Production and Extraction of Commercially Important Ergot Alkaloids from Penicillium Species by Fermentation Process

PRODUCTION AND EXTRACTION OF COMMERCIALLY IMPORTANT ERGOT ALKALOIDS FROM PENICILLIUM SPECIES BY FERMENTATION PROCESS

BY MEMUNA GHAFOOR SHAHID REGISTRATION NO. 14-PHD-BOT-GCU-08

GOVERNMENT COLLEGE UNIVERSITY, LAHORE

PRODUCTION AND EXTRACTION OF COMMERCIALLY IMPORTANT ERGOT ALKALOIDS FROM PENICILLIUM SPECIES BY FERMENTATION PROCESS

Submitted to the Government College University, Lahore in partial fulfillment of the requirements for the award of degree of

Ph.D.

BOTANY BY

MEMUNA GHAFOOR SHAHID REGISTRATION NO. 14-PHD-BOT-GCU-08

DEPARTMENT OF BOTANY GOVERNMENT COLLEGE UNIVERSITY, LAHORE

DEDICATION

I DEDICATE THIS WORK TO MY PARENTS WHO ARE THE SOURCE OF MY INSPIRATION AND ENCOURAGEMENT IN ACHIEVING THE ACADEMIC TITLE OF ’DOCTOR OF PHILOSOPHY’.

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RESEARCH COMPLETION CERTIFICATE

Certified that the research work contained in this thesis, titled “Production and Extraction of Commercially Important Ergot Alkaloids from Penicillium species by Fermentation Process” has been carried out in FBRC, PCSIR Laboratories, Complex, Lahore and completed by Miss Memuna Ghafoor Shahid, Registration No. 0014-PHD-GCU-BOT-08, under my supervision during her Ph.D. studies in the subject of Botany.

Dated: 22-09-2014

Supervisor

Dr. Safdar Ali Mirza Assistant Professor (TTS) Department of Botany GC University, Lahore

Submitted through:

Dr. Ghazala Yasmeen Butt Controller of Examinations Chairperson & Associate Professor GC University, Lahore Department of Botany GC University, Lahore

DECLARATION

I, Memuna Ghafoor Shahid, Registration No. 0014-PHD-GCU-BOT-08, student of Ph.D. in the subject of Botany declare that the work contained in this thesis titled “Production and Extraction of Commercially Important Ergot Alkaloids from Penicillium species by Fermentation Process” is my work and has not been printed, published and submitted as research work, thesis or publication in any form in any university and research institute etc. within or without Pakistan.

Dated: 22-09-2014 Memuna Ghafoor Shahid

Table of Contents

Chapters Title/Subheads Page No. List of Tables viii List of Figures xi List of Plates xiv List of Abbreviations xv Acknowledgements xvi Abstract xviii 1 I Introduction

2 Review of Literature 6 2.1 Ergot and Ergotism 6 2.2 Biosynthesis of Ergot Alkaloids 7 2.3 Statistical Optimization of Culture Conditions 13 2.4 Structural Diversity of Ergot Alkaloids 15 2.5 Ergot Alkaloids Pharmacodynamics 16 2.6 Ergot Alkaloids and Analytical Methods 17

3 Materials and Methods 20 Section-I Optimization of Culture Conditions by OFAT 20 Method Section-II Response Surface Methodology 30 Section-III Strain Improvement 35 Section-IV Analytical Studies 42

4 Results 47

Section-I Optimization of Culture Conditions by OFAT 47 Method Section-II Response Surface Methodology 83 Section-III Strain Improvement 104 Section-IV Analytical Studies 116

5 Discussion 150 Section-I Optimization of Culture Conditions by OFAT 151 Method Section-II Response Surface Methodology 156 Section-III Strain Improvement 159 Section-IV Analytical Studies 162

Conclusions 165

6 References 167

Annexure-I 180

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List of Tables

Table Title Page No. No.

3.1 Composition of different fermentation media for screening purpose 22 3.2 Composition of screened M5 fermentation medium 23 3.3 Composition of optimized fermentation medium and conditions 29 for Penicillium commune and Penicillium sp. IIB 3.4 Plackett-Burman Design experimental range and level for 31 screening of variables 3.5 Plackett-Burman experimental design for screening of variables 32 3.6 Experimental range and levels for optimization of significant 33 variables 3.7 Experimental design for optimization of significant variables 34 3.8 Composition of fermentation medium for the production of ergot 37 alkaloids by mutated strains of Penicillium commune and Penicillium sp. IIB 3.9 Absorbance of the dilutions of bromocriptine mesylate (BCM) and 39 dihydroergotamine methane sulfonate (DMS) salts 3.10 Screening of TLC mobile phases for the determination of ergot 43 alkaloids 3.11 Selection of TLC mobile phases for the determination of ergot 44 alkaloids 4.1 Screening of fungal organisms and fermentation medium for 48 production of ergot alkaloids 4.2 Effect of different carbon sources on the production of ergot 50 alkaloids 4.3 Analysis of variance for the effect of carbon sources 50 4.4 Effect of different nitrogen sources on the production of ergot 52 alkaloids 4.5 Analysis of variance for the effect of nitrogen sources 52 4.6 Effect of concentration levels of sucrose on the production of ergot 54 alkaloids 4.7 Analysis of variance for the effect of concentration levels of sucrose 54 4.8 Effect of concentration levels of yeast extract on the production of 56 ergot alkaloids 4.9 Analysis of variance of the effect of concentration levels of yeast 56 extract 4.10 Effect of concentration levels of KH2PO4 on the production of ergot 58 alkaloids 4.11 Analysis of variance for the effect of concentration levels of 58 KH2PO4

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4.12 Effect of the different concentration levels of tryptophan on the 60 production of ergot alkaloids 4.13 Analysis of variance of effect of concentration levels of tryptophan 60 4.14 Effect of different concentrations of asparagine on the production 62 of ergot alkaloids 4.15 Analysis of variance for the effect of concentration levels of 62 asparagine 4.16 Effect of concentration levels of succinic acid on the production of 64 ergot alkaloids 4.17 Analysis of variance for the effect of concentration levels of 64 succinic acid 4.18 Effect of concentration levels of NH4Cl on the production of ergot 66 alkaloids 4.19 Analysis of variance of the effect of concentration levels of NH4Cl 66 4.20 Effect of the concentration levels of MgSO4.7H2O on the production 68 of ergot alkaloids 4.21 Analysis of variance for the effect of concentration levels of MgSO4. 68 7H2O 4.22 Effect of concentration levels of FeSO4.7H2O on the production of 70 ergot alkaloids 4.23 Analysis of variance for the effect of concentration levels of FeSO4. 70 7H2O 4.24 Effect of concentration levels of ZnSO4 on the production of ergot 72 alkaloids 4.25 Analysis of variance for the effect of concentration levels of ZnSO4 72 4.26 Effect of pH on the production of ergot alkaloids on the production 74 of ergot alkaloids 4.27 Analysis of variance of the effect of various pH levels 75 4.28 Effect of incubation temperature on the production of ergot 76 alkaloids 4.29 Analysis of variance for the effect of different incubation 77 temperatures 4.30 Effect of different incubation time periods on the production of 78 ergot alkaloids 4.31 Analysis of variance for the effect of incubation times 79 4.32 Effect of different size of inoculum on the production of ergot 80 alkaloids 4.33 Analysis of variance for the effect of various inoculum sizes 81 4.34 Production of ergot alkaloids in fermentor 82 4.35 Screening of variables for ergot alkaloids production by Penicillium 84 commune using PBD 4.36 Analysis of variance for ergot alkaloids yield by Penicillium 85 commune using PBD

4.37 Observed and predicted values of ergot alkaloids yield by 87 Penicillium commune using BBD 4.38 Observed and predicted values of ergot alkaloids yield by 87 Penicillium sp. IIB using BBD 4.39 Analysis of variance for alkaloids production by Penicillium 94 commune using BBD 4.40 Analysis of variance for ergot alkaloid production by Penicillium 95 sp. IIB using BBD 4.41 Survival rate of colonies of UV mutated strains of Penicillium 105 commune and Penicillium sp. IIB 4.42 Survival rate of colonies of EMS mutated strains of Penicillium 108 commune and Penicillium sp. IIB 4.43 Screening of UV mutant and comparison of ergot alkaloids 111 production with wild strains 4.44 Screening of EMS mutants and comparison of ergot alkaloids 113 production with wild strains 4.45 Comparison of the ergot alkaloids yield of selected mutant and 115 wild strains 4.46 values of culture liquid and mycelial filtrate extracts of 117 Penicillium commune 4.47 푅푓 values of culture liquid and mycelial filtrate extracts of 118 Penicillium sp. IIB 4.48 푅푓 values of selected culture liquid and mycelial filtrate extracts of 119 Penicillium commune 4.49 푅푓 values of selected culture liquid and mycelial filtrate extracts of 120 Penicillium sp. IIB 4.50 Retention푅푓 times of reference salts of ergot alkaloids 124 4.51 Retention time of culture liquid and mycelial filtrate extracts of 125 Penicillium commune 4.52 Retention time of culture liquid and mycelial filtrate extracts of 126 Penicillium sp. IIB

List of Figures

Fig. No. Title Page No.

