 Fig (2-5): Structures of inhibitors of GA biosynthesis that act 17 as growth retardants. Fig (2-6): Chemical conversion of chitin. 42 Fig (4-1): HPLC analysis of standard gibberellic acid. 69 Fig (4-2): HPLC analysis of extracted gibberellic acid from the 69 local isolate of F. moniliforme . Fig (4-3): FT-IR spectra of gibberellic acid (A): Standard 70 gibberellic acid, (B): Extracted gibberellic acid from the local isolate of F. moniliforme . Fig (4-4): Gibberellic acid production by G.fujikuroi (local 72 isolate) grown in various media. Fig (4-5): Effect of incubation time on gibberellic acid 75 production by F. moniliforme grown on GPI medium. Fig (4-6): Effect of different incubation temperatere (20-40 ºC) 77 on gibberellic acid production by F. moniliforme incubated for 6 days. Fig (4-7): Effect of different pH values (3-7) on gibberellic 79 acid production by F. moniliforme incubated for 6 days at 30 ºC. Fig (4-8): Gibberellic acid production by G.fujikuroi at 82 different aeration regimes (growth conditions: temp. 30 ºC, pH 5 and incubation time 6 days). Fig (4-9): Effect of different carbon sources on gibberellic acid 85 production by F. moniliforme (growth condition temp. 30 ºC, pH 5, 6 days, 200 rpm).

vii List of Figures

Fig (4-10): Effect of different fructose concentractions (w/v) on 87 gibberellic acid production by F. moniliforme (growth conditions as in Table 4-6). Fig (4-11): Effect of different nitrogen sources on gibberellic 90 acid production by F. moniliforme (growth conditions as in Table 4-6 at 6% w/v fructose conc.). Fig (4-12): Effect of different ammonium sulfate concentrations 92 on gibberellic acid production by F. moniliforme (growth conditions as in Table 4-8). Fig (4-13): Effect of different conc. of rice flour on gibberellic 94 acid production by F. moniliforme .

Fig (4-14): Effect of different conc. of Mg SO4.7H2O on 95 gibberellic acid production by F. moniliforme .

Fig (4-15): Effect of different conc. of KH2PO4 on gibberellic 97 acid production by F. moniliforme (growth conditions as in Table 4-9). Fig (4-16): Effect of type of F. moniliforme inocula on 99 gibberellic acid production (under OMPM). Fig (4-17): Effect of inoculum (seed culture) age of F. 100 moniliforme on gibberellic acid production (under OMPM). Fig (4-18): Effect of inoculum density of F. moniliforme on 102 gibberellic acid production (under OMPM). Fig (4-19): Effect of working volume of production medium on 104 gibberellic acid production by F. moniliforme (under OMPM). Fig (4-20): Effect of different doses of gamma radiation on the 107 surviving of F. moniliforme spores. Fig (4-21): Effect of different doses of gamma radiation of F. 109 moniliforme for gibberellic acid production (under OMPM).

Fig (4-22): Gibberellic acid production by immobilized gamma 112 irradiated (0.5 kGy) cells, 24 h age of F. moniliforme (growth conditions as in Table 4-8). Fig (4-23): Effect of inoculum age of gamma irradiated (0.50 114 kGy) immobilized cells of F. moniliforme (0.5g cubes/flask) on gibberellic acid production (under OMPM).

viii List of Figures

Fig (4-24): Effect of inoculum density of gamma irradiated 115 (0.50 kGy) immobilized cells of F. moniliforme on gibberellic acid production (under OMPM). Fig (4-25): Time course of gibberellic acid production by 118 gamma irradiated (0.50 kGy) immobilized cells of F. moniliforme (under OMPM). Fig (4-26a): Gibberellic acid production from milk permeate in 121 repeated batch process by 0.50 kGy gamma irradiated immobilized cells of F. moniliforme under optimized fermentation conditions. Fig (4-26b): Gibberellic acid production from milk permeate in 121 repeated batch process by 0.50 kGy gamma irradiated free cells of F. moniliforme under optimized fermentation conditions. Fig (4-27): Effect of gibberellic acid produced by F. 123 moniliforme on chicken embryos. Fig (4-28): Effect of type of media on chitosan production. 124 Fig (4-29): Effect of Addition of gibberellic acid on chitosan 126 production. Fig (4-30): Effect of incubation time on chitosan production 128 (MSM medium, 5% molasses, 3 mg GA, 120 rpm). Fig (4-31): Effect of addition of different concentrations of GA 130 in chitosan production: (MSM medium, 5% molasses, 48 h, 120 rpm). Fig (4-32): FT-IR of different fungal chitosan along with 131 chitosan from Sigma. (A) Chitosan obtained from Sigma; (B) Citosan obtained from A. niger in MSM; (C) Chitosan obtained from A. niger in MSM with the addition of gibberellic acid.

ix List of Abbreviations

List of Abbreviations

AEA Atomic Energy Authority ATCC American Type Culture Collection CFU/ml Colony forming unit per ml CMV Culture medium volume CPS/KS ent-copalyl diphosphate synthase/ ent-kaurene synthase D Day FAO Food Agriculture Organization FV Flask volume GAs Gibberellins GA Gibberellic acid Gy Gray J/kg Joule / kilogram Kg Kilogram kGy Kilo Gray Krad Kilo rad NCRRT National Center For Radiation Research and Technology ND Not Detected OMPM Optimized modified production medium PDA Potato dextrose agar pH Hydrogen ion concentration rpm Rotation per minute SE Standard Error SmF Submerged Fermentation SSF Solid-State Fermentation UV Ultra violet v/v Volume / Volume WHO World Health Organization w/v Weight per volume

Abstract

Gibberellic acid is a natural plant growth hormone which is gaining much more attention all over the world due to its effective use in agriculture and brewing industry. At present gibberellic acid is produced throughout the world by fermentation technique using the fungus Gibberella fujikuroi (recently named Fusarium moniliforme). The aim of the current study is the isolation of local F. moniliforme isolate have the ability to produce gibberellic acid on specific production media. The submerged fermentation technique for the production of gibberellic acid is influenced to a great extent by a variety of physical factors (incubation time, temperature, pH, agitation speed) also, gibberellic acid production by F. moniliforme depends upon the nature and concentrations of carbon and nitrogen sources. The optimization of these factors is prerequisite for the development of commercial process. The addition of some elements in a significant quantities to the production media stimulate gibberellic acid production. The use of seed culture inocula (24 h) age at rate of (2% v/v) also enhance the production. Working volume 50 ml in 250 ml Erlenmeyer flask was found to be the best volume for the production. Low doses of gamma radiation (0.5 kGy) stimulate gibberellic acid production and microbial growth by the local F. moniliforme isolate. Immobilized cell fermentation technique had also been developed as an alternative to obtain higher yield of gibberellic acid. Milk permeate (cheap dairy by- product) was found suitable to used as main production medium for gibberellic acid production by the fungus under investigation. The influence of gibberellic acid on enhancement growth of Aspergillus niger and chitosan production was also studied, the addition of 2 mg/l of gibberellic acid to chitosan production medium stimulate its production in comparison with media without gibberellic acid.

Introduction

1. Introduction

Plant hormones are involved in several stages of plant growth and development, it is essential for plant defense (Costacurta and Vanderleyden 1995). Until recently it was assumed that the main plant growth processes were controlled by 5 types of phytohormones, namely the auxins, cytokinins, gibberellins, abscisic acid, and ethylene. Gibberellins (GAs) are a large family of structurally related diterpenoid acids that occur in green plants and some microorganisms. Gibberellic acid (GA) is an important member of gibberellin family and acts as a natural plant growth hormone, controlling many developmental processes such as the induction of hydrolytic enzyme activity during seed germination, stem elongation, induction of flowering, improvement of crop yield, overcoming dwarfism, elimination of dormancy, sex expression, enzyme induction and leaf and fruit senescence, etc (Rangaswamy 2012; Rios-Iribe, et al., 2010; Bruckner and Blechschmidt 1991 & Kumar and Lonsane 1989). Due to these properties, this hormone (GA) is of great importance in agriculture, nurseries, tissue culture, tea garden, viticulture, acceleration of germination in the brewery industry, etc (Lu et al., 1995; Kumar and Lonsane 1990 & Martin 1983). This is due to its fast and strong effects at low concentrations (µg) on the above processes, this will led to obtaining better harvests in the agriculture area and by extension improving the food industry . In addition, GA was also used in a variety of activities related to research work and pharmacological applications (Kumar and Lonsane 1989). Gibberellins are distributed widely through the plant kingdom where they play an important role in plant growth and development. They have also been isolated from fungi and bacteria. There are l3l presently known naturally occurring GAs (Mander 2003, Leitch et al., 2003 and Crow et al., 2006).

1 Introduction

There are three routes to obtaining GA have been reported: extraction from plants, chemical synthesis and microbial fermentation. The latter being the most common method used to produce GA. The filamentous fungus Gibberella fujikuroi recently named Fusarium moniliforme was investigated as the main producer of gibberellins. These hormones are widely used to optimize growth in several products, particularly seedless grapes. A mong the gibberellins, the most important form on industrial perspective is gibberellic cid (GA3), which can be produced by fermentation at relatively high concentration (Kumar and Lonsane 1988).

The commercial source of bio-active GAs, particularly GA3 is by fermentation of the fungus Gibberella fujikuroi.( Fusarium moniliforme) from which the GAs were originally discovered (Rangaswamy., 2012).

The industrial process currently used for the production of GA3 is based on submerged fermentation (SmF) techniques, using Gibberella fujikuroi. The cost of GA3 has restricted its use to preclude application for plant growth promotion, except to certain high value plants. Reduction in its production costs could lead to wider application fields (Shukla, et al 2005). Gibberellic acid yields in SmF are highly affected by different environmental and nutritional factors (incubation time, incubation temperature, pH, agitation speed, carbon and nitrogen source, bioelements and other factors). Gibberellic acid can also be produced by the solid-state fermentation (SSF),These traditional fermentation process is labour-intensive, time consuming and requires large cultivation areas, therefore the utilization of submerged fermentation (SmF) technique for the production of GA has been studied to overcome the problems of space, scale-up and process control of SSF.

2 Introduction

Aim of the work The objective of this study is to investigate GA production by the most producer local isolate in SmF conditions. The following points were investigated:- 1-Screening of the most potent local fungal isolate for gibberellic acid production. 2-Optimization of different growth parameters for gibberellic acid production by the most potent producer fungal isolate. 3-Enhancement of gibberellic acid production by gamma radiation treatment as a mutagenic agent. 4-Continous production of gibberellic acid from milk permeate by immobilization techniques. 5-Influence of gibberellic acid on the growth of Aspergillus niger and chitosan production.

3 Literature Review

2. Literature Review

2.1. Discovery of Gibberellin: A disease of rice known as Bakanae or " foolish seedling " disease in which plants grew in ordinately long , became weak , and eventually fell over and died was known in Japan since the early 1800s (Fig. 2-1). The disease was attributed to infection by a fungus, Gibberella fujikuroi recently named (Fusarium moniliforme) , an ascomycete, and Kurosawa in 1926 showed that the fungal extract applied to healthy plants produced the same symptoms as in Bakanae disease. In 1930s, Yabuta and Hayashi purified the active compound from the fungal extract and called it gibberellin. This major discovery went un- noticed in western countries until after the end of World war Π. In 1950s, researchers in England and United Stated perfected their own methodologies to isolate the active compound from Gibberella cultures and named the compound gibberellic acid. Because gibberellic acid application could induce elongation growth in genetic dwarfs of pea and maize, it was surmised that gibberellic acid must also occur in higher plants, a surmise proven correct by isolation of the first gibberellin from higher plants by MacMillan and Suter in 1957 .

Fig (2-1): Bakanae disease of rice. Rice plants infected with Gibberella fujikuroi are yellow and elongated relative to shorter and greener healthy plants (Srivastava, 2002).

4 Literature Review

Discovery of a gibberellin in higher plants led to a flurry of activity not only in analysis of more and more plant extracts for identification and purification of other gibberellins (GAs), but also into discovery of various biological responses, other than stem elongation, elicited by GAs. As more GAs became known from fungi as well as higher plants. 2.2. Chemistry and Structure of gibberellic acid (GA):

Gibberellic acid (C19H22O6), chemically characterized as a tetracarbocyclic dihydroxy-g-lactonic acid containing two ethylene bonds and one free carboxylic acid group (Cross, 1954). It is a white crystalline powder, with a melting point of 233-235 ºC, soluble in alcohols, acetone, ethyl acetate, butyl acetate, while is soluble with difficulty in petroleum ether, benzene and chloroform. The product cannot be decomposed in dry condition and it is rapidly decomposed in hot condition and in aqueous solutions. It's half live in diluted aqueous solutions is about 14 days at 20°C (O'Neil, 2001). Gibberellins are defined by their structure rather than their biological activity. They are all cyclic diterpenes with an ent-gibberellane ring structure. Two main types of GAs are recognized, those with the full complement of 20 carbon atoms, the C20- GAs, and the C19-GAs, which have lost one C atom and possess a lactone (Fig. 2-2 ). Biologically active GAs in higher plants are C19 compounds.

5 Literature Review

Fig (2-2): ent-Gibberellane skeleton and structures of C20- and C19-GAs. Four rings, A-D, and a carbon numbering system are shown for ent-gibberellane (Srivastava, 2002).

2.3. Properties and uses of gibberellic acid:

The gibberellins are tetracyclic diterpenoid acids strictly related, representing an important group of plant growth hormones. known gibberellins are identified by subscribed numbers GAn where "n" corresponds, approximatly, the order of discovery. Gibberellic acid, which was the first gibberellin to be structurally characterized, is GAЗ (Arteca, 1995). All gibberellins have an ent–gibberellane ring system and are divided in two main types based on the number of carbon atoms, the C20 GAs which have a full complement of 20 carbon atom and C19 GAs in which the twentieth carbon atom has been lost by metabolism. Many structural modifications can be made to the ent-gibberellane ring system. This diversity accounts the large number of known GAs (Kumar and Lonsane 1989 & Sponsel and Hedden 2004). Considering the numerous effects of gibberellins, it seems logical that they would be used in commercial application (Martin 1983; Taiz and Zeiger 1991 and Mander 2003). Their major uses are:- * Management of fruit crops

6 Literature Review

* Most seedless table grapes are now grown with the application of GAЗ, inhibiting senescence of citrus fruit, maintaining the rind in better condition; controlling skin disorder in golden delicious apples. * Production of ornamental plants–inducing to flower either earlier than usual, or in off- seasons. Sporadic flowering in some plants is often a problem with plant breeders, but may be ameliorated with GA applications.

* Malting of barley: 2-3 days may be saved by the addition of 25-500 µg of GA3 for each Kg of barley. * GAs used in malting of barley for increasing the yield of malt and in reducing the steeping time. * Extension of sugarcane: increase in grown and sugar yield. * Its uses in grapes resulted in an increased size of the berry as well as the cluster weight. * Also used in a variety of research projects and for pharmacological applications in animals. * GAs are used at ppm levels and their use results in a number of physiological effects such as: - Elimination of dormancy in seeds. - Acceleration of seed germinations. - Improvement in crop yields. - Marked stem elongation. - Promotion of fruit setting. - Overcoming of dwarfism.

2.4. Improvement of the fruit quality by gibberellic acid: A fruit is a living, respiring and edible organ, which once detached from the parent plant, will undergo significant post-harvest changes. The term ‘shelf- life’ or the synonyms ‘storage life’ and ‘storability’, can be defined as the time periods that a fruit can be expected to maintain a predetermined level of quality

7 Literature Review under specified storage conditions. Fruit shelf-life can be extended by optimization of environmental conditions, minimization of mechanical damage, application of food additives in the form of chemical sprays or dips, or by ionizing radiation. The use of post-harvest chemicals, ranging from fungicides and fumigants to sprout suppressants and antioxidants, has allowed a considerable extension of the storage life of many fruits and vegetables. This, in turn has extended the geographical range from which produce is sourced and enabled year-round supply of many perishable commodities to the consumer. However, consumer desires for food free of synthetic chemical residues is driving a trend towards reduced use of post-harvest chemicals. Thus, the need for effective development of non-chemical treatments for produce has never been greater. Progress has been made in the use of controlled atmosphere storage and research has shown the potential for post-harvest disease control using biological agents. Other approaches have been to try and identify effective natural chemicals (i.e. those present in plant extracts), which may be more acceptable to consumers than those that are synthetically produced. Thus, much research has been focused on extending post-harvest fruit storage life by pre- or post-harvest treatments with gibberellins. Considerable published data exists concerning the effect of gibberellins (GA) on senescence, particularly on the ability of GA to inhibit chlorophyll loss in a variety of tissues, including leaves, fruits, cotyledons and flowers (Nooden, 1988). However, there are only a few reports on the effect of GA treatments on post-harvest fruit, most treatments being applied to trees before harvesting. Pre- harvest GA treatment is effective in delaying fruit ripening on the tree for cherries (Facteau et al., 1985), citrus fruit, apricots (Southwick and Yeager, 1995) and nectarines (Zilkah et al., 1997), and pre-harvest GA applications also inhibited fruit ripening and softening during storage in mangos and nectarines (Khader, 1991 and Zilkah et al., 1997).

8 Literature Review

The most remarkable effect of GA treatments (normally as GA3), in both, pre and post-harvest applications, is a net reduction in fruit colour changes. Thus, carotenoid synthesis and chlorophyll breakdown in mandarins (Garcia- Luis et al., 1992 and Nagar, 1993), lemons (El-Zeftawi, 1980) and mangos (Khader, 1992), as well as anthocyanin synthesis in strawberries (Martinez et al., 1994), can be delayed by GA treatments. In other research, Valero et al.

(1998) found that post-harvest infiltration with 100 ppm GA3 of lemon fruit harvested at colour break reduced weight loss during storage and delayed colour development in the flavedo. This process could be due to the inhibition of carotenogenesis and to the decline in the rate of chlorophyll loss, as has been shown to occur in lemons sprayed with GA3 before harvesting (El-Zeftawi, 1980).

Post-harvest treatment of lemons with GA3 was also effective in reducing weight loss during storage, improving fruit quality and storage life (Valero et al., 1998), and increasing fruit firmness after treatment, thus maintaining high firmness levels during storage. Likewise, it has been found that pre-harvest GA3 treatments also increase fruit firmness in apricots (Southwick and Yeager, 1995) and peaches (Southwick et al., 1995). Another effect of the postharvest application of GA is a delay in the degradation ascorbic acid and of the activities of both, α-amylase and peroxidase (Khader, 1992). Thus, in general, it could be concluded that GA treatment of fruit after harvesting might extend the fruit shelf-life, improving fruit quality and storability by delaying colour changes, weight loss, softening.

GA3 applied to peach trees at the end of pit hardening inhibited fruit maturation on the tree, delayed harvest and reduced flesh browning after storage. Thus the marketing season of this fruit was prolonged by four weeks, and fruit quality also improved due to a significant increase in fruit weight and soluble solids content (Zhiguo et al., 1999). Preharvest treatment of fruit with

GA3 also delayed colour changes. Thus, GA3 sprays on cactus pear fruit 10 9 Literature Review weeks after full bloom delayed the appearance of full orange colour of the skin and reduced the rate of colour change during postharvest storage at low temperature as well as fruit softening (Schirra et al., 1999). GA3 treatment of young strawberry plants improved the weight size and the colour of strawberry fruits and enhanced anthocyanin content and phenylalanine ammonialyase activity (Montero et al., 1998), a key enzyme in controlling anthocyanin biosynthesis from phenyl-alanine. In plum, GA3 pre-harvest treatment has also been recommended to produce better colour and larger, firmer fruit (Boyhan et al., 1992).

2.5. Gibberellin Biosynthesis: Terpenes, or terpenoids, are a large class of plant secondary products with a major role in defense against plant-feeding insects and herbivores (Gershenzon and Croteau, 1991). However, not all terpenoids act as secondary products. Many have important roles in primary processes, such as photosynthesis, stability of cell membranes, signaling, and as source compounds for several plant hormones. Terpenes are characterized by their basic structural element, the five-carbon isoprene unit:

All terpenes are formed by condensation of two or more isoprene units and are classified on the basis of the number of isoprene units they contain. Isopentenyl diphosphate (IPP) is the five-carbon activated building block of terpenes, which isomerizes to dimethylallyl diphosphate (DMAPP). DMAPP and IPP are also the starting points for a series of head-to- tail condensations and cyclization to yield geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP) (Domenech et al., 1996 and Linnemannstons et al., 2002). GGPP undergoes cyclization in two closely linked steps to give rise to the first fully cyclized compound, ent-kaurene Two

10 Literature Review enzymes sequentially catalyze this reaction—copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS)—and commit GGPP on the pathway to GA biosynthesis. These enzymes occur in plastids. a series of oxidations at C-19 lead to the formation of ent-kaurenoic acid, which is hydroxylated at C-7 to yield ent-7α-hydroxykaurenoic acid. This latter compound yields GA12-aldehyde by contraction of the B ring and a further oxidation at C-6. Enzymes involved in these oxidations, ent-kaurene oxidase, ent-kaurenoic acid hydroxylase, and

GA12-aldehyde synthase, are membrane-bound cytochrome P450 monooxygenases, enzymes are believed to be located on the endoplasmic reticulum. (Bomke and Tudzynski 2009 and Tudzynski, 2005). These first steps of the pathway are identical in the higher plants

(Fig 2-3) and in the fungus (Fig 2-4). After GA12-aldehyde, the pathways in higher plants and Gibberella fujikuroi differ. In G. fujikuroi, GA12-aldehyde is first 3β- hydroxylated to GA14-aldehyde, which is then oxidized at C-7 to form

GA14 (Hedden et al. 1974 and Urrutia et al. 2001).

The subsequent conversion of GA14 to GA4 by 20-oxidation is analogous to the formation of GA9 and GA20 in higher plants.

11 Literature Review

Fig (2-3) The gibberellin biosynthesis pathways in plants. Bioactive GAs found in a wide variety of plant species are highlighted in grey. In each metabolic reaction, the enzymes are highlighted in color. Abbreviations: 2ox, GA 2-oxidase (Class I and II); 2ox*, GA 2- oxidase (Class III); 3ox, GA 3-oxidase; 13ox, GA 13-oxidase; 20ox, GA 20-oxidase; GGDP, geranylgeranyl diphosphate; ent-CDP, ent-copalyl diphosphate; CPS, ent- copalyl diphosphate synthase; KS, ent-kaurene synthase; KO, ent-kaurene oxidase; KAO, ent-kaurenoic acid oxidase (Bomke and Tudynski, 2009).

Desaturation of GA4 at C-1,2 results in the formation of GA7, which is converted to the main product in G. fujikuroi, GA3, by 13-hydroxylation. GA1 is 12 Literature Review formed as a minor product by 13-hydroxylation of GA4 and is not converted to

GA3 (MacMillan, 1997).

In plants, GA12-aldehyde is converted to GA12, which is either oxidised at C-20 to form the 19-carbon gibberellin, GA9, or is first 13-hydroxylated to

GA53, which is then oxidized at C-20 to yield GA20 (Fig. 2-3). GA9 and GA20 are formed in parallel pathways, both involving oxidation of C-20 to alcohol and aldehyde, and the final formation of biological active 19-carbon GAs by loss of C-20. In plants, the oxidation and removal of C-20 is catalysed by a multifunctional GA 20-oxidase (Lange et al., 1994 and Phillips et al., 1995).

At the end of the pathway, both GA9 and GA20 are converted to GA4 and GA1, respectively, by introduction of a 3β-hydroxyl group. Thus, a major difference between the GA pathways in G. fujikuroi and plants is the stage at which the hydroxyl groups are introduced. In the fungus, GA12-aldehyde is 3β- hydroxylated to GA14-aldehyde, whereas in plants GA12-aldehyde is converted to GA12, which is then 13- hydroxylated to GA53 (Fig 2-3). In fungi, 13- hydroxylation takes place only in the final step to form GA3 from GA7, whereas in plants the final step is the 3β-hydroxylation of GA9 and GA20 to GA4 and

GA1, respectively (Fig 2-4). Gibberellin biosynthesis in G. fujikuroi differs from that in higher plants in several respects: a single enzyme (CPS/KS) catalyzes both steps in the formation of ent-kaurene from GGPP, 3β-hydroxylation occurs early in the pathway as one of the reactions catalyzed by GA14 synthase, a highly multifunctional cytochrome P450 monoxygenase that converts ent-kaurenoic acid to GA14 (3β-hydroxy GA12), and 13-hydroxylation is the final step in the pathway to GA3. Furthermore, removal of C20 is accomplished by a cytochrome P450 rather than a dioxygenase. The genes for the GA-biosynthetic enzymes are clustered in G. fujikuroi, whereas they are dispersed throughout the genome of the higher plant A. thaliana.

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Fig (2-4) The gibberellin biosynthesis pathways in fungi. Differences in the fungal pathways and the final products of the pathway in F. fujikuroi, S. manihoticola and Phaeosphaeria sp., as shown. Enzymes common in all three fungi are marked in grey, enzymes present in F. fujikuroi and Sphaceloma manihoticola are marked in blue, enzymes present only in F. fujikuroi or Phaeosphaeria are marked in dark green and light green, respectively (Bomke and Tudynski, 2009).

A very few patents are available in the literature which reported that the addition of intermediate compound of gibberellin pathway (e.g. mevalonnic acid, ent-kaurene as precursors in the fermentation medium have greatly improved the yield of GA3 (Birch et al., 1960 and Tachibana et al., 1994).

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2.6. GA3 formation physiology in fermentative process:

Fermentative production of gibberellins is a classic example of secondary metabolite production as the phases of growth can be clearly distinguished and related to nutritional and environmental states operating in the fermentor. Borrow et al., (1964a,b) have exhaustively studied this process and established producing and non-producing phases of the gibberellin fermentation process. The conventional lag phase in nitrogen-limited medium is undetectable as the strain requires little or no adaptation and growth in the fermentor starts quickly due to the use of vigorous mycelial cells as inoculum. Growth during the balanced phase is initially exponential and subsequently becoming linear. The uptake of glucose, nitrogen and other nutrients remains almost constant per unit increase in dry weight. This phase extends until exhaustion of one of the nutrients occurs subjecting the cells to a deceleration stage, no GA is produced in this phase. The following storage phase occurs with the presence of excess glucose and the exhaustion of nitrogen, causing an increase of dry weight due to accumulation of lipids (45%), carbohydrates (32%) and polyols. In this phase the production of gibberellins and other secondary metabolites begins and is continued in the presence of available glucose. The next maintenance phase, is operative between the maximum mycelial formation and the onset of terminal breakdown of mycelial components. Because it is the main gibberellin producing phase, its continuation, even for several hundred hours, if glucose is present in excess, is of industrial importance. Except for the continued uptake of glucose, the cells take up no other nutrients and their dry weight remains constant. Finally, in the terminal phase the mycelial cells undergo many changes due to no availability of sources of utilizable carbon. This phase is not allowable to occur in fermentations, as the fermentor run for production of GAs is terminated just prior to the onset of this phase (Borrow et al., 1961, 1964 a,b; Kumar and Lonsane 1989 & Bruckner and Blechschmidt 1991).

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2.7. Chemical Regulation of GA Biosynthesis: Chemical inhibitors of GA biosynthesis are used extensively in agriculture and horticulture as growth retardants, primarily to control plant stature . Three classes of inhibitors have been developed, differing in their site of action in the GA biosynthetic pathway. Examples of each type are shown in Fig (2-5). The ‘‘onium’’ inhibitors, exemplified by chlormequat chloride and AMO-1618, contain a permanent positive charge and inhibit the conversion of GGPP to CPP by CPS. They are thought to act by mimicking a charged transition state in the cyclization. Their high water solubility allows for ease of application and efficient transport within the plants. However, they are relatively inefficient inhibitors, particularly chlormequat chloride, which is the most widely used retardant, mainly on cereals. Potential side effects of these compounds are inhibition of epoxy squalene cyclization and transmethylation reactions in steroid biosynthesis. A large group of compounds with a nitrogen containing heterocyclic ring target ent-kaurene oxidase. prominent members of this group are the triazoles, paclobutrazol, and uniconazole.

Chlormequat chloride (CCC)

Paclobutrazol

16 Literature Review

Prohexadione calcium exo-16,17-dihydro-GA5 13-acetate

Fig (2-5) Structures of inhibitors of GA biosynthesis that act as growthretardants (Hedden, 2003).

The heterocyclic compounds inhibit cytochrome P450 monooxygenases, with highest affinity for methyl hydroxylases. Thus, they are active only against ent-kaurene oxidase in the GA-biosynthetic pathway, but some of these inhibitors have activity also against sterol 14-demethylase and abscisic acid 8- hydroxylase. They are highly efficient inhibitors that function at low concentrations, but have low water solubility and are transported poorly in the plant, primarily in the xylem. The third group of retardants are the acylcyclo- hexanediones, such as prohexadione, which act on the dioxygenases of GA- biosynthesis, with highest activity against GA 3β-hydroxylases. They are also effective against 2-oxidases, but have relatively little activity against GA 20- oxidases. They act as mimics of 2-oxoglutarate and block binding of this cosubstrate to the enzyme active site. Although less efficient as inhibitors, they have higher water solubility than the nitrogen-containing heterocyclic compounds and are transported more readily throughout the plant. As side effects, they may inhibit other 2-oxoglutarate-dependent dioxygenases. For example, inhibition of flavanone 3β-hydroxylase can result in reduced anthocycanin production and therefore loss of color in flowers and fruit. A new group of retardants that is still being developed for commercial application comprises 16, 17-dihydroGAs, such as exo-16,17-dihydroGA5 13-acetate. These

17 Literature Review compounds, which are thought to inhibit GA 3β-hydroxylation, are extremely potent growth retardants on members of the Poaceae, but are much less effective on dicotyledonous plants.

2.8. Fermentation techniques: Fermentation is the industrial method practiced for the manufacture of

GA3 preferentially with G.fujikuroi (Borrow et al., 1955). Gibberellic acid production is possible either by chemical synthesis (Hook et al., 1980) or extraction from plants (Kende 1967) but these methods are not economically feasible. Liquid surface fermentation (LSF) was employed in earlier years for the production of GAs and its use was continued until 1955. Although, due to disadvantages inherently present in LSF as production of a wide range of by- products, very low yield (40 to 60 mg GAs. L substrate-1), prolonged incubation time (10-30 days) and prone to contamination this technique was abandoned for

GA3 production being substituted by submerged fermentation (SmF) (Kumar and Lonsane 1989 & Bruckner and Blechschimidt, 1991). Recently different studies (Uthandi et al., 2010 and Rangaswamy,

2012) have been carried out to decrease GA3 production costs using several approaches as screening of fungi, optimization of the nutrients and culture conditions, use of agro-industrial residues as substrate, development of new processes (immobilizes cells, fed-batch culture) and minimization of the cost of the extraction procedure.

2.9. Parameters conditions for Gibberellic acid production: Studies were carried out on optimization of nutritional and physical factors, such as supplementation of minerals and nitrogen to the substrate, inital pH and incubations temperature, for improved yields of GA3 (Machado et al., 2002 & Soccol and Vandenberghe, 2003).

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2.9.1. Physical conditions: 2.9.1.a. Incubation periods: The time course for growth and gibberellic acid production in G.fujikuroi liquid media cultures was examined by Bu'Lock et al. (1974) who reported that growth as well as gibberellic acid content increased through the 5 d of his study, with the linear phase of growth preceding the period of rapid accumulation. Significant amounts of gibberellic acid were found in the cultures after 3 days. These patterns for growth and gibberellic acid production are similar to those observed by Johnson and Coolbaugh. (1990) who’s found that 6-days time course was optimum incubation time for gibberellic acid accumulation. Whereas, Munoz and Agosin. (1993) reported that gibberellic acid was detected after 72 h in the extracellular medium in shake flask cultures.

The production of gibberellic acid GA3 by Fusarium moniliforme M- 7121 in solid state culture was evaluated in flask culture as well as in 3-L horizontal rotary reactors (Qian et al., 1994). The authors revealed that a low O2 concentration resulted in a much decreased GA3 yield and the appearance of yellow to reddish pigmentation in the mycelium. Furthermore, the data cleared that the lag phase was short and rapid growth continued for up to 2 days in the rotary reactor and the maximum rate of GA3 production occurred during the subsequent 3 to 10 days of incubation and the final GA3 concentration reached was 18.7 to 19.3 mg g-1 dry culture. Pervious work with G. fujikuroi has indicated that gibberellic acid production occurs from the beginning of fermentation, Shukla et al., (2005) was found that gibberellic acid synthesis started after 40 h of cultivation while the optimum incubation period for gibberellic acid production was 170 h using spore suspension of G. fujikuroi as inoculum in a 3 l fermentor.

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Meleigy and khalaf, (2009) reported that 6 days incubation time was optimum for gibberellic acid accumulation by a mutant Fusarium moniliforme cells immobilized on loofa sponge using milk permeate as production medium. The production of gibberellins began after 20 h when Lale and Gadre (2010) used Fed-batch fermentation for gibberellic acid production by the mutant G. fujikuroi Mor-189. 2.9.1.b. Temperature: The effect of temperature on the GA3 production is dependent on the strain employed. The optimum temperatures reported for the production of GA, using G. fujikuroi or F. moniliforme include 25°C (Kahlon and Malhotra ,1986), 27°C (Gohlwar et al., 1984), 28°C (Darken et al., 1959), 28.5-29.5°C (Borrow et al., 1964b) 29 ± 0.5°C (Holme and Zacharias, 1965), 30°C (Sanchez-Marroquin, 1963; Maddox and Richert, 1977) and 34°C. Kinetic studies showed that variation in the temperature of the fermentation broth affects the process both qualitatively and quantitatively (Borrow et al., 1964a,b).

A process for the production of GA3 by G. fujikuroi in higher yields by maintaining the temperature at 27.5-30°C in the final stage of fermentation was patented by Borrow et al. (1959). This process emanated from the findings that optimum temperature for growth of the strain was between 31 and 32°C, while the production of GA3 was maximal at 29°C (Jefferys, 1970). At temperatures above 29°C, the reduction in the production of GA3 was rather rapid. Kahlon and Malhotra (1986) found that the maximum yield of GA production by F.moniliforme entrapped in alignate gel, was recorded at 25±1 °C. Also, Escamilla et al. (2000) recorded that the greatest production of GA in fluidized bioreactors by immobilized G. fujikuori mycelium in Ca-polygalate was at 30 °C. Meleigy and Khalaf (2009) studied the effect of different temperature levels (20-40 °C) on the ability of F.moniliforme γ-14 isolate to transform milk

20 Literature Review permeate sugars in to GA, the maximum GA (1.84 gl-1) was produced at 30°C and the yield of GA production decreased by increasing the temperatures and complete inhibition of GA synthesis was recorded at 40 °C .

2.9.1.c. Initial pH: pH variation is one of the most influent factors in the composition of the produced gibberellin mixture. The initial pH values generally employed by various workers were either around 5.5 or within the range 3.5-5.8 (Borrow et al., 1964a). These were usually not controlled during fermentation (Holme and Zacharias, 1965) and thus resulted in a final pH of 3.9-5.2 or 1.8-1.9 or in slight alkalinization (Sanchez-Marroquin, 1963). The effects of the pH of the fermentation medium on growth of the strain and production of GAs include (1) no significant differences in product yield and growth were reported when initial pH was in the range of 3.5-5.5 (Jefferys, 1970 and Gohlwar et al., 1984); (2) the pattern of pH changes during fermentation were similar in media containing glycerol or glucose, although these patterns were different when the complete dose of glucose was added initially (Darken et al., 1959); (3) the specific growth rate was fairly constant in ammonium tartrate media in the pH range of 3.5-6.3 but it decreased beyond this range (Borrow et al., 1964a); (4) the inhibition of NH3-N assimilation in presence of N03-N at pH 2.8-3.0 was observed until the pH increased to a value at which ammonia assimilation was resumed (Borrow et al., 1961, 1964a); (5) the yield constant for glucose was not affected by initial pH, although the yield constant for nitrogen was found to increase with increasing pH value (Borrow et al., 1964a); (6) the rate of production decreased when the pH was outside the range of 3.0-5.5 (Borrow et al., 1964a); (7) concomitant production of GA, and GA3 was reported at low initial pH values (Fuska et al., 1961).

Qian et al. (1994) studied the effect of pH on GA3 production by Fusarium moniliforme, and the data revealed that at an initial substrate pH 4

21 Literature Review

-1 GA3, accumulation was slow, reaching a final value of 10.9 mg g dry culture was obtained at an initial pH of 5 after 18 days of incubation. Kahlon and Malhotra (1986) observed the high yield of GA production by immoblilized F. moniliforme at initial pH 5.5. Meanwhile, Escamilla et al. (2000) reported that the initial pH 5.0 was recorded as more suitable pH range for GA production by immobilized G. fujikuroi. Furthermore Meleigy and Khalaf (2009) indicated that there was significant increase of GA production by F. moniliforme γ-14 isolate (2.25 gL-1) at pH 5 after 6 days of incubation and lower yields of GA production were obtained at pH 3.0 (0.36 gL-1) and pH 7.0 (0.21 gL-1).

2.9.1.d. Agitation: Since biosynthesis of gibberellic acid involves many oxidative steps, a good aeration of fermentors is critical for an optimal production process. In fact, since the value of oxygen consumption for a growing mycelium in the exponential phase of growth remains constant, the demand of oxygen increases more or less exponentially (Tudzynski 1999). Jeffers (1970) suggests that the aeration must be as vigorous as possible, being reported, for SmF rotations between 150-1400 rpm. It is well recognized that a continuous supply of oxygen is required for production of GA3 (Jefferys, 1970), as the biosynthesis progresses through compounds of increasing levels of oxidation (Geissman et al., 1966). The interactions of different aeration and agitation rates and the consequent changes in oxygen and gas transfer processes were reported by some workers (Borrow et al., 1964a and Jefferys, 1970). These were caused mainly by the lower rate of oxygen supply or the conditions causing oxygen restriction and are (1) lower yields of acidic compounds, (2) diversion of metabolic pathways, (3) production of a new range of compounds, (4) development of estery smell in the medium, (5) unchanged yield constants

22 Literature Review for nitrogen, (6) introduction of linear growth phase, (7) lower utilization of glucose, (8) lower productivity, and (9) changes in biomass formation.

2.9.2. Nutritional conditions: The conditions for optimal production of GAs by G. fujikuroi are of biotechnological interest and have been studied intensively over the last 50 years (Darken et al., 1959; Borrow et al. 1955, 1964b; Geissman et al., 1966; Bruckner and Blechschmidt 1991; Candau et al., 1992; Avalos et al., 1997, 1999 and Tudzynski 1999).

2.9.2.a. Carbon source: Different workers have used a wide variety of carbon sources for the production of GAs. A distinction is made in slowly and readily utilizable carbon sources, and both are often used at various ratios. The salient features of the effect of carbon sources on fermentative production of GAs include (1) an inhibitory effect of a higher concentration of glucose on the specific growth rate of G. fujikuroi strain ACC 917 was found (Borrow et al., 1964a); (2) a decreased rate of production of GA, and overall productivity with an increase in the initial concentration of glucose and its level at the time of exhaustion of nitrogen during the course of fermentation was found (Borrow et al., 1964a); (3) a combination of readily and slowly metabolizable carbon sources gave a higher yield of GA3 (Darken et al., 1959); (4) there was a 300-559% increase in the yield of GAs with the use of natural oils such as linseed oil, sunflower oil, olive oil, cottonseed oil, and ethyl palmitate as compared to the yields on sucrose and a reported improvement in yield of 16.7% with linseed oil (Kumar and Lonsane, 1987); (5) there was a 49% decrease in the yield of GAs with stearic acid as compared to sucrose; (6) there was improved yield of GAs with the use of carbohydrate polymers such as plant meals or wheat bran in the media (Kumar and Lonsane, 1987); (7) the yield with glucose was better than that with sucrose (Darken et al., 1959) but was equal to that with lactose (Holme 23 Literature Review and Zacharias, 1965); (8) the improvement of yield was marginal when 3% glucose in the medium was supplemented with a 3% methanol, 3.5% ethanol, 1% malt extract (Sanchez-Marroquin, 1963); (9) the yield of GAs was ~ 0.5 g/liter in molasses residue, sulfite liquor, or skimmed milk media; (10) use of dairy waste as a carbon source resulted in 0.75 g GA,/liter in 1 2 days (Maddox and Richert, 1977); (11) production of GA, was inhibited by rice bran oil, although the rate of GA, formation was slightly enhanced for up to 5 days of fermentation (Kumar and Lonsane, 1987); and (12) the production was completely inhibited by 1 ppm geraniol due to total inhibition of cell growth (Kumar and Lonsane, 1987). Glucose and sucrose have often been used as carbon source, but concentrations above 20% of glucose at the beginning of the fermentation should be avoided, as it causes catabolic repression (Borrow et al., 1964a). Many workers have used alternative carbon sources, such as maltose, mannitol, starch and plant meals or mixtures of fast and slowly utilized carbon sources, e.g. glycerol, glucose and galactose (Darken et al., 1959; Sanchez-Marroquin, 1963; Maddox and Richert 1977; Kahlon and Malhotra 1986; Kumar and Lonsane 1989; Pastrana et al., 1995; Cihangir and Aksoza 1997; Tomasini et al., 1997 & Machado et al., 2001). The biosynthesis of gibberellins was indicated to be suppressed by high amount of glucose (> 20%), which had been the most commonly used carbon source for GA3 production (Kumar and Lonsane , 1989 and Burckner 1992). Alternative carbon sources, such as maltose, mannitol, glycerol and galactose increased the GA3 production rate (Darken et al., 1959 & Gancheva and Dimova, 1991). Gonzalez et al., 1994 found that the mixture of sucrose- starch was identified as the best carbon source for the GA3 production. Some oils such as sunflower oil and cooking oil have also been successfully used to enhance GA3 production (Gancheva et al., 1984 and

Hommel et al., 1989). The biosynthesis of GA3 is based on acetate and follows 24 Literature Review the isoprenoid pathway (Tudzynski 1999 & Kawanabe et al., 1983). Therefore, plant oil, as a carbon source is not only inert for carbon catabolite repression but also makes available a pool of acetyl CoA and additionally might contain natural precursors for GA3 biosynthesis. Several industrial residues such as milk whey, molasses, sugar beet pulp and hydrol has also been used as carbon sources. These residues give low but economically useful yield (Gohlwar et al., 1984 & Cihangir and Aksoz, 1996).

2.9.2.b. Nitrogen source: The involvement of nitrogen in growth, metabolism, and product formation is well known (Ribbons, 1970). A variety of organic and inorganic nitrogen sources, including those from plant and animal origin, were evaluated by different workers to study their effect on the production of GAs. Although ammonia was shown to be utilized in preference to nitrate, no information is available on its use in the fermentation processes for the production of GAs. Some of the important results on the efficiency of different nitrogen sources in the production of GAs include (1) among various nitrogen sources tested, ammonium nitrate at 1.0-4.0 g/liter concentration was the most adequate and higher yields were obtained by supplementation with corn steep liquor (Sanchez-Marroquin, 1963); (2) incorporation of nitrogenous compounds in whey medium resulted in higher biomass formation, but yields of GA3 were lower (Gohlwar et al., 1984); (3) increased total production of GAs was observed when corn extract in Raulin-Thom medium was replaced with soybean flour or any one of the nine fractions of soybean flour (Fuska et al., 1961); (4) addition of soybean and arachis flour to nutrient medium enhanced the production of GAs and shortened the fermentation period (Fuska et al., 1961); (5) the use of ammonium acetate resulted in poor productivity (Borrow et al., 1964a,b), however, good yields with ammonium acetate have also been reported (Nestyouk et al., 1961); (6) production of metabolites other than GAS was at a

25 Literature Review higher level in glycine-based media (Cross et al., 1963); (7) addition of thiourea in the medium of Borrow et al. (1955) gave slightly higher yields (Sanchez- Marroquin, 1963); (8) peanut or soybean meal were better sources than corn steep liquor; (9) GA3 was the only member of GAs to be synthesized in corn steep liquor medium; (10) substitution of corn steep liquor by soybean flour led to production of GA3 and GAI at 1 : 1 ratio and the concentration of GA3 thus produced was equal to that produced in corn steep liquor medium; (11) plant seed meals may contain some precursors for GAs); (12) higher yields of GAs were obtained by substituting ammonium succinate for ammonium nitrate and by adding corn steep liquor to the medium; and (13) a combination of ammonia nitrogen and natural plant meals, as well as the selection of their proper concentrations to match the gas transfer characteristics of the fermentor vessel, led to improved yield of GA3 (Jefferys, 1970). The most obvious regulatory principle is the strong repression of GA production by high amounts of nitrogen (e.g. ammonium, glutamine, glutamate, asparagine, nitrate) in the culture medium (Munoz and Agosin 1993& Bruckner and Blechschmidt 1991). The quality and quantity of nitrogen are very important for gibberellin fermentation because of the ammonium regulation of this process. All described media that guarantee high yields of GAs are low nitrogen media, as gibberellic acid production begins at, or soon after, nitrogen exhaustion (Borrow 1964a; Bruckner and Blechschmidt 1991 & Tudzynski 1999). Favorable nitrogen sources are ammonium sulfate, ammonium chloride and slowly assimilable sources as glycine, ammonium tartarate, plant meals and corn step liquor (Kumar and Lonsane, 1989).

The kinetics of growth and GA3 production in nitrogen limited medium was established. After the period of exponential growth, when the uptake of glucose, nitrogen, and phosphate remained constant, no GA3 was produced (Borrow et al., 1964a). All literature reported media yielding high amount of 26 Literature Review

GA3 contained low concentrations of nitrogen sources e.g. ammonium nitrate and ammonium chloride (Silva et al., 1999). Besides media with low ammonium or nitrate concentrations, complex ingredients such as corn steep liquor (Sanchez-Marroquin 1963 & Darken et al 1959), peanut meals (Fuska et al., 1961) and soya meal positively affected GA3 biosynthesis. It was suggested that plant extracts might be containing precursors or inducers of the

GA3 pathway. Nitrogen repression is a well regulatory principle of secondary metabolite formation (Munoz and Agosin 1993). In a mutant strain of G. fujikuroi, ammonium or nitrate ions was known to effect the production of GA3 while phosphate does not influence the biosynthesis of GA3 (Bruckner and Blechschmidt 1991 & Sanchez-Fernandez et al., 1997). It was found that the negative effect of ammonium or nitrate ions was due to both the inhibition of activity and the repression of de novo synthesis of specific gibberellin producing enzyme (Bruckner and Blechschmidt 1991). Candau et al. (1992) found that nitrate, ammonium and L-glutamine blocked gibberellin biosynthesis independently of whether present from the beginning or added to a producing culture. Also, Sanchez-Fernandez et al. (1997) investigated that all nitrogen sources tested including urea and many amino acids are effective inhibitors for gibberellin production in G. fujikuori. Munoz and Agosin (1993) investigated that when the fungus G. fujikuroi ATCC12616 was grown in fermentor cultures extracellular GA3 accumulation reached high levels when exogenous nitrogen was depleted in the culture and when ammonium or glutamine was added to hormone- producing cultures, extracellar GA3 did not accumulate. Sanchez-Fernandez et al., (1997) reported that gibberellin production in G. fujikuori starts up on exhaustion of nitrogen source and the authors concluded that nitrate inhibited gibberellin biosynthesis by itself, independently of the intra-cellular signal that conveys nitrogen availability.

27 Literature Review

Several enzymatic activities reached to nitrogen metabolism were expressed simultaneously with the onest of GA3 synthesis. This strongly suggests that all of these activities are nitrogen regulated and may be subject to the same type of control. The development of formamidase and urease activities was similar to that reported in other fungi (Munoz and Agosin, 1993). Kim et al. (2006) also showed that in the repeated batch cultures of immobilized G. fujikuroi cells in the bioreactor, the sole nitrogen of NH4No3 was replaced by cotton seed flour (CSF), the later was subsequently found to be an improved source for GA3 production than NH4No3. This finding was supported by reports that plant extracts in general, such as corn steep extracts, soybean, peanuts meals and rice flour, positively affect GA3 biosynthesis (Tudzynski, 1999 & Burckner, 1992).

2.9.2.c. Bio-elements: In spite of the pronounced effect of minerals and trace elements in the biosynthesis of secondary metabolites, negligible information is available on these aspects in the microbial production of GAs. The requirement for salts of Mg, K, and P in the production of GAs is well recognized, and in most cases the requirement was efficiently met by using the salt combinations from Czapek- Dox, Raulin-Thom or modified Raulin media (Borrow et al., 1955). Different combinations of KH2PO4, K2HPO4.H2O, KCI, &SO4, and MgSO4.7H2O along with various ratios of glucose and NH4NO3 were evaluated extensively by Borrow et al. (1961) to study the effect on growth and metabolism of G. fujikuroi in stirred culture. The data were analyzed in terms of morphology of mycelia, changes in pH, uptake of nutrients, and accumulation of mycelial fat, phospholipids, carbohydrates, and phosphorus-containing compounds. The results indicated the importance of mineral salts in the fermentative production of GAs.

28 Literature Review

It was reported that GA3 production was not induced in spite of phosphate depletion in the presence of excess nitrogen in a suspended- cell culture of G. fujikuroi (Candau et al., 1992 and Bruckner, 1992). No significant adverse effect of high concentrations of phosphate (KH2PO4) on GA3 had been reported for immobilized cell cultures (Escamilla et al., 2000).

2.9.3. Inoculum: Despite the importance of inoculum development, little work has been published that addresses the problem of inoculum optimization (DeTilly et al., 1983). Kahlon and Malhotra (1986) entrapped F. moniliforme in various alginate gel concentrations and obtained low yield of GA with increase in gel concentrations. Similar results were observed for sorbitol and clavulanic acid production by microbial immobilized cells on loofa sponge (Saudagar et al., 2008). Meleigy and Khalaf (2009) tested the immobilized inoculum (48 h old) in varying concentration (2-8 discs) to find the optimum immobilized fungal mycelium for GA production by F. moniliforme γ-14 isolate and the authors reported that the best yield of GA (1.9 gL-1) was obtained with 4 immobilized discs, at immobilized inoculum greater than 4 discs (6-8) the production of GA was inferior.

2.9.4. Working volume: Formation of gibberellic acid by a wild type of Gibberella fujikuroi strain IMI58289 was carried out in 500 ml flasks containing 250 ml of fermentation medium (Candau et al., 1992 and Sanchez-Fernandez et al., 1997). The fermentation experiments for GA production was carried out in 500 ml Erlenmeyer flasks, each containing 100 ml of the fermentation medium (Munoz and Agosin, 1993). Also, the initial working volume of the

29 Literature Review fermentation medium was 4.1L in 6L fermentor was recommended by Hollman et al., (1995) for gibberellic acid production by G. fujikuroi. Batch cultures from G.fujikuroi NRRL2284 were performed in 3 l fermentors containing 1.8 l optimizing medium for gibberellic acid production (Shukla et al., 2005). Gibberella fujikuroi cultures were grown in 250 ml flasks containing 50 ml of production medium for gibberellic acid synthesis (Johnson and Coolbaugh 1990 & Meleigy and Khalaf 2009).

2.10. Gibberellic acid improvement: The development of highly productive microbial strains is a prerequisite for efficient biotechnological processes. Strain improvement was mainly based on induced mutagensis. Mutagenic agents commonly used to induce mutagenesis include chemical mutagenic agents, ionizing radiation and other agents. Microbial strain for use in fermentation should be genetically stable, except when treated with mutagenic agents. Mutagenic agents act in a variety of ways, and the susceptibility of DNA to the different agents will depend upon the nucleotide sequence. Hence with a given mutagenic agent some genes will be mutated readily and others less often (JunShe et al., 2002). Mutagenic agents in common use include X-rays, UV, gamma radiation and chemical mutagen. Mutagen agents kill cells as well as mutating them, so investigations have to be carried out to determine the dose that gives the highest proportion of mutant cells among the survivors.

2.10.1. Effect of gamma irradiation on fungi secondary metabolites: It is known that all environments have a natural radioactivity of their own. Natural sources of radiation, over which little or no control can be exerted, are cosmic rays, radioisotopes generated in air envelope, radiant from earth and

30 Literature Review building material as well as radio active substances regularly found as natural constituents of the living body. On the other hand, artificial radiation can be attributed to radioactive material produced by nuclear explosions and radioactive wastes.

A. Ionizing radiation: Radiation is defined as a physical phenomenon in which energy travel through space without aid of a material medium. The radiant energy is a form of energy that travels through space in a wave motion (Ingram and Robert, 1980). Ionizing radiation includes x-rays and γ-rays. It also includes all the atomic and subatomic particles, such as electrons, positrons, protons, alphas, neutrons, heavy ions and mesons (Tsoulfanidis, 1983). Ionizing radiation is characterized by a very high energy content and great penetrating power. Ionizing radiation usually used in various peaceful applications, i.e. biological application, food irradiation, sterialization of chemical and pharmaceutical products. Ionizing radiation losses its energy in passing through materials. This energy may displace electrons from one orbital shell to another of higher energy to produce excited atoms. Ionizing radiation causes the loss of one or more orbital electrons, the atom is left positively charged and this makes it ionized, this is the reason for changes exerted by ionizing radiation (Silliker et al., 1980). Excited nuclei or other processes involving subatomic particles emit gamma radiation. In selecting one or another of these sources, the limitation of the use of elecrtones, due to their poor penetration should be considered. Of the irradiation process parameters, the most important is the amount of ionizing energy absorbed by the target material. This is termed "absorbed dose". The unit of absorbed dose was the Rad (1 rad= 10-2 J/kg).

31 Literature Review

Now Gray (Gy) is used instead of rad, which equal to 100 rad. One Gy is equal to the absorption of one joule per kg. The dose of radiation recommended by Food and Agriculture Organization / World Health Organization (FAO/WHO), Codex Alimentaris Commission for use in food irradiation does not exceed 10000 Gy (10 kGy). This is actually a very small amount of energy. Food receiving this amount of radiation is considered safe for human consumption (WHO, 1988). The dose employed is dependent on the level of the initial contamination (number of organisms), the kind of organisms, and the purpose of the treatment (FAO/IAEA, 1991). Ionzing radiation effect on living cells by direct and indirect effects. Direct effects are changes that appear because of the adsorption of radiant energy by the molecules being studied "targets". The indirect effect is occurred when target molecules are present in solution. The changes in the target molecules in the solution caused by the products of radiation decomposition (radiolysis) of water or other solutes. In the indirect effect, the process of radiolysis of water, which is the bulk (up to 90%) of the matter in cells, a number of atoms, free radicals and molecules are generated. They are atomic hydrogen, hydroxyl radical, solvated proton, hydrated electron, hydroxyl ion, + - - hydrogen molecule and hydrogen peroxide (H, OH, H3O , e aq, OH , H2, H2O2). The free radicals contained unpaired electrons and are therefore distinguished by exceedingly high reactivity. Their life time in water does not exceed 10-5 second. During this time, they either combine with one another to react with biological important molecules to possess the indirect action of radiation (Puchala and Schuessler, 1993 & Kovacs and Keresztes, 2002). Therefore, microorganisms are more resistant to radiation in the dry state than in the aqueous state. The effect of gamma radiation on microorganisms leads to different effects, i.e. growth inhibition, alteration in nutrient requirements, gene mutation, and changes in membrane permeability (Desrosier, 1970).

32 Literature Review

The descending order of resistance for various microorganisms was as follows. Virus > Bacteria spores > Yeast > Mould > Gram +ve bacteria > Gram –ve (Stegeman, 1981). Viruses are the most minute living entities and radiation resistant (FAO/IAEA, 1982). Although the molecular mode of action of some mutagens is quite well known, what can ever be predicted the effect of mutagen on a specific cell. In addition to the strain-specific cell, the treatment conditions including the mutagen concentration, exposure time, and growth phase of the organism may greatly effect on the efficiency of the mutagenesis process. By plotting dose- response curves, all these factors may be optimized (Adrio et al., 1993). The dose response curve represents the relation between the variation dose (kGy) and the number of microorganisms surviving. To obtain this relation, plot doses versus log N/No. wher N, represents the numbers of microorganisms have survived after irradiation. No, represents the initial number of microorganisms before irradiation. The radiosensitity of microoganisms is expressed in terms of D10 values. D10 values represent the dose of radiation required to reduce the viable count by a factor of 10 and can be obtained from the shape of the dose response curve by dividing dose by log N/No (Sztanyik, 1974) or calculated from the regression linear equation (Lawrence, 1971a). These values are useful in calculating sub-lethal doses and to know the relative sensitivity of microorganisms to gamma radiation.

B. Non-ionizing radiation: Non-ionizing radiation is electromagnetic radiation with wavelength (λ) of about 1.0 nm or longer. That part of the electromagnetic spectrum includes radiowaves, microwaves, visible light (λ = 390 to 770 nm) and ultraviolet light (λ = 5 to 400 nm). Ultraviolet radiation is a component of the sun radiation (less than 5%) and is also produced artificially in arc lamps e.g. in mercury arc lamp.

33 Literature Review

The ultraviolet radiation in sunlight is divided into three bands, UVA (320-400 nm), UVB (280-320 nm) and UVC below 280 nm (Halliday, 2005). A small amount of sunlight is necessary for good health; vitamin D is produced by the action of ultraviolet radiation on ergosterol, a substance present in human skin. The ultraviolet radiation also kills germs; it is widely used to sterilize rooms, exposed body tissues, blood plasma and vaccines. On the others hand ultraviolet radiation causes DNA damage, inflammation erythema sunburn, immunosuppressive, photoaging, gene mutations in skin and skin cancer (Melnikova and Anathaswamy, 2005). Several previous studies recorded that the low doses of gamma radiation may stimulate the microbial metabolic activities (El-Batal and Khalaf, 2003 & Khalaf and Khalaf, 2005). Improvement of the microbial producer strain (through subjecting the genetic material to physical and chemical mutagenic agents) offers the greatest opportunity for cost reduction without significant capital outlay (Stanbury et al., 1995 and Lotfy et al., 2007). This is achieved when a selected strain can synthesize a higher proportion of the product using the same amount of raw materials. One of the methods for increasing gibberellic acid production is the isolation of a mutant induced by various mutation methods. Meleigy and Khalaf (2009) exposured the spore suspension of Fusarium moniliforme to gamma radiation and isolated hyper gibberellic acid productive mutant γ-14. GA production by the mutagenized γ-14 isolate was approximately 2.1 fold increase when compared to the wild type. A high yield mutant producing gibberellic acid was obtained by the mutagenic treatment of UV irradiation (Lale et al., 2006). They isolate a mutant of G.fujikuroi Mor-25 that led to lower viscosity in fermentation broth resulted in increased production of gibberellic acid twofold more than the parent.