3.1 Standard curve of BCM salt 39

3.2 Standard curve of DMS salt 40 4.1 Mycelial growth of all fungal species in M5 fermentation medium 48 4.2 Effect of different carbon sources on the mycelial growth of 51 Penicillium commune and Penicillium sp. IIB 4.3 Effect of different nitrogen sources on the mycelial growth of 53 Penicillium commune and Penicillium sp. IIB 4.4 Effect of different concentrations of sucrose on the mycelial growth 55 of Penicillium commune and Penicillium sp. IIB 4.5 Effect of different concentrations of yeast extract on the mycelial 57 growth of Penicillium commune and Penicillium sp. IIB 4.6 Effect of different concentrations of KH2PO4 on the mycelial 59 growth of Penicillium commune and Penicillium sp. IIB 4.7 Effect of different concentrations of tryptophan on the mycelial 61 growth of Penicillium commune and Penicillium sp. IIB 4.8 Effect of different concentrations of asparagine on the mycelial 63 growth of Penicillium commune and Penicillium sp. IIB 4.9 Effect of different concentrations of succinic acid on the mycelial 65 growth of Penicillium commune and Penicillium sp. IIB 4.10 Effect of different concentrations of NH4Cl on the mycelial growth 67 of Penicillium commune and Penicillium sp. IIB 4.11 Effect of different concentrations of MgSO4.7H2O on the mycelial 69 growth of Penicillium commune and Penicillium sp. IIB 4.12 Effect of different concentrations of FeSO4.7H2O on the mycelial 71 growth of Penicillium commune and Penicillium sp. IIB 4.13 Effect of different concentrations of ZnSO4 on the mycelial growth 73 of Penicillium commune and Penicillium sp. IIB 4.14 Effect of different pH levels on the mycelial growth of Penicillium 75 commune and Penicillium sp. IIB 4.15 Effect of incubation temperature on mycelial growth of Penicillium 77 commune and Penicillium sp. IIB 4.16 Effect of different incubation times on mycelial growth of 79 Penicillium commune and Penicillium sp. IIB 4.17 Effect of different inoculum size on mycelial growth of Penicillium 81 commune and Penicillium sp. IIB 4.18 Mycelial growth of Penicillium commune and Penicillium sp. IIB in 82 fermentor 4.19 Pareto chart showing the significant variables i.e. sucrose, yeast 85

extract and FeSO4 influencing the production of ergot alkaloid yield by Penicillium commune 4.20 Observed and predicted values of ergot alkaloid production by 89 Penicillium commune using BBD 4.21 Observed and predicted values of ergot alkaloids production by 91 Penicillium sp. IIB using BBD 4.22 Desirability chart of the optimized factors i.e. sucrose, yeast extract 92 and FeSO4 showing their predicted values for the production of ergot alkaloids by Penicillium commune 4.23 Desirability chart of the optimized factors i.e. sucrose, yeast extract 93 and FeSO4 showing their predicted values for the production of ergot alkaloids by Penicillium sp. IIB 4.24 Response surface graph showing combined interaction effect of 98 sucrose and yeast extract on ergot alkaloid production by Penicillium commune 4.25 Response surface graph showing combined interaction effect of 99 sucrose and FeSO4 on ergot alkaloids production by Penicillium commune 4.26 Response surface graph showing combined interaction effect of 100 Yeast extract and FeSO4 on ergot alkaloids production by Penicillium commune 4.27 Response surface graph showing combined interaction effect of 101 sucrose and yeast extract on ergot alkaloid production by Penicillium sp. IIB 4.28 Response surface graph showing combined interaction effect of 102 sucrose and FeSO4 on ergot alkaloid production by Penicillium sp. IIB 4.29 Response surface graph showing combined interaction effect of 103 Yeast extract and FeSO4 on ergot alkaloid production by Penicillium sp. IIB 4.30 Comparison of mycelial growth of wild and UV mutated strains of 112 Penicillium commune and Penicillium sp. IIB 4.31 Comparison of mycelial growth of wild and EMS mutated strains 114 of Penicillium commune and Penicillium sp. IIB 4.32 Comparison of mycelial growth of wild and selected UV and EMS 115 mutated strains of Penicillium commune and Penicillium sp. IIB 4.33 Chromatogram of ergotamine standard (1 mg/ml) with retention 128 time of 3.69 min. 4.34 Chromatogram of Dihydroergotamine Methane Sulfonate Salt 129 (standard salt 2) (1 mg/ml) with retention time of 4.01 min. 4.35 Chromatogram of Bromocriptine Mesylate Salt (standard 3) 130 (1mg/ml) with retention times of 3.66 min and 5.89 min. 4.36 Chromatogram of Mixture of Ergotamine (1 mg/ml), DMS Salt 131

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(1mg/ml) and BCM Salt (1mg/ml) with retention times of 3.64, 4.89 and 6.36 min. 4.37 Chromatogram of Penicillium commune culture liquid filtrate extract 132 No. 1 (1mg/ml) with retention time of 3.71 min. 4.38 Chromatogram of Penicillium commune culture liquid filtrate extract 133 No. 2 (1mg/ml) with retention time of 3.68 min. 4.39 Chromatogram of Penicillium commune culture liquid filtrate extract 134 No. 9 (1mg/ml) with retention times of 3.73 min. 4.40 Chromatogram of Penicillium commune culture liquid filtrate extract 135 No. 12 (1mg/ml) with retention time of 3.61 min. 4.41 Chromatogram of Penicillium commune mycelial filtrate extract No. 136 1 (1mg/ml) with retention times of 3.69 min. 4.42 Chromatogram of Penicillium commune mycelial filtrate extract No. 137 2 (1mg/ml) with retention times of 3.71 min. 4.43 Chromatogram of Penicillium commune mycelial filtrate extract No. 138 4 (1mg/ml) with retention times of 3.74 min. 4.44 Chromatogram of Penicillium commune mycelial filtrate extract No. 139 8 (1mg/ml) with retention time of 3.60 min. 4.45 Chromatogram of Penicillium sp. IIB culture liquid filtrate extract 140 No. 1 (1mg/ml) with retention time of 3.69 min. 4.46 Chromatogram of Penicillium sp. IIB culture liquid filtrate extract 141 No. 2 (1mg/ml) with retention time of 3.60 min. 4.47 Chromatogram of Penicillium sp. IIB culture liquid filtrate extract 142 No. 8 (1mg/ml) with retention time of 3.63 min. 4.48 Chromatogram of Penicillium sp. IIB culture liquid filtrate extract 143 No. 9 (1mg/ml) with retention time of 3.61 min. 4.49 Chromatogram of Penicillium sp. IIB culture liquid filtrate extract 144 No. 12 (1mg/ml) with retention time of 3.62 min. 4.50 Chromatogram of Penicillium sp. IIB mycelial filtrate extract No. 1 145 (1mg/ml) with retention time of 3.62 min. 4.51 Chromatogram of Penicillium sp. IIB mycelial filtrate extract No. 2 146 (1mg/ml) with retention time of 3.63 min. 4.52 Chromatogram of Penicillium sp. IIB mycelial filtrate extract No. 6 147 (1mg/ml) with retention time of 3.59 min. 4.53 Chromatogram of Penicillium sp. IIB mycelial filtrate extract No. 8 148 (1mg/ml) with retention time of 3.60 min. 4.54 Chromatogram of Penicillium sp. IIB mycelial filtrate extract No. 9 149 (1mg/ml) with retention time of 3.60 min.

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List of Plates

Plate No. Title Page No.

1 Effect of UV mutagen on the development of colonies of Penicillium 106 commune

2 Effect of UV mutagen on the development of colonies of Penicillium 107 sp. IIB

3 Effect of EMS mutagen on the development of colonies of 109 Penicillium commune

4 Effect of EMS mutagen on the development of colonies of 110 Penicillium sp. IIB

5 Screening of mobile phase for TLC analysis of Penicillium commune 121 and Penicillium sp. IIB filtrates.

6 Selection of suitable mobile phase for TLC analysis of Penicillium 122 commune culture liquid filtrate extracts in phase 2 using mobile phases A1 (Chloroform: Methanol: Ammonia soln.) and H2 (Chloroform: Isopropanol: Water), (S: Standard Salt spot, PC: Penicillium commune, 1, 4 and 8: Filtrate No., M: Mycelial filtrate extract).

7 TLC analysis of Penicillium sp. IIB extracts in phase 2 mobile phases 123 A1 (Chloroform: Methanol: Ammonia soln.) and H2 (Chloroform: Isopropanol: Water), (S: Standard Salt spot, P: Penicillium sp. IIB, 1, 4, 9 and 12: Filtrate No., M: Mycelial filtrate extract).

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List of Abbreviations

°C Degree centigrade (Celsius) % Percentage min Minutes w/v Weight/Volume g Gram mg Milligram ml Milliliter mg/ml Milligram/Milliliter L Liter v/v Volume/Volume OD Optical Density CLFE Culture Liquid Filtrate Extract MFE Mycelial Filtrate Extract EMS Ethyl Methane Sulfonate UV Ultraviolet RSM Response Surface Methodology PBD Plackett-Burmann Design BBD Box-Behnken Design CCD Central Composite Design OFAT One Factor At a Time ANOVA Analysis of Variance

ACKNOWLEDGEMENTS

Before embarking upon my Ph.D. studies I have been relishing the idea that it will be easy to earn. But the thing was not as simple as it had been visualized. With the start of my study a plethora of work unfolded before me. I had to expend hours in Lab and to remain vigilant of my experiments installed for the growth of fungi for many days. Many a time I had to face failure and difficulties were as hard as I had to start my experiments anew and fresh. Had all these hardships and difficulties made me a failed student, if I would have not been helped by my supervisor, Dr. Safdar Ali Mirza, Assistant Professor, GC University, Lahore. He very kindly put his efforts best in the removal of all obstacles that raised their head to hamper my way and to streamline my research work.

I am indebted to Dr. Shahjehan Baig, Chief Scientific Officer (Retd.) and Dr. Muhammad Nadeem, Senior Scientific Officer, Food and Biotechnology Research Center, PCSIR Laboratories Complex, Lahore for providing me all the Lab facilities during my research work. I am beholden to them and shall remain forever. Owing to their timely guidance without which it would have been very difficult to accomplish research work.

I am grateful to Dr. Quratulain, Principal Scientific Officer, Dr. Robina Nelofar, Senior Scientific Officer and Dr. Yasar Saleem, Senior Scientific Officer, FBRC, PCSIR Laboratories Complex, Lahore for their cooperation and the services they rendered to me.

I am, particularly, very grateful to Dr. Ghazala Yasmeen Butt, Chairperson, Department of Botany, GC University, Lahore who supported me during my PhD tenure.

I am very grateful to Dr. Khalid Hamid Skeikh, Professor Emeritus, Department of Botany, GC University, Lahore, Dr. Ikram-ul-Haq, Director, ORIC, GC University,

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Lahore, Dr. Bushra Munir, Assistant Professor, Institute of Industrial Biotechnology, GC University, Lahore, Dr. A. R. Shakoori, Professor Emeritus, School of Biological Sciences and Dr. Muhammad Saleem, Professor, Department of Botany, University of the Punjab, New Campus, Lahore for their help and support.

I also salute my friends and colleagues specially Saira Atta who had been a fountain head of encouragement and buoyancy and their tension free help.

I also don’t forget to express my heart-deep thanks to Mehwish, Huma, Umar (Research associates) and Azhar (Lab attendant) FBRC, PCSIR Laboratories, Lahore who helped me and remained watchful to my all wants while working in the lab. I am very thankful to my sisters (Samina, Naghmana, Misbah and Zareen) and brother (Zubair) for their support during my research work.

In the end, being a Muslim, I am very grateful to Allah Almighty who granted me the courage and the will to complete the research work.