34 Literature Review

2.11. Immobilizaton: The immobilization of enzymes or whole cells offers many advantages, chiefly repetitive use, improved stability, enzyme contamination-free product, and easy stoppage of the reaction by removing the enzyme or the cells from the reaction mixture. The successful use of immobilized enzymes and immobilized whole cells in laboratory- and industrial-scale processes has evoked worldwide attention, leading to application of these techniques to other products as well as to improve the techniques (Kumar and Lonsane, 1988 and Silva et al., 2000). Among the immobilized systems, immobilization of whole cells provides a means for entrapment of multistep and cooperative enzyme systems present in the intact cell. Three different types of microbial cells are employed in whole cell immobilization; these include dead or treated cells, resting cells, and growing cells. The latter is of commercial importance, as the cells are kept in the growing state within a gel matrix by constantly supplying suitable nutrients. Immobilized growing cells have been shown to offer advantages, such as (1) superior stability due to protection of cells by physicochemical interactions between gels and cells; (2) protection of growing cells against unfavorable environmental factors; (3) changed permeability of cells favoring high penetration of substrate; (4) faster removal of end products from fermentation vessels; and (5) the renewable, self-regenerating, or self-proliferating nature of the biocatalytic system (Kahlon and Malhotra, 1986).

Reports on the continuous fermentation for the production of GA3 are available (Holme and Zacharias 1965 and Bu`Lock et al., 1974). There are many published reports of immobilized biocatalysts such as enzymes, microorganisms, organelles, and plant and animal cells (Kumakura and Kaetsu, 1983, Kumakura et al., 1989 and Lu et al., 1995) that being explored in the search for yield increase in the biotechnology processes. Recently, the immobilization of whole growing cells has been studied, mainly because of its economical potential. Advantages of an immobilized system based on living 35 Literature Review cells include their active metabolic ability to synthesize various useful and complicated bioproducts using the multi-enzyme steps, and the regeneration capability to prolong their catalytic life (Vignoli et al., 2006). Immobilization of microorganisms with a number of techniques including encapsulation, entrapment in polymer gels, and adhesion onto the surface of carriers has been reported (Aykut et al., 1988; Mozes and Rouxhet, 1984). Of these various methods adhesion to carriers has the advantage of simple preparation involving low cost procedures and the preservation of viability and activity. When compared with encapsulation or entrapment in polymer gel matrices, the immobilization by cell adhesion also has advantages in a lower diffusion limitation in the transport of substrates, products, and oxygen (Saudagar et al., 2008). The major disadvantages of the encapsulation and entrapment techniques are release of the cells due to weak binding to carriers and are expensive (Hermesse et al., 1987). Numerous matrices constructed from synthetic polymers or biological materials have been used for the immobilization of biocatalysts (Furusaki and Seki, 1992). Some of the synthetic matrices may be toxic (Macaskie et al., 1987). Biogels on the other hand, lack open spaces to accommodate new cell growth which then result in their rupture and the release of cells into the medium, also their efficiency is limited by diffusion restrictions, and decreased enzyme activity beside it is expensive (Iqbal et al., 1993). A matrix for immobilization should ideally be strong, resistant to operating conditions, and preferably have an open structure as well as inexpensive. The plant-derived loofa sponge (Luffa cylindrica) is an inexpensive and easily available biological, and therefore, renewable, matrix produced in most of the tropical and subtropical countries. Merits of the loofa sponge as biomatrix include freedom from materials that might be toxic to microbial cells, simple application and operation technique, and a high stability during long-term 36 Literature Review repeated use (Iqbal et al., 1993 and Ogbonna et al., 1997). The high void volume, permeability, and low cost of fibrous matrices make it particularly attractive. The simplicity of the immobilization technique, the strong binding and the low cost of the loofa sponge can help to find future applications for whole cells immobilization for various applications. Recently, the viability of using loofa sponge as a carrier for microbial cells was studied successfully (Vignoli et al., 2006 and Saudagar et al., 2008). Immobilization of G.fujikuroi has been reported, (Heinrich and Rehin, 1981, Surinder and Malhotra, 1986, Kumar and Lonsane, 1988 and Jones and Pharis, 1987, Saucedo et al., 1989 ). The GA3 is commercially produced by the fermentation of G. fujikuroi (Tudzynski, 1999 and Kim et al, 2006). Efforts for process optimization and strain improvement to enhance commercial

GA3 production by using suspended- cell cultures approached a saturation point (Kumar and Lonsane, 1988).

Lu et al., (1995) reported that the GA3 production by G. fujikuroi immobilized on polymeric fibrous carriers was maintained at a constant value of about 210 mg L-1 during 12 consecutive batch fermentation cycles over an 84 day period in flask cultures. Escamilla et al. (2000) optimized the pH, C:N ratio, rice flour concentration and temperature for GA3 production by immobilized G.fujikuroi in Ca-polygalacturonate in a batch fluidized bioreactor and obtained a product concentration 3 fold more greater than those previously reported for either suspended and solid culture. Kim et al., (2006) studies the performance of immobilized G. fujikuroi for gibberellic acid production on celite beads and the authors investigated that repeated incubations of immobilized fungal cells increased cell concentrations and volumetric productivity and they concluded that the maximum volumetric productivity obtained in the immobilized cell culture was 3 fold greater than that in suspended-cell culture. 37 Literature Review

The immobilized-cell culture systems are known to achieve high cell density while maintaining high mass transfer rates and thereby offer the advantage of high productivity (Gbewonyo and Wang, 1983, Na et al., 2005, Lim et al., 2006 and Ibrahim et al., 2006). With immobilized- cell cultures, cells can be used repeatedly there by minimizing substrate consumption for cell growth, and the substrate feeding process can be optimally controlled further enhancing substrate utilization efficiencies (Deo and Gaucher, 1985; Lu et al, 1995 and Sarra et al 1997).

Kim et al (2006) observed that GA3 production began only in the later stage of cultivation in suspended- cell cultures and alternatively, the immobilized cells started to produce GA3 day 1. The same physiological characteristics of GA3 biosynthesis have been observed in immobilized- cell cultures (Duran-Parampo et al., 2004 and Nava Saucedo et al., 1989). Meleigy and Khalaf (2009) used loofa sponge for immobilization of F.moniliforme γ-14 cells for GA production and the authors reported that the best yield of GA (1.2 gl-1) with conversion rate 2.54 % and productivity rate 8.33 mg1-1h-1 were obtained by immobilized cells after 6 days. Kahlon and Malhotra (1986) and Lu et al. (1995) investigated that the yield of GA production by immobilized F.moniliforme cells in sodium alginate gel was more than that produced by free cells. Gibberellic acid productivity by Gibberella fujikuroi cells immobilized with the carrier covered copolymer of hydrophilic hydroxyl ethyl acrylate and hydrophobic trimethylpropane was higher than that in the free cells (Lu et al., 1995 and Saudagar et al., 2008). The higher yield with an immobilized system depends up on the nature of material matrix, which affects the permeability of the cell towards high penetration of the substrate, as well as faster removal of the end products from the fermentation sites, due to protection of the mycelium from micro

38 Literature Review environmental changes by the material matrix (Lu et al, 1995; Saudagar et al., 2008 and Meleigy and Khalaf, 2009).

2.12. Gibberellic acid production from different wastes: In Egypt, milk permeate is a cheap carbon source available in plenty as a dairy by-product, which has not been put to any economical use and it is discharged into dairy effluents. Inspection of a typical composition of milk permeate reveals that it is reasonably high in lactose and contains minerals necessary for microbial growth, but it is low in nitrogen (Yellore and Desai, 1998). Maddox and Richert (1977) reported that the lactic casein whey filtrate could be used as a basal medium for gibberellic acid production. Lactic casein whey is the material remaining after the removal of casein from whole milk. The typical composition of whey filtrate reveals that it is reasonably high in lactose and contains minerals necessary for microbial growth and it is low in nitrogen content, thus it could be suitable for the production of fungal secondary metabolites. Meleigy and Khalaf (2009) reported that milk permeate is a suitable medium for gibberellic acid production in repeated batch operation by a mutant F. moniliforme cells immobilized on loofa sponge.

2.13. Fungal chitosan production: Chitosan is a natural polysaccharide synthesized by a great number of living organisms. Chitosan [poly-(β-1/4)-2-amino-2-deoxy-D-glucopyranose] is a collective name for a group of partially and fully deacetylated chitin compounds (Tikhonov et al., 2006). Chitosan is derived by deacetylation of naturally occurring biopolymer chitin, which is present in the exoskeleton of crustacea such as crab, shrimp, lobster, crawfish and insects, and is considered to be the second most abundant polysaccharide in the world after cellulose.

39 Literature Review

Chitosan can also be found in the cell wall of certain groups of fungi, particularly zygomycetes. It is a straight chain natural hydrophilic polysaccharide having a three dimensional α-helical configuration stabilized by intramolecular hydrogen bonding (Kas, 1997)

The basic structure of chitin Fully deacetylated chitosan

Chitosan is obtained by chemical conversion of chitin fig (2-6), which is a constituent of the exoskeleton of crustacea and insects. An alternative source of chitosan is the cell wall of fungi. Shell waste from shrimp, crab and lobster processing industries is the traditional source of chitin. However, commercial production of chitosan by deacetylation of crustacean chitin with strong alkali appears to have limited potential for industrial acceptance because of seasonal and limited supply, difficulties in processing particularly with the large amount of waste of concentrated alkaline solution causing environmental pollution and inconsistent physico-chemical properties. However, uniform deacetylation is a prerequisite to specific industrial applications.

40 Literature Review

Fig (2-6): Chemical conversion of chitin.

With advances in fermentation technology chitosan preparation from fungal cell walls becomes an alternative route for the production of this polymer in an ecofriendly pathway (Rane and Hoover, 1993; Crestini, et al., 1996; Tan et al., 1996). Fungal culture media and fermentation condition can be manipulated to provide chitosan of more consistent physico-chemical properties compared to that derived chemically from chitin. However, new research has been carried out on the use of alternative sources for chitosan, the studies were focused mainly on chitosan from fungi. Fungi are easily grown, produce high yield of biomass and extensively used in a variety of industrial fermentation processes. The production and purification of chitosan, from the cell walls of fungi grown under controlled conditions, offer a greater potential for more consistent products. Nowadays, Aspergillus niger is almost exclusively used for industrial scale production of citric acid. More than 600,000 metric tons are produced annually worldwide (Anastassiadia, et al., 2002). The fungal mycelia produced as by-products of fermentation industries could be considered as a promising potential source for the isolation of chitin and/or chitosan.

41 Literature Review

The ideal antimicrobial polymer should possess the following characteristics: (1) easily and inexpensively synthesized, (2) stable in long-term usage and storage at the temperature of its intended application, (3) not soluble in water for a water-disinfection application, (4) does not decompose to and/or emit toxic products, (5) should not be toxic or irritating to those who are handling it, (6) can be regenerated upon loss of activity, and (7) biocidal to a broad spectrum of pathogenic microorganisms in brief times of contact (Kenawy et al., 2007). As a natural poly-aminosaccharide, chitosan possesses many of these attributes. Antimicrobial activity of chitosan has been demonstrated against many bacteria, filamentous fungi and yeasts (Kong et al., 2010, Sajomsang, et al., 2012 and Martinez-Camacho et al., 2010). Chitosan has several advantages over other chemical disinfectants since it possesses a stronger antimicrobial activity, a broader spectrum of activity and high killing rate against Gram-positive and Gram-negative bacteria, but lower toxicity toward mammalian cells (Franklin and Snow, 1981 andTakemono et al., 1989).

2.13.1. Uses of chitosan: Chitosan being polycationic, nontoxic, biodegradable as well as antimicrobial finds numerous applications especially in the agriculture, food and pharmaceutical industries, such as food preservation (Xia et al., 2011; Kean and Thanou 2010; Kong et al 2010; Dutta et al., 2009), fruit juice clarification (Rungsardthong et al., 2006) water purification particularly for removal of heavy metal ions (Bhatnagar and Sillanpaa 2009); sorption for dyes and flocculating agent (Srinivasan and Viraraghavan 2010). Chitosan can also be used as a biological adhesive for its hydrogel-forming properties (Ono, et al., 2000), wound healing accelerator (Paul and Sharma 2004), also in cosmetics industries, nutrition (emulsifying, thickening and stabilizing agent, packaging

42 Literature Review membrane, antioxidant and dietary supplement), biotechnology (enzyme immobilization), water engineering (flocculants, chelating agent for metals), medical applications (artificial skin, blood anticoagulants, drug-delivery systems) and, recently, in gene therapy (Sandford, 1989; Li, et al., 1997 and Rinaudo, 2006). Fungal chitosan possesses two advantageous properties for medical applications- a lower molecular weight and lower contents of heavy metals. Production of fungal chitosan from food processing by-products such rinse washes from distilleries, molasses, or soybean and mungbean residues has also been reported. ( Suntornsuk et al., 2002).

2.13.2. Influence of gibberellic acid on the growth of Aspergillus niger and chitosan production: It is known that plant hormones are involved in several stages of plant growth and development, but only a few conflicting reports are available regarding their effect on growth of microorganisms. Makarem and Aldridge 1969 found that gibberellic acid at an optimum concentration of 10 mg/L increased cell division rate of different strains of Hansenula wingei. -4 Tomita et al.,1984 showed that GA3 at the concentration of 10 M helped better growth and development of N. crassa Also, Michniewicz and -9 Rozej 1988 reported that GA3 at the concentration of 10 helped better growth and development of F.culmorum. Paul et al., 1994 observed stimulation of growth of food yeast, K. fragilis, in whey medium supplemented with gibberellic acid (10mg/l). Chatterjee et al., (2005) stated that the growth of M.rouxii under submerged fermentation condition in molasses salt medium was maximum at sucrose concentration 4% and further increase in sucrose concentration did not improve the growth. Chatterjee et al., (2008) showed that maximum enhancement of growth and chitosan production by Rhizopus oryzae in whey medium was observed with

43 Literature Review gibberellic acid. The author found that fifty percent more chitosan could be obtained from 1 L of whey containing 0.1 mg/L gibberellic acid. Meanwhile, hormone, at higher dose, instead of stimulation inhibited both growth and mycelial chitosan content. Chatterjee et al., (2009) investigated that supplementation of molasses salt medium with plant growth hormone (gibberellic acid), increased chitosan production by Mucor rouxii as well as its growth at different optimum concentrations. The increase in yield of chitosan was found to range from 34% to 69% and mycelia growth from12% to 17.4%. Also the authors reported that the optimum GA3 concentration was 3 mg/l, give the highest stimulation, in both mycelia growth (9.3 g/l) and chitosan production (11.2% of mycelia).The author added that, it may be surmised that the addition of plant growth hormones to the fermentation medium of M. rouxii might be an economical way of enhancing chitosan production by this fungus. Tayel et al., (2011) mentioned that the waste biomass of Aspergillus niger, following citric acid production, was used as a source for fungal chitosan extraction. The authors added that The A. niger productivity of mycelia dry weight, after the completion of fermentation process, was recorded to be 11.34 g/l, whereas the produced chitosan weight was 1.32 g/l of fermentation medium. The chitosan yield (g of chitosan/g of dry biomass×100), however, was calculated to be of 11.64%.

44 Material and Methods

3. Material and Methods

3.1. Material: 3.1.1. Samples: Soil and grain samples were used in the present study for isolation of gibberellic acid (GA) producing fungi. The soil samples were collected from the rhizosphere soil of different plants name: beans, egg plant, tomato, potato and onion. The grain samples (white corn, yellow corn, barely and wheat) were collected from the local market. 3.1.2. Standard chemical used: Gibberellic acid and chitosan were purchased from Sigma Company, USA. 3.1.3. Media used: Several media were prepared for isolation, maintenance, and gibberellic aicid production of fungi: all the following components for broth media are in g/L. For solidification of media 15 g/L of agar were added. All media were sterilized by autoclaving at 121 ºC for 15 min. 3.1.3.a. Isolation and maintenance media: Medium 1: Potato Dextrose PD (Oxoid, 1981): Potato 200.0 Dextrose 20.0 pH 5.6

Medium 2: GPI (Silva, et al., 2000) Glucose 80.0 NH4NO3 0.75 MgSO4.7H2O 1.5 KH2PO4 3.0 Rice flour 2.0 pH 5.5

45 Material and Methods

3.1.3.b. Gibberellic acid production medium: Medium 3: GPI (see 3.1.3.a.) Medium 4: Czapek`s-Dox medium (Uthandi et al., 2010): Yeast extract 5.0 Sucrose 30.0 Sodium nitrate 30.0 Magnesium sulphate 0.5 Potassium chloride 0.5 Ferrous sulphate 0.01 Dipotassium hydrogen phosphate 1.0 pH 6.4

Medium 5: GPII (Atez et al., 2006) Glucose 4.0 NH4Cl 1.0 Rice flour 2.0 KH2PO4 3.o MgSO4 1.5 pH 5.0

Medium 6: Whey medium: Whey was obtained from Agriculture Research Center (Giza, Egypt). The pH of whey was adjusted to 4.5 by adding 1N HCl to remove the excessive proteins in it (Yellore and Desai, 1998). After filteration, the solution was boiled and cooled, excess protein was removed. The clear solution formed (which containing total sugar 67 gL-1 and nitrogen content 3.8 gL-1) was adjusted to pH 5.0 with 1N NaOH and divided into 50 ml in 250 ml Erlenmeyer flasks. The whey in flasks was autoclaved at 121 ºC for 10 min and it was used as complete production medium of GA. 3.1.3.c. Seed culture medium: (Sanchez-Marroquin, 1963) Medium 7: Glucose 30.0 NH4NO3 1.65 MgSO4.7H2O 5.0 Corn steep liquor 1.5 ml pH 5.0

46 Material and Methods

3.1.3.d. Chitosan production media: Medium 8: Molasses salt medium: (Chatterjee et al., 2005) Molass 70 NaNO3 2 K2HPO4 0.1 FeSO4 0.01 MgSO4 0.01 Yeast extract 2 pH 5.0

Medium 9: Yeast peptone glucose medium (YPG): (Chatterjee et al., 2005) yeast extract 3 Peptone 10 Glucose 20 pH 5.0

Medium 10: Potato dextrose broth (PDB): (See Medium 1)

3.2. Methods: 3.2.1. Isolation of gibberellic acid producer fungi on solid medium: Ten grams of each sample were put into 250 ml Erlenmeyer flasks containing 90 ml sterile saline solution (0.85% NaCl). The flasks were shaken on an electric shaker for 5 min. Serial dilutions were made for each flask, then 1 ml from the dilutions (10-4-10-6) was transferred to sterile plates. The sterilized specific gibberellic acid production medium GPI (3.1.3.b) was cooled to 45 ºC before pouring on the plates. The solidified plates were incubated at 28 ºC for 7 days. The emerged fungi were selected and purified on the same medium using single spore technique (Smith, 1961), then, the pure colonies were inoculated on PDA (potato dextrose agar) slants to make stock cultures. The fungal isolates were identified according to the key of Gilman (1957) and Pitt and Hocking (1985)

47 Material and Methods

3.2.2. Isolation of gibberellic acid producer fungi on broth medium: The purified fungal isolates were screened for gibberellic acid production on GPI broth medium. Each isolate was grown on PDA agar flasks for 10 days at 28 ºC. After incubation period, about 10 ml of sterile saline solution containing 0.1% tween 80 were added to each flask and the spores were scratched by sterile needle, then the suspension was centrifuged twice at 3000 rpm for 15 min. The suspended spores for each isolate were collected and adjusted to 5x106 CFU/ml by sterile saline solution. Erlenmeyer flasks (250 ml) each containing 50 ml of sterile GPI medium (initial pH 5.5) were inoculated with 1 ml spore suspension from each tested isolate. The inoculated flasks were incubated at 30 ºC for 7 days at 150 rpm. After incubation period, the fermented media were filtered and the filtrate was used to determine the gibberellic acid content according to Kahlon and Malhotra (1986) using spectrophotometer (ATI Unicam, 5600 Series UV/VIS) at 254 nm. Also, microbial growth was determined by drying the filter paper for 24h at 60 ºC to a constant weight. The highly producer gibberellic acid isolate was selected and used for further investigation in this study. 3.2.3. Factors affecting gibberellic acid Production by Fusarium moniliforme: The highly gibberellic acid producer fungal isolate Fusarium moniliforme (previously named Gibberella fujikuroi) was selected for the investigation of some conditions affecting gibberellic acid production in flask batch cultures. These factors include different environmental and nutritional conditions.

48 Material and Methods

3.2.3.a. Influence of specific media: Different media for gibberellic acid production (GPI, GPII, Czapek`s yeast extract) were used in this study. Erlenmeyer flasks (250 ml) each containing 50 ml of each medium were autoclaved at 121 ºC for 15 min. The pH of the media was adjusted by 1 N NaOH or HCl at 5.5, 5.0, 6.4, respectively. Each flask was inoculated by 1 ml of F.moniliforme spore suspension (5x106 CFU/ml). The inoculated flasks were incubated at 30 ºC for 7 days under shaking condition (150 rpm). After incubation period, the fermented media were filtered through Whatman filter paper No.1 and the supernatants were used to estimate gibberellic acid content (3.3.1) and residual sugar. Also, microbial growth was determined as described above. 3.2.3.b. Influence of incubation periods: Fifty milliliters of sterile GPI medium (the best gibberellic acid production medium) in 250 ml Erlenmeyer flasks at initial pH 5.5 were inoculated by F.moniliforme spore suspension as described above, and incubated at 30 °C with shaking (150 rpm) for 9 days. Samples were withdrawn at regular intervals 24 hr for estimation of gibberellic acid content, residual sugar and microbial growth. 3.2.3.c. Influence of incubation temperature: Different incubation temperatures were studied for their effect on gibberellic acid production. Fifty milliliters of sterile GPI medium (initial pH 5.5) in 250 ml Erlenmeyer flasks, were inoculated as above and incubated at different temperatures (20, 25, 30, 35 and 40 °C) with shaking at 150 rpm. After 6 days (the optimal incubation period for gibberellic acid production), mycelia were removed by filtration and the filtrates were used to estimate the gibberellic acid content and residual sugar.

49 Material and Methods

3.2.3.d. Influence of initial medium pH: The initial pH of the GPI medium was adjusted with 1 N NaOH or 1 N HCl to different values ranging from 3 to 7.5, then the flasks inoculated and incubated at 30 °C (the best incubation temperature for gibberellic acid production) for 6 days with shaking at 150 rpm. Gibberellic acid content and microbial dry weight were determined as described above. 3.2.3.e. Influence of different aeration regimes: Fifty milliliters of sterile GPI medium (pH 5.0, the best pH degree for gibbe F.moniliforme rellic acid production) were inoculated by 1 ml spore suspension of G. fujikuroi and incubated at 30 °C for 6 days, pH 5.0 under different agitation speeds (0, 50, 150, 200, 250 and 300 rpm). The gibberellic acid content and microbial growth were estimated as described above. 3.2.3.f. Influence of carbon sources: Different carbon sources namely: glucose, fructose, galactose, lactose, sucrose, manitol, maltose, starch, xylose and glycerol, were separately added to the GPI medium in a concentration of 8 % (w/v). The inoculated flasks (initial pH 5.0) were incubated under shaking (200 rpm, the best shaking rate for gibberellic acid production)) at 30 °C. After 6 days of incubation, the mycelia were removed by filtration and estimated, also the filtrates were used to determine gibberellic acid content. 3.2.3.g. Influence of fructose concentration: Different concentrations of fructose (the best carbon source for gibberellic acid production) 2, 3, 4, 5, 6, 7, 8 and 9 % (w/v) were added separately to 50 ml of GPI medium (without glucose) in 250 ml Erlenmeyer flasks. The flasks were inoculated and incubated as described above. The produced gibberellic acid and microbial dry weight were determined after 6 days of incubation.

50 Material and Methods

3.2.3.h. Influence of nitrogen sources: Various sterile solutions of organic and inorganic nitrogen sources (malt extract, yeast extract, tryptone, beef extract, soy peptone, soyabean, pea nut, glycine, ammonium sulfate, ammonium phosphate, ammonium chloride, ammonium nitrate, potassium nitrate and ammonium tartarate) were added separately to the GPI medium (containing 6 % w/v fructose as the best concentration of carbon source for gibberellic acid production) in amounts calculated to give equal final nitrogen concentration (0.75 g/l). The inoculated flasks (initial pH 5.0) were incubated at 30 °C with shaking at 200 rpm. After 6 days of incubation, the gibberellic acid concentration in the filtrate was estimated. Also, the biomass content was determined. 3.2.3.i. Influence of different ammonium sulfate concentration:

Sterile (NH4)2SO4 solution, the best nitrogen source for gibberellic acid production, was added to 50 ml of sterile GPI medium, as only sole of nitrogen source, in 250 ml Erlenmeyer flasks, to make final concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9 g/L as nitrogen. The inoculated flasks, at initial pH 5.0, were incubated at 30 °C with shaking at 200 rpm for 6 days. The gibberellic acid production and microbial dry weight were estimated at the end of incubation period. 3.2.3.j. Influence of different rice flour concentration: Different concentrations of rice flour 1, 2, 3 and 4 g/L were added to GPI medium (containing 6 % fructose as carbon source and 0.06 % ammonium sulfate as nitrogen source). The flasks were inoculated and incubated as described above. The gibberellic acid produced and microbial dry weight were determined after 6 days of incubation. 3.2.3.k. Influence of different magnesium sulfate concentration:

Different concentrations of MgSO4.7H2O 0.5, 1.0, 1.5, 2 and 2.5 g/L were added separately to Erlenmeyer flasks containing 50 ml of GPI medium

51 Material and Methods

(fructose 6%, (NH4)2SO4 0.06%) . The flasks were inoculated and incubated at 30 °C under shaking at 200 rpm. After the end of incubation period (6 days), the gibberellic acid content and mycelium dry weight were determined. 3.2.3.l. Influence of different potassium dihydrogen phosphate concentration:

Different concentrations of KH2PO4 up to 5 g/L were used to study their effect on gibberellic acid production by F.moniliforme. The gibberellic acid content and microbial growth were determined as mentioned before. 3.2.3.m. Influence of inoculum type: Different types of F.moniliforme inoculum (spore suspension, seed culture and mycelium disc) were prepared to study their effect on gibberellic acid production. For preparation of seed culture 1.0 ml of fungal spore suspension (5 x 106 CFU/ ml) was inoculated in seed culture medium (3.1.3.c) and incubated under shaking (150 rpm) at 30 °C for 2 days. After 2 days, 1 ml of the fungal growth was used as inoculum. For preparation of mycelium fungal growth, the fungus was inoculated on PDA plates at 28 °C for 7 days. After that the fungal growth was cut into 8 mm discs by sterile cork porer, and one disc was used as inoculum. Erlenmeyer flasks containing 50 ml of sterile optimized

GPI medium (fructose 6%, (NH4)2SO4 0.06%, MgSO4 0.15%, KH2PO4 0.1%) were separately inoculated by 1 ml spore suspension, 1 ml of 48 h age seed culture and/or one mycelium disc (8mm) from the F.moniliforme. The inoculated flasks (initial pH 5.0) were incubated at 30 °C with shaking at 200 rpm for 6 days, and then analyzed for gibberellic acid production and biomass content. 3.2.3.n. Influence of inoculum age: The effect of seed culture, the best type of inoculum, age on gibberellic acid production was studied by growing the spore suspension of the fungus on seed culture medium for different incubation periods (12-48 h). One ml from

52 Material and Methods each seed culture was inoculated separately in Erlenmeyer flasks (250 ml) containing 50 ml of sterile optimized GPI medium at pH 5.0. The inoculated flasks were incubated at 30 °C with shaking at 200 rpm. At the end of incubation periods (6 days), the culture media were filtered and the gibberellic acid content and fungal dry weight were determined. 3.2.3.o. Influence of inoculum density: Fifty milliliters of sterile optimized modified GPI medium (initial pH 5.0) in 250 ml Erlenmeyer flasks were inoculated by 24 h seed culture, the best inoculum age for gibberellic acid production, with different inoculum sizes (1, 2, 3 and 4 %) and incubated at 30 °C with skaking at 200 rpm for 6 days. At the end of incubation time the gibberellic acid production and mycelium dry weight were estimated. 3.2.3.p. Influence of working volume: Erlenmeyer flasks (250 ml) containing different volumes of sterilized optimized modified GPI medium (25-150 ml) were inoculated by 24 h seed culture (at rate 2 %, the best inoculum density) and incubated at 30 °C, with skaking at 200 rpm for 6 days. After incubation time, the culture media were filtered and the gibberellic acid and fungal biomass were determined as described above. 3.2.4. Effect of gamma radiation on F.moniliforme activity: The effect of different doses of gamma radiation on fungus growth and its gibberellic acid production were investigated. Irradiation was carried out at the National Center for Radiation Research and Technology (NCRRT), using 60Co gamma irradiation source of Indian facility with a dose rate (3.056 kGy/h) at the time of experiments.

3.2.4.a. Determination of D10-value: Five milliliters of the F.moniliforme spore suspension (5 x 106 CFU/ml) was transferred to sterile tubes and exposed to increase doses of gamma

53 Material and Methods radiation (0.0, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0,3.5, 4.0, 4.5 and 5.0 kGy) at ambient temperature (in triplicates for each dose). Serial dilutions of 1 ml from each tube were made in 9 ml sterile saline solution. Then, 1 ml from the dilutions (10-1-10-6) was transferred to sterile Petri dishes in triplicates. The pre- cooled GPI medium (45 °C) was poured on the plates mixed well and then incubated at 28 °C for 5 days. The log number of the fungal counts was determined and the dose response curve was plotted. D10-value was calculated from regression line equation (Wholesomeness of Irradiated Food, 1981) as follow: Y = a + bx −1 ∑ xy − nx`y` D-10 value = b = b ∑ x 2 − nx`2 Where, b = regression factor x = dose level (kGy) y = log microbial count n = number of calculated points x y x` = ∑ y`= ∑ n n Also, the correlation coefficient (r) of this equation can calculated as follow: ∑ y ∑ xy − ∑ x r = n 2 2 ⎡ ()x ⎤ ⎡ ()y ⎤ ⎢ x 2 − ∑ ⎥ ⎢ y 2 − ∑ ⎥ ∑ n ∑ n ⎣⎢ ⎦⎥ ⎣⎢ ⎦⎥ 3.2.4.b. Effect of gamma irradiation on gibberellic acid production: The remaining irradiated spore suspension of each dose was used for preparation of 24 h inoculum age in seed culture medium. Sterile 50 ml of optimized GPI medium in 250 ml Erlenmeyer flasks were inoculated with 1 ml fungal seed culture inoculum prepared above. Control treatment was carried out

54 Material and Methods with fungal inoculum (24 h age in seed culture medium) prepared from non irradiated spore suspension. The inoculated flasks (at initial pH 5.0) were incubated at 30 °C for 6 days under shaking at 200 rpm. The gibberellic acid and biomass production were estimated as described before. 3.2.5. Effect of immobilization on gibberellic acid production by gamma irradiated spores: According to methods described by West and Strohfus (1996), the sponge (which derived from Egyptian Company of sponge, located in 6th Oct. City) was cut into cubes (1 cm3) and soaked in water for a minimum of 10 times with continual rinsing, then dried at 80 ºC over night. The dried cubes (0.5 g) were added to Erlenmeyer flasks (250 ml) and autoclaved. Then 50 ml of sterile seed culture medium and 1 ml of spore suspension (5x106 CFU/ml) exposured to 0.5 kGy were added. The flasks were incubated at 30 °C under shaking at 150 rpm. After 24h of incubation, the culture medium was removed and the cubes were washed twice with 50 ml of sterile NaCl solution (0.85 %), then 50 ml of sterile optimized GPI medium (pH 5) were added to each flask containing the immobilized sponge cubes (0.5 g). The flasks were incubated at 30 °C with shaking at 200 rpm. Control treatment was carried out with non immobilized irradiated cells (free seed culture cells). After 6 days of incubation, the gibberellic acid was determined as described before, and the sponge cubes were dried at 50 °C to a constant weight to determine the fungus biomass. 3.2.6. Effect of immobilized inoculum age: Fifty milliliters of seed culture medium were added to Erlenmeyer flasks (250 ml) containing 1 ml of gamma irradiated (0.5 kGy) F.moniliforme spores and 0.5 g of sterile sponge cubes. The flasks were incubated at 30 °C with shaking 150 rpm. Three flasks were taken from the shaker after 12, 24, 36 and 48 h and the culture media were removed and the sponge cubes were washed twice. To each flask, 50 ml of sterile optimized modified GPI medium (initial

55 Material and Methods pH 5.0) were added, and then incubated at 30 °C with shaking at 200 rpm for 6 days. After incubation period, the gibberellic acid and biomass were determined. 3.2.7. Effect of inoculum density of immobilized cells: Erlenmeyer flasks containing different weight of sponge cubes, 0.25, 0.5, 0.75 and 1.0 g were autoclaved. Sterile 50 ml of seed culture medium and 1 ml of gamma irradiated F.moniliforme spores were added to each flask, then the flasks were incubated for 24 h (the best inoculum age of immobilized cells) at 30 °C, under shaking (150 rpm). After that the culture media were removed and sponge cubes were washed. To each flask 50 ml of sterile optimized mdified medium (initial pH 5.0) were added and then incubated at 30 °C with shaking (200 rpm). After 6 days of incubation, the gibberellic acid and biomass content were determined. 3.2.8. Time profile of gibberellic acid production by immobilized cells: Fungal inoculum was prepared by adding 1 ml of gamma irradiated (0.5 kGy) F.moniliforme spores to Erlenmeyer flasks containing 50 ml of sterile seed culture medium and 0.5 g of sterile sponge cubes (the best inoculum density of immobilized cells). The flasks were incubated at 30 °C under shaking (150 rpm) for 24 h. Then the culture medium were removed and the cubes were washed, and 50 ml of sterile optimized GPI medium (initial pH 5.0) were added to each flask. The flasks were incubated at 30 °C with shaking at 200 rpm for 9 days. Three flasks were taken from the shaker every day and filtered to determine the gibberellic acid content and microbial biomass. 3.2.9. Recycling batch fermentation using milk permeate as production medium: Gamma irradiated (0.5 kGy) fungal inoculum preparation (immobilized cells in 0.5 g sponge cubes for 24 h age) was carried out as described before. After washing immobilized and free seed culture cells, sterile 50 ml of milk

56 Material and Methods permeate (initial pH 5.0) medium were added to each flask as production medium (instead of optimized GPI medium). The flasks were incubated at 30 °C with shaking at 200 rpm. When the maximum production of gibberellic acid was achieved (after 6 days of incubation), the fermented milk permeate medium was removed, and the sponge cubes or free cells (control treatment) were washed with sterile NaCl solution. Fresh sterile milk permeate medium (50 ml) were added to each flask and incubated at 30 °C for 6 days under shaking (200 rpm). The above batch fermentations were repeated 6 cycles, and the gibberellic acid content and the amount of biomass were determined after each cycle (6 days). Also, the residual sugar in the fermented milk permeate was estimated after each cycle. 3.2.10. Toxicological evaluation of the produced gibberellic acid: Fertile eggs were employed in testing for the presence of toxic substances in both the culture filtrate of production medium cultivated with F.moniliforme and the simple purified gibberellic acid dissolved in ethanol (1 mg/1ml). The sterile production medium and ethanol, as well as, sterile distilled water were used as controls. One tenth ml of each treatment was used to inoculate the air cells in fertile eggs. One day-old fertile eggs were obtained from local poultry farm. As mentioned by Ye and Fields (1989) and Martinkova, et al. (1995), the eggs were candled to eliminate eggs with cracks or other imperfections and to find the location of air cell. The non inoculated fertile eggs were incubated at 37 °C with a relative humidity of about 65%. The eggs were incubated for 5 days and were turned daily so that the embryos would not stick to the shell. After incubation, the eggs were candled and the healthy embryos were selected for the assay. A hole was made in the shell opposite the air cell with sterile needle. Prior to inoculation the shell area around the air cell was treated with Merthiolate. One tenth ml of each treatment and the controls was injected into the inner membrane of the air cell with a sterile hypodermic needle. Eggs were 57 Material and Methods incubated for an additional 3 days after inoculation with each treatment. To determine if the embryos were living, the eggs were cracked and the embryos were examined. 3.2.11. Effect of gibberellic acid on fungal chitosan production: Aspergillus niger used in this study was isolated from soil and was identified according to the key of Gilman (1957) and Pitt and Hocking (1985). 3.2.11.a. Preparation of inoculum and fermentation: Inocula were prepared by growing Aspergillus niger in potato dextrose agar (PDA) plates at 30 °C for 3 days. Flasks containing chitosan production medium were inoculated with 1 ml spore suspension 6×107 spores/ml (prepared as F.moniliforme, see 3.2.2). The inoculated flaskes were incubated at 30 °C under shaking condition (120 rpm) for 72 h. At the end of the desired incubation period mycelia were harvested by filtration, dried by lyophilization and weighted. Mycelia and culture filtrates were stored at 4 °C until use. 3.2.11.b. Influence of Specific media: Different media for chitosan production (MSM, PDB and YPG, Chatterjee et al., 2005) were used in this study. Erlenmeyer flasks (250 ml) each containing 50 ml of each medium were autoclaved at 121 °C for 15 min. The pH of the media was adjusted at 5.0, 5.6, 5.0, respectively. Each flask was inoculated by 1 ml of A. niger spore suspension (6 x 107 CFU/ml). The inoculated flasks were incubated at 30 °C for 72 h under shaking condition (120 rpm). After incubation period, the fermented media were filtered, and the mycelia were used to determine chitosan content and microbial growth (see 3.4.1). 3.2.11.c. Influence of addition of gibberellic acid on growth of A. niger and chitosan production: Fifty milliliters of molasses salt medium (MSM), the best chitosan production medium, pH 5.0, in 250 ml Erlenmeyer flasks were inoculated by

58 Material and Methods

1ml spore suspension of Aspergillus niger, and 3 mg/l gibberellic acid. The flasks were incubated at 30°C for 72h with shaking (120 rpm). The mycelia were removed by filtration, then used to determine chitosan content and microbial growth. Control treatment was carried out without addition of gibberellic acid. 3.2.11.d. Effect of incubation time: Erlenmyer flasks (250 ml) containing 50 ml of MSM medium (pH 5.0) were inoculated separately with 1ml spore suspension of A. niger and 3 mg/l gibberellic acid. The flasks were incubated at 30 °C with shaking at 120 rpm for 4 days. Three flasks were taken from the shaker every day and filtered. The mycelia were used to determine the microbial growth and chitosan content. 3.2.11.e. Effect of addition of different concentrations of gibberellic acid on chitosan production: Different concentrations of gibberellic acid 1, 2, 3, 4, 5 mg/l were added separately to 50 ml of MSM medium in 250 ml Erlenmeyer flasks. The flasks were inoculated and incubated as described above. The chitosan content and microbial growth were determined after 2 days of incubation (the best incubation time for chitosan production).

3.3. Chemical analysis: 3.3.1. Estimation of gibberellic acid: The gibberellic acid was extracted and estimated by the method of Kahlon and Malhotra (1986). Filtered fermented production medium (10 ml) was transferred to a centrifuge tube and 0.5 ml zinc acetate (1 M) solution was added and shaken for 3 min, followed by addition of 0.5 ml potassium ferrocyanide solution (1 M) and the mixture was centrifuged for 15 min. Following centrifugation, 2.5 ml supernatant was transferred to a 250 ml flask containing 8 ml absolute ethanol and 90 ml HCl (30%). For control, 35 ml HCl solution (5%) was taken in a 250 ml flask and the volume made to 100 ml with

59 Material and Methods distilled water. The flasks were incubated at 20 °C in a water bath for 75 min and the absorbance was read at 254 nm. The GA concentration was obtained from a standard GA curve. 3.3.2. Extraction of gibberellic acid: The filtrate of the fermentation broth was acidified to pH 2.0-2.5 with 18 % HCl and extracted twice with ethyl acetate. The combined organic layers were concentrated to 1/5 of their volume under reduced pressure at 50 °C and then re-extracted with 1N NH4OH. These extracted were acidified with HCl as described above and extracted again with ethyl acetate. The upper organic phases were dried over anhydrous Na2SO4 and then concentrated. The concentrate was stored at 8° C for crystallization (Rachev et al., 1993). 3.3.3. Isolation and identification of gibberellic acid produced by isolated F.moniliforme: Gibberellic acid produced by the local isolate of F.moniliforme was extracted according to Rachev et al., 1993. The produced gibberellic acid was identified and compared to standard gibberellic acid using HPLC and FT-IR. 3.3.3.a. HPLC analysis: Convenient method of analysis of formulations containing gibberellic acid involves reversed phase HPLC with UV detection at 210 nm. The HPLC system consisted of the following components from Philips analytical (Cambridge, UK): a pump (PU 4100). The injector was a Rheodyne (Cotati, CA,

USA) Model 7125. The column was a reversed-phase C18 column Spherisorb S5ODS1 (25 cm x 4.6 mm I.D., 5 µm, Philips Scientific, Cambridge, UK). The mobile phase consisted of 60% acetone, The detection took place at 210 nm at a flow rate 0.6 ml / min. 3.3.3.b. Infra-red Spectroscopy (FT-IR): Infrared spectrum of fungal gibberellic acid was used to monitor the extracted gibberellic acid by comparing it with the standard spectrum of sigma

60 Material and Methods gibberellic acid.The transmittance was carried out in the form of KBr pellets in the range of 400-4000 cm-1 using infra red spectrophotometer FT-IR 6300 JASCO, Japan. 3.3.4. Determination of mycelium dry weight (Biomass): Culture growth was filtered through pre-weighted filter paper. The mycelial mat was dried at 60 °C until a constant weight. Cooled in a desiccator and weighted. The same procedure was used in case of immobilized sponge cubes. 3.3.5. Residual sugar: Residual sugar was measured by the phenol sulphuric method (Southgate, 1976) using glucose as the standard. One ml of fermented filtrate or milk permeate was mixed with 1 ml of phenol solution (50 g phenol in one liter distilled water) in a test tube. Five ml of pure H2SO4 were added to the mixture with shaking. After 10 min the tube was shaken again and placed in a water bath at 30 °C for 20 min. The developed colour was measured at 490 nm. A blank and a series of glucose standards were carried out at the same conditions. 3.4. Chemical analysis for chitosan production: 3.4.1. Fungal chitosan extraction: Lyophilized mycelia were autoclaved at 121 °C for 15 min after homogenizing in a warring blender with 1N NaOH (1:40, w/v). Alkali insoluble mass was thoroughly washed with water and refluxed with 100 volumes of 2% acetic acid (v/v) for 24 h at 95°C. The slurry was centrifuged at 6,000 rpm in a MIKRO 22R centrifuge, for 15 min, chitosan was precipitated out from supernatant by adjusting the pH to 13.0 with 1N NaOH. The solution was recentrifuged and the precipitated chitosan was washed with distilled water, followed by 95% ethanol and acetone then dried at 60 °C to a constant weight (Chatterjee et al., 2009 and Tayel et al., 2011).

61 Material and Methods

3.4.2. Infrared spectrum Infrared spectrum of fungal chitosan was used to monitor the chitosan extraction by comparing it with the standard spectrum of Sigma chitosan. The transmittance was carried out in the form of KBr pellets in the range of 400- 4000 cm-1 using infra red spectrophotometer JASCO FT-IR 6300, Japan. 3.4.3. Degree of deacetylation The potentiometry method reported by Lin et al., (1992) was followed. Dried chitosan of 0.5g were accurately weighed and dissolved in 0.1 mol-1 HCl. The solution was titrated with 0.1 mol-1 NaOH. The degree of deacetylation was calculated as follows:

(C1 V1 −C2 V2 )0.16 x100 NH2 % = G (100 −W )

-1 where C1 is the concentration of HCl (mol l ); C2, the concentration of NaOH -1 (mol l ); V1, the volume of HCl (ml); V2, the volume of NaOH (ml); G, the sample weight (g); W, the water percentage of sample (%) and 0.16 is the -1 weight of NH2 equal to 1 ml 0.1 (mol l ) HCl (g).

Degree of deacetylation (%) = NH2 % / 9.94% x 100%

Where: 9.94% is the theoretical NH2 percentage. 3.4.4. Determination of chitosan molecular weight: The average molecular weight of chitosan was determined according to the viscometric method using an aqueous solution of 0.2 M acetic acid, 0.1 M sodium chloride and 4 M urea. Computed from the Mark-Houwink equation, η a -4 = k Mv where η is The intrinsic viscosity using the constants K = 8.93x10 and a = 0.71 (Roberts, 1992), K and a are polymer-solvent interaction constants at a given temperature, Mv is the viscosity-average molecular weight of the polymer. The viscosity measurement was taken at a temperature of 25 °C using a Brookfield digital Rheometer (Model DV-II+Pro, Brookfield Engineering Laboratories, Inc., Stoughton, MA, USA). The presence of the urea in the

62 Material and Methods solvent helped to prevent any decrease in chitosan molecular weight due to acid- catalysed hydrolysis in solution. 3.5. Definitions: Conversion percentage for biomass and/or gibberellic acid was calculated as: Amount of product per g Conversion % = x100 Consumed sugar per g Gibberellic acid productivity (GA. Productivity) was calculated as: GA productivity (mg/l/h) = amount of gibberellic acid in mg per l / incubation time per h. Consumed sugar = The difference in weight between the original sugar content and that obtained after a given time of incubation of the microorganism culture indicated to amount of consumed sugar. 3.6. Statistical analyses: The means of three replicates and standard error (SE) were calculated for all the results of the gibberellic acid obtained, and the data were subjected to analysis of Variance (Spatz, 1993).

63 Results and Discussion

4. Results and Discussion

Microbial products such as gibberellins, pigments, alkaloids, toxins and antibiotics that serve no obvious function in the life of microorganisms that produced them, are all called secondary metabolites. The production of secondary metabolites is not universal among microbes; their formation is generally repressed during logarithmic growth or depressed during the suboptimal and stationary growth phases (Deacon, 2001).

Gibberellic acid (GA3), an important plant growth regulator, which gain much more attention all over the world, is used extensively in agriculture, nurseries, viticulture and tea garden for a variety of economic benefits. Its use, at present, is limited to high premium crops mainly because of its cost. Reduction in cost will lead to its wider application to a variety of crops and also to the harvest of innumerable industrial and economic benefits. It is produced by submerged fermentation using Gibberella fujikuroi (recently named Fusarium moniliforme). The submerged fermentation technique for the production of GA3 is influenced to a great extent by a variety of physical (pH, temperature, agitation rate) and nutritional factors ( carbon and nitrogen sources), the optimization of these factors is prerequisite for the development of commercial process. 4.1. Isolation of gibberellic acid producer fungi: Ten rhizosphere soil samples from different cultivated areas and one sample each from white corn, yellow corn, barley and wheat were used for the isolation of gibberellic acid producer fungi on specific gibberellic acid production solid medium (GPI). The results obtained in Table (4-1) indicated that there were 28 fungal isolates belonging to 3 genera namely: Fusarium (14 isolates), Aspergillus (8 isolates) and Penicillium (6 isolates) were isolated from different sources on

64 Results and Discussion

GPI medium. Also, from the results in Table (4-1), it could be observed that, Fusarium contributed the greatest number of isolates. 4.2. Screening of isolated fungi for gibberellic acid production: The aim of the present experiment is to demonstrate the ability of the isolated fungi to grow on specific gibberellic acid liquid media (GPI) as pure culture. The experiments were designed to select the most active fungi for gibberellic acid production. From each of the isolated fungi (1 ml spore suspension, 5x106 CFU/ml) was grown in Erlenmeyer flasks containing 50 ml GPI medium (pH 5.5). The inoculated flasks were incubated at 30 ºC for a period of 7 days on a shaker incubator (150 rpm). Biomass and gibberellic acid concentration were determined and the results were shown in Table (4-1). The results obtained in Table (4-1) showed that ten of the fungal isolates can grow in the broth medium with production of gibberellic acid. The concentration of produced gibberellic acid by these 10 isolates were in the range of 0.02-0.270 g/l. The highest gibberellic acid concentration was produced by Fusarium isolate number 10 which identified according to Barron (1968); Booth, (1977); Domsch et al, (1980); Nelson et al, (1983) and Pitt and Hocking, (1985) as Fusarium moniliforme previously named (Gibberella fujikuroi) that produce (0.270 g/l). Curtis (1957) studied fungi and 500 actinomycetes for their ability to produce GAs, and concluded that G. fujikuroi was the only microorganism that can produce GAs. Sanchez-Marroquin (1963) tested about 43 strains of Fusarium sp. and reported that F. moniliforme was able to give higher yields of GA3 on a variety of media.

65 Results and Discussion

Table (4-1): Screening of different fungal strains isolated from different sources for their potentiality of gibberellic acid production. Strain Source of Biomass Gibberellic acid Fungal sp. No. isolation (g/l) (g/l)* A Fusarium

1 Soil 6.78 ND** 2 Soil 5.12 ND 3 Soil 5.44 ND 4 Barley 6.65 0.06 ± 0.005 5 Soil 6.34 ND 6 Soil 7.00 0.150 ± 0.003 7 Barley 5.99 0.209 ± 0.004 8 Soil 6.03 ND 9 Yellow corn 6.88 0.131 ± 0.002 10 White corn 7.52 0.270*** ± 0.003 11 Soil 6.25 ND 12 Soil 5.89 0.141 ± 0.004 13 Yellow corn 6.55 ND 14 Soil 6.30 ND B Aspergillus 1 Soil 7.82 ND 2 Yellow corn 5.69 ND 3 Soil 6.88 0.05 ± 0.003 4 Barley 6.91 ND 5 Wheat 5.79 ND 6 Soil 6.49 ND 7 White corn 7.12 0.09 ± 0.006 8 Soil 7.31 0.04 ± 0.003 C Penicillium 1 White corn 7.64 ND 2 Soil 6.54 ND 3 Yellow corn 6.00 0.02 ± 0.002 4 Soil 8.24 ND 5 Soil 6.80 ND 6 Soil 7.12 ND * Mean ±SE **ND = Not Detected ***Significant from all values (p <0.05)

66 Results and Discussion

4.3. Isolation and identification of gibberellic acid produced by isolated Fusarium moniliforme: Gibberellic acid produced by the local isolate of G. fujikuroi was extracted according to Rachev et al., (1993). The extracted gibberellic acid was identified by comparing with standard gibberellic acid (GA3) using HPLC and FT-IR. 4.3.1. HPLC analysis: Convenient method of analysis of formulations containing gibberellic acid involves reversed phase HPLC with UV detection at 210 nm.

Fig (4-1): HPLC analysis of standard gibberellic acid (GA3).

67 Results and Discussion

Fig (4-2): HPLC analysis of extracted gibberellic acid from the local isolate of F. moniliforme

4.3.2. Infra-red Spectroscopy (FT-IR): Infrared spectrum of the extracted gibberellic acid was compared with the standard spectrum of sigma gibberellic acid.The transmittance was carried out in the form of KBr pellets in the range of 400-4000 cm-1 using infra red spectrophotometer FT-IR 6300 JASCO, Japan. The FT-IR spectra for gibbberellic acid from F.moniliforme in comparison with standared gibberellic acid are illustrated in Fig (4-3). The main characteristic peaks in (A) curve, standared gibberellic acid, are at 1177 -1 -1 -1 -1 cm (C=C), 1747 cm (C=O), 2965 cm (CH3 group), 3449 cm (OH group). In curve (B), extracted gibberellic acid from the local isolate F.moniliforme culture, there is no change in the main characteristic absorption bands of the standard gibberellic acid

68 Results and Discussion

A %

T r a n s m i t t a n c e B

4000 3500 3000 2500 2000 1500 1000 500 Wavenumbers

Fig (4-3): FT-IR spectra of gibberellic acid (A): Standard gibberellic acid (GA3), (B): Extracted gibberellic acid from the local isolate of F.moniliforme. 4.4. Optimization of batch culture conditions: Biotechnology is an old field that deals with the use of living organisms or their products in large scale industrial processes. The use of microbiology, biotechnology and engineering is an integrated fashion with the goal of using microorganisms to manufacture useful products. Variation in biotechnology processes can pose a significant problem because slight change in operating conditions or producers can have large effects on production and product quality. F.moniliforme growth and gibberellic acid accumulation were studied in the present investigation in flask batch cultures under different growth conditions. These conditions include incubation time, incubation temperature, pH, aeration, different carbon and nitrogen sources, inoculum density and

69 Results and Discussion age. The aim of these experiments is the optimization of gibberellic acid production by the strain under investigation. 4.4.1. Influence of specific media: The fungus was cultured on different specific gibberellic acid production media (GPI, GPII and Czapek`s-Dox), in 250 ml Erlenmyer flasks, each containing 50 ml medium. The flasks were inoculated with 1 ml spore suspension (5 x 106) and incubated at 30 ºC under shaking (150 rpm) for 7 days. The results in Table (4-2) and Fig (4-4) showed variation in both growth and gibberellic acid production among the tested media. The highest gibberellic acid concentration was obtained by GPI medium which recorded 0.272 g/l. while the lowest gibberellic acid concentration 0.103 g/l was obtained when grown on Czapek`s-Dox broth medium. These results indicated that the GPI medium was the best medium for gibberellic acid production by this F.moniliforme isolate. Table (4-2): Gibberellic acid production by F.moniliforme (local isolate) grown in various media. Consumed Biomass Gibberellic Biomass GA GA Type of Sugar, B Acid, GA Conversion Conversion Productivity medium CS (g/L) (g/L) (g/L)* (%) (%) (mg/l/h) GPI 36.58 7.52 0.272** 20.56 0.744 1.62 ±0.001 Czapek 23.93 8.71 0.103 36.40 0.430 0.613 `s-Dox ±0.005 GPII 30.26 8.04 0.185 26.57 0.611 1.10 ±0.002 *values are means of 3 replicates ± SE. **Significant from all GA values (p < 0.05)

70 Results and Discussion

Consumed sugar g/L Biomass g/L Gibberellic acid g/L GA Conversion (%) GA Productivity mg/l/h

40 1.8

35 1.6

l 1.4 30

1.2 25 1 20 0.8 15 0.6 Productivity mg/l/h

10 Consumed sugar Biomassg/l, g/ 0.4

5 0.2 GAGibberellic acid Conversion g/l, (%), GA

0 0 GPI Czapex`s- Dox GPII Type of inocula Fig (4-4): Gibberellic acid production by F.moniliforme (local isolate) grown in various media. 4.4.1. Influence of incubation period: The following experiment was carried out to determine the rate of growth and gibberellic acid production on GPI medium (the best gibberellic acid production medium) by F.moniliforme at different time intervals, the inoculated flasks were incubated at 30 ºC, pH 5.5 under 150 rpm, in order to obtain the maximum gibberellic acid production at a proper time. As shown in Table (4-3) and Fig (4-5), gibberellic acid production started after 48 h and increased gradually reaching its maximum value (0.314 g/l) after 6d with conversion content (0.90 %) and productivity rate (2.19 mg/l/h), then decreased there after. The biomasses were increased as the incubation periods increased, the maximum biomass content (8.61 g/l) and conversion value (24.52%) were obtained after 6d of incubation then decline slowly. Our results was contrasted with that reported by Lale et al. (2006) who reported that the best incubation period for gibberellic acid production was 5 days.

71 Results and Discussion

Pervious work with G. fujikuroi has indicated that gibberellic acid production occurs from the beginning of fermentation, Lale and Gadre (2010) showed that gibberellic acid production began after 20h of incubation when they used Fed-batch fermentation for gibberellic acid production by the mutant G. fujikuroi Mor-189. Shukla et al. (2005) was found that gibberellic acid synthesis started at 40 h of cultivation while the optimum incubation period for gibberellic acid production was 170 h using spore suspension of G. fujikuroi as inoculum in a 3 l fermentor. Whereas, Munoz and Agosin (1993) reported that gibberellic acid was detected after 72 h in the extracellular medium in shake flask cultures. Table (4-3): Effect of incubation time on gibberellic acid production by F.moniliforme grown on GPI medium. Consumed Gibberellic Biomass GA GA Time Biomass Sugar, CS Acid, GA Conversion Conversion Productivity /day B (g/L) (g/L) (g/L)* (%) (%) (mg/l/h) 1 6.54 2.63 - 40.21 - - 2 11.31 3.91 0.080 34.57 0.707 1.67 ±0.002 3 19.79 6.01 0.141 30.36 0.712 1.96 ±0.001 4 24.38 7.25 0.201 29.73 0.824 2.09 ±0.002 5 28.17 8.00 0.256 28.39 0.908 2.13 ±0.003 6 35.10 8.61 0.314** 24.52 0.900 2.19 ±0.003 7 37.39 7.64 0.263 20.43 0.703 1.57 ±0.002 8 40.52 6.11 0.189 15.08 0.466 0.98 ±0.001 9 41.93 4.56 0.070 10.88 0.166 0.32 ±0.004 *values are means of 3 replicates ± SE. **Significant from all GA values (p <0.05)

72 Results and Discussion

The time course for growth and gibberellic acid production in G.fujikuroi liquid media cultures was examined by Bu'Lock et al. (1974) who reported that Growth as well as gibberellic acid content increased through the 5 d of his study, with the linear phase of growth preceding the period of rapid accumulation. Significant amounts of gibberellic acid were found in the cultures after 3 days.

These patterns for growth and gibberellic acid production are similar to those observed by Johnsen and Codbaugh (1990) whose found that 6- days time course was optimum incubation time for gibberellic acid accumulation. Also, Meleigy and khalaf, (2009) reported that 6 days incubation time was optimum for gibberellic acid accumulation by a mutant Fusarium moniliforme cells immobilized on loofa sponge using milk permeate as production medium.

Consumed sugar g/l Biomass g/l Gibberellic acid g/l GA Conversion (%) GA Productivity mg/l/h 45 2.5

l 2 35

30 1.5 25

20 1

15 mg/l/h Productivity

ConsumedBiomass g/ sugar g/l, 10 0.5

5 GA Conversion g/,l (%) acid ,GA Gibberellic

0 0 123456789 Incubation time

Fig (4-5): Effect of incubation time on gibberellic acid production by F.moniliforme grown on GPI medium.