Memuna Ghafoor Shahid

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ABSTRACT

Production of ergot alkaloids was achieved in culture liquid (extracellular) and mycelial (intracellular) filtrate extracts of Penicillium commune and Penicillium sp. IIB in M5 fermentation medium using surface culture fermentation technique. Various species of genus Penicillium were screened for their ability to produce ergot alkaloids from their culture liquid (extracellular) and mycelial (intracellular) filtrate extracts of five different fermentation media. Among all the species tested, Penicillium commune (CLFE=1.359±0.002 mg/ml; MFE=0.958±0.001 mg/ml) and Penicillium sp. IIB (CLFE=1.154±0.002 mg/ml; MFE=0.635±0.001 mg/ml) produced maximum ergot alkaloids after 21 days of incubation at initial pH 5.2 and at 25°C in M5 fermentation medium. Optimization of culture conditions such as effect of different substrates (carbon and nitrogen sources), culture medium ingredients (tryptophan, asparagine, succinic acid, KH2PO4, NH4Cl, MgSO4.7H2O, FeSO4.7H2O and ZnSO4), and various process parameters (pH, incubation temperature, incubation time and size of inoculum) were optimized using one factor at a time technique (OFAT). Maximum yield of ergot alkaloids in extracellular filtrate extracts of Penicillium commune (4.38 mg/ml) and Penicillium sp. IIB (5.51 mg/ml) was achieved at optimum levels of sucrose (35%), yeast extract (30%), KH2PO4 (2%), tryptophan (2%), asparagines (2% to 2.5%), succinic acid

(2%), NH4Cl (1.5% to 2%), MgSO4.7H2O (1.5%) , FeSO4.7H2O (1.0%), ZnSO4 (1%, 1.5%) at pH 5.0 and 25°C after 21 days of incubation. Statistical designs of response surface methodology (RSM) such as Plackett-Burman Design (PBD) and Box-Behnken Design (BBD) were used for screening and optimization of different factors for ergot alkaloids production for the enhanced production of ergot alkaloids by Penicillium commune and

Penicillium sp. IIB. Among various factors, sucrose, yeast extract and FeSO4.7H2O were selected due to their significant positive effects on ergot alkaloids yield. Maximum response for ergot alkaloids production was observed from experimental run 6 and 13 of Penicillium commune and Penicillium sp. IIB with a maximum yield of 14.64 mg/ml

xviii and 35.60 mg/ml respectively, using Box-Behnken Design (BBD) as compared to their predicted values. The high correlation between the predicted and observed values indicated the validity of RSM. Strains of Penicillium commune and Penicillium sp. IIB were subjected to various mutagens to improve the yield of ergot alkaloids. Wild strains were treated with UV irradiations and EMS (ethyl methane sulfonate) for different time intervals (1-150 min). It was found that mutant strains such as PCUV-4 and PCEMS-3 showed maximum ergot alkaloids yield as compared to wild strain of Penicillium commune in pre- optimized culture conditions. During the scale up process, it was found that PCUV-4 mutant strain of Penicillium commune showed 12.32 mg/ml yield which was 3-fold higher than the wild strain. This strain was designated as the best positive mutant for the enhanced production of ergot alkaloids. The further analysis of ergot alkaloids from filtrate extracts was performed through analytical techniques such as TLC and HPLC. It was found that large Rf values for the extracellular and intracellular ergot alkaloids of Penicillium commune and Penicillium sp. IIB were obtained in mobile phase A1 and H2. PCCLFE9, PCMFE9 of Penicillium commune and PCLFE9, PMFE9 of Penicillium sp. IIB exhibited maximum colored spots and large Rf values revealing pinkish purple and blue colors indicating the presence of ergotamine, agroclavine and ergocriptine alkaloids. The chromatographic separation of ergot alkaloids present in filtrates was achieved on HPLC using chloroform:isopropanol (80:20 v/v) as mobile phase. Maximum retention time (3.83 min) was recorded from the mycelial (intracellular) filtrate (PCMFE12 sample) of Penicillium commune which indicated the presence of ergocriptine. Highest concentration of ergot alkaloids (ergotamine) i.e. 20.12 mg/ml was investigated from the culture liquid (extracellular) filtrate of sample PCLFE9 of Penicillium sp. IIB. The ergot alkaloids yield from wild and mutant strain of Penicillium commune and Penicillium sp. IIB appeared as the potential source for up-scaling the process at required levels for the commercial production of ergot alkaloids for pharmaceutical purposes. Protocol improvement in this regard is strongly

xix recommended for uplifting the ergot alkaloids production to fulfill the indigenous demands.

Chapter 1 INTRODUCTION ______

Natural products synthesized by microorganisms have played a major role in the discovery and manufacturing of drugs which are in-use for the treatment of several human ailments. These natural products are commonly called as secondary metabolites, and these constitute an important group of bioactive compounds that can be used in pharmaceutical, cosmetic and food industry (Devi and Prabakaran, 2014).

Alkaloids are the largest group of natural products synthesized as secondary metabolites in plants, animals and in fungi (Polak and Rompala, 2007). Alkaloids have been reported as a group of organic substances of plants, containing at least one nitrogen atom in the ring structure of a molecule. Alkaloids in microbes such as fungi were initially recognized in Claviceps purpurea, the agent causing ergot of rye. These alkaloids so discovered were isolated from the sclerotia formed after the infection of ovary of carpel by ascospores or conidia of Claviceps. Ergot is common name of disease- forming sclerotia by fungi of genus Claviceps, which produces ergot alkaloids. The sclerotium is a dark colored compact mass that virtually replaces seeds (or kernel) of plants with a black dark infected part (Burfening, 1973). Many types of commercially important ergot alkaloids have been found from the sclerotia of various species of genus Claviceps. Besides the sclerotia of Claviceps, other fungi such as Blansia, Epichole, Penicillium and Aspergillus and several higher plants also harbor some quantity of ergot alkaloids (Zafar et al., 2010).

Generally, the members of Clavicipitaceae family infest grass species, including cereal grains, and these members are capable of producing a number of different important ergot alkaloids (Danicke and Diers, 2013). These compounds have proven to be

1 important both pharmacologically and agriculturally (Wallwey and Shu-Ming, 2011; Ryan et al., 2013).

Various members of the Clavicipitaceae family produce ergot alkaloids, for instance, all the species of genus Claviceps, Epichole and Balansia. The most prominent ergot alkaloids produced by Claviceps species are ergometrine, ergotamine, ergosine, ergocristine, ergocriptine and ergocornine. The amount and the productivity of ergot alkaloids vary in different species that depend upon the host and organism’s ecological relationships. It has been discovered that ergot alkaloids could be produced by different members other than genus Claviceps, i.e. from genus Penicillium. Many species of genus Penicillium and Aspergillus are the potential candidates for the production of ergot alkaloids, including Penicillium sizovae, Penicillium chermisinum, Penicillium roquefortii, Penicillium corylophilum, Penicillium regulosum (Moussa, 2003) Aspergillus fumigatus, Aspergillus flavus and Aspergillus tamari, respectively (Flieger et al., 1997).

Ergot alkaloids are usually classified in accordance with their structures, e.g. clavine- type alkaloids commonly called clavines, ergoamides and ergopeptines. Clavine alkaloids simply consist of tetracyclic ergoline ring structure (Schardl et al., 2006). Ergoamides and ergopeptines are amides and peptides of D-lysergic acid, respectively (Flieger et al., 1997). These compounds have the same ergoline ring structure as in clavine-type alkaloids. Ergot alkaloids can interact with receptors of central nervous system (CNS). Ergot alkaloids generally cause adverse effects on the health of human beings and animals. Human intoxications and ergot poisoning of farm animals particularly in cattle, horses, sheep, pigs, chicken and even in wild animals have been reported to be effected by ergot alkaloids (Mavungu et al., 2011). Their effects on CNS are due to the structural similarity of ergot alkaloids with nor-adrenaline, dopamine and serotonine (Katzung, 2009). Some ergot alkaloids act as agonists whereas other act as antagonists (Sinz, 2008). The differences in the physiochemical, physiological and pharmacological properties of ergot alkaloids are caused by a range of different substituents attached to the carboxyl group of D-lysergic acid (Flieger et al., 1997).

Penicillium is well known all over the world, for producing secondary metabolites and commercially valued extracellular enzymes (Gulliamon et al., 1998; Tiwari et al., 2007, 2011). The secondary metabolites produced by Penicillium include alkaloids, antibiotics, hormones and mycotoxins, etc. Each species of the genus Penicillium produces different types of physiologically active compounds and have different growth peculiarities (Kozlovskii et al., 2013). The genus Penicillium synthesizes clavine alkaloids, and these alkaloids are of three types: i) 6-N Methylergoline derivatives such as festuclavine, epicostaclavine, fumigaclavine and isofumigaclavine that have a complex and saturated structure of ring D, ii) ergolenes derivatives which include agroclavine kind of alkaloids, iii) clavine alkaloids that have a modification in the ring C and D, e.g. rugulovasines A & B, and α-cyclopiazonic acid alkaloids (Kozlovsky et al., 2013).

The profiles of ergot alkaloids have been studied for decades mainly due to their deleterious effects in the contaminated foods and feeds, and also for their useful applications in pharmaceutical and agricultural processes (Neilsen et al., 2014). Historically, the preparations of ergot alkaloids in small doses were employed by the midwives to produce strong uterine contractions, and it became very popular in France, Germany and United States. Afterwards, the use of ergot alkaloids was recommended to control the post-partum hemorrhage in United States and in Europe. Ergotamine, dihydroergotamine and bromocriptine are very significant peptide alkaloids that are generally used for the treatment of vascular, hepatic, renal, coronary artery and hypertension diseases. These also act as a therapeutic agent to cure hyperprolactinemis and pituitary prolactinoma and also for the treatment of moderate to severe migraine (Lüllmann et al., 2000; Katzung and Julius, 2001; King and Herndon, 2005).

Biosynthesis of ergot alkaloids is being carried out commercially for manufacturing various pharmaceuticals. Three types of fermentation techniques are being employed for the biosynthesis of alkaloids, i.e. solid state, submerged state and surface state culture fermentation. Alkaloid synthesis has also been motivated and regulated with the addition of different organic and inorganic compounds in the fermentation medium

3 to enhance the yield of ergot alkaloids. The traditional methods used for the optimization of fermentation process include various parameters; usually one factor is employed at a time. However, the single variable optimization methods are not only tedious but may lead to misinterpretation of results, especially when the concerted effects among different factors are overlooked (Wenster-Botz, 2000). Unfortunately, this approach has been failed to identify the variables which have optimum response because the interaction of variables was not taken into account in these simple procedures (Deepak et al., 2008). Statistical procedures allow rapid screening of a number of factors at one time and their interactions to get the maximum yield of the product. These also reflect the role of every individual variable in a particular fermentation process. Response Surface Methodology (RSM), which has been used in the present study, comprises of mathematical and statistical techniques for building of empirical modeling. It is used as a powerful tool for optimizing conditions for producing industrially and commercially important products (Khurana et al., 2007). The application of response surface methodology (RSM) is to develop a low-cost methodology by reducing the cost of expensive analysis of fermentation experiments.