73 Results and Discussion

4.4.2. Effect of incubation temperature: Temperature is one of the most important parameters regulating the activity of microorganisms in natural environments. Generally, there is an optimal temperature for the activity of enzymes produced by different microorganisms which responsible for the biosynthesis or degradation of compounds. This optimal temperature may be similar or different from the optimal temperature of the microbial growth. To determine the effect of incubation temperature on mycelial growth and gibberellic acid production, F.moniliforme was cultivated at optimum incubation time (6 d) under various temperature degrees ranging from 20 to 400C at pH value 5.5 for 6d and 150 rpm. The results in Table (4-4) and Fig (4-6) showed that the optimum incubation temperature for fungus growth was observed at 300C (8.53 g/l) with conversion value (24.04%). Also, gradual increase of the incubation temperature from 20 to 30°C enhanced gibberellic acid production by the tested fungus. The maximum gibberellic acid content (0.307 g/l) was obtained at incubation temperature of 30°C with conversion content (0.865%) and productivity rate (2.131 mg/l/h). The yield of gibberellic acid production decreased at 20, 25 and 35 °C to 0.121, 0.232 and 0.199, respectively. This may be due change in enzyme activity at different temperatures or denaturation of the enzyme at high temperatures. Our results is in accordance with Meleigy and Khalaf, (2009), whose found that maximum gibberellic acid production (1.84 gl-1) was produced at 30ºC.

74 Results and Discussion

Table (4-4): Effect of different incubation temperatere (20-40 ºC) on gibberellic acid production by F.moniliforme incubated for 6 days. Consumed Gibberellic Biomass GA GA Time Biomass Sugar, CS Acid, GA Conversion Conversion Productivity /day B (g/L) (g/L) (g/L)* (%) (%) (mg/l/h) 20 21.31 5.13 0.122 24.07 0.572 0.847 ±0.004 25 33.11 7.92 0.232 23.92 0.700 1.611 ±0.001 30 35.47 8.53 0.307** 24.04 0.865 2.131 ±0.003 35 29.18 4.12 0.199 14.12 0.681 1.381 ±0.001 40 17.25 2.05 0.066 11.88 0.383 0.458 ±0.002 *values are means of 3 replicates ± SE. **Significant from all GA values (p < 0.05)

Kahlon and Malhotra (1986) found that the maximum yield of GA production by F. moniliforme, entrapped in alginate gel, was recorded at 25±1°C. According to the same author the proteins fold tightly at lower temperatures and thus decrease the catalytic activity. Also, Escamilla et al. (2000) recorded that the greatest production of GA in fluidized bioreactors by immobilized G. fujikuroi mycelium in Ca-polygalacturonate was at 30°C. Jeferys (1970), reported that the optimum temperature for growth of the strain G. fujikuroi is between 31-32 ºC while the production of GA3 was maximized at 29 °C. Negligible production of gibberellic acid in our study was recorded at 40°C.

75 Results and Discussion

Consumed sugar g/L Biomass g/L Gibberellic acid g/L GA Conversion (%) GA Productivity mg/l/h 40 2.5

l 2 30

25 1.5

1 15 Productivity mg/l/h Productivity

Consumed Biomass sugar g/ g/l, 0.5 5 G ibberellic acid g/l, G A Conversion (% ), G A

0 0 20 25 30 35 40 Incubation temperature (C)

Fig (4-6): Effect of different incubation temperatere (20-40 ºC) on gibberellic acid production by F.moniliforme incubated for 6 days.

4.4.3. Effect of initial pH: Hydrogen ion concentration (pH) of the medium is considered one of the most important factors, which not only affected the growth of microorganisms but also has a great influence on their physiological activity. The effect of initial culture pH on biomass yield and gibberellic acid production was studied at different pH values in order to reach the maximum gibberellic acid accumulation. The present experiment aimed to investigate the influence of pH on gibberellic acid production by F.moniliforme. Data presented in Table (4-5) and Fig (4-7) show that F.moniliforme was able to grow with different degrees in all pH values. The maximum growth was obtained with an initial pH value of 5.5, which recorded 8.46 g/l. The extreme acidic or alkaline pH resulted in lower growth (3.52 and 4.17 g/l, respectively). On the other hand, the results indicated that the maximum gibberellic acid production was achieved at pH 5.0 reaching to 0.380 g/l with conversion content (1.03%) and productivity rate (2.64 mg/l/h)

76 Results and Discussion

Table (4-5): Effect of different pH values (3-7) on gibberellic acid production by F.moniliforme incubated for 6 days at 30 ºC. Consumed Gibberellic Biomass GA GA Initial Biomass Sugar, CS Acid, GA Conversion Conversion Productivity pH B (g/L) (g/L) (g/L)* (%) (%) (mg/l/h) 3 13.16 3.52 0.115 26.75 0.873 0.798 ±0.001 3.5 17.52 5.19 0.156 29.62 0.890 1.08 ±0.001 4 24.17 6.77 0.223 28.00 0.923 1.55 ±0.002 4.5 26.35 7.05 0.253 26.76 0.960 1.76 ±0.003 5 36.83 7.91 0.380** 21.48 1.03 2.64 ±0.002 5.5 35.32 8.46 0.321 23.95 0.908 2.23 Control ±0.003 6 24.12 7.21 0.216 29.89 0.895 1.50 ±0.002 6.5 23.51 6.57 0.192 27.95 0.816 1.33 ±0.002 7 16.75 5.03 0.122 30.03 0.728 0.847 ±0.003 7.5 13.17 4.17 0.091 31.66 0.690 0.632 ±0.001 *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05) Whereas, lower concentration of gibberellic acid production (0.091 g/l) was recorded at pH 7.5. Our results is in agreement with that reported by Shukla et al ., (2005) whose found that pH 5.0 was the optimum for gibberellic acid production.

77 Results and Discussion

Consumed sugar g/L Biomass g/L Gibberellic acid g/L GA Conversion (%) GA Productivity mg/l/h 40 3

35 2.5

l 30

2 25

20 1.5

15 Productivity mg/l/h 1

Consumed Biomass sugar g/ g/l, 10

0.5 GA Conversion acid g/l, Gibberellic (%), GA 5

0 0 33.544.555.566.577.5 Initial pH Fig (4-7): Effect of different pH values (3-7) on gibberellic acid production by F.moniliforme incubated for 6 days at 30 ºC.

Meleigy and Khalaf, 2009 studied the effect of initial milk permeate pH on GA production, the authors recorded significant increase of gibberellic acid production (2.25 gl-1) with conversion 3.64% and productivity rate 15.63 mgl-1 h-1 were obtained at pH 5 after 6 days of incubation. Lower yields of gibberellic acid production were obtained at pH 3.0 (0.36 gl-1) and 8 (0.21 gl-1). Also, initial pH 5.0 was recorded as more suitable pH range for gibberellic acid production by immobilized G. fujikuroi mycelium (Escamilla et al., 2000). Meanwhile, Kahlon and Malhotra (1986) observed the high yield of gibberellic acid production by immobilized F. moniliforme at initial pH 5.5. The lower yield of gibberellic acid production at extreme pH values (pH 3 and 7.5, in our study) may be due to conformational changes and biocatalyst denaturation. 4.4.4. Effect of agitation speed: The primary objective of agitation is to supply the necessary oxygen to the microorganisms in order to achieve the proper metabolic activities. A

78 Results and Discussion secondary function is to keep the microorganisms in suspension. These two objectives are accomplished by the rotary shaker apparatus (Richard, 1961). The effect of agitation on growth and gibberellic acid production was studied by incubating the inoculated flasks (pH 5.0) at different agitation speeds (0 to 300 rpm) on a rotary shaker at 30°C for 6d. From the data represented in Table (4-6) and Fig (4-8), it was noticed that the fungus strain under investigation could grow and synthesis gibberellic acid under static and shaking conditions. The cell growth increased by increasing the agitation speed up to 150 rpm reaching the maximum dry weight (8.51 g/l) with biomass conversion value (23.48%). Similarly, gibberellic acid production increased by increasing agitation speed but reached its maximum at 200 rpm (0.462 g/l) with conversion content (1.21%) and productivity rate (3.21 mg/l/h). While, cells dry weight and gibberellic acid production decreased with further increase in agitation speed. This may be due to the morphological changes that results from high agitation speed which related to physiological changes resulting in a lower production of secondary metabolites. On the other hand, lower concentration of gibberellic acid production (0.116 g/l) was observed under static condition. Aerobic fermentations involving fungi, their filamentous nature commonly leads to excessive viscosity in the fermentation broth and demands higher agitation and aeration to maintain satisfactory levels of dissolved oxygen (Lale et al ., 2006). Lale and Gadre (2010) found that 220 rpm was the best agitation for gibberellic acid production. When they used a mutant of G. fujikuroi Mor- 189, for the production of GA, they reported that on the use of morphological mutants that have short mycelial lengths in liquid cultures, which led to better oxygen transfer and increased production of gibberellic acid and has the advantages of a low viscosity because of the short length of its mycelium.

79 Results and Discussion

Table (4-6): Gibberellic acid production by F.moniliforme at different aeration regimes (growth conditions: temp. 30 ºC, pH 5 and incubation time 6 days). Consumed Biomas Gibberellic Biomass GA GA Agitation Sugar, CS s B Acid, GA Conversion Conversion Productivity rate (rpm) (g/L) (g/L) (g/L)* (%) (%) (mg/l/h) 0 17.73 5.55 0.116 31.30 0.654 0.805 Static ±0.003 50 20.42 6.25 0.137 30.61 0.670 0.951 ±0.001 100 25.60 7.11 0.182 27.77 0.710 1.26 ±0.002 150 36.25 8.51 0.380 23.48 1.05 2.64 Control ±0.002 200 38.13 6.99 0.462** 18.33 1.21 3.21 ±0.001 250 19.54 3.64 0.168 18.63 0.859 1.17 ±0.003 300 13.32 1.92 0.095 14.41 0.713 0.660 ±0.001 *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05)

Giordano and Domench (1999) described how oxygen availability causes differences in the biosynthesis of fats, pigments and gibberellins by G. fujikuroi. They reported that increased oxygen transfer increased the biosynthesis of gibberellins by G. fujikuroi, as the use of mutants with short mycelial lengths and low viscosities permits the utilization of more concentrated media. It was found that high aeration could result in decreased production of fatty acids and fusarin C but it stimulated the growth and production of both gibberellic acid and bikaverin simultaneously (Shukla et al., 2003). Meleilgy and Khalaf (2009) mentioned that the production of gibberellic acid was high on the use of agitation speed 150 rpm. In the studies of growth and gibberellic acid accumulation, the cultures contained 50 mL of liquid media and were grown at 250 rpm on an orbital shaker. These

80 Results and Discussion conditions resulted in high levels of gibberellic acid (Johnson and Coolbaugh, 1990).

Consumed sugar g/L Biomass g/L Gibberellic acid g/L GA Conversion (%) GA Productivity mg/l/h 45 3.5

40 3 l 35 2.5 30

25 2

20 1.5

15 Productivity mg/l/h 1 10 Consumed Biomass sugar g/ g/l, 0.5

5 GA Conversion g/l, acid (%),Gibberellic GA

0 0 0 50 100 150 200 250 300 Agitation rate (rpm)

Fig (4-8): Gibberellic acid production by F.moniliforme at different aeration regimes (growth conditions: temp. 30 ºC, pH 5 and incubation time 6 days).

4.4.5. Effect of carbon sources: To investigate the effect of carbon sources on gibberellic acid production by the local isolate of F.moniliforme, the fungus was cultured on GPI medium (pH 5.0) containing different carbon sources (8% w/v) at 30°C for 6 days under shaking (200 rpm). The data presented in Table (4-7) and Fig (4-9) showed that the maximum cell growth obtained using starch and glucose was 8.63 and 8.31 g/l, respectively, followed by galactose (7.32 g/l). Also, the results revealed that the best carbon sources for gibberellic acid production were fructose and sucrose that recorded 0.609 and 0.525 g/l, respectively. Whereas, starch and glycerol represented the poorest carbon sources for gibberellic acid production (0.293 and 0.331 g/l, respectively). Also the highest value of gibberellic acid productivity (4.23 mg/l/h) was recorded when fructose was

81 Results and Discussion used as the sole carbon source. The biosynthesis of gibberellic acid was indicated to be suppressed by high amount of glucose (> 20%), which had been the most commonly used carbon source for gibberellic acid production (Bruckner, 1992). Also, Kumar and Lonsane, 1989) reported that low glucose concentration (< 4%) is necessary for the production of gibberellic acid and maintenance of biomass in the production phase. Alternative carbon sources, such as maltose, manitol, glycerol and galactose increased the gibberellic acid production rate (Gancheva and Dimova, 1991). Rangaswamy, (2012) reported that of all the carbon sources, sucrose was the best carbon source under optimized conditions for gibberellic acid production.

Gonzalez et al. (1994) found that the mixture of sucrose- starch was identified as the best carbon source for the GA3 production. Some oils such as sunflower oil and cooking oil have also been successfully used to enhance

GA3 production (Gancheva et al., 1984 and Hommel et al., 1989).

82 Results and Discussion

Table (4-7): Effect of different carbon sources on gibberellic acid production by F.moniliforme (growth condition temp. 30 ºC, pH 5, 6 days, 200 rpm). Carbon Consumed Gibberellic Biomass GA GA Biomass B sources Sugar, CS Acid, GA Conversion Conversion Productivity (g/L) (8% w/v) (g/L) (g/L)* (%) (%) (mg/l/h) Glucose 38.41 8.31 0.462 21.63 1.20 3.21 (control) ±0.001 36.52 7.13 0.609** 19.52 1.67 4.23 Fructose ±0.002 30.16 7.32 0.352 24.27 1.17 2.44 Galactose ±0.004 28.93 6.11 0.370 21.12 1.28 2.57 Lactose ±0.002 33.51 6.90 0.525 20.59 1.57 3.65 Sucrose ±0.001 27.31 3.52 0.401 12.89 1.47 2.78 Manitol ±0.011 26.42 4.85 0.387 18.36 1.46 2.69 Maltose ±0.003 20.13 8.63 0.293 42.87 1.45 2.03 Starch ±0.004 29.65 5.03 0.420 16.96 1.42 2.92 Xylose ±0.004 19.95 4.15 0.331 20.80 1.66 2.30 Glycerol ±0.001 *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05).

The biosynthesis of GA3 is based on acetate and follows the isoprenoid pathway (Tudzynski 1999 and Kawanabe et al., 1983). Therefore, plant oil, as a carbon source is not only inert for carbon catabolite repression but also makes available a pool of acetyl CoA and additionally might contain natural precursors for GA3 biosynthesis. Borrow et al. (1955) reported that, the yield of gibberellic acid was 40 mg/l when sucrose used as carbon source but it was lower when glucose used.

83 Results and Discussion

10 0.7 Biomass g/L Gibberellic acid g/L 9 0.6 8

7 0.5

6 0.4

5 llic acid g/L

0.3 Biomass g/L 4 Gibbere

3 0.2

2 0.1 1

0 0 xylose galactose fructose glucose maltose lactose sucrose manitol glycerol starch Carbon sources (8% w /v)

45 4.5 Consumed sugar g/L GA Productivity mg/l/h 40 4

35 3.5

30 3

25 2.5

20 2

Consumed sugar g/L 15 1.5 GAmg/L/h Productivity

10 1

5 0.5

0 0 xylose galactose fructose glucose maltose lactose sucrose manitol glycerol starch Carbon sources (8% w /v) Fig (4-9): Effect of different carbon sources on gibberellic acid production by F. moniliforme (growth condition temp. 30 ºC, pH 5, 6 days, 200 rpm). Glucose was chosen as the best carbon source, as its use resulted in the production of considerably higher levels of total gibberellin (422 mg l-1)

84 Results and Discussion when Lale, and Gadre (2010) used mutant of Gibberella fujikuroi in wheat gluten medium for production of gibberellin.

4.4.6. Effect of fructose concentration: Since fructose was the best carbon source for gibberellic acid production by the microorganism under investigation, an experiment was performed at various fructose concentrations ranged from 2-9% (w/v) to determine the optimum fructose concentration for both growth and gibberellic acid production. Table (4-8): Effect of different fructose concentractions (w/v ) on gibberellic acid production by F. moniliforme (growth conditions as in Table 4-7). Fructose Consumed Gibberellic Biomass GA GA Biomass conc. Sugar, CS Acid, GA Conversion Conversion Productivity B (g/L) (% w/v) (g/L) (g/L)* (%) (%) (mg/l/h) 17.23 4.15 0.120 24.09 0.696 0.833 2 ±0.001 26.37 4.68 0.319 17.75 1.21 2.22 3 ±0.001 28.73 5.32 0.402 18.52 1.40 2.79 4 ±0.003 30.26 6.63 0.457 21.91 1.51 3.17 5 ±0.002 33.01 7.11 0.682** 21.54 2.07 4.74 6 ±0.002 30.72 7.23 0.620 21.11 1.78 4.31 7 ±0.001 36.75 7.21 0.615 19.62 1.67 4.27 8 ±0.004 29.13 6.15 0.420 21.11 1.44 2.92 9 ±0.003 *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05) As illustrated in Table (4-8) and Fig (4-10) the biomass content was increased by increasing the fructose concentration, and the maximum mycelial growth (7.23 g/l) was recorded at 7% fructose concentration with biomass conversion (21.11%). On the other hand 6% of fructose

85 Results and Discussion concentration gave the best production of gibberellic acid (0.682 g/l) with conversion and productivity rate (2.07% and 4.74 mg/l/h, respectively), and increasing the fructose above this limit did not stimulate gibberellic acid production.

Consumed sugar g/L Biomass g/L Gibberellic acid g/L GA Conversion (%) GA Productivity mg/l/h 40 5

4.5 35

l 4 30 3.5 25 3

20 2.5

2 15 Productivity mg/l/h 1.5 10 Consumed Biomass sugarg/ g/l, 1

5 GA Conversion g/l, acid %,GA Gibberellic 0.5

0 0 23456789 Fructose con. %

Fig (4-10): Effect of different fructose concentractions (w/v) on gibberellic acid production by F. moniliforme (growth conditions as in Table 4-6). 4.4.7. Effect of nitrogen source: Nitrogen is present in cells predominantly as reduced compound, namely as amino group. Most microorganisms can assimilate this element as oxidized compounds and can reduce nitrates. The most common sources of nitrogen for microorganisms are ammonium salts. A few prokaryotes can reduce molecular nitrogen (N2). Other microorganisms may require amino acids as nitrogen sources in which the nitrogen is already present in an organic form (Schlegel, 1995). The efficiency of gibberellic acid synthesis is obviously depends on the type of nitrogen source used (Lale and Gadre, 2010).

86 Results and Discussion

In order to increase gibberellic acid production by F. moniliforme in this study, several types of both organic and inorganic nitrogen sources (at concentration 0.75 g/l, as N2) were used. The data represented in Table (4-9) and Fig (4-11) showed that the biomass concentrations ranged from 4.21 to7.82 g/l and from 4.14 to 8.62 g/l when organic and inorganic nitrogen sources were used, respectively. It is clear that yeast extract was the best nitrogen source for growth of F. moniliforme (8.62 g/l). Also, the present results indicated that the maximum gibberellic acid production (0.797 g/l) was obtained when ammonium sulfate used as sole nitrogen source, while peanut showed lower gibberellic acid production (0.367 g/l). Gibberellic acid production began at or soon after the depletion of the nitrogen source and is inhibited when this source is renewed (Borrow et al., 1964a). Candau et al. (1992) reported that ammonium nitrate was the best nitrogen source for gibberellic acid production. Nitrogen repression is a well- known regulatory principle of secondary metabolite formation (Munoz and Agosin, 1993). In a mutant strain of G. fujikuroi, ammonium or nitrate ions was known to affect the production of gibberellic acid (Bruckner and Blechschmidt, 1991; Sanchez-Fernandez et al., 1997)

87 Results and Discussion

Table (4-9): Effect of different nitrogen sources on gibberellic acid production by F. moniliforme (growth conditions as in Table 4-7 at 6% w/v fructose conc.) Consumed Gibberellic Biomass GA GA Nitrogen Biomass Sugar, CS Acid, GA Conversion Conversion Productivity sources B (g/L) (g/L) (g/L)* (%) (%) (mg/l/h) Malt 29.51 8.21 0.557 27.82 1.89 3.87 extract ±0.002 Yeast 28.23 8.62 0.522 30.53 1.85 3.63 extract ±0.002 Tryptone 22.75 6.11 0.500 26.86 2.20 3.47 ±0.001 Beef 20.64 6.53 0.398 31.64 1.93 2.76 extract ±0.003 Soy 24.17 4.35 0.488 18.00 2.02 3.39 peptone ±0.002 Soybean 23.40 4.14 0.471 17.69 2.01 3.27 ±0.004 Pea nut 19.80 5.27 0.367 26.62 1.85 2.55 ±0.003 Glycine 20.95 4.65 0.403 22.20 1.92 2.80 ±0.002 (NH4)2 36.27 7.11 0.797** 19.60 2.20 5.53 SO4 ±0.001 NH4 31.35 7.63 0.610 24.34 1.95 4.24 HPO4 ±0.001 NH4Cl 32.01 7.82 0.621 24.43 1.94 4.31 ±0.001 NH4NO3 33.51 7.21 0.680 21.52 2.03 4.72 (control) ±0.002 KNO3 21.50 4.21 0.515 19.58 2.40 3.56 ±0.003 Amm. 24.67 6.15 0.540 24.93 2.19 3.75 Tartarate ±0.002 *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05) Lale and Gadre (2010) observed that, amongst all organic and inorganic nitrogen sources studied, the highest level of gibberellic acid was produced by G. fujikuroi mutant using wheat gluten as the nitrogen source.

88 Results and Discussion

40 6

Consumed sugar g/L GA Productivity mg/l/h 35 5

4 25

20 3

15 Consumed sugarg/l 2 GA mg/l/h Productivity

1 5

0 0 4 4 L 3 3 ut O O C O e ract n ne S P 4 N rat ract tone a 2 H H 4 KNO lyci ) 4 N ext ext Pe G 4 H arta lt Tryptone pep oyabean H N t a S N NH M ( Beef Soy ium Yeast extract on m m A Nitrogen sources

10 0.9 Biomass g/L Gibberellic acid g/L 9 0.8

8 0.7 7 0.6 6 0.5 5 0.4

Biomass g/l 4

0.3 Gibberellic acid g/l 3 0.2 2

1 0.1

0 0

t 4 4 3 3 e ne O CL ac o act one ean cine S 4 NO r t b )2 HPO H 4 KNO pt ly 4 4 N H t extr extract y ext Pea nut G H N t Tr N Soya (NH Mal Beef Yeas Soy pep

Ammonium tartarat Nitrogen sources

Fig (4-11): Effect of different nitrogen sources on gibberellic acid production by F. moniliforme (growth conditions as in Table 4-6 at 6% w/v fructose conc.).

89 Results and Discussion

4.4.8. Effect of ammonium sulfate concentrations: Studying the effect of different nitrogen sources revealed that ammonium sulfate was the best nitrogen source for gibberellic acid production, therefore an experiment was performed with various concentrations of ammonium sulfate ranged from 0.1 to 0.9 g/l, as N2 to determine the optimum concentration for gibberellic acid production. Table (4-10): Effect of different ammonium sulfate concentrations on gibberellic acid production by F. moniliforme (growth conditions as in Table 4-9). Consumed Gibberellic Biomass GA GA Amm. Biomass Sugar, CS Acid, GA Conversion Conversion Productivity Sulfate B (g/L) (g/L) (g/L)* (%) (%) (mg/l/h) 0.0 16.18 3.21 0.208 19.84 1.29 1.44 ±0.001 0.1 19.23 3.95 0.324 20.54 1.68 2.25 ±0.002 0.2 21.09 4.53 0.397 21.48 1.88 2.76 ±0.002 0.3 23.19 4.91 0.444 21.17 1.91 3.08 ±0.001 0.4 26.53 5.65 0.530 21.30 1.99 3.68 ±0.003 0.5 28.46 6.24 0.581 21.93 2.04 4.03 ±0.002 0.6 38.00 6.87 0.841** 18.08 2.21 5.84 ±0.003 0.7 36.11 7.25 0.738 20.08 2.04 5.13 ±0.001 0.8 32.56 7.43 0.627 22.82 1.93 4.35 ±0.001 0.9 30.25 6.31 0.395 20.86 1.31 2.74 ±0.002 *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05)

Table (4-10) and Fig (4-12) illustrated that the biomass increased gradually by increasing the amount of ammonium sulfate reaching its

90 Results and Discussion maximum (7.43 g/l) at 0.8 g/l ammonium sulfate concentration. While, the maximum gibberellic acid production (0.841 g/l) was obtained at 0.6 g/l ammonium sulfate concentration. In addition, the best concentration of ammonium sulfate for gibberellic acid productivity (5.84 mg/l/h) was 0.6 g/l. Hence it was selected to be used in the following experiments.

Consumed sugar g/L Biomass g/L Gibberellic acid g/L GA Conversion (%) GA Productivity mg/l/h 40 7

35 6

30 5

25 4 20 mg/l/h 3 15

2 10 Consumed sugar g/l,Biomass g/l 1 5 Gibberellic acid g/l, GA acid Conversion g/l, Gibberellic %, GA Productivity

0 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

(NH4)2SO4 g/L

Fig (4-12): Effect of different ammonium sulfate concentrations on gibberellic acid production by F. moniliforme (growth conditions as in Table 4-8).

A maximum yield of 1.16 g/l gibberellic acid was predicted for supplementation of the basal medium with 0.1% ammonium sulfate (Maddox and Richert, 1977). Kumar and Lonsane (1989) reported that production of 389 mg GA. Kg. dry matter-1 (mixed substrate containing coffee and cassava bagasse, 7:3 dry wt) was achieved with the saline solution, consisting of 10 mg of -1 (NH4)SO4 100 ml . Candau et al. (1992) showed that 1 g/l of NH4NO3 was the best concentration for gibberellic acid production.

91 Results and Discussion

4.4.9. Effect of different concentrations of rice flour: To investigate the effect of the addition of different concentrations of rice flour on gibberellic acid production by the isolate under investigation, an experiment was performed at various rice flour concentrations ranged from zero to 4 g/l to determine the optimum rice flour concentration for both growth and gibberellic acid production. As illustrated in Table (4-11) and Fig (4-13) the biomass content was increased by increasing the rice flour concentration, and the maximum mycelial growth (7.37 g/l) was recorded at the addition of 3 g/l of rice flour to the production media, also the highest value of biomass conversion (24.97 %) was recorded at the same concentration. On the other hand, media without rice flour (zero concentration) gave the best production of gibberellic acid (0.992 g/l) with conversion and productivity rate (2.81 % and 6.89 mg/l/h, respectively), as the experiment showed that the addition of rice flour to the media did not stimulate the gibberellic acid production. Our results is in contrast with Escamilla et al. (2000) who reported that the addition of 2 g/l rice flour to GA production medium stimulate its production. Table (4-11): Effect of different conc. of rice flour on gibberellic acid production by F. moniliforme (growth conditions as in Table 4-9). Rice Consumed Gibberellic Biomass GA GA Biomass B flour Sugar, CS Acid, GA Conversion Conversion Productivity (g/L) g/l (g/L) (g/L)* (%) (%) (mg/l/h) 0 35.25 6.10 0.992** 17.30 2.81 6.89 ±0.001 1 31.96 6.53 0.850 20.43 2.65 5.90 ±0.001 2 31.18 6.92 0.849 22.19 2.72 5.90 control ±0.002 3 29.51 7.37 0.772 24.97 2.62 5.36 ±0.003 4 29.15 7.00 0.698 24.01 2.39 4.85 ±0.001 *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05).

92 Results and Discussion

Consumed sugar g/L Biomass g/L Gibberellic acid g/L GA Conversion (%) GA Productivity mg/l/h 40 8

35 7

30 6 l

25 5

20 4 mg/l/h

15 3 Consumed sugar g/l, Biomass g/ Consumed sugar g/l, 10 2

5 1 GA acidConversion g/l, Gibberellic %, GA Productivity

0 0 01234 rice flour g/l

Fig (4-13): Effect of different conc. of rice flour on gibberellic acid production by F. moniliforme.

4.4.10. Effect of different concentrations of magnesium sulfate: The effect of the addition of different concentrations of magnesium sulfate on mycelial growth and gibberellic acid production was studied by the addition of different concentrations of magnesium sulfate ranged from (0 to 2.5 g/l) to the production medium. As shown in Table (4-12) and Fig (4-14) the mycelium growth increased by increasing magnesium sulfate concentration reached its maximum value (7.45 g/l) at the addition of 2 g/l of magnesium sulfate. On the other hand, the highest gibberellic acid production (0.989 g/l) was recorded at (1.5 g/l) of magnesium sulfate with gibberellic acid conversion and productivity rate ( 2.69 % and 6.87 mg/l/h respectively). A maximum production of 0.83 g/l of gibberellic acid predicted when the medium was

93 Results and Discussion supplemented only with 0.001% magnesium sulfate (Maddox and Richert, 1977).

Table (4-12): Effect of different conc. of Mg SO4.7H2O on gibberellic acid production by F. moniliforme (growth conditions as in Table 4-9). Consumed Biomass Gibberellic Biomass GA GA MgSO4 Sugar, CS B Acid, GA Conversion Conversion Productivity .7H2O (g/L) (g/L) (g/L)* (%) (%) (mg/l/h) 0.0 23.10 6.00 0.450 25.97 1.95 3.13 ±0.001 0.5 26.50 6.37 0.635 24.04 2.40 4.41 ±0.002 1.0 30.71 6.80 0.792 22.14 2.58 5.50 ±0.002 1.5 36.73 7.01 0.989** 19.09 2.69 6.87 Contro ±0.003 l 2.0 33.62 7.45 0.813 22.16 2.42 5.65 ±0.001 2.5 29.27 6.35 0.675 21.69 2.30 4.69 ±0.001 *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05).