Statistical procedures, such as Plackett-Burman Design (PBD) and Box-Behnken Design (BBD), have shown to be very efficient and significant approaches to target the factors that influence the growth of the product. PBD is an effective tool for screening of individual variables it considerably reduces the number of experiments and identifies the significant variables as much as possible. Hence, through this tool, only the most effective and significant factors can be selected for further optimization studies (Venil and Lakshmanaperumalsamy, 2009a). Similarly, the less significant or negative effect producing factors can be omitted from further experiments (Plackett and Burman, 1946). PBD has been applied in many fields, such as in medium optimization and formulation of multicomponent designs (Loukas, 2001; Naveena et al., 2005). After screening the entire process, Box-Behnken Design (BBD) and Central Composite Design (CCD) can be used to optimize targeted individual variables and their combined effects

4 to get the desirable yields of the products. Hence, such useful tools may help efficiently in optimization of the laboratory procedures to produce valuable products in a very cost effective way.

Taking into consideration the above-mentioned scenario and the day by day increasing demand of ergot alkaloids as pharmacological and therapeutic agents, a strong need has been felt to develop a cost-effective process for the biosynthesis of ergot alkaloids for commercial use in Pakistan using some novel sources. Hence, the present study was designed and conducted in pursuit of the following main objectives.

1.1. Objectives

1. Screening and selection of Penicillium species for the production of ergot alkaloids using surface culture fermentation technique.

2. Optimization of fermentation medium and various culture conditions for the synthesis of ergot alkaloids by employing statistical tools, such as Plackett- Burman Design (PBD) and Box-Bhenken Design (BBD).

3. Improvement of Penicillium species by employing chemical and physical mutagenesis such as Ethyl Methane Sulfonate (EMS) and UV irradiations.

4. The qualitative and quantitative evaluation of ergot alkaloids produced by the selected species of Penicillium by Thin Layer Chromatography (TLC), Spectrophotometry and High Performance Liquid Chromatography (HPLC).

Chapter 2 REVIEW OF LITERATURE ______

The name "alkaloids" derived from a German word “Alkaloide” was introduced in 1819 by the German chemist Carl Friedrich Wilhelm Meisner. This word is also derived from Latin word “alkali” which, in turn, comes from the Arabic word “al-qalwī” means "ashes of plants". However, the term was widely used as it was published in a Chemical Dictionary of Albert Ladenberg after 1880. Alkaloids are basically a group of naturally occurring chemical compounds (natural products) which contain nitrogen in their structures. These compounds have some neutral or slightly acidic properties. Some compounds have been synthesized in laboratories having similar structures with naturally occurring compounds. These compounds usually contain oxygen, sulfur and rarely chlorine, bromine and phosphorus in their structures (Manske, 1965).

2.1. Ergot and Ergotism

Fungi of family Clavicipitaceae infect several types of grasses, and transform seeds of the grasses into a compact mass of sclerotia that is commonly called as ergot. The sclerotium is 1 to 3 cm long cylindrical brown or blackish dense tissue of hyphae that forms ascocarp (fruiting body) containing ascospores. The ascospores are dispersed by wind and infect other healthy seeds, replacing the seed with a new sclerotium (Ryman and Holmasen, 1992). Ergot of rye has been very common in Europe, as the cool damp growing conditions favor the growth of fungi on the grasses promoting the ergot infection (Hart, 1999). The consumption of ergot-contaminated bread was found responsible for ergotism in many countries. In the past, two main types of ergotism were reported namely “gangrenous” and “convulsive”. The symptoms of both the ergotisms are quite similar. It begins from the vague illness with some gastrointestinal illness followed by the abnormal sensations in limbs that feel like ants crawling over

6 the affected part of the skin. This is followed by loss of sensation in limbs by an intense burning pain called St. Anthony’s fire. In severe cases, the affected tissue becomes dry and black and sometimes the limbs are dropped off without bleeding. It is also followed by secondary infections in which death rate is too high (De-Costa, 2002; Eadie, 2003).

2.2. Biosynthesis of Ergot Alkaloids

The clinical usage of ergot alkaloids is widespread today and more than 50 formulations contain natural and synthetic ergot alkaloids. These are significant for the treatment of uterine atonia, post-partum bleeding, migraine, hypertension, hyperprolactinemia and Parkinson disease. Recently, new therapeutic applications of ergot alkaloids have been found against schizophrenia. These physiological effects of ergot alkaloids are based on their binding with the neurotransmitter receptors (Kren, 1997).

A large amount of ergot alkaloids have been found in the fungal species belonging to the genus Claviceps, and the widely occurring ergot alkaloids source is Claviceps purpurea. Another well-known species that has ability to produce ergot alkaloids is Claviceps paspali. A lower concentration of ergot alkaloids has also been detected in higher plants, e.g. Ipomea genus of family Convolvulaceae (Komarova and Tolkachev, 2001).

Several species of genus Claviceps are involved in the development of ergotism in cattle grazing on ergot-infected grasses. Other members of family Clavicipitaceae causing infections of grasses are Epichloe, Typhina, Myrogenospora atramentosa and Balansia. Four species of genus Balansia, isolated from infected pastures, were grown in artificial culture media by Bacon et al. (1979) using submerged state fermentation conditions. Glucose, sorbitol and inorganic salts were added in the fermentation medium and the production of ergot alkaloids was investigated by Balansia epichloe, Balansia claviceps, Balansia henningsiana and Balansia strangulans. It was found that ergonovine was produced by Balansia claviceps, while Balansia epichloe produced chanoclavine, agroclavine, penniclavine and ergonovine alkaloids.

Biosynthesis of ergot alkaloids in laboratories was attempted in saprophytic conditions. Fermentative production of ergot alkaloids was attempted many times using different Claviceps spp. The experiments were performed under saprophytic conditions. Various organic and inorganic compounds were added in the fermentation medium to obtain enhanced yield of ergot alkaloids. Desai et al. (1983) reported that ergot alkaloids yield was increased when biotin was added in the culture medium during the submerged cultivation of Claviceps sp. The yield of ergot alkaloids was further enhanced when leucine was incorporated in the culture medium.

The process of fermentative production of ergot alkaloids was improved periodically with the passage of time. Efforts were put up in the past to enhance the productivity through the optimization of fermentation technology, genetic improvement of strain, semi-continuous fermentation processes with immobilized cells and the use of protoplasts of the cultures. Solid state fermentation and submerged state fermentation processes were compared by Trejo et al. (1992) for the synthesis of ergot alkaloids by Claviceps fusiformis and Claviceps purpurea. The process of solid state fermentation was not explored earlier for the production of ergot alkaloids.

Trejo et al. (1993) described that minor alteration in the production medium of ergot alkaloids significantly influenced the ergot alkaloids spectra specifically in the solid state fermentation process. A total of 16 different combinations of nutrient media impregnated with sugar-cane pith bagasse, used for the production of ergot alkaloids by culturing Claviceps purpurea in a solid state fermentation experiment and to evaluate the effect of the media alterations on the profile of ergot alkaloids. They investigated a clear difference in the spectra of alkaloids produced in solid state fermentation process as compared to submerged state fermentation process.

Ergot alkaloids presence in the cereals were identified as a serious disease problem that out-broke in cattle, as well as in human beings in the past. But one can’t forget the beneficial aspects of ergot alkaloids to cure different ailments. Hence, with the increased demand for new and advanced medicines for the treatment of various diseases, a need

8 to find out new species was felt other than the genus Claviceps, Balansia and Epichole, for the production of ergot alkaloids. Screening studies were carried out in the past in view of dramatic discoveries where ergot alkaloids could be produced by various species of fungi other than the genus Claviceps and Balansia spp. Flieger et al. (1997) reported that ergot alkaloids could be produced by fungi belonging to the genus Penicillium e.g. Penicillium aurantivoirens, Penicillium kapuscinkii, Penicillium regulosum and Penicillium concavorugulosum.

Moussa (2003) conducted an extensive work on the screening of 30 species of genus Penicillium, having the potential of ergot alkaloids production. He screened different Penicillium species to find-out the best producer of ergot alkaloids and described Penicillium corylophilum as the best producer because this produced 750 mg ergot alkaloids per liter of the fermentation medium. He optimized the various process parameters, such as effect of nitrogen and carbon sources, pH, incubation temperature and addition of phosphates in the fermentation medium to enhance the ability of Penicillium corylophilum to produce maximum ergot alkaloids. He also analyzed the ergot alkaloids profile and identified ergocriptine and agroclavin in the alkaloids spectra of Penicillium corylophilum.

Penicillium being the important member of class ascomycetes, is also known for the production of secondary metabolites and extracellular enzymes. That is why the mycologists preferred to use Penicillium species for the synthesis of such useful compounds. Some of the well-known secondary metabolites such as penicillin have been produced by Penicillium chrysogenum. Similarly, Frisvad et al. (2004) reported the presence of bioactive secondary metabolites including mycotoxins in different species of genus Penicillium. They identified some unique secondary metabolites that were confined to some specific species of Penicillium. The most widespread secondary metabolite, roquefortine C produced by 25 species of Penicillium. Moreover, the neprotoxic mycotoxin ochratoxin A was produced by Penicillium verrucosum and Penicillium nordicum. Another mycotoxin, citrinin was produced by Penicillium

9 expansum. The mutagenic mycotoxin, botrydiplodin, was produced by Penicillium brevicompactum and Penicillium paneum. Such secondary metabolites were produced by almost all of the Penicillium species which they isolated in their experiment. Moreover, it was also found that the antibiotic, penicillin, was produced by all of the 25 species of genus Penicillium.

Penicillium roquefortii strain was subjected to mutation by ultraviolet irradiation (UV) and ethyl methane-sulfonate (EMS) for strain improvement by El-Bondkly and Abeer (2007). They measured the ability of these mutated strains under various conditions to produce ergot alkaloids, i.e. synthesis of clavin, dihydro-roquefortine and some other ergot alkaloids compounds. Their findings indicated that production of ergot alkaloids was enhanced by mutated strains of Penicillium. They also produced lipase in low quantity, when olive oil was added in the fermentation medium. Less lipase and ergot alkaloids production was found when cotton seed-oil was used by mutated strains in the fermentation medium.