94 Results and Discussion

Consumed sugar g/L Biomass g/L Gibberellic acid g/L GA Conversion (%) GA Productivity mg/l/h 40 8

35 7

30 6

25 5

20 4 Consumed sugar g/l mg/l/h, Biomassg/l

15 3

10 2 Gibberellic acid g/l, GA Conversion (%), g/l, acid GA Productivity Gibberellic 5 1

0 0 00.511.522.5 MgSO4.7H2O g/L

Fig (4-14): Effect of different conc. of Mg SO4.7H2O on gibberellic acid production by F. moniliforme.

4.4.11. Effect of addition of different concentrations of potassium dihydrogen phosphate:

The effect of the addition of different concentrations of potassium dihydrogen phosphate on mycelial growth and gibberellic acid production was studied by the addition of different concentrations of potassium dihydrogen phosphate ranged from (1.0 to 5.0 g/l) to the production medium. Table (4-13) and Fig (4-15) illustrated that the biomass increased gradually by increasing the amount of potassium dihydrogen phosphate reaching its maximum (7.48 g/l) at 2.0 g/l potassium dihydrogen phosphate concentration. While, the maximum gibberellic acid production (1.35 g/l) was obtained at 1.0 g/l potassium dihydrogen phosphate concentration. In addition, the best concentration of potassium dihydrogen phosphate for gibberellic acid productivity (9.38 mg/l/h) was 1.0 g/l.

95 Results and Discussion

Table (4-13): Effect of different conc. of KH2PO4 on gibberellic acid production by F. moniliforme. (growth conditions as in Table 4-9). Consumed Gibberellic Biomass GA GA Biomass KH PO Sugar Acid, GA Conversion Conversion Productivity 2 4 B (g/L) , CS (g/L) (g/L)* (%) (%) (mg/l/h) 1 42.25 7.14 1.35** 16.90 3.20 9.38 ±0.002 2 40.35 7.48 1.030 18.54 2.55 7.15 ±0.002 3 40.75 6.93 0.995 17.01 2.44 6.90 contro ±0.001 l 4 33.64 6.75 0.804 20.07 2.39 5.58 ±0.003 5 28.13 5.96 0.692 19.78 2.29 4.81 ±0.001 *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05).

In a mutant strain of G. fujikuroi phosphate does not influence the biosynthesis of gibberellic acid (Bruckner and Blechschmidt, 1991; Sanchez-Fernandez et al., 1997). Whereas, Candau et al. (1992) reported that gibberellic acid didn’t produced upon depletion of phosphate.

Consumed sugar g/L Biomass g/L Gibberellic acid g/L GA Conversion (%) GA Productivity mg/l/h 45 10

40 9

l 8 35

7 30 6 25 5 20 4

15 Productivity mg/l/h 3 Consumed Biomass sugar g/ g/l, 10 2 Gibberellic acid g/l, GA Conversiong/l, acid (%),Gibberellic GA 5 1

0 0 12345 KH2PO4 g/L

Fig (4-15): Effect of different conc. of KH2PO4 on gibberellic acid production by F. moniliforme (growth conditions as in Table 4-9).

96 Results and Discussion

From the above results we can concluded that the optimum conditions for gibberellic acid production by the local isolate F. moniliforme. are ( 6d, 30 °C, pH 5.0, 200 rpm, 6% fructose, 0.06% ammonium sulfate, 0.15% magnesium sulfate and 0.1% potassium dihydrogen phosphate). These conditions will be called optimized modified GPI production medium (OMPM). 4.4.12. Effect of inoculum type: In this experiment the effect of different types of F. moniliforme. inocula (spore suspension, seed culture and mycelium disc) on the biomass and gibberellic acid production from OMGPI medium was studied. The results presented in Table (4-14) and Fig (4-16) showed that the seed culture inoculum (spore suspension incubated for 48 h in seed culture before used as inoculum) was found to be the best inocula type for fungus growth (8.43 g/l) and gibberellic acid production (1.50 g/l). Whereas, reduction in biomass content and gibberellic acid production (7.00 g/l and 0.872 g/l, respectively) was obtained when vegetative mycelium disc was used as inoculum comparing with seed culture inoculum. Also maximum gibberellic acid conversion (3.43%) and productivity (10.42 mg/l/h) were recorded when seed culture was used as inoculum.

97 Results and Discussion

Table (4-14): Effect of type of F. moniliforme. inocula on gibberellic acid production (under OMPM). Consume Gibberellic Biomass GA GA Type of Biomass d Sugar, Acid, GA Conversi Conversio Productivity inocula B (g/L) CS (g/L) (g/L)* on (%) n (%) (mg/l/h) Control (spore 1.29 42.68 7.25 16.99 3.02 8.96 suspension, ±0.004 2%) Seed culture 1.50** 43.73 8.43 19.28 3.43 10.42 age, 48h, 2% ±0.003 Disc 8 mm 0.872 34.15 7.00 20.50 2.55 6.06 7 day old ±0.003 *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05)

Silva et al. (1999) investigated that strain H-984 of G. fujikuroi grown for 38 h in a seed culture medium containing 20 g glucose l-1, 3 g yeast extract l-1, -1 -1 -1 -1 2.5 g NH4NO3 l , 0.5 g KH2PO4 l , 0.1 g MgSO4 l , 1 g CaCO3 l , and inoculated into a bioreactor with medium containing 60 g glucose l-1; 1 g -1 -1 -1 NH4Cl l ; 3 g KH2PO4 l and 1.5 g MgSO4 l produced 1100 mg gibberellic acid l-1.

98 Results and Discussion

Consumed sugar g/L Biomass g/L Gibberellic acid g/L GA Conversion (%) GA Productivity mg/l/h

50 12

45 10 40 l

35 8 30

25 6

4 Produc tiv ity mg/l/h 15 Consumed Biomass sugar g/ g/l, 10 2 Gibberellic acid g/l, GA Conversion g/l, acid Gibberellic (%), GA 5

0 0 Control (spore suspention, 2%) Seed culture age, (48h, 2%) Disc 8mm, (7 day old)

Type of inocula Fig (4-16): Effect of type of F. moniliforme. inocula on gibberellic acid production (under OMPM).

4.4.13. Effect of inoculum age: The effect of inoculum (seed culture) age on growth and gibberellic acid production of the local isolate of F. moniliforme. was studied. The optimized modified production medium (50 ml with initial pH 5) were inoculated with different inoculum ages (0, 12, 24, 36 and 48 h) and incubated at 200 rpm and 30°C for 6 days. The data represented in Table (4-15) and Fig (4-17) indicated that the inoculum (12 and 24 h age) resulted in nearly similar cell concentration ranging from (7.61 and 7.88 g/l, respectively). While the inoculum age (0 h) gave the lowest growth (7.46 g/l). On the hand, the highest gibberellic acid production (1.80 g/l) was obtained by (24 h) inoculum age with high gibberellic acid conversion and productivity (4.24 % and 12.50 mg/l/h, respectively). Whereas, the inoculum age (0 h) recorded lower gibberellic acid concentration (1.17 g/l). As the inoculum age (24 h) was the best for

99 Results and Discussion gibberellic acid, subsequently it was selected to carry out the next experiments. Table (4-15): Effect of inoculum (seed culture) age of F. moniliforme. on gibberellic acid production (under OMPM). Consumed Biomass GA GA Inoculum Biomass Gibberellic Acid, Sugar, CS Conversion Conversion Productivity age/h B (g/L) GA (g/L)* (g/L) (%) (%) (mg/l/h) 0 40.30 7.46 1.17±0.002 18.51 2.90 8.13 (spore suspention) 12 40.96 7.61 1.21 ±0.004 18.58 2.95 8.40 24 42.50 7.88 1.80**±0.004 18.54 4.24 12.50 36 41.63 8.17 1.49 ±0.003 19.63 3.58 10.35 48 38.51 8.35 1.47 ±0.001 21.68 3.82 10.21 *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05)

Consumed sugar g/L GA Productivity mg/l/h Biomass g/L Gibberellic acid g/L GA Conversion (%) 45 9

40 8 h

35 7

30 6

25 5 g/l 20 4

15 3

10 2

Consumed sugar GA Productivity g/l, mg/l/ 5 1 Gibberellic acid g/l, GA Conversion (%), Biomass 0 0 0 12243648 Inoculum age (h)

Fig (4-17): Effect of inoculum (seed culture) age of F. moniliforme. on gibberellic acid production (under OMPM).

100 Results and Discussion

Silva et al. (1999) investigated that strain H-984 of G. fujikuroi grown for 38 h in a seed culture with medium containing 20 g glucose l-1, 3 g yeast extract -1 -1 -1 -1 -1 l , 2.5 g NH4NO3 l , 0.5 g KH2PO4 l , 0.1 g MgSO4 l , 1 g CaCO3 l , and inoculated into a bioreactor with medium containing 60 g glucose l-1; 1 g -1 -1 -1 NH4Cl l ; 3 g KH2PO4 l and 1.5 g MgSO4 l produced 1100 mg gibberellic acid l-1. 4.4.14. Effect of inoculum density: As the effect of inoculum age was studied, the effect of inoculum density on growth and gibberellic acid accumulation have to be studied as well. The production medium was inoculated by inoculum age 24 h (which represented the highest gibberellic acid accumulation from the last experiment) with different concentrations (1, 2, 3 and 4 % v/v). As shown in Table (4-16) and Fig (4-18) the mycelium growth increased from (6.38 to 8.75 g/l) by increasing the inoculum density from (1.0 to 4.0 %) with maximum value of biomass conversion (17.94 %) at 3 % inoculum density. Regarding gibberellic acid production, the maximum accumulation (1.85 g/l) was recorded at inoculum density 2.0 % then gibberellic acid synthesis begin to decrease sharply by further increase of inoculum density reaching to 0.401 g/l at 4.0 % inoculum density. Also, the highest conversion of gibberellic acid production (4.19 %) and productivity (12.85 mg/l/h) were recorded at inoculum density 2.0%. Meleigy and Khalaf (2009) inoculated milk permeate medium (gibberellic acid production medium) with 2% (v/v) of inoculum in shake flasks in the production of gibberellic acid.

101 Results and Discussion

Table (4-16): Effect of inoculum density of F. moniliforme. on gibberellic acid production (under OMPM). Inoculum Consumed Gibberellic Biomass GA GA Biomass density Sugar, CS Acid, GA Conversion Conversion Productivity B (g/L) (%v/v) (g/L) (g/L)* (%) (%) (mg/l/h) 1 41.57 6.38 0.916 15.35 2.203 6.36 ±0.001 2 44.20 7.69 1.85** 17.40 4.19 12.85 ±0.003 3 47.16 8.46 0.637 17.94 1.35 4.42 ±0.004 4 49.14 8.75 0.401 17.81 0.82 2.78 ±0.001 *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05).

Consumed sugar g/L Biomass g/L Gibberellic acid g/L GA Conversion (%) GA Productivity mg/l/h 60 14

12 50 l 10 40

6 Productivity mg/l/h 20 4 Consumed sugar g/l, BiomassConsumed g/ sugar g/l,

10 GA Conversion (%), g/l, acid GA Gibberellic 2

0 0 1234 Inoculum density (%v/v)

Fig (4-18): Effect of inoculum density of F. moniliforme on gibberellic acid production (under OMPM).

102 Results and Discussion

4.4.15. Effect of working volume: From the previous result the maximum gibberellic acid accumulation was achieved at agitation speed 200 rpm, so the present experiment aimed to illustrate the proper working volume for maximum gibberellic acid accumulation. The effect of different working volumes of GPI medium (25, 50, 75, 100, 125 and 150 ml) on growth and gibberellic acid accumulation was studied in 250 ml Erlenmeyer flask at initial pH 5 and 30 °C for 6d, under shaking conditions (200 rpm). The data represented in Table (4-17) and Fig (4-19) indicated that the maximum growth (7.95 g/l) was obtained at working volume 75 ml with biomass conversion value (17.40 %), then decreased gradually by increasing the volume of the medium reaching (3.91 g/l) at 150 ml. Whereas, the maximum gibberellic acid concentration (1.88 g/l) was obtained at 50 ml medium, with conversion and productivity content (4.25% and 13.06 mg/l/h, respectively). On the other hand, sharp decrease of gibberellic acid production (0.392 and 0.250 g/l) were obtained at working volumes 125 and 150 ml, respectively. Table (4-17): Effect of working volume of production medium on gibberellic acid production by F. moniliforme (under OMPM). Working Consumed Biomass GA GA Biomass Gibberellic Acid, volume/ Sugar, CS Conversio Conversion Productivity B (g/L) GA (g/L)* ml (g/L) n (%) (%) (mg/l/h) 25 40.36 6.47 0.550 ±0.004 16.03 1.36 3.82 50 44.20 7.73 1.88**±0.00217.49 4.25 13.06 75 45.70 7.95 1.03 ±0.003 17.40 2.25 7.15 100 39.22 6.42 0.830 ±0.004 16.37 2.12 5.76 125 31.83 4.38 0.392±0.005 13.76 1.23 2.72 150 29.19 3.91 0.250 ±0.003 13.39 0.856 1.74 *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05).

103 Results and Discussion

Consumed sugar g/L Biomass g/L Gibberellic acid g/L GA Conversion (%) GA Productivity mg/l/h 50 14

45 12 40

35 10

30 8 25 6 20 Consumed sugar g/l

15 4 Productivity mg/l/h, Biomass g/l 10

2 G ibberellic ac id g/l, G A C onv ers ion (% ), G A 5

0 0 25 50 75 100 125 150

Working volume (ml)

Fig (4-19): Effect of working volume of production medium on gibberellic acid production by F. moniliforme (under OMPM).

During aerobic viscous fermentation, oxygen supply is very important because an insufficient oxygen supply can lead to suboptimal productivity rates, as well as product of low quality (Shu and yang, 1990 and Lee et al., 1995). The effect of oxygen [culture medium volume, (CMV), per flask volume, (FV)] on gibberellic acid production by G. Fujikuroi strain under investigation showed that the highest gibberellic acid accumulation was obtained at higher oxygen percentage studied namely 80 % (50 ml CMV/ 250 ml FV). This finding was supported by (Meleigy and khalaf, 2009). On contrast Lale et al. 2006 reported that the best working volume for gibberellic acid production from G. fujikuroi mutant was 30 ml in 250 ml Erlenmeyer flask. 4.5. Gamma irradiation study: Radiation is a physical phenomenon in which energy travels through space as a wave motion without the aid of traveling medium. Gamma rays is an ionizing radiation which have short wave lengths and contain enough

104 Results and Discussion energy to ionize the molecules in their paths. The exposure of materials like living cells, food and medical products to gamma radiation in such a way that a precise and specific dose is absorbed is termed "gamma radiation". Gamma radiation from 60Co are the most widely used in practices because of costs and high penetration. On one hand, the depth of penetration of an ionizing radiation depends on the nature of the radiation, the charge of the particles forming it and their energy. On the other hand, it depends on the composition and density of the irradiated substance (Ivanov et al., 1986). Among the methods which can be used for changing metabolic activities of living cells is ionizing radiation (Alabostro et al., 1978). Ionizing radiations impart their energy to molecules in a manner that depends on the atomic number of constituent atoms and not on the molecular configuration as in case of UV radiation in which interaction of radiation with matter occurs in a more or less random manner. In a biological material, in living cells it is to be expected that certain sites or systems will be more readily damaged than others (IAEA, 1973 and Giusti et al., 1988). 4.5.1. The effect of gamma irradiation on the survival of F. moniliforme: The spore suspension (5 x 106 CFU /ml) of the higher producer gibberellic acid strain, F. moniliforme, was exposed to increasing doses of gamma irradiation (0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 kGy, using 60Co gamma source at dose rate of 3.056 kGy/h. The dose response curve of the survivors was drawn, and the D10- values (the dose required to reduce the initial population by 90% or by one loge cycle) was determined. Table (4-18) and Fig (4-20) showed the effect of different doses of gamma radiation on survival of F. moniliforme. From these results, it was noticed that the number of viable cells was greatly decreased with increasing the irradiation doses. In addition, dose survival curve for F. moniliforme

105 Results and Discussion

illustrated in Fig (4-20) followed a straight line relationship. The D10- value was calculated from the regression line equation as illustrated before, −1 ∑ xy − nx`y` D-10 value = b = b ∑ x 2 − nx`2

The D-10 value of F. moniliforme obtained from this equation was 1.1 kGy, with correlation coefficient value, - 0.971. This means that the correlation between x and y in this study was very strong. Table (4-18): Effect of different doses of gamma radiation on the surviving of F. moniliforme spors. Doses Average count Log count (kGy) cfu/ml 0.0 3.5×106 6.54 0.25 1.41×105 5.15 0.50 1.09×105 5.04 0.75 5.0×104 4.70 1.0 3.9×104 4.60 1.5 2.14×104 4.33 2.0 2.0×104 4.30 2.5 3.5×103 3.54 3.0 2.0×103 3.30 3.5 3.0×102 2.48 4.0 1.0×102 2.00 4.5 2.0×101 1.30 5.0 0.0 0.0

106 Results and Discussion

Log count count Log 3

0 0123456 Dos e (kGy ) Fig (4-20): Effect of different doses of gamma radiation on the surviving of F. moniliforme spores. 4.5.2. Effect of gamma irradiation on the production of gibberellic acid: The present experiment was carried out to investigate the effect of gamma radiation on the activity of F. moniliforme towards gibberellic acid production. The irradiated spore suspensions were used for preparation of seed culture inocula (24 h – old). The inoculated OMGPI medium pH 5.0 was incubated at 30°C for 6 days under shaking (200 rpm), the cell dry weight and gibberellic acid contents were investigated. The data in Table (4-19) and Fig (4-21) showed that the low doses of gamma radiation from 0.25 to 0.75 kGy stimulate both growth content and gibberellic acid production in comparison with the control (non irradiated inoculum).

107 Results and Discussion

Table (4-19): Effect of different doses of gamma radiation of F. moniliforme for gibberellic acid production (under OMPM). Consumed Gibberellic Biomass GA GA Dose Biomass Sugar, CS Acid, GA Conversion Conversion Productivity (kGy) B (g/L) (g/L) (g/L)* (%) (%) (mg/l/h) 0.0 44.26 7.69 1.70 17.37 3.84 11.80 ±0.004 0.25 47.01 7.90 1.87 16.80 3.98 12.98 ±0.003 0.5 48.37 8.43 2.36** 17.43 4.88 16.38 ±0.003 0.75 48.09 8.24 1.68 17.13 3.49 11.66 ±0.002 1.0 44.36 6.45 1.43 14.54 3.22 10.63 ±0.001 1.5 36.51 4.00 0.730 10.96 2.00 5.07 ±0.001 2.0 30.18 1.74 0.571 5.77 1.89 3.96 ±0.003 2.5 27.32 0.820 0.325 3.00 1.19 2.25 ±0.002 3.0 26.25 0.410 0.212 1.56 0.807 1.47 ±0.002 3.5 20.93 0.132 0.109 0.631 0.520 0.756 ±0.001 4.0 ------*values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05)

108 Results and Discussion

The highest cell content and gibberellic acid concentration (8.24 g/l and 2.36 g/l, respectively, were achieved at the dose 0.50 kGy with gibberellic acid conversion of 4.88 % and productivity rate of 16.38 mg/l/h. While, a decrease in the biomass was recorded by the increasing doses of gamma radiation (> 0.75 kGy) compared with control. Also, decreasing in gibberellic acid production was achieved by the increasing doses (> 1.0 kGy) in comparison with control. Furthermore, the data revealed that little amount of biomass and gibberellic acid content was obtained at a dose 3.5 kGy. In addition, no growth of fungus was obtained at a dose 4.0 kGy.

Consumed sugar g/L Biomass g/L Gibberellic acid g/L GA Conversion (%) GA Productivity mg/l/h 60 18

16 50 14

40 12

10 30 8 Consumed sugar g/l

20 6 mg/l/h, Biomass g/l

4 10 2 Gibberellic acid g/l, GA Conversion acid g/l, Gibberellic (%), GA Productivity 0 0 0 0.25 0.5 0.75 1 1.5 2 2.5 3 3.5

Dose (kGy)

Fig (4-21): Effect of different doses of gamma radiation of F. moniliforme for gibberellic acid production (under OMPM).

Several studies recorded that the low doses of gamma radiation may stimulate the microbial growth and metabolic activities (El-Batal and Khalaf, 2003; Khalaf and Khalaf, 2005). Helal et al. (1987) recorded that the maximum metabolic activity of Trichoderma koningii and Aspergillus niger were obtained after exposure to irradiation doses of 0.1 and 0.25 kGy respectively. Meanwhile, high doses of

109 Results and Discussion gamma radiation were proved to be inhibitory for both growth and enzymatic activities of microorganisms (Sadi, 1987). The exposure of cells to ionizing radiation sets off a chain of reactions giving rise to chemical and then to metabolic or physiological changes. The irradiation presents an additional stress to the cell which tends to disturb their organization (Lawrence, 1971a). Irradiation effects have been shown to occur with proteins, enzymes, nucleic acid, lipids and carbohydrates, all which have marked effects on the cell (Habbs and Macellan, 1975). Generally, Lawrence (1971b) mentioned that the loss of proliferate capacity is usually caused by damage within DNA molecules which controlling all aspects of metabolism, structure and development. It has been reported that a number of intracellular constituents may be responsible for the high radiation resistance in some radio-resistant strains. This may include certain chemical compounds such as mercaptoalkyl-amine (Anderson et al., 1956), sulphydryl compounds (Bridges, 1964), amino acids (Work, 1964) and proteins (Mareson and Stelow, 1987). 4.6. Immobilization study: Conventional methods of fermentation that use free cells in batch processes have several limitations. The use of immobilized cells offers several advantages over free cells. The immobilized cell culture system achieve high cell density while maintaining high mass transfer rates and thereby offer the advantage of high productivity (Ibrahim et al., 2006). Offer superior stability due to protection of cells by physico-chemical interactions between substrate and cells, changed permeability of cells towards high penetration of substrate and renewable or self- generating or self- proliferating nature of biocatalytic system (Kahlon and Malhorta 1986), also decrease gibberellic acid production costs.

110 Results and Discussion

4.6.1. Gibberellic acid production by sponge-immobilized cells of gamma irradiated F. moniliforme: In this experiment, production of gibberellic acid by irradiated cells entrapped in sponge cubes was investigated. The irradiated spores (0.5 kGy) of F. moniliforme were immobilized in sponge cubes (for 24 h) and the batch cultures (50 ml) of the production medium pH 5.0 with sponge cubes (0.5 g) containing irradiated cells were incubated under obtained optimized cultural conditions. The results present in Table (4-20) and Fig (4-22) showed that using of immobilized cells as inoculum lead to increasing of biomass and gibberellic acid production (8.54 and 2.51 g/l, respectively) comparing with free cells inoculum (8.33 and 2.30 g/l, respectively). Also, the conversion of gibberellic acid production and its productivity reached to their maximum values (5.21 % and 17.43 mg/l/h, respectively) with immobilized cells. Table (4-20): Gibberellic acid production by immobilized gamma irradiated (0.5 kGy) cells, 24 h age of F. moniliforme (under OMPM). Consumed Biomass Gibberellic Biomass GA GA State of Sugar, CS B Acid, GA Conversion Conversion Productivity inoculum (g/L) (g/L) (g/L)* (%) (%) (mg/l/h)

Free cells 2.30 ± 48.5 8.33 17.18 4.74 15.97 (2% v/v) 0.004 Immobilized cells 2.51** 48.2 8.54 17.72 5.21 17.43 (0.5g ±0.004 cubes/flask) *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05).

111 Results and Discussion

Meleigy and Khalaf (2009) reported that best yield of gibberellic acid (2.40 gl-1) was recorded by immobilized cells under optimized cultural conditions (4 immobilized discs, 30°C and pH 5). The yield of GA production by immobilized F. moniliforme cells in sodium alginate gel was more than that produced by free cells (Kahlon and Malhotra, 1986). The higher yield with an immobilized system depends upon the nature of material matrix, which affects the permeability of the cell towards high penetration of the substrate, as well as faster removal of the end products from the fermentation sites, due to protection of the mycelium from micro environmental changes by the material matrix (Kahlon and Malhotra, 1986; Lu et al., 1995). Gibberellic acid productivity by G. fujikuroi cells immobilized with the carrier-covered copolymer of hydrophilic hydroxyethyl acrylate and hydrophobic trimethylpropane triacrylate (HEA-A-TMPT) was higher than that in the free cells (Lu et al., 1995; Saudagar et al., 2008). The results observed also explain that in contrast to the sodium alginate immobilization which requires more sophisticated equipments involving high cost, the more robust immobilized loofa sponge systems can be made simply by adding spore suspension to the medium containing the inexpensive sponge disc without any prior chemical treatment.

112 Results and Discussion

Consumed sugar g/L Biomass g/L GA Productivity mg/l/h Gibberellic acid g/L GA Conversion (%)

60 6 h

50 5

40 4

30 3

20 2 G ibberellic ac id g/l, G A Conversion (% )

10 1 Consumed GA Biomass sugar Productivity g/l, g/l, mg/l/

0 0 Free cells(2% v/v) Immobilized cells (0.5 g cubes /flask)

State of inoculum

Fig (4-22): Gibberellic acid production by immobilized gamma irradiated (0.5 kGy) cells, 24 h age of F. moniliforme (growth conditions as in Table 4-8).

4.6.2. Effect of immobilized inoculum age: To investigate the effect of the age of immobilized cells on growth and gibberellic acid production from the medium, irradiated spore suspension with 0.5 kGy, was immobilized (0.5g sponge cubes) for different time ranging from 12 to 48 h, and used as inocula. The data represented in Table (4-21) and Fig (4-23) indicated that the immobilized cells age (24 h) gave the highest biomass (8.62 g/l) and gibberellic acid content (2.53 g/l). While, the lowest growth (5.38 g/l) and gibberellic acid (1.32 g/l) amounts were recorded with immobilized cells age (48 h). Also, the maximum gibberellic acid conversion (5.20 %) and its productivity rate (17.57 mg/l/h) were recorded with immobilized cell age (24 h).

113 Results and Discussion

Table (4-21): Effect of inoculum age of gamma irradiated (0.5 kGy) immobilized cells of F. moniliforme (0.5g cubes/flask) on gibberellic acid production (under OMPM). Age of Consumed Gibberellic Biomass GA GA Biomass immobilized Sugar, Acid, GA Conversion Conversion Productivity B (g/L) cells/h CS (g/L) (g/L)* (%) (%) (mg/l/h) 2.17 12 46.91 8.29 17.67 4.63 15.07 ±0.004 2.53** 24 48.63 8.62 17.73 5.20 17.57 ±0.003 1.79 36 39.05 7.01 17.95 4.58 12.43 ±0.004 1.32 48 30.72 5.38 17.51 4.30 9.17 ±0.002 *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05). Meleigy and Khalaf (2009) reported that 48 h old F. moniliforme γ- 14 immobilized loofa sponge discs were used for the production of gibberellic acid. They added that there was no increase in the immobilized loofa sponge disc weight in seed culture medium after 48 h. This may be indication of the saturation of space for further immobilization on the disc. While the immobilized cells are packed tightly within the sponge, there remain large number of micro channels for free movement of the solute during the fermentation process (Iqbal et al., 2005), thus making the conditions favorable for diffusion of media nutrients to the immobilized cells.

114 Results and Discussion

Consumed sugar g/L Biomass g/L GA Productivity mg/l/h Gibberellic acid g/L GA Conversion (%) 60 6

50 5

40 4

30 3 mg/l/h

20 2 Gibberellic acid g/l, GA Conversion acid g/l, (%)Gibberellic 10 1 Consumed BiomassGA sugar g/, Productivity g/l,

0 0 12 24 36 48 Age of immobilized cells /h Fig (4-23): Effect of inoculum age of gamma irradiated (0.50 kGy) immobilized cells of F. moniliforme (0.5g cubes/flask) on gibberellic acid production (under OMPM).

4.6.3. Effect of immobilized inoculum density: The aim of this experiment is to determine the best inoculum density of immobilized cells for growth and gibberellic acid production. Erlenmeyer flasks containing 50 ml OMGPI production medium (pH 5.0) were inoculated with different weight of immobilized sponge cubes (24 h age) ranging from 0.25 to 1.0 g/flask, and incubated under optimized fermentation conditions. The data represented in Table (4-22) and Fig (4-24) illustrated that the maximum gibberellic acid production (2.57 g/l) was obtained at inoculum size of 0.5 g (% w/v) with conversion value (5.30 g/l) and productivity rate (17.85 mg/l/h). Regarding microbial growth the maximum biomass (8.57 g/l) was obtained at immobilized cells density 0.5 g (% w/v). Meanwhile, increasing inoculum size lead to decreasing in biomass content.