Many alkaloids producing fungi pertaining to the genus Penicillium accumulate ergot alkaloids in liquid culture medium (extracellular) and in the mycelium (intracellular). However, the phenomenon of the accumulation of ergot alkaloids in the mycelium is yet poorly understood. This accumulation and the production of ergot alkaloids is dependent upon the age of the culture of fungus, the composition of the fermentation medium and the culture conditions of the fungal organism. Zhelifonova and Kozlovskii (2007) studied some aspects of the transport of ergot alkaloids and quinocitrinins in Penicillium citrinum at young and old stage of its growth period in the fermentation medium under different culture conditions. They reported a decrease in the concentration of ergot alkaloids in the culture medium of Penicillium citrinum when there was an uptake of quinocitrinins by its cells. Furthermore, it was found that the ability of mycelium to take up the quinocitrinins was not dependent on its age where-as the ability to take up other ergot alkaloids was higher in young mycelium rather than the old mycelium. The uptake ability of exogenously added quinocitrinins by the young

10 mycelium inhibited the secretion of ergot alkaloids in fermentation medium, however, the secretion of old mycelium occurred throughout the cultivation period of Penicillium citrinum.

The genus Penicillium is also significant for the production of enzymes of commercial importance such as pectinase that is utilized in medicines and in fruit juice industry. Cardoso et al. (2007) selected two species of genus Penicillium, i.e. Penicillium expansum and Penicillium griseos-roseum and used them for in vitro production of pectinase enzyme. They found that various culture conditions such as inoculum age, percentage of inoculum, incubation time period, substrate concentration, addition of various carbon and nitrogen sources influenced the production of pectinase in the fermentation medium. The pectinase activity of Penicillium griseos-roseum was higher than the Penicillium expansum. The two species were then characterized on using RAPD technique for their molecular studies. The monomorphic fragments such as 600 bp of Penicillium expansum and 594 bp of Penicillium griseos-roseum were amplified using RAPD technique. The telomeric fingerprinting analysis indicated the polymorphic relationship between these two species.

Coyle et al. (2007) investigated the production of ergot alkaloids by Aspergillus fumigatus that also causes various health problems exclusively by contaminating the food stuff by shedding its conidia. Aspergillus fumigatus is virtually a grass endophyte that produces mycotoxins in grasses and can affect nervous and reproductive system of the animals that graze on such infected grasses. It was found that the ergot alkaloids such as festuclavine and fumigaclavine were produced by Aspergillus fumigates.They also worked on conidia producing genes and to create conidia-deficient strains of Aspergillus fumigatus, they manipulated the bristle A gene (brlA) that controlled the vesicle formation and that thought to be necessary for the conidial development in the fungal strain. Disturbance in this gene stimulated the development of a conidia-deficient mutant strain that was producing only bristle-like structures instead of developing conidia. However, it was found that the mutated strain failed to produce ergot alkaloids

11 in the fermentation medium. The findings indicated that the vegetative mycelium of the fungus could not produce ergot alkaloids because it was directly associated with the formation of conidia on the mycelium.

Torres et al. (2008) analyzed various ecological habits such as free living nature, insect- parasite relationship, habitat conditions and mode of nutrition of various species of family Clavicipitaceae. They found their phylogenetic relationships, using subunit rDNA sequence data to represent a large range of ecological habitats. Their capacity to produce ergoline was also evaluated in the culture medium. It was investigated that the ergoline formation was associated with the plant biotrophy and they distributed various species belonging to the family Clavicipitaceae in two clades. It was also noted that ergot alkaloids produced on the surfaces of plants reduced the vulnerability of plants to herbivory.

Sreedevi et al. (2011) mutated Aspergillus terreus by ultraviolet irradiations (UV) and ethyl methane-sulphonate (EMS) treatments for the enhanced production of lovastatin. The best UV mutant strain of Aspergillus terreus exhibited 32.78% lovastatin productivity in the fermentation medium that was higher than the wild strain. The EMS mutant produced large quantity of lovastatin, which was 38.87% higher than the UV mutant and 84.17% higher than the wild strain of Aspergillus terreus, respectively. Their results indicated that UV and EMS were effective mutagenic agents for strain improvement of Aspergillus terreus for an enhanced production of lovastatin in the fermentation medium.

Peter and Shu-Ming (2013) indicated that ergot alkaloids are secondary metabolites of significant toxicological and pharmacological importance. Lysergic acid amides or peptides produced by Claviceps purpurea included a significant profile of alkaloids e.g. ergometrine, ergotamine, ergotoxine and their semisynthetic derivatives which are widely used in modern medicine for the treatment of various diseases. Large-scale production of ergot alkaloids for pharmaceutical applications was achieved through biotechnological processes, including cultivation of Claviceps purpurea on rye. Some

12 members of the family of trichocomaceae e.g. Aspergillus fumigatus and Penicillium commune were identified as the potential candidates for production of clavine-type ergot alkaloids.

Kozlovskii et al. (2013) reported that the genus Penicillium isolated from wild habitats was able to synthesize physiologically active compounds of alkaloid nature, i.e. diketopiperazines, quinolines, quinazolines and polyketides. They used the ergot alkaloid and secondary metabolite profiles for the classification of ergot alkaloids. Moreover, they found that the addition of tryptophan always help in obtaining the maximum yield of ergot alkaloids in the fermentation medium.

2.3. Statistical Optimization of Culture Conditions

Owing to the potential usefulness of ergot alkaloids there is an increasing demand to develop cost-effective bioprocess for the synthesis of ergot alkaloids. Several studies have been carried out for the optimization of various factors that influence the production of ergot alkaloids in the fermentation medium. The composition of fermentation medium greatly influences the growth of the organism and production of ergot alkaloids. Hence, the optimization of medium composition and the culture conditions have been the primary focus in any biological procedure (Djekrif- Dakhmouche et al., 2006). The main strategy adopted in the past was the optimizing one parameter at one time while keeping the others at a constant level. However, such optimization studies did not consider the combined effects of the process parameters as these processes could be influenced by several factors simultaneously (Silva and Roberto, 2001). The limitation of the single factor at one time can be eliminated by following Response Surface Methodology (RSM) which is a better option to explain the combined effect of various factors at one time. RSM comprises of a collection of mathematical and statistical techniques for empirical model building. Statistical methodologies such as Plackett-Burman Design (PBD) and Box-Behnken Design (BBD), have shown to be efficient and effective approaches to identify the significant factors, or parameters. PBD is an effective screening design that reduces the number of

13 experiments and help rapid identification of significant factors. The factor having negative effects can easily be omitted from the design. The significant factors are thus selected for the optimization studies using BBD models. In BBD model, the combined effects of all the significant factors can be studied through various mathematical models.

Venil and Lakshmanaperumalsamy (2009a) used Plackett-Burman Design (PBD) and Box-Behnken Design (BBD) for the optimization of various independent variables for the production of prodigiosin by Serratia marcescens. Among various independent variables; incubation temperature, effect of concentration levels of (NH4)2PO4 and trace salts were selected due to their significant influence on the yield of prodigiosin. The screening of important factors was done using PBD and the optimization of selected variables was done using Box-Behnken Design (BBD). The media formulations were optimized having the independent variables such as 30°C incubation temperature, 6.0 g/L (NH4)2PO4 and 0.6g/L trace salts. The maximum response was 1397.96 mg/L where-as the predicted value was 1394.26 mg/L. The correlation between the predicted and observed values indicated the validity of the statistical models used for this study.

Guo et al. (2010) combined response surface methodology statistical design with Artificial Neural Network Genetic Algorithm (ANN-GA) to optimize the process parameters for the nisin production. Plackett-Burman Design was used for the identification of significant independent variables that influenced the nisin production. RSM combined with the ANN-GA was used for analyzing the impact of individual variables and their combined effects on the nisin production. Glucose 15.92 g/L, peptone 30.57 g/L, yeast extract 39.07 g/L and NaCl 5.25 g/L were optimized as significant factors influencing the production of nisin. The average yield of nisin was estimated at 21423 IU/ml in the fermentation medium.

Zhang et al. (2010) investigated Jiean-peptide production by Bacillus subtilis using response surface methodology. Initially, eight factors were evaluated including six factors were of culture conditions of fermentation medium and two of cell adsorption

14 conditions. Soybean hydrolysate and MgSO4.7H2O were screened as the significant factors for the production of Jiean-peptide. The concentrations of these significant factors were further optimized using Central Composite Design (CCD). Soybean hydrolysate and MgSO4.7H2O were optimized at 24% and 0.38% respectively that increased the production of Jiean-peptide upto 41%.

Wu et al. (2011) improved One Factor at a Time (OFAT) optimization technique combing with the techniques of RSM. These statistical procedures were used for the optimization of fermentation medium for the production of fumigaclavin C (FC) and helvolic acid (HA) by Aspergillus fumigatus. Moreover, the impact of various carbon and nitrogen sources on the production of ergot alkaloids was analyzed by OFAT technique. Various parameters such as Inorganic phosphate, pH and inoculum size were screened by Plackett-Burman Design (PBD) and further optimization was done by Central Composite Design (CCD). Various parameters, i.e. mannitol 50 g/L, sodium succinate

5.4 g/L, NaNO3 2 g/L, MgSO4.7H2O 0.3 g/L, KH2PO4 0.67 g/L, FeSO4 0.01 g/L, pH 4.2, incubation temperature (28°C) and 19 days incubation time were also optimized for the production of FC and HA. The highest yield of FC and HA were 17.26 mg/L and 16.8 respectively, achieved by using the above-mentioned fermentation conditions.