115 Results and Discussion

Table (4-22): Effect of inoculum density of gamma irradiated (0.50 kGy) immobilized cells of F. moniliforme on gibberellic acid production (under OMPM). Inoculum Consumed Gibberellic Biomass GA GA density (g Biomass Sugar, CS Acid, GA Conversion Conversion Productivity cubes/flas B (g/L) (g/L) (g/L)* (%) (%) (mg/l/h) k) 1.67 0.25 42.36 7.29 17.21 3.94 11.60 ±0.003 2.57** 0.50 48.50 8.57 17.67 5.30 17.85 ±0.004 1.69 0.75 49.91 7.85 15.73 3.39 11.74 ±0.003 1.17 1.00 48.70 6.38 13.10 2.40 8.13 ±0.002 *values are means of 3 replicates ± SE. **Significant from all GAvalues (p< 0.05).

Consumed sugar g/L Biomass g/L GA Productivity mg/l/h Gibberellic acid g/L GA Conversion (%) 60 6

50 5

40 4

30 3 mg/l/h

20 2

10 1 Gibberellic acid g/l,G A Conversion(%) Consumed sugar Biomassg/l, GA g/, Productivity

0 0 0.25 0.5 0.75 1 Inoculum density (g cubes/flask)

Fig (4-24): Effect of inoculum density of gamma irradiated (0.50 kGy) immobilized cells of F. moniliforme on gibberellic acid production (under OMPM).

116 Results and Discussion

Meleigy and Khalaf (2009) reported that the best yield of gibberellic acid (1.90 gl-1) with conversion level 3.38% and productivity rate 13.20 mgl-1 h-1 were obtained with 4 immobilized discs and any increased in disc number did not stimulate growth. Kahlon and Malhotra (1986) entrapped F. moniliforme in various alginate gel concentrations and obtained low yields of GA with increase in gel concentrations due to limited transportation of oxygen to the inside and biocatalyst, thus lowering the efficiency of biocatalyst. Similar results were observed for sorbitol and clavulanic acid production by microbial immobilized cells on loofa sponge (Vignoli et al., 2006 and Saudagar et al., 2008). In high inoculum density the utilization of sugar was high but the yield of gibberellic acid was low, this means that the microorganism utilized the sugars for vegetative growth.

4.6.4. Time course of gibberellic acid production by gamma irradiated (0.5 kGy) immobilized cells of F. moniliforme: In this experiment, the inoculated flasks containing 50 ml of OMGPI production medium (pH 5.0) and 0.5 g of immobilized sponge cubes (24 h age) were incubated at various cultivation times under optimized fermentation conditions. From the results in Table (4-23) and Fig (4-25), it is clear that the gibberellic acid production was increased rapidly for the first 6 days then decreased. The amount of gibberellic acid reached its maximum concentration (2.63 g/l) after 6 days of cultivation, comparing with (2.30 g/l) for gibberellic acid produced by free irradiated cells at the same time (Table 4-20). Thereafter, the amount of produced gibberellic acid decreased to be (0.852 g/l) after 9 days of incubation. In addition, the maximum values of gibberellic acid conversion and productivity (5.46 % and 18.26 mg/l/h,

117 Results and Discussion respectively) were recorded after 6 days of incubation. On the other hand, the mycelium growth was increased rapidly with increasing incubation period till reached its maximum (8.63 g/l) after 7 days of cultivation. In addition, little decrease in biomass production was occurred with increasing incubation time after 7 days. Table (4-23): Time course of gibberellic acid production by gamma irradiated (0.5 kGy) immobilized cells of F. moniliforme (under OMPM). Consumed Gibberellic Biomass GA GA Incubation Biomass Sugar, CS Acid, GA Conversion Conversion Productivity time/days B (g/L) (g/L) (g/L)* (%) (%) (mg/l/h) 1 29.00 4.52 - 15.59 - - 2 32.42 6.05 0.516 18.66 1.59 3.58 ±0.004 3 36.57 6.41 0.735 17.53 2.01 5.10 ±0.003 4 41.02 7.25 1.51 17.67 3.68 10.49 ±0.001 5 47.61 8.13 2.06 17.08 4.33 14.31 ±0.001 6 48.13 8.49 2.63** 17.64 5.46 18.26 ±0.002 7 49.51 8.63 1.67 17.43 3.37 11.60 ±0.003 8 50.39 8.00 1.33 15.88 2.64 9.24 ±0.002 9 51.00 7.83 0.852 15.35 1.67 5.92 ±0.002 *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05).

118 Results and Discussion

Consumed sugar g/L Biomass g/L Gibberellic acid g/L GA Conversion (%) GA Productivity mg/l/h 60 20

50 16

14 40 12

30 10

8 mg/l/h, Biomass g/l Consumed sugar g/l 20 6

4 10

2 G ibberellic ac id g/l, G A Conv ers ion (% ), G A Produc tiv ity

0 0 123456789 Incubation time/ days Fig (4-25): Time course of gibberellic acid production by gamma irradiated (0.50 kGy) immobilized cells of F. moniliforme (under OMPM).

4.6.5. Effect of repeated batch fermentation: The study was directed to use whey (cheap dairy by-product), as the main medium for gibberellic acid production to substitute the optimized modified production medium under repeated batch fermentation. It is reasonably high in lactose and contains minerals necessary for microbial growth and it is low in nitrogen (Yellore and Desai, 1998). Each 50 ml of prepared milk permeate (pH 5.0) were inoculated by 0.5 g of immobilized sponge cubes (24 h age) and incubated at 30 °C and 200 rpm (optimized fermentation conditions) for 6 cycles, (each consisting of 6 days). As shown in Table (4-24) and Fig (4-26), the amount of gibberellic acid produced by free cells reached its maximum (1.63 g/l) in the first batch and decreased markedly in the subsequent batches. With immobilized cells, the gibberellic acid production was increased from the first batch (1.93 g/l) to the fourth (1.61 g/l), in comparison with maximum free cells production, with

119 Results and Discussion maximum concentration (2.20 g/l) at the second batch. In addition, the highest gibberellic acid conversion and productivity (4.28 % and 15.28 mg/l/h, respectively) were observed by immobilized cells at the second batch. Similarly, biomass production by immobilized cells was fairly highly after 4 batches, in comparison with maximum free cells production, with maximum content (8.67 g/l) at the second batch. On the other hand, the maximum biomass produced by free cells (6.61 g/l) was obtained in the first batch, then sharp decrease was occurred. Several industrial residues such as milk, whey, molasses, sugar beet pulp and hydrol had also been used for gibberellic acid production, these residues give low but economically useful yield (Sastry et al., 1988; Cihangir and Aksoz (1996). Lu et al. (1995) found that during 12 batch fermentation cycles over 84 days, GA productivity by immobilized G. fujikuroi cells with copolymer HEA-A-TMPT, was maintained at a constant value. This suggested that the immobilized cells produced a stable system for GA production. On the other hand,

120 Results and Discussion

Table (4-24): Gibberellic acid production from milk permeate in repeated batch process1 by 0.50 kGy gamma irradiated free2 or immobilized cells3 of F. moniliforme under optimized fermentation conditions4. Consumed Gibberellic Biomass GA GA Batch Biomass Sugar, CS Acid, GA Conversion Conversion Productivity No. B (g/L) (g/L) (g/L)* (%) (%) (mg/l/h) 1 48.5 8.45 1.93 17.42 3.98 13.40 (46.63) (6.61) ±0.002 (24.81) (3.52) (11.39) (1.64) ±0.004 2 51.40 8.67 2.20** 16.87 4.28 15.28 (35.50) (4.62) ±0.003 (13.01) (2.79) (6.88) (0.99) ±0.003 3 46.20 7.19 1.94 15.56 4.20 13.47 (20.36) (0.85) ±0.004 (4.17) (1.03) (1.46) (0.21) ±0.003 4 40.65 6.75 1.61 16.61 3.96 11.18 ND*** ±0.004 5 37.83 5.93 1.30 15.68 3.44 9.03 ±0.004 6 30.19 3.82 0.85 12.65 2.82 5.90 ±0.003 1= 6 days for each run, 2=1% v/v, 24 h age, 3= 0.5g cubes/flask, 24h age, 4= initial pH 5.0, incubation temp. 30 ºC, working volume medium 50 ml/flask and agitation rate 200 rpm. *values are means of 3 replicates ± SE. **Significant from all GA values (p< 0.05). *** ND = Not Detected. Data between brackets were values of free cells.

121 Results and Discussion

Meleigy and Khalaf (2009) reported that, when they studied gibberellic acid production in four reuse cycles, during the first reuse the GA production increased gradually with the increase in time of fermentation and reached a maximum of 2.40 gl-1 at the end of 8 d. For the second reuse, the cells were already in the late exponential phase and hence a faster initiation of the GA production was observed. Levels of GA produced in second ruse were 0.60 and 1.62 gl-1 at the end of 2 and 4 d, respectively, as compared to 0.45 and 1.36 gl-1 for similar time intervals in the first reuse. These results illustrated that the mutant γ-14 isolate immobilized on loofa sponge discs can be repeatedly reused under the fermentation conditions. Although, a decrease in the final production level of GA was observed with every reuse, the time for initiation of GA production was decreased as the cells are already in the production stage after the first cycle. The decrease in the final production level may be as a result of increase in the number of immobilized dead cells with reuse.

Consumed sugar g/L Biomass g/L Gibberellic acid g/L GA Conversion (%) GA Productivity mg/l/h 60 18

16 50

14 oductivity

40 12

10 30 8 mg/l/h, Biomass g/l Consumed sugarg/l 20 6

4 10 2 G ibberellic ac id g/l, G A Convers ion (% ), G A Pr

0 0 123456 Batch number (Immobilized cells)

Fig (4-26a): Gibberellic acid production from milk permeate in repeated batch process by 0.50 kGy gamma irradiated immobilized cells of F. moniliforme under optimized fermentation conditions.

122 Results and Discussion

Consumed sugar g/L Biomass g/L Gibberellic acid g/L GA Conversion (%) GA Productivity mg/l/h 50 12

10 40

35 8 30

25 6

20 mg/l/h, Biomass g/l Consumed sugarg/l 4 15

10 llic ac id g/l, G A Conv ers ion(% ),G AProduc tiv ity 2

5 Gibbere

0 0 123 Batch number (free cells)

Fig (4-26b): Gibberellic acid production from milk permeate in repeated batch process by 0.50 kGy gamma irradiated free cells of F. moniliforme under optimized fermentation conditions.

4.7. Toxicological evalution: Fertile eggs were employed in testing for toxins, especially mycotoxins, since chicken embryos are very sensitive to aflatoxins and other mycotoxins. In this regard, the same method was used to determine the possible toxicity of the F. moniliforme culture filterate and evaluate the presence of toxic substances contaminating the gibberellic acid recovered from ethanol extraction of the culture filtrate. As shown in Table (4-25) and Fig (4-27), the data revealed that survival of chicken embryos inoculated with gibberellic acid dissolved in ethanol indicated that the trace amount of soluble toxic substance in the sample lead to only 8 % death of chicken embryo. However, little toxic substance in the culture filtrate of production medium cultivated with F. moniliforme caused a moderate death rate (20%) among tested chicken

123 Results and Discussion embryos, compared with the control treatment (sterile production medium) which caused 19% death. Table (4-25): Effect of gibberellic acid produced by F. moniliforme on chicken embryos. Dead chicken embryos/ Treatments Dead % total inoculated eggs Control eggs inoculated with: Sterile distilled water 0/25 0 Sterile production medium 4/25 19 Sterile ethanol 1/25 4 Culture filtrate of production 5/25 20 medium cultivated with F. moniliforme Gibberellic acid dissolved in 2/25 8 ethanol (1mg/ml)

Inoculated eggs Dead embryos % Dead 30 25

25 20

20 15

15 Dead (%) 10 10 Inoculated eggs, DeadInoculated embryos

5 5

0 0 H2O Production medium Ethanol Filterate Gibberellic acid Treatments

Fig (4-27): Effect of gibberellic acid produced by F. moniliforme on chicken embryos.

124 Results and Discussion

4.8. Chitosan stydy: The application of fungal chitosan in many fields could verify its biosafety and suitability for human use, and that was reported by many researchers (Alishahi and Aider, 2012; Kong et al., 2010 and Sandford, 1989). The present investigation was undertaken to ascertain whether gibberellic acid play any beneficial role on biomass and chitosan production by Aspergillus niger in the best fermentation medium and the optimum incubation time and the optimum gibberellic acid concentration. 4.8.1. Influence of type of media: The fungus (A .niger) was cultured on different specific chitosan production media (MSM, PDB and YPG), in 250 ml Erlenmyer flasks, each containing 50 ml medium. The flasks were inoculated with 1 ml spore suspention (6 x 107 CFU/ml) and incubated at 30 °C under shaking (120 rpm) for 72 h. Growth of A. niger under submerged fermentation condition in three different media is presented in Table (4-26) and Fig (4-28). It appear from the result that the production of chitosan has found to be influenced by composition of the growth medium, as the highest amount of chitosan (0.83 g/l) was obtained with MSM with productivity rate 11.53 mg/l/h and chitosan content 0.136 in g/g of mycelia. These results is in accordance with the result reported by Chatterjee et al. (2005) whose found that MSM was the best production medium for chitosan production.

125 Results and Discussion

Table (4-26): Effect of different media on mycelial growth and chitosan production growth conditions (72 h, 120 rpm, 30 °C). Type of Mycelial growth Chitosan Chitosan Chitosan media (Dry weight) production (g/l) productivity content (g/g g/l mg/l/h of mycelia) MSM 6.10 0.83 11.53 0.136 PDB 4.09 0.39 6.66 0.095 YPG 5.31 0.62 8.61 0.117

Dry weight Chitosan productivity Chitosan production Chitosan content 14 0.9

0.8 12 0.7 10 0.6

8 0.5

6 0.4 mycelia

0.3 4 0.2

Dry weight g/l,Chitosan productivity mg/l/h productivity Dry g/l,Chitosan weight 2 0.1 Chitosan production g/l,Chitosan content g/g of of g/g content g/l,Chitosan production Chitosan

0 0 MSM PDB YPG Type of media

Fig (4-28): Effect of type of media on chitosan production. Also Arcidiacono and Kaplan (1992) showed that medium composition affected biomass production and molecular weight of chitosan in case of Mucor rouxii.

Sucrose concentration of MSM (the best fermentation medium) was standardized and maximum growth and chitosan production was obtained at 5%, further increase in sucrose concentration did not improve the growth or chitosan production (data not shown). Chatterjee et al. (2005) reported that

126 Results and Discussion the optimum concentration of sucrose in molasses is 4% when MSM used in chitosan production from Mucor roxii 4.8.2. Influence of addition of gibberellic acid: To study the effect of gibberellic acid on chitosan production from A. niger, fifty milliliter of MSM (pH 5.0) was added to 250 ml Erlenmyer flask. The flasks were inoculated with 1 ml spore suspension and 3 mg/l gibberellic acid. The flasks were incubated at 30 °C for 72h. The effect of GA on growth and chitosan production by A. niger is presented in Table (4-27) and Fig (4-29), it appear from the results that the addition of gibberellic acid increased mycelial growth as well as chitosan production by 34.9 and 27% respectively with productivity chitosan rate 15.41 mg/l/h. In our opinion the increase in the chitosan content in the media supplement with gibberellic acid may be due to increase in biomass production and increase in protein content of the biomass. Table (4-27): Influence of gibberellic acid on growth of Aspergillus niger and chitosan production. (MSM medium, 5% molasses, 3 mg GA, 120 rpm, 72 h). Mycelial Increase Increase in Chitosan growth Chitosan in Chitosan GA chitosan content (Dry production mycelial productivity addition production (g/g of weight) (g/l) growth mg/l/h % mycelia) g/l % Control 6.92 0.874 - - 0.126 12.14 media ±0.17 ±0.06 ±0.03 Media + 9.34 1.13 34.97 27.00 0.121 15.41 GA ±0.12 ±0.08 ±0.18 ±0.11 ±0.01

Chatterjee et al. (2009) reported that supplementation of molasses– salt medium with gibberellic acid at a concentration of 3 mg/l, increased mycelial growth to 9.3 g/l and chitosan production to 11.2% of mycelia

127 Results and Discussion during study the influence of gibberellic acid on the growth of Mucor rouxii and chitosan production. Whereas Brain et al. (1954) did not find any effect on the growth of a number of bacteria and molds.

Dry weight Chitosan productivity Chitosan production Chitosan content

18 1.2

16 1 14

12 0.8

10 0.6

8 mycelia

6 0.4

4 Dry productivity mg/l/h g/l,Chitosan weight 0.2 Chitosan production g/l,Chitosan content Chitosan of g/g production g/l,Chitosan 2

0 0 control medium+GA Addtion of GA

Fig (4-29): Effect of Addition of gibberellic acid on chitosan production. 4.8.3. Effect of incubation time: The effect of incubation time on the mycelial growth and chitosan production was measured during 96 h, dry weight determination at time interval of 24 h. The MSM was inoculated and incubated at 30 °C and 120 rpm for 24, 48, 72 and 96 h. The data in Table (4-28) and Fig (4-30) revealed that the mycelial growth increased by increasing the incubation time reaching its maximum (10.95 g/l) after 48 h, also chitosan production reaches its maximum (1.30 g/l) after 48 h of incubation with an increase in mycelial growth and chitosan production 71.63 and 73.33%, respectively. Productivity chitosan rate was 27.08 mg/l/h. Our results was contradicted with that obtained by Chatterjee et al. (2009) who mentioned that the best incubation

128 Results and Discussion time for both mycelial growth and chitosan production from Mucor rouxxi was 32 h. Also, Chatterjee et al., (2008) showed that the optimum mycelial growth and chitosan production was obtained after 72 h of incubation when Rhizopus oryzae used in chitosan production from whey medium. Table (4-28): Effect of incubation time on chitosan production (MSM medium, 5% molasses, 3 mg GA, 120 rpm). Mycelial Chitosan Increase Increase in Chitosan Chitosan growth production in chitosan content productivity Time (Dry (g/l) mycelial production (g/g of mg/l/h weight) growth % mycelia) g/l % 6.38 0.75 - - 0.118 31.25 24 h ±0.11 ±0.09 ±0.17 10.95 1.30 71.63 73.33 0.119 27.08 48 h ±0.19 ±0.08 ±0.13 ±0.07 ±0.15 9.40 1.18 47.34 57.33 0.126 16.39 72 h ±0.14 ±0.07 ±0.15 ±0.09 ±0.13 7.45 0.83 16.77 10.67 0.111 8.65 96 h ±0.18 ±0.12 ±0.17 ±0.09 ±0.14

129 Results and Discussion

Mycelial growth Chitosan productivity Chitosan production Chitosan content g/g 35 1.4

30 1.2

25 1

20 0.8

15 0.6

10 0.4 Dry weight g/l,Chitosan productivity productivity mg/l/h Dry g/l,Chitosan weight 5 0.2 Chitosan production g/l,Chitosan content g/g of mycelia of g/g content g/l,Chitosan production Chitosan

0 0 24 h 48 h 72 h 96 h Incubation time

Fig (4-30): Effect of incubation time on chitosan production (MSM medium, 5% molasses, 3 mg GA, 120 rpm).

4.8.4. Effect of addition of different concentrations of GA in chitosan production: To determine the optimum GA concentration for chitosan production, different concentrations of GA acid (1,2,3,4,5 mg/l ) were added to MSM, the media was inoculated and incubated for 48 h at 120 rpm. The results in Table (4-29) and Fig (4-31) showed that the optimum GA concentration is 2 mg/l, with an increase in mycelial growth and chitosan production by 24 and 26.09 % respectively. The chitosan productivity rate of this concentration was 30.21 mg/l/h. Chatterjee et al. (2009) reported that supplementation of molasses salt medium with gibberellic acid at a concentration of 3 mg/l, increased mycelial growth (9.3 g/l) and chitosan production (11.2% of mycelia) during study the influence of gibberellic acid on the growth of Mucor rouxii and chitosan production. Also Chatterjee et al. (2008) showed that when GA3

130 Results and Discussion added to the whey medium at a concentration of 0.1 mg/L increased both mycelial growth and chitosan content by 32 and 14.3%, respectively. Table (4-29): Effect of addition of different concentrations of GA in chitosan production: (MSM medium, 5% molasses, 48 h, 120 rpm). Mycelial Increase Increase in Chitosan GA growth Chitosan in Chitosan chitosan content Concentra (Dry production mycelial productivity production (g/g of -tions mg weight) (g/l) growth mg/l/h % mycelia) g/l % 9.25 1.15 - - 0.124 23.96 1 ±0.04 ±0.06 ±0.07 11.47 1.45 24.00 26.09 0.126 30.21 2 ±0.06 ±0.07 ±0.09 ±0.05 ±0.09 10.87 1.33 17.51 15.65 0.122 27.71 3 ±0.09 ±0.11 ±0.07 ±0.04 ±0.05 8.00 0.95 - - 0.119 19.79 4 ±0.17 ±0.13 ±0.12 5.52 0.67 - - 0.121 13.96 5 ±0.11 ±0.09 ±0.07 3.93 0.63 - - 0.160 13.13 6 ±0.05 ±0.06 ±0.08

131 Results and Discussion

Mycelial grow th Chitosan productivity Chitosan production Chitosan content g/g

35 1.6

30 1.4

1.2 25 1 20

mg/l/h 0.8 15 0.6

10 mycelia of g/g content 0.4 Dry weight g/l,Chitosan productivity productivity Dry g/l,Chitosan weight Chitosan production g/l,Chitosan g/l,Chitosan production Chitosan 5 0.2

0 0 1 mg 2 mg 3 mg 4 mg 5 mg GA Concentration

Fig (4-31): Effect of addition of different concentrations of GA in chitosan production : (MSM medium, 5% molasses, 48 h, 120 rpm).

Paul et al. (1994) observed stimulation of growth of food yeast, K. fragilis, in whey medium supplemented with gibberellic acid (10 mg/L). Makarem and Aldridge (1969) found that gibberellic acid at an optimum concentration of 10 mg/L increased cell division rate of different strains of Hansenula wingei Our findings although contradicted the previous report of

Evans (1984) that GA3 had no significant effect on the growth of microorganisms but corroborated other findings in this regard, showing better growth and development of fungi in presence of GA3 (Tomita et al., 1984 and Michniewicz and Rozej ,1988). The produced fungal chitosan was characterized with deacetylation degree of 81.3 % and molecular weight of 24.2 kDa. The FT-IR spectra for chitosan from A. niger in comparison with standard chitosan from Sigma are illustrated in Fig (4-31). The main characteristic peaks of chitosan were observed at (A) curve, standared chitosan, are at 1099 cm-1 (-C-O saccharide ring), 1642 cm-1 (C=O residue of

132 Results and Discussion acetyl group), 1401 cm-1 (NH stretching), 2928 cm-1 (C-H stretch) and -1 3100-3350 cm (broad band for OH and NH2 group). In (B) curve, chitosan from A. niger, there is no change in the main characteristic absorption bands of chitosan in compared to A curve, the main difference only the intensity of C=O appeared at 1642 cm-1 decreased than that of A. In (C) curve, chitosan from A. niger after the addition of GA, there is a new carbonyl group formed at 1661 cm-1 during the modification and some broadness for OH group occurred at 3160- 3267 cm-1 .

% A

T r a n s B m i t t a n c e C

4000 3500 3000 2500 2000 1500 1000 500 Wavenumbers Fig (4-32): FT-IR of different fungal chitosan along with chitosan from Sigma. (A) Chitosan obtained from Sigma; (B) Citosan obtained from A. niger in MSM; (C) Chitosan obtained from A. niger in MSM with the addition of gibberellic acid.

133 Summary

Summary

Gibberellic acid, an important growth regulator, is used extensively in agriculture, nurseries, viticulture and tea garden for a variety of economic benefits. Its use, at presents, is limited to high premium crops mainly because of its cost. Reduction in cost will lead to its wider application to avariety of crops and also to the harvest of innumerable industrial and economic benefits. In this regard, the present study has been devoted to investigate the effect of different parameters on gibberellic acid production by the local isolated strain of F. moniliforme, in an attempt to maximize the production, the study included isolation and selection of most efficient gibberellic acid producing fungi from different soil and grain samples, nutritional and environmental factors supporting their efficiencies to produce gibberellic acid. A part of this study concentrated on the use of milk permeate ( cheap by- product) as main gibberellic acid production medium. Also study the enhancement of chitosan production from Aspergillus niger by the addition of gibberellic acid. The results can be summarized in the following points: 1- Out of 28 fungal isolates previously isolated from different sources, only 10 isolates showed the ability to produce gibberellic acid in the broth medium, with F. moniliforme isolate the most producer strain (0.299 g/L).

2- The produced gibberellic acid was extracted and identified by comparing it with standard sigma gibberellic aicd using HPLC and FT-IR.

3- For improving gibberellic production by F. moniliforme, optimization of environmental and cultural fermentation conditions were carried out: a- The incubation period of 6 days was the optimum time for highest gibberellic acid concentration and productivity (0.314 g/l and 2.19 mg/l/h, respectively).

134 Summary b- The optimum incubation temperature for both gibberellic acid production and mycelial growth of F. moniliforme was 30 °C. c- Different levels of initial pH values were investigated. pH 5.0 was the most favorable one for gibberellic acid production (0.380 g/L), whereas the highest biomass (8.46 g/l) was noticed at pH 5.5. d- Gibberellic acid was increased by increasing agitation speed up to 200 rpm, at which the fungus produced 0.462 g/L and further increase did not improve the production. e- Ten carbon sources were investigated for gibberellic acid production by F. moniliforme isolate, the highest gibberellic acid concentration ( 0.609 g/l) was obtained when fructose used as main carbon source. f- Eight concentrations of fructose ranged from 2.0 to 10% were added to the production medium for gibberellic acid fermentation. Maximum gibberellic acid concentration and productivity (6.82 g/l and 4.74 mg/l/h, respectively) were obtained at 6% g/l of fructose. g- Supplementation of the production medium with ammonium sulfate (0.6 g/L) as nitrogen source was superior for the production of gibberellic acid (0.841 g/L) by this fungus. h- Elimination of rice flour from the production medium was stimulate increase in gibberellic acid production (0.992 g/L). i- Different concentrations of MgSO4.7H2O ranged from zero to 2.5 g/l were added to the production medium and the best concentration for gibberellic acid production was 1.5 g/l, where as 2.0 g/l was the best concentration for biomass production. j- The addition of 1 g/L KH2PO4 to the production medium increase gibberellic acid content as the production reached (1.35 g/L). k- The inoculum type, seed culture, at age 24 h and density 2% (v/v) gave significant increase in gibberellic acid production (1.85 g/L).

135 Summary l- Also, the maximum gibberellic acid concentration was obtained at 50 ml CMV/250 ml FV.

4- Exposuring the spore suspension of F. moniliforme to different doses of gamma radiation showed the sensitivity of this fungus to gamma radiation

with D10 value of 1.1 kGy. Furthermore, the results showed that low doses of gamma radiation stimulate the gibberellic acid production, and the highest gibberellic acid accumulation (2.36 g/L) was achieved at radiation dose of 0.5 kGy.

5- Immobilized irradiated spores (in sponge cubes) of F. moniliforme (24 h age and 0.5 g cubes/50 ml medium) produced amount of gibberellic acid reached up to 2.5 g/L, after 6 days of incubation, compared with the amount of gibberellic acid produced by the free cells (2.30 g/L).

6- Whey (cheap dairy by-product) was examined as the main culture medium for gibberellic acid production by this fungus under optimizing culture conditions for repeated batches. The results showed that with irradiated immobilized cells, the maximum amount of gibberellic acid production (2.20 g/L) was recorded at the seconed batch.

7- For agriculture, nurseries, tissue culture and tea garden applications the possible toxicity of the produced gibberellic acid was investigated. The data revealed that very little amount of soluble toxic substances in the extracted sample leading to only 8 % dead chicken embryos.

8- Studying the influence of gibberellic acid on enhancement growth of Aspergillus niger and chitosan production.

9- The results showed that the addition of 2 mg/L gibberellic acid to the best chitosan production medium under optimum conditions (MSM, 30 ºC, 48 h, 120 rpm) increase the production to 1.45 g/L in comparing to 0.874 g/L chitosan from the medium didn't supplemented with gibberellic acid.

136 Conclusion

Conclusion

From the previous results, it could be concluded that the local isolate Fusarium moniliforme (previously named Gibberella fujikuroi) isolated from white corn could be effectively used for the production of gibberellic acid and the optimization of fermentation condition will a cheive a great increase in the production. The highest gibberellic acid production in shake flask culture was obtained in medium containing fructose, ammonium sulfate, potassium dihydrogen phosphate and magnesium sulfate at pH 5.0, 30 ºC, 200 rpm for 6 days. The exposure of F. moniliforme isolate to low doses of gamma radiation (0.5 kGy) would stimulate the production. The use of immobilization technique (0.5 g / 24 h age seed culture) will enhance the production that will reach 2.57 g/l. Milk permeate (cheap dairy by-product) might be used as whole production medium of gibberellic acid. The addition of gibberellic acid (2 mg/l) to chitosan production medium will stimulate both growth of Aspergillus niger and chitosan production (1.45 g/l).