2.4. Structural Diversity of Ergot alkaloids

The important structural feature of all ergot alkaloids is the specific tetracyclic ergoline ring (Schardl et al., 2006). In various ergot alkaloids, the ergoline ring is methylated on nitrogen at position 6 and also substituted on carbon 8. Most of the ergot alkaloids contain double bond of carbon in 8, 9 or may be in position 10. The ergot alkaloids can be classified on the basis of substitution at carbon 8 and are divided into following four types:

1. Clavine alkaloids 2. Lysergic acid derivatives 3. Ergopeptine alkaloids 4. Ergopeptam alkaloids

Clavines are hydroxyl and dehydro derivatives of 6, 8-dimethylergolenes and this group includes chanoclavines, agroclavines, festuclavine and pyroclavine. These have been identified in various species of fungi (Leistner and Steiner, 2009). The lysergic acid alkaloids are basically the amides that produce amide linkage by a small peptide or an alkylamide. These are naturally occurring compounds of this group, and are collectively called ergometrine. In these compounds lysergic acid is amidated with 2- aminopropanol, or 2-aminobutanol. The ergopeptines are also called cyclol ergot alkaloids and are composed of lysergic acid and a tripeptide moiety. Ergopeptams are non-cyclol Lactam Ergot Alkaloids (LEA). Their structure is similar to ergopeptines except that the amino acid-III is proline and the tripeptide chain is a non-cyclol lactam. The ergopeptams are further classified as ergotamams, ergoxams, ergotoxams, and ergoannams (Rajan and Wing, 2010).

2.5. Ergot Alkaloids Pharmacodynamics

Ergot alkaloids are very significant in pharmaceutical industry but their effects are very complex and variable. Lysergic acid alkaloids are therapeutically very significant and this major group includes ergonovine, methylergonovine and methysergide. Ergonovine also known as ergometrine, ergotocine, ergosterine and ergobasin, is a sensitive water soluble compound, and commercially it is available as the water soluble maleate salt. Ergonovine stimulates the smooth muscles of uterus resulting in the increased rhythmical contractions of uterus. Methyl egonovine is not produce naturally but synthetically in laboratories. This is sparingly soluble in water and used in the same manner as ergonovine. This is also used in the management of post-partum uterine atony and hemorrhage after the child birth. The drug is marketed as Methergine tablets (Cvak, 1999). Similarly, Methysergide is also a synthetic compound, and it is used to treat the patients who suffer frequent or severe vascular headaches. However, this compound is not used in the symptomatic acute headache. The methysergide drug can be taken as the oral tablet and it should not be taken for more than 6 months.

Peptide alkaloids are also therapeutically very significant compounds and this group includes ergotamine, dihydroergotamine and bromocriptine. All types of peptide alkaloids play an effective role in the treatment of migraine headaches. Generally, peptide alkaloids are taken in a very small amount but in high concentrations these compounds may cause ergotism, gangrene or severe peripheral vasoconstriction problem. Bromocriptine is very useful to cure hyperprolactinemia and pituitary prolactinemia. Pergolidemesylate is also a closely related compound to ergotamine and that is useful in the treatment of Parkinson disease (Sheehan et al., 2005).

In Medieval ages, midwives had been using ergot for labour room applications where only a small amount of ergot was given to induce labour and to prevent post-partum bleeding. The extracts of ergot alkaloids were used in Germany to treat the vascular headaches, such as migraine. Meanwhile some people in USA also began to use the extracts of ergot for obstetric purposes (Tudzynski et al., 2001). Later, it was found that it had increased still-birth risks and prevented hemorrhages after delivery (Eadie, 2003).

2.6. Ergot Alkaloids and Analytical Methods

Various analytical techniques have been used in the past to identify the ergot alkaloids spectra and its profiles. Adriana and Godoy (2001) developed a simple and inexpensive Thin Layer Chromatographic (TLC) process for the determination of ergovaline in leaf sheaths of tall fescue (Festuca arundinacea). In the first step, all the leaf samples were ground and dipped in methanol for 24 hours. The extracts were filtered and the methanol was allowed to evaporate. The aqueous residues were extracted again with chloroform at pH 9. These extracts were concentrated and further purified on a silica gel TLC plate developed with toluene: ethylacetate: acetonitrile in 50:10:40 ratio. Ergovaline bands were eluted with methanol. Another TLC plate was developed with the mobile phase containing chloroform: acetone: acetic acid in 90:10:5 ratios. Ergoline TLC plate was sprayed with p-dimethylaminobenzyldehyde and sulfuric acid. The bands of ergoline were compared with the standard reference of ergotamine and that the same Rf values were achieved. The TLC study was applied to 15 samples of tall

17 fescue grass, and it was found that all contaminated samples showed the presence of ergovaline. They also reported that the TLC method could be applicable for the identification of ergovaline in the seeds of tall fescue, where-as ergovaline contents in seeds were usually higher than the leaf. Two methods of thin layer chromatography (TLC) are in-use these days, instrumental TLC and non-instrumental TLC. TLC is often used as a pilot method followed by HPLC or simply to achieve separation of ergot alkaloids (Mroczek et al., 2006).

The effect of chromatographic conditions on TLC behavior of ergot alkaloids has been investigated in many studies. Polak and Rompala (2007) investigated the behavior of various acids on the ergot alkaloids poured on silica gel plates. The effect of acids and their derivatives i.e. hydrogen di-(2-ethylhexyl) orthophosphoric, chloroacetic, dichloroacetic, tri-chloroacetic, and trifluoroacetic acids, on the retention time of ergot alkaloids was investigated in this study. Rf values of all the ergot alkaloid samples were measured and it was investigated that the higher concentration of acid in the mobile phase reduced the adsorption of alkaloids on silica gel plates and facilitate in increasing the Rf values.

Aranda and Morlock (2007) developed a new method to identify and quantify the caffeine, ergotamine and metamizol in different standard drugs. These compounds were separated on silica gels using High Performance Thin Layer Chromatography (HPTLC) plates with the mobile phase containing, ethylacetate: methanol: ammonia in 90:15:1 ratio. Detection of the compounds was done in UV at 274 nm for caffeine and metamizol and ergotamine was detected by fluorescence at 340 nm. Recovery of these compounds was also investigated from 95% to 102% at three different concentration levels. They also reported the use of mass spectrometry positive electrospray ionization process for the detection of caffeine and ergotamine and a negative electrospray ionization process for metamizol detection. Their results proved that this technique is an alternative and cost-effective for the detection of ergot alkaloid compounds.

Ashour and Soulafa (2013) developed a selective, sensitive and simple Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method for the determination of Ergotamine Tartrate (ET) in pharmaceutical dosage forms. All variables were studied to optimize the chromatographic conditions. The chromatographic separation of Ergotamine Tartarate (ET) and Bromo-Criptine Mesylate (BCM) was done on a reversed phase BDS Hypersil C8 column (250×4.6 mm i.e. 5 cm particle size) with a mobile phase consisted of MeOH-HCOOH 0.1 M (70:30, v/v), pumped at a flow rate 1.0 ml per minute and detected under UV at 320 nm. The retention time was 8.30 and 10.93 minutes for ET and BCM, respectively.

Chapter 3 MATERIALS AND METHODS ______

The present study comprises of screening of Penicillium species, optimization of culture conditions and some fermentation parameters using One Factor at a Time (OFAT) technique and Response Surface Methodology (RSM) by employing statistical tools such as PBD and BBD. Improvement of the selected Penicillium strains was achieved by employing chemical and physical mutagenesis. The qualitative and quantitative determination of ergot alkaloids was accomplished using various analytical techniques such as Thin Layer Chromatography (TLC), Spectrophotometer and High Performance Liquid Chromatography (HPLC). The details of the methods are given in the subsequent sections below.

SECTION 1: OPTIMIZATION OF CULTURE CONDITIONS BY OFAT METHOD

3.1. Procurement of Fungal Cultures

Different species of Genus Penicillium were collected from the Fungal Culture Bank (FCB), Institute of Agricultural Sciences, University of the Punjab, New Campus, Lahore and Institute of Industrial Biotechnology, GC University Lahore, as test species for the production of ergot alkaloids. The following five species of genus Penicillium were used in the present study:

1. Penicillium commune 2. Penicillium italicum 3. Penicillium digitatum 4. Penicillium oxalicum 5. Penicillium sp. IIB

3.1.1. Maintenance of fungal cultures

Fungal cultures were maintained on malt extract agar (MEA) medium slants. Slants were prepared by taking 2 g of malt extract and 2 g of agar in 250 ml Erlenmeyer flask and dissolved in 100 ml of distilled water. The medium was sterilized in autoclave at 121°C for 15 min. After sterilization, 5-7 ml of MEA medium was transferred to the sterilized test tubes and placed in slanting position at room temperature for solidification. A loopful of growth from pure cultures was transferred aseptically to the slants and incubated at 25°C for five days. The slants having proper growth after incubation were stored in refrigerator at 4°C for further studies.

3.2. Screening of Fungal Organism and Fermentation Medium For this purpose, each species of genus Penicillium as mentioned under 3.1 subsection was cultured on five different fermentation media to find out the best ergot alkaloid producer among the test species and the most suitable fermentation medium for the production of ergot alkaloids using surface culture fermentation process.

3.2.1. Preparation of inoculum for screening purpose

Spore suspensions of each Penicillium species i.e. Penicillium commune, Penicillium italicum, Penicillium digitatum, Penicillium oxalicum and Penicillium sp. IIB were prepared separately by scrapping the surface of the fungal colonies with inoculating needle and releasing the scrapped fungal spores in separate flasks containing 50 ml of distilled water under aseptic conditions. These were vortexed for 2-3 minutes to break the mycelial fragments. The number of spores was adjusted at 10 6-7spores/ml using hemocytometer.

3.2.2. Composition of fermentation media

Five different self formulated fermentation media i.e. M1, M2, M3, M4 and M5 were designed for screening purpose for the production of ergot alkaloids. The composition of fermentation media is given in the Table 3.1.

Table 3.1: Composition of different fermentation media for screening purpose Ingredients (g/100 ml) M1 M2 M3 M4 M5 Sucrose - 5 5 - 5 Mannitol 5 - - 5 - Peptones - 0.1 - - - NH4OH - 0.01 - - - NH4Cl - - - 0.2 0.2 Succinic Acid 0.5 - - 0.54 0.5 Yeast Extract - - 0.3 - 0.5 Tryptophan - - - 0.025 0.5 Asparagine - - - - 0.5 K2HPO4 - 0.1 - - - KH2PO4 - - 0.1 0.5 0.5 MgSO4. 7H2O 0.01 0.15 0.001 0.03 0.03 FeSO4 0.01 0.001 0.001 0.001 0.01 ZnSO4 0.004 0.002 0.001 0.001 0.002 pH = 5.2, Incubation temperature = 25°C

All the ingredients of each medium were poured separately in 250 ml Erlenmeyer flasks and volume of the medium was raised upto 100 ml with distilled. pH of all the fermentation media was adjusted at 5.2 with 0.1N HCl and ammonia solution prior to sterilization. All the flasks were sterilized in autoclave at 121°C for 15 min. After sterilization, inoculation was done by adding 5 ml of spore suspension of each Penicillium species separately under aseptic conditions in each flask. After inoculation all the flasks were incubated at 25°C for 21 days. After 21 days of incubation period, the fermented broth and mycelia were separated in glass bottles and checked for the presence of ergot alkaloids.