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163 اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻰ

اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻰ

ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ ﻣﻨﻈﻢ ﻧﻤﻮ ﻧﺒﺎﺗ ﻰ هﺎم ، ﻳﺴﺘﺨﺪم ﺑﺸﻜﻞ آﺒﻴﺮ ﻓﻰ اﻟﺰراﻋﺔ ، واﻟ ﻤﺸﺎﺗﻞ وﻣﺰارع اﻟﺸﺎى وﻧﺒﺎﺗﺎت اﻟﺰﻳﻨﺔ وذﻟﻚ ﻷﻏﺮاض اﻗﺘﺼﺎدﻳﺔ آﺜﻴﺮة . وﻟﻘﺪ ﻗﺼﺮ اﺳﺘﺨﺪاﻣ ﻪ ﻓﻰ اﻟﻮﻗﺖ اﻟﺤﺎﻟﻰ ﻋﻠﻰ اﻟﻨﺒﺎﺗﺎت ذات اﻟﻘﻴﻤﺔ اﻻﻗﺘﺼﺎدﻳﺔ اﻟﻌﺎﻟﻴﺔ ﺧﺼﻮﺻﺎ ﺑﺴﺒﺐ إرﺗﻔﺎع ﺛﻤﻨﻪ . ﺗﻘﻠﻴ ﻞ اﻟﺘﻜﺎ ﻟﻴﻒ ﺳﻮف ﻳﺆدى اﻟﻰ زﻳﺎدة ﺗﻄﺒﻴﻖ اﺳﺨﺪاﻣﻪ ﻟﻤﺤﺎﺻﻴﻞ آﺜﻴﺮة وﻣﺘﻌﺪدة وﺑﺎﻟﺘﺎﻟﻰ ﺳﻮف ﻳﻌﻮد هﺬا ﺑﻤﻨﺎﻓﻊ اﻗﺘﺼﺎدﻳﺔ وﺻﻨﺎﻋﻴﺔ آﺜﻴﺮة.

وﻣﻦ هﺬا اﻟﻤﻨﻄﻠﻖ ﻓﺎن هﺬا اﻟﺒﺤﺚ ﻳﻬﺪف اﻟﻰ اﻧﺘﺎج ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ ﺑﻮاﺳﻄﺔ ﻋﺰﻟﺔ ﻣﺤﻠﻴﺔ ﻣﻦ ﻓﻄﺮ اﻟﺠﻴﺒﺮﻳﻼ ﻓﻴﻮﺟﻴﻜﻴﻮرى. وآﺬﻟﻚ دراﺳﺔ ﺗﺄﺛﻴﺮ ﺑﻌﺾ اﻟﻌﻮاﻣﻞ اﻟﻤﺨﺘﻠﻔﺔ ﻋﻠﻰ اﻧﺘﺎج هﺬا اﻟﺤﻤﺾ آﻤﺤﺎوﻟﺔ ﻟﺘﻌﻈﻴﻢ اﻻﻧﺘﺎج. اﻟﺪراﺳﺔ ﺗﺸﻤﻞ ﻋﺰل اﻟﻔﻄﺮﻳﺎت ﻣﻦ ﻣﺼﺎدر ﻣﺨﺘﻠﻔﺔ ﻣﻦ اﻟﺘﺮﺑﺔ واﻟﺤﺒﻮب واﺧﺘﻴﺎر أﻓﻀﻞ ﻋﺰﻟﺔ ﻻﻧﺘﺎج ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻠﻚ وآﺬﻟﻚ دراﺳﺔ ﺗﺄﺛﻴﺮ اﻟﻌﻮاﻣﻞ اﻟﺒﻴﺌﻴﺔ وﻋﻮاﻣﻞ اﻟﺘﻐﺬﻳﺔ ﻋﻠﻰ اﻧﺘﺎج ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻠﻚ . وآﺬﻟﻚ ﺗﻄﻮﻳﺮ ﻋﻤﻠﻴﺔ اﻻﻧﺘﺎج ﻣﻦ ﺧﻼل اﺳﺘﺨ ﺪام ﻣﺨﻠﻒ ﺷﺮش اﻟﻠﺒﻦ آﺒﻴﺌﺔ اﻧﺘﺎج رﺧﻴﺼﺔ اﻟﺜﻤﻦ. واﻳﻀﺎ دراﺳﺔ ﺗﺄﺛﻴﺮ اﺿﺎﻓﺔ ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ ﻋﻠﻰ اﻧﺘﺎج اﻟﻜﻴﺘﻮزان ﺑﻮاﺳﻄﺔ ﻓﻄﺮ اﻻﺳﺒﺮﺟﻠﺲ ﻧﻴﺠﺮ.

وﻳﻤﻜﻦ ﺗﻠﺨﻴﺺ اﻟﻨﺘﺎﺋﺞ اﻟﺘﻰ ﺗﻢ اﻟﺘﻮﺻﻞ اﻟﻴﻬﺎ ﻓﻴﻤﺎ ﻳﻠﻰ:

١- ﺗﻢ اﻟﺤﺼﻮل ﻋﻠﻰ ١٠ ﻋﺰﻻت ﻣﻦ ﺑﻴﻦ ٢٨ ﻋﺰﻟﺔ ﻓﻄﺮﻳﺔ ﻟﻬﺎ اﻟﻘﺪرة ﻋﻠﻰ اﻧﺘﺎج ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ ﻓﻰ اﻟﺒﻴﺌﺔ اﻟﺴﺎﺋﻠﺔ. وﻗﺪ ﺗﻢ اﺧﺘﻴﺎر اﻟﻌﺰﻟﺔ اﻷآﺜﺮ اﻧﺘﺎﺟﺎ (٠.٢٩٩ ﺟﺮام / ﻟﺘﺮ ) وﻋﺮﻓﺖ ﻋﻠﻰ أﻧﻬﺎ ﺗﻨﺘﻤﻰ ﻟﺠﻨﺲ اﻟﻔﻴﻮزارﻳﻮم ﻣﻮﻧﻴﻠﻴﻔﻮرم.

٢- ﺗﻢ اﻟﺘﻌﺮف ﻋﻠﻰ ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ اﻟﻤﻨﺘﺞ ﺑﻤﻘﺎرﻧﺘﺔ ﺑﺤﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ اﻟﻘﻴﺎﺳﻰ ﺑﺎﺳﺘﺨﺪام ﺟﻬﺎزHPLC وﺟﻬﺎز FT-IR.

٣- وﻟﺘﺤﺴﻴﻦ اﻧﺘﺎج ﺣﻤﺾ اﻟﺠﺒﺮ ﻳﻠﻚ اﻟﻤﻨﺘﺞ ﺑﻮاﺳﻄﺔ هﺬا اﻟﻔﻄﺮ (اﻟﻔﻴﻮزارﻳﻮم ﻣﻮﻧﻴﻠﻴﻔﻮرم ) ﺗﻢ دراﺳﺔ ﺗﺄﺛﻴﺮ ﺑﻌﺾ ﻋﻮاﻣﻞ اﻟﻨﻤﻮ اﻟﻤﺨﺘﻠﻔﺔ، وأﻇﻬﺮت اﻟﻨﺘﺎﺋﺞ اﻵﺗﻰ:

أ- ﻓﺘﺮة ﺗﺤﻀﻴﻦ ٦ أﻳﺎم هﻰ اﻷﻧﺴﺐ ﻻﻧﺘﺎج اﻟﺤﻤﺾ ٠.٣١٤ ﺟﺮام/ﻟﺘﺮ.

ب- درﺟﺔ ﺣﺮارة اﻟﺘﺤﻀﻴﻦ ٣٠ °م هﻰ اﻷﻓﻀﻞ ﻟﻨﻤﻮ اﻟﻔﻄﺮ واﻧﺘﺎج اﻟﺤﻤﺾ.

اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻰ

ت- ﺗﻢ دراﺳﺔ رﻗﻢ اﻷس اﻟﻬﻴﺪروﺟﻴﻨﻰ اﻷوﻟﻰ ﻣﻦ (٣-٧.٥) ووﺟﺪ أن رﻗﻢ اﻷس اﻟﻬﻴﺪروﺟﻴﻨﻰ ٥ أﻋﻄﻰ أﻓﻀﻞ اﻧﺘﺎﺟﻴﺔ ﻟﺤﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ ٠.٣٨٠ ﺟﺮام/ﻟﺘﺮ أﻣﺎ أﻓﻀﻞ أس هﻴﺪروﺟﻴﻨﻰ ﻟﻨﻤﻮ اﻟﻔﻄﺮ هﻮ ٥.٥ ﺣﻴﺚ أﻋﻄﻰ8.46 ﺟﺮام/ﻟﺘﺮ.

ث- ﺳﺠﻠﺖ اﻻﻧﺘﺎﺟﻴﺔ اﻷﻓﻀﻞ ﻟﺤﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ ﻓﻰ ﻇﺮوف اﻟﺘﺨﻤﺮ اﻟﻤﻐﻤﻮر ﺑﻤﻌﺪل رج ٢٠٠ ﻟﻔﺔ / دﻗﻴﻘﺔ ﺣﻴﺚ أﻧﺘﺞ اﻟﻔﻄﺮ ٠.٤٦٢ ﺟﺮام/ﻟﺘﺮ.

ج- ﺗﻢ دراﺳﺔ ١٠ ﻣﺼﺎدر آﺮﺑﻮن ﻣﺨﺘﻠﻔﺔ ﻻﻧﺘﺎج ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ ﺑﻮاﺳﻄﺔ اﻟﻌﺰﻟﺔ اﻟﻤﺤﻠﻴﺔ اﻟﻔﻴﻮزارﻳﻮم ﻣﻮﻧﻴﻠﻴﻔﻮرم ووﺟﺪ أن أﻓﻀﻞ ﻣﺼﺪر آﺮﺑﻮن هﻮ اﻟﻔﺮآﺘﻮز ﺣﻴﺚ وﺻﻞ ﻣﻌﺪل اﻻﻧﺘﺎج اﻟﻰ ٠.٦٠٩ ﺟﺮام/ﻟﺘﺮ.

ح- ﺗﻢ دراﺳﺔ ٨ ﺗﺮآﻴﺰات ﻣﺨﺘﻠﻔﺔ ﻣﻦ اﻟﻔﺮآﺘﻮز (٢- ١٠%) ﻋﻠﻰ اﻧﺘﺎج ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ ووﺟﺪ أن أﻓﻀﻞ اﻧﺘﺎج ﻋﻨﺪ ٦ % ﺟﺮام/ﻟﺘﺮ ﻣﻦ اﻟﻔﺮآﺘﻮز.

خ- ﻋﻨﺪ اﺳﺘﺨﺪام آﺒﺮﻳﺘﺎت اﻷﻣﻮﻧﻴﻮم ﺑﺘﺮآﻴﺰ ٠.٦ ﺟﺮام / ﻟﺘﺮ آﻤﺼﺪر ﻧﻴﺘﺮوﺟﻴﻨﻰ زاد اﻧﺘﺎج ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ اﻟﻰ (٠.٨٤١ ﺟﺮام /ﻟﺘﺮ).

د- أدى اﺳﺘﺒﻌﺎد دﻗﻴﻖ اﻷرز ﻣﻦ ﺑﻴﺌﺔ اﻻﻧﺘﺎج اﻟﻰ زﻳﺎدة اﻻﻧﺘﺎج ﺣﻴﺚ وﺻﻞ ﻣﻌﺪل اﻻﻧﺘﺎج اﻟﻰ (٠.٩٩٢ ﺟﺮام /ﻟﺘﺮ).

ذ- ﺗﻢ دراﺳﺔ اﺿﺎﻓﺔ ﺗﺮآﻴﺰات ﻣﺨﺘﻠﻔﺔ ﻣﻦ آﺒﺮﻳﺘﺎت اﻟﻤﺎﻏﻨﺴﻴﻮم ﺗﺘﺮاوح ﻣﻦ (٠-٢.٥ ﺟﺮام / ﻟﺘﺮ ) ﺑﺎﺿﺎﻓﺘﻬﺎ اﻟﻰ ﺑﻴﺌﺔ اﻻﻧﺘﺎج ووﺟﺪ أن أﻓﻀﻞ ﺗﺮآﻴﺰ ﻻﻧﺘﺎج ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ هﻮ ١.٥ ﺟﺮام/ﻟﺘﺮ ﺑﻴﻨﻤﺎ أﻓﻀﻞ ﺗﺮآﻴﺰ ﻟﻨﻤﻮ اﻟﻔﻄﺮ هﻮ ٢ ﺟﺮام /ﻟﺘﺮ . وآﺬﻟﻚ اﺿﺎﻓﺔ اﻟﺒﻮﺗﺎﺳﻴﻮم داى هﻴﺪروﺟﻴﻦ ﻓﻮﺳﻔﺎت ﺑﺘﺮآﻴﺰ ١ ﺟﺮام /ﻟﺘﺮ اﻟﻰ ﺑﻴﺌﺔ اﻻﻧﺘﺎج زاد ﻣﻦ اﻧﺘﺎج ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ ﺣﻴﺚ وﺻﻞ اﻻﻧﺘﺎج اﻟﻰ ١.٣٥ ﺟﺮام /ﻟﺘﺮ.

ر- اﺳﺘﺨﺪام ﻻﻗﺤﺔ ﻣﻦ اﻟﻔﻄﺮ ﻋﻤﺮهﺎ ٢٤ ﺳﺎﻋﺔ ﺑﺘﺮآﻴﺰ ٢% ادى اﻟﻰ زﻳﺎدة واﺿﺤﺔ ﻓﻰ اﻧﺘﺎج ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ وﺻﻠﺖ اﻟﻰ ﻣﻌﺪل ١.٨٥ ﺟﺮام /ﻟﺘﺮ.

ز- آﻤﺎ ﺗﺒﻴﻦ أن أﻓﻀﻞ اﻧﺘﺎج ﻟﺤﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ اﻟﻤﻔﺮز ﻳﺘﻢ ﻋﻨﺪ ﺗﻨﻤﻴﺔ اﻟﻔﻄﺮ ﻋﻠﻰ ٥٠ ﻣﻞ ﻣﻦ ﺑﻴﺌﺔ اﻻﻧﺘﺎج.

٤ - وﻟﺘﻌﻈﻴﻢ اﻧﺘﺎﺟﻴﺔ ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ ﺑﻮاﺳﻄﺔ ا ﻟﻌﺰﻟﺔ اﻟﻤﺬآﻮرة أﺟﺮى ﺗﻌﺮﻳﺾ ﺑﻌﺾ ﺟ ﺮاﺛﻴﻢ هﺬة اﻟﻌﺰﻟﺔ ﻟﺠﺮﻋﺎت ﻣﺨﺘﻠﻔﺔ ﻣﻦ أﺷﻌﺔ ﺟﺎﻣﺎ وأوﺿﺤﺖ اﻟﻨﺘﺎﺋﺞ أن اﻟﺠﺮﻋﺎت اﻟﻤﺨﻔﻀﺔ ﻟﻬﺎ ﺗﺄﺛﻴﺮ ﻣﺨﻔﺰ ﻻﻧﺘﺎج ﺣﻤﺾ اﻟﺠﻴﺮﻳﻠﻚ ﺣﻴﺚ ﺣﻘﻘﺖ اﻟﺨﻼﻳﺎ اﻟﻤﻌﺮﺿﺔ ﻟﺠﺮﻋﺔ اﺷﻌﺎﻋﻴﺔ ٠.٥٠

اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻰ

ك ﺟﺮاى أﻋﻠﻰ اﻧﺘﺎج ﻟﺤﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ ٢.٣٦ ﺟﺮام /ﻟﺘﺮ. آﻤﺎ ﺗﻢ رﺳﻢ ﻣﻨﺤﻨﻰ اﻟﺒﻘﺎء ﻟﻠﻔﻄﺮ

وﺗﺤﺪﻳﺪ ﻗﻴﻤﺔ D10 ﺣﻴﺚ آﺎﻧﺖ ١.١ ك ﺟﺮاى.

٥- ﻋﻨﺪ اﺳﺘﺨﺪام ﺧﻼﻳﺎ هﺬا اﻟﻔﻄﺮ اﻟﻤﻌﺎﻟﺠﺔ ﺑﺄﺷﻌﺔ ﺟﺎﻣﺎ (٠.٥٠ ك ﺟﺮاى) واﻟﻤﺤﻤﻠﺔ داﺧﻞ ﻣﻜﻌﺒﺎت اﻻﺳﻔﻨﺞ آﺎن اﻧﺘﺎج ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ ٢.٥٧ ﺟﺮام /ﻟﺘﺮ ﺑﻌﺪ ٦ أﻳﺎم ﻣﻦ ﺣﻘﻦ اﻟﺨﻼﻳﺎ اﻟﻤ ﺤﻤﻠﺔ ذات اﻟﻌﻤﺮ ٢٤ ﺳﺎﻋﺔ ﺑﻤﻌﺪل ٠.٥ ﺟﺮام / ٥٠ ﻣﻞ ﻣﻦ ﺑﻴﺌﺔ اﻟﻨﻤﻮ.

٦- ﺗﻢ دراﺳﺔ اﻣﻜﺎﻧﻴﺔ اﺳﺘﺨﺪام ﻣﺨﻠﻒ ﺷﺮش اﻟﻠﺒﻦ ﻓﻰ اﻧﺘ ﺎج ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ ﺑﻮاﺳﻄﺔ هﺬا اﻟﻔﻄﺮ . وﻗﺪ أﺛﺒﺘﺖ اﻟﻨﺘﺎﺋﺞ ﻗﺪرة ﺧﻼﻳﺎ اﻟﻔﻄﺮ اﻟﻤﺸﻌﻌﺔ (٠.٥٠ ك ﺟﺮاى ) واﻟﻤﺤﻤﻠﺔ داﺧﻞ ﻣﻜﻌﺒﺎت اﻻﺳﻔﻨﺞ ﻋﻠﻰ اﻧﺘﺎج ﺣﻤﺾ اﻟﺠﻴﺮﻳﻠﻚ ﻓﻰ دﻓﻌﺎت ﻣﺘﻜﺮرة وﺳﺠﻠﺖ أﻋﻠﻰ اﻧﺘﺎﺟﻴﺔ ٢.٢ ﺟﺮام /ﻟﺘﺮ ﺑﻌﺪ اﻧﺘﻬﺎء اﻟﺪرورة اﻟﺜﺎﻧﻴﺔ ﻣﻦ اﻟﻨﻤﻮ.

٧- ﺗﻢ دراﺳﺔ اﻟﺘﺄﺛﻴﺮ ا ﻟﺴﺎم ﻟﺤﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ اﻟﻤﻔﺮز ﺑ ﻮاﺳﻄﺔ هﺬا اﻟﻔﻄﺮ ﻋﻠﻰ اﻷ ﺟﻨﺔ اﻟﻤﺨﺼﺒﺔ وذﻟﻚ ﺑﺤﻘﻨﻪ ﻓﻰ ﺑﻴﺾ اﻟﺪﺟﺎج اﻟﻤﺨﺼﺐ . وأﻇﻬﺮت اﻟﻨﺘﺎﺋﺞ أن ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ اﻟﻤﻨﺘﺞ ﺑﻮاﺳﻄﺔ هﺬة اﻟﺴﻼﻟﺔ ﻟﻔﻄﺮ اﻟﻔﻴﻮزارﻳﻮم ﻣﻮﻧﻴﻠﻴﻔﻮرم ﺗﺤﺘﻮى ﻋﻠﻰ ﻧﺴﺒﺔ ﺿﺌﻴﻠﺔ ﻣﻦ ﻣﻮاد ذات ﺗﺎﺛﻴﺮ ﺿﺎر أدت اﻟﻰ ﻣﻮت ٨% ﻷﺟﻨﺔ اﻟﺪﺟﺎج.

٨- ﺗﻢ دراﺳ ﺔ ﺗﺄﺛﻴﺮ اﺿﺎﻓﺔ ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ ﻋ ﻠ ﻰ ﺗﺤﻔﻴﺰ اﻧﺘﺎج اﻟﻜﻴﺘﻮزان ﻣﻦ ﻓﻄﺮ اﻷ ﺳﺒﺮﺟﻠﺲ ﻧﻴﺠﺮ. وﻟﻘﺪ أﻇﻬﺮت اﻟﻨﺘﺎﺋﺞ أن اﺿﺎﻓﺔ ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ ﺑﺘﺮآﻴﺰ ٢ ﻣﻠﻠﻴﺠﺮام / ﻟﺘﺮ اﻟﻰ ﺑﻴﺌﺔ اﻧﺘﺎج اﻟﻜﻴﺘﻮزان ﺗﺤﺖ اﻟﻈﺮوف اﻟﻤﺜﻠﻰ ( ﺑﻴﺌﺔ أﻣﻼح اﻟﻤﻮﻻس , ٣٠ مº ، ٤٨ ﺳﺎﻋﺔ ﺗﺤﻀﻴﻦ ، ١٢٠ ﻟﻔﺔ /دﻗﻴﻘﺔ) ﺗﺰﻳﺪ اﻻﻧﺘﺎج اﻟﻰ ١.٤٥ ﺟﺮام /ﻟﺘﺮ ﺑﺎﻟﻤﻘﺎرﻧﺔ ﺑﺎﻟﺒﻴﺌﺔ ﺑﺪون اﺿﺎﻓﺔ ﺣﻤﺾ اﻟﺠﺒﺮﻳﻠﻚ ﺗﻨﺘﺞ ٠.٨٧٤ ﺟﺮام/ ﻟﺘﺮ.

ﺟﺎﻣﻌـﺔ ﺑﻨﻬــﺎ آﻠﻴــﺔ اﻟﻌﻠــﻮم ﻗﺴــﻢ اﻟﻨﺒــﺎت

ﺩﺭﺍﺳﺎﺕ ﻋﻠﻰ ﺍﻻﻧﺘﺎﺝ ﺍﳊﻴﻮﻯ ﳊﻤﺾ ﺍﳉﱪﻳﻠﻚ ﻣﻦ ﺍﻟﻔﻄﺮﻳﺎﺕ

رﺳﺎﻟﺔ ﻣﻘﺪﻣﺔ ﻟﻠﺤﺼﻮل ﻋﻠﻰ درﺟﺔ دآﺘﻮراة اﻟﻔﻠﺴﻔﺔ ﻓﻲ اﻟﻌﻠﻮم ﻓﻰ اﻟﻨﺒﺎت (اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻰ)

ﻣﻦ

ﺩﻋﺎء ﻋﺒﺪ ﺍﳌﻨﻌﻢ ﺍﻣﺎﻡ ﺳﻠﻴﻢ اﻟﻤﺪرس اﻟﻤﺴﺎﻋﺪ ﺑﻘﺴﻢ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻰ – اﻟﻤﺮآﺰ اﻟﻘﻮﻣﻰ ﻟﺒﺤﻮث وﺗﻜﻨﻮﻟﻮﺟﻴﺎ اﻻﺷﻌﺎع

هﻴﺌﺔ اﻟﻄﺎﻗﺔ اﻟﺬرﻳﺔ

ﺗﺤﺖ إﺷﺮاف

ﺃ.ﺩ/ ﳏﻤﻮﺩ ﳏﻤﺪ ﻫﺰﺍﻉ ﺃ.ﺩ/ ﺳﻬﺎﻡ ﳏﻤﺪ ﺷﺎﺵ

أﺳﺘﺎذ ورﺋﻴﺲ ﻗﺴﻢ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻲ أﺳﺘﺎذ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻲ آﻠﻴﺔ اﻟﻌﻠﻮم – ﺟﺎﻣﻌﺔ ﺑﻨﻬﺎ آﻠﻴﺔ اﻟﻌﻠﻮم – ﺟﺎﻣﻌﺔ ﺑﻨﻬﺎ

ﺃ.ﺩ/ ﻫﺸﺎﻡ ﳏﻤﻮﺩ ﺳﻮﻳﻠﻢ ﺃ.ﺩ / ﻧﺎﺟﻰ ﺣﻠﻴﻢ ﻋﺰﻳﺰ

أﺳﺘﺎذ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻲ - اﻟﻤﺮآﺰ اﻟﻘﻮﻣﻰ أﺳﺘﺎذ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻲ - اﻟﻤﺮآﺰ اﻟﻘﻮﻣﻰ ﻟﺒﺤﻮث وﺗﻜﻨﻮﻟﻮﺟﻴﺎ اﻻﺷﻌﺎع ﻟﺒﺤﻮث وﺗﻜﻨﻮﻟﻮﺟﻴﺎ اﻻﺷﻌﺎع هﻴﺌﺔ اﻟﻄﺎﻗﺔ اﻟﺬرﻳﺔ هﻴﺌﺔ اﻟﻄﺎﻗﺔ اﻟﺬرﻳﺔ

2013

ﺟﺎﻣﻌﻪ ﺑﻨﻬﺎ آﻠﻴــﺔ اﻟﻌﻠــﻮم ﻗﺴــﻢ اﻟﻨﺒــﺎت

ﺩﺭﺍﺳﺎﺕ ﻋﻠﻰ ﺍﻻﻧﺘﺎﺝ ﺍﳊﻴﻮﻯ ﳊﻤﺾ ﺍﳉﱪﻳﻠﻚ ﻣﻦ ﺍﻟﻔﻄﺮﻳﺎﺕ

اﺳﻢ اﻟﺒﺎﺣﺜﻪ : دﻋﺎء ﻋﺒﺪ اﻟﻤﻨﻌﻢ إﻣﺎم ﺳﻠﻴﻢ

ﺍﻟﻤﺩﺭﺱ ﺍﻟﻤﺴﺎﻋﺩ ﺒﻘﺴﻡ ﺍﻟﻤﻴﻜﺭﻭﺒﻴﻭﺠﻰ ﺍﻟﻤﺭﻜﺯ ﺍﻟﻘﻭﻤﻰ ﻟﺒﺤﻭﺙ ﻭﺘﻜﻨﻭﻟﻭﺠﻴﺎ ﺍﻻﺸﻌﺎﻉ - ﻫﻴﺌﺔ ﺍﻟﻁﺎﻗﺔ ﺍﻟﺫﺭﻴﺔ

ﻟﺠﻨﺔ اﻹﺷﺮاف

م اﻻﺳـــــــــــــــﻢ اﻟﻮﻇﻴﻔـــــــــﺔ اﻟﺘﻮﻗﻴــــــــﻊ أﺳﺘﺎذ ورﺋﻴﺲ ﻗﺴﻢ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻲ ١ ﺃ.ﺩ/ ﳏﻤــــﻮﺩ ﳏﻤــــﺪ ﻫــــﺰﺍﻉ آﻠﻴﺔ اﻟﻌﻠﻮم – ﺟﺎﻣﻌﺔ ﺑﻨﻬﺎ أﺳﺘﺎذ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻲ ٢ ﺃ.ﺩ/ ﺳﻬﺎﻡ ﳏﻤﺪ ﺷﺎﺵ آﻠﻴﺔ اﻟﻌﻠﻮم – ﺟﺎﻣﻌﺔ ﺑﻨﻬﺎ أﺳﺘﺎذ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻰ -اﻟﻤﺮآﺰ اﻟﻘﻮﻣﻰ ٣ ﺃ.ﺩ/ﻫﺸـــﺎﻡ ﳏﻤـــﻮﺩ ﺳـــﻮﻳﻠﻢ ﻟﺒﺤﻮث وﺗﻜﻨﻮﻟﻮﺟﻴﺎ اﻻﺷﻌﺎع هﻴﺌﺔ اﻟﻄﺎﻗﺔ اﻟﺬرﻳﺔ أﺳﺘﺎذ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻰ -اﻟﻤﺮآﺰ اﻟﻘﻮﻣﻰ ٤ ﺃ.ﺩ/ﻧـــــﺎﺟﻰ ﺣﻠـــــﻴﻢ ﻋﺰﻳـــــﺰ ﻟﺒﺤﻮث وﺗﻜﻨﻮﻟﻮﺟﻴﺎ اﻻﺷﻌﺎع هﻴﺌﺔ اﻟﻄﺎﻗﺔ اﻟﺬرﻳﺔ

ﺟﺎﻣﻌﻪ ﺑﻨﻬﺎ آﻠﻴــﺔ اﻟﻌﻠــﻮم ﻗﺴــﻢ اﻟﻨﺒــﺎت

ﺩﺭﺍﺳﺎﺕ ﻋﻠﻰ ﺍﻻﻧﺘﺎﺝ ﺍﳊﻴﻮﻯ ﳊﻤﺾ ﺍﳉﱪﻳﻠﻚ ﻣﻦ ﺍﻟﻔﻄﺮﻳﺎﺕ

اﺳﻢ اﻟﺒﺎﺣﺜﻪ : دﻋﺎء ﻋﺒﺪ اﻟﻤﻨﻌﻢ إﻣﺎم ﺳﻠﻴﻢ

ﺍﻟﻤﺩﺭﺱ ﺍﻟﻤﺴﺎﻋﺩ ﺒﻘﺴﻡ ﺍﻟﻤﻴﻜﺭﻭﺒﻴﻭﻟﻭﺠﻰ

ﺍﻟﻤﺭﻜﺯ ﺍﻟﻘﻭﻤﻰ ﻟﺒﺤﻭﺙ ﻭﺘﻜﻨﻭﻟﻭﺠﻴﺎ ﺍﻻﺸﻌﺎﻉ –ﻫﻴﺌﺔ ﺍﻟﻁﺎﻗﺔ ﺍﻟﺫﺭﻴﺔ ﻟﺠﻨﺔ اﻟﺤﻜﻢ واﻟﻤﻨﺎﻗﺸﻪ

م اﻻﺳـــــــــــــــﻢ اﻟﻮﻇﻴﻔـــــــــﺔ اﻟﺘﻮﻗﻴــــــــﻊ

ﺗﺎرﻳﺦ اﻟﻤﻨﺎﻗﺸﻪ / /٢٠١٣