3.3. Selection of Penicillium species and Fermentation Medium

Penicillium commune and Penicillium sp. IIB produced maximum ergot alkaloids in M5 fermentation medium among the media tested (Table 3.1). So these two species and M5 fermentation medium were selected for the further optimization studies.

3.3.1. Composition of the M5 fermentation medium

The composition of selected M5 fermentation medium used for the further optimization studies is given in the Table 3.2.

Table 3.2: Composition of screened M5 fermentation medium Ingredients g/100 ml Sucrose 5 NH4Cl 0.2 Succinic Acid 0.5 Yeast Extract 0.5 Tryptophan 0.5 Asparagine 0.5 KH2PO4 0.5 MgSO4. 7H2O 0.03 FeSO4 0.01 ZnSO4 0.002 pH = 5.2, Incubation temperature = 25°C

3.4. Optimization Studies of Fermentation Medium

Various fermentation conditions were optimized using one factor at a time (OFAT) technique, to obtain the enhanced production of ergot alkaloids. The various parameters influencing the growth of mycelia and production of ergot alkaloids were as follows:

 Optimization of substrate o Effect of different carbon and nitrogen sources o Effect of concentration levels of selected carbon and nitrogen sources  Optimization of medium ingredients

o Effect of concentration levels of KH2PO4 o Effect of concentration levels of Tryptophan o Effect of concentration levels of Asparagine o Effect of concentration levels of Succinic acid

o Effect of concentration levels of NH4Cl

o Effect of concentration levels of MgSO4.7H2O

o Effect of concentration levels of FeSO4.7H2O

o Effect of concentration levels of ZnSO4  Optimization of process parameters/fermentation conditions o Effect of initial pH o Effect of cultivation temperature o Effect of incubation period o Effect of size of inoculum

3.4.1. Optimization of substrate for ergot alkaloids production Different carbon and nitrogen sources were used as substrate for the production of ergot alkaloids by Penicillium commune and Penicillium sp. After maintaining the pH of the fermentation medium at 5.2 with 0.1N HCl and ammonia solution, 5 ml of spore suspension (106-7spores/ml) was added in the fermentation medium and flasks were incubated at 25°C for 21 days.

3.4.1.1. Screening of different carbon sources

Different carbon sources e.g. glucose, fructose, sucrose, maltose, mannose and mannitol were employed in the fermentation medium to find the suitable carbon source for mycelium growth and ergot alkaloids production by Penicillium commune and Penicillium sp. IIB. All these sources were studied at 5% (w/v) concentration level.

3.4.1.2. Screening of different nitrogen sources

Different nitrogen sources e.g. yeast extract, peptones, malt extract, meat extract, ammonium chloride and urea were employed in the fermentation medium to find out the suitable nitrogen source for mycelium growth and ergot alkaloids production by Penicillium commune and Penicillium sp. IIB. All these sources were studied at 5% (w/v) concentration level.

3.4.2. Optimization of concentration levels of selected carbon and nitrogen sources Different concentration levels of selected carbon and nitrogen sources were optimized for mycelium growth and production of ergot alkaloids by Penicillium commune and Penicillium sp. IIB. After maintaining the pH of the fermentation medium at 5.2, 5 ml of spore suspension (10 6-7 spores/ml) was added in the fermentation medium and flasks were incubated at 25°C for 21 days.

3.4.2.1. Effect of concentration levels of sucrose

Effect of different concentration levels of sucrose i.e. 10, 15, 20, 25, 30, 35 and 40% were studied to find out the suitable sucrose concentration level for the growth of mycelium and maximum yield of ergot alkaloids by Penicillium commune and Penicillium sp. IIB.

3.4.2.2. Effect of concentration levels of yeast extract

Effect of different concentration levels of yeast extract i.e. 5, 10, 15, 20, 25 and 30% were evaluated to find out the suitable yeast extract concentration level for the maximum mycelial growth and yield of ergot alkaloids by Penicillium commune and Penicillium sp. IIB.

3.4.3. Optimization of medium ingredients

Different ingredients of fermentation medium were evaluated to determine the significance of each ingredient for maximum mycelial growth and production of ergot alkaloids by Penicillium commune and Penicillium sp. IIB. The pH of the fermentation medium was maintained at 5.2.

3.4.3.1. Effect of concentration levels of KH2PO4

Effect of different concentration levels of KH2PO4 i.e. 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 % were evaluated to find out the suitable KH2PO4 concentration level for maximum mycelial growth and yield of ergot alkaloids by Penicillium commune and Penicillium sp. IIB.

3.4.3.2. Effect of concentration levels of tryptophan

Effect of different concentration levels of tryptophan i.e. 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 % were evaluated to find out the suitable tryptophan concentration level for maximum growth of mycelium and production of ergot alkaloids by Penicillium commune and Penicillium sp. IIB.

3.4.3.3. Effect of concentration levels of asparagine

Effect of different concentration levels of asparagine i.e. 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 % were studied to find out the suitable asparagine concentration level for maximum mycelial growth and yield of ergot alkaloids by Penicillium commune and Penicillium sp. IIB.

3.4.3.4. Effect of concentration levels of succinic acid

Effect of different concentration levels of succinic acid i.e. 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 % were estimated to find out the best succinic acid concentration level for the growth of mycelium and maximum yield of ergot alkaloids by Penicillium commune and Penicillium sp. IIB.

3.4.3.5. Effect of concentration levels of NH4Cl

Different concentration levels of NH4Cl at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 % were used in fermentation medium to determine the suitable NH4Cl concentration level for the enhanced growth of mycelium and production of ergot alkaloids by Penicillium commune and Penicillium sp. IIB.

3.4.3.6. Effect of concentration levels of MgSO4.7H2O

Effect of different concentration levels of MgSO4.7H2O i.e. 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 % were evaluated to find out the most suitable MgSO4.7H2O concentration level for the mycelial growth and maximum yield of ergot alkaloids by Penicillium commune and Penicillium sp. IIB.

3.4.3.7. Effect of concentration levels of FeSO4.7H2O

Different concentration levels of FeSO4.7H2O i.e. 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 % were employed in fermentation medium to find out the suitable FeSO4.7H2O concentration level for maximum growth of mycelium and maximum yield of ergot alkaloids by Penicillium commune and Penicillium sp. IIB.

3.4.3.8. Effect of concentration levels of ZnSO4

Effect of different concentration levels of ZnSO4 i.e. 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 % was evaluated to find out the best ZnSO4 concentration level for the maximum mycelial growth and yield of ergot alkaloids by Penicillium commune and Penicillium sp. IIB.

3.4.4. Optimization of process parameters

Optimization of process parameters such as pH, incubation temperature, incubation time period and size of inoculum were studied to evaluate the effect of these parameters on the growth of mycelium and yield of ergot alkaloids by Penicillium commune and Penicillium sp. IIB.

3.4.4.1. Effect of pH

The optimum pH level was evaluated for the growth of mycelium and production of ergot alkaloids by Penicillium commune and Penicillium sp. IIB by adjusting the pH of the fermentation medium at different pH levels i.e. 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0. The pH of each growth medium was adjusted before sterilization with 0.1N HCl and ammonia solution.

3.4.4.2. Effect of incubation temperature

Effect of incubation temperature ranging from 23°C to 33°C was determined on growth of mycelium and production of ergot alkaloids by Penicillium commune and Penicillium sp. IIB by incubating the fermentation media on above mentioned range in incubator.

3.4.4.3. Effect of incubation time period

The effect of incubation time period was evaluated on growth of mycelium and yield of ergot alkaloids by Penicillium commune and Penicillium sp. IIB. The best optimum time period was determined by incubating the fermentation medium for 7, 14, 21 and 30 days.

3.4.4.4. Effect of size of inoculum

Different sizes of inoculum i.e. 5 ml, 10 ml, 15 ml, 20 ml, 25 ml and 30 ml were evaluated to find out the optimum inoculum size for the maximum mycelial growth and production of the ergot alkaloids by Penicillium commune and Penicillium sp. IIB.

3.5. Production of Ergot Alkaloids in Fermentor Using Optimum Fermentation Medium and Conditions After optimization of all parameters as mentioned above, the production of ergot alkaloids was carried out using optimum fermentation conditions in one liter fermentor by Penicillium commune and Penicillium sp. IIB separately. The pH of the fermentation medium was maintained using 0.1N HCl and ammonia solution. After inoculation with the optimized spore suspension volume of the Penicillium commune and Penicillium sp. IIB, the fermentation media were incubated at 25°C for 21 days. The fermentation medium containing the optimum ingredients is given in Table 3.3.

Table 3.3: Composition of optimized fermentation medium and conditions for Penicillium commune and Penicillium sp. IIB Medium Ingredients Penicillium commune Penicillium sp. IIB (g/L) Sucrose 35 35 NH4Cl 1.5 2 Succinic Acid 2 2 Yeast Extract 30 30 Tryptophan 2 2 Asparagine 2 2 KH2PO4 2 2 MgSO4. 7H2O 1.5 1.5 FeSO4 1 1 ZnSO4 1.5 1 Fermentation conditions Inoculum Size (ml) 15 20 pH 5 5 Incubation 25 25 Temperature (°C) Incubation Time Period 21 21 (Days)

3.6. Statistical Analysis

For the authentication of results, the data of OFAT technique, various experiments were tested statistically to draw a reasonable and logical conclusion. For this, standard deviation and one way ANOVA was applied after Steel and Torrie (1996).

SECTION-II: RESPONSE SURFACE METHODOLOGY

3.7. Response Surface Methodology (RSM)

After the optimization of culture conditions by OFAT method, statistical tools were used to optimize culture conditions for mycelial growth and production of ergot alkaloids from wild selected species of Penicillium. For this purpose, two statistical designs of response surface methodology (RSM) were used such as Plackett-Burman Design (PBD) and Box-Behnken Design (BBD) for the screening of the significant ingredients of fermentation medium and the optimization and estimation of the interaction effects of selected ingredients, respectively, for the enhanced growth of mycelium and maximum production of ergot alkaloids following Venil and Lakshmanaperumalsamy (2009a) and Nelofar et al. (2011). This optimization process involved three major steps such as performing the statistically designed experiments, estimating the coefficients in a mathematical model and predicting the response or adequacy of the statistical model. 3.7.1 Penicillium species and their maintenance Penicillium commune and Penicillium sp. IIB screened in the section-I were used for further studies. These were maintained on malt extract agar (MEA) medium slants. The slants were prepared by taking 2 g of malt extract and 2 g of agar in 250 ml Erlenmeyer flask and dissolved in 100 ml of distilled water. The medium was sterilized in autoclave at 121°C for 15 min. A loopful of growth from pure cultures was transferred aseptically to the slants containing 7 ml MEA medium, incubated at 25°C for five days. The slants having proper growth were stored at 4°C for further studies.

3.7.2. Preparation of inoculum for screening purpose

Spore suspensions of Penicillium species i.e. Penicillium commune and Penicillium sp. IIB were prepared separately by scraping the surface of the fungal colonies with inoculating needle and releasing the scrapped fungal spores in separate flasks containing 50 ml of distilled water under aseptic conditions. These were vortexed for 2-

3 minutes to break the mycelial fragments. The number of spores was adjusted at 10 6- 7spores/ml using hemocytometer.

3.7.3. Response Surface Methodology Experimental Designs Two statistical approaches were used for the statistical optimization of variables used in fermentation medium such as Plackett Burmann Design (PBD) and Box-Behnken Design (BBD). The detail of these methods is present in the subsequent subsections. 3.7.3.1 Screening of variables using Plackett-Burman Design (PBD) The Plackett-Burman experimental design identified the critical physico-chemical parameters required for enhanced ergot alkaloid production by screening “x” variables in “x+1” experiment (Plackett and Burman, 1946). The variables chosen for the present study were Sucrose, Yeast Extract, Succinic acid, Asparagine, Tryptophan, KH2PO4,

MgSO4.7H2O, FeSO4, ZnSO4 and pH in the culture medium. All the variables are denoted as numerical factors and investigated at two levels, designated as -1 (low level) and +1 (high level) for the growth of mycelium and production of ergot alkaloids. The experimental range and levels are given in the Table 3.4 and PBD experimental design used for screening of significant variables is presented in Table 3.5.

Table 3.4: Plackett-Burman Design experimental range and level for screening of variables Independent Variable Range and Level -1 +1 Sucrose, X1 5 35 Yeast Extract, X2 5 30 Succinic acid, X3 0.1 1 Asparagine, X4 0.1 1 Tryptophan, X5 0.1 1 KH2PO4, X6 0.1 1 MgSO4, X7 0.25 0.625 FeSO4, X8 0.01 0.1 ZnSO4, X9 0.02 0.2 pH, X 10 3 5 X1, X2……X10 are the independent variables and the important ingredients of fermentation media.

Table 3.5: Plackett-Burman experimental design for screening of variables Runs Variables (x) X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 Sucrose Yeast Succinic Asparagine Tryptophane MgSO4 KH2PO4 ZnSO4 FeSO4 pH Extract Acid 1. 35 5 0.1 0.1 1 0.625 0.1 0.2 0.1 5 2. 35 30 0.1 0.1 0.1 0.625 1 0.02 0.1 5 3. 5 5 0.1 0.1 0.1 0.25 0.1 0.02 0.01 3 4. 5 30 0.1 1 1 0.25 0.1 0.02 0.1 5 5. 35 5 0.1 1 0.1 0.25 1 0.2 0.01 3 6. 35 5 1 1 1 0.25 1 0.02 0.1 3 7. 5 5 1 0.1 1 0.625 1 0.02 0.01 3 8. 5 30 0.1 1 1 0.625 1 0.2 0.01 3 9. 35 30 1 0.1 1 0.25 0.1 0.2 0.01 5 10. 5 30 1 0.1 0.1 0.25 1 0.2 0.1 3 11. 5 5 1 1 0.1 0.625 0.1 0.2 0.1 5 12. 35 30 1 1 0.1 0.625 0.1 0.02 0.01 5

In the initial screening step, the factors influencing the yield of ergot alkaloids were studied at the two different levels (High level +1, Low level -1), in 12 experimental runs as shown in Table 3.5. according to Plackett and Burman (1946). All the experiments were done in triplicate and the average production was taken as a response. The effect of individual parameters on the alkaloids production was calculated using the following equation:

Y = βo + ∑ βXi

Where Y is the yield of ergot alkaloids, βo is the intercept and βXi is the linear coefficient and independent variables. The pH of all runs was maintained with the help of 0.1 N HCl and Ammonia solution and these were autoclaved at 121°C for 15 min. All of these flasks were incubated at 25°C for 21 days.

3.7.3.2. Optimization of significant factors using Box-Behnken Design (BBD) After screening of various variables using PBD, three variables were selected for the further optimization and for the estimation of their interaction effects for the production of ergot alkaloids using Box-Behnken Design (BBD) by following Box and Behnken (1960). In this step, the experiments consisted of 13 trials and independent variables were studied at three different levels i.e. low (-1), medium (0) and high (+1). The Box- Behnken experimental design (BBD) used for this study is presented in Table 3.6. All the experiments were done in triplicate and the average of ergot alkaloids production was taken as the response or yield (Y). The general form of the polynomial equation was as follows:

Y = βo + ∑ βii + X ∑ βii x i2 + ∑ βij Xi Xj

Where Y is the predicted response or yield, XiXj are the input variables which influence the response Y; βo is the intercept coefficient, βii is the linear coefficient; βii x i2 is the quadratic coefficient and βij is the interaction of these variables. X1, X2 and X3 are the independent variables, selected after screening of all the factors used in the first step of RSM. Experimental design for the optimization of significant factors and their influence on the production of ergot alkaloids is presented in Table 3.7. The pH of the all runs was maintained at 5 with the help of 0.1 N HCl and Ammonia solution and these flasks were autoclaved at 121°C for 15 min under 15lb/cm2 pressure. All flasks were incubated at 25°C for 21 days.

Table 3.6: Experimental range and levels for optimization of significant variables Independent Range and Level Variable -1 0 +1 Sucrose, X1 5 23 41 Yeast Extract, X2 5 22 39 FeSO4, X3 0.01 0.06 0.11

Table 3.7: Experimental design for optimization of significant variables Runs Variables X1 X2 X3 Sucrose Yeast Extract FeSO4 1 41 5 0.06 2 41 39 0.06 3 41 22 0.01 4 41 22 0.11 5 5 5 0.06 6 5 39 0.06 7 5 22 0.01 8 5 22 0.11 9 23 5 0.01 10 23 39 0.01 11 23 5 0.11 12 23 39 0.11 13 23 22 0.06

3.7.3. Statistical analysis of RSM

Statistical analysis of RSM was performed using the analysis of variance (ANOVA) test. This analysis included F-test (Fisher’s test), t-test (overall model significance), probability test (p), correlation coefficient (R), determination coefficient (R2) that were measured by the regression model. For each significant variable, the quadratic models were generated as 3-D counter plots and response surface curves were generated using STATISTICA version 7 (Stat-Ease Inc., Minneapolis, USA) software.

SECTION-III: STRAIN IMPROVEMENT

3.8. Strain Improvement

The wild strains of Penicillium commune and Penicillium sp. IIB after optimization were subjected to physical and chemical mutagenesis i.e. Ultraviolet Irradiations (UV) and Ethyl Methane Sulfonate (EMS) to improve the growth of mycelium and the production of ergot alkaloids.

3.8.1. Mutagenesis by UV irradiations

UV irradiations were used to induce mutagenesis in both species of Penicillium i.e. Penicillium commune and Penicillium sp. IIB. For this purpose, spore suspension cultures of both strains were prepared in 50 ml distilled water and diluted by serial dilution method. After serial dilutions, from 7th dilution, 1 ml of the spore suspension was poured in the sterilized Petri plates. These plates were placed under UV lamp to induce mutation in the Penicillium strains at 254 nm for 15, 30, 45, 60, 75, 90, 105, 120, 135 and 150 minutes respectively. The UV exposed Petri plates with spore suspensions were kept in dark for overnight. Next morning, malt extract agar (MEA) medium was poured to each of the UV exposed Petri plate. These batches were incubated at 25°C. After 10 days, survival of colonies was compared with the colonies of control (wild) species. This method was performed after Sreedevi et al. (2011).

3.8.2. Mutagenesis by Ethyl Methane Sulfonate (EMS)

Penicillium test species were exposed to chemical mutagenesis i.e. Ethyl Methane Sulfonate. For this purpose, spore suspension cultures of both strains were prepared in 50 ml distilled water and diluted by following serial dilution method. After a series of serial dilutions, from the last dilution, 2.5 ml of the spore suspension was poured in the separate test tube and 1 ml of EMS (0.3 ml of EMS was dissolved in 0.7 ml of double distilled water to make 1 ml solution of EMS) was mixed in it. It was vortexed well and 1 ml of this was transferred to already sterilized Petri plates. These Petri plates were

35 exposed to EMS solution for 10, 15, 20, 25 and 30 minutes respectively. Malt extract agar (MEA) medium was poured in Petri plate after each exposure and after exposure, these Petri plates were incubated at 25°C for 10 days. After 10 days, the survival of colonies of mutated strains was compared with the colonies of control (wild) strains. This method was also done after Sreedevi et al. (2011). Survival percentage of colonies was calculated using the following formula:

x 100 No. of Mutated Colonies 3.8.3. Maintenance of Survival mutant ratestrains (%) = No. of Wild Colonies After the above mentioned mutagenic treatments with UV and EMS, the survived colonies of mutated strains (UV and EMS treated) of Penicillium commune and Penicillium sp. IIB were streaked on MEA medium slants and incubated at 25°C for 10 days. After the incubation period the mutated strains were kept in refrigerator at 4°C for the further studies. The mutant strains were identified on the basis of their colony appearance and ergot alkaloids yield. 3.9. Production of Ergot Alkaloids by Mutated Strains 3.9.1. Screening of best UV mutant for the production of ergot alkaloids

The survived colonies of Penicillium commune and Penicillium sp. IIB after 135 and 150 min of exposure under UV were streaked on MEA slants. These were named as PCUV- 1, PCUV-2, PCUV-3, PCUV-4 and PCUV-5 for Penicillium commune and PUV-1, PUV-2, PUV-3 and PUV-4 for Penicillium sp. IIB respectively. These mutants were grown on already optimized M5 fermentation medium to find out the best mutated strain for the production of ergot alkaloids and also compared with the ergot alkaloids production of wild strain. The composition of fermentation medium is given in the Table 3.8.

3.9.2. Screening of best EMS mutant for the production of ergot alkaloids

The survived colonies of Penicillium commune and Penicillium sp. IIB after 25 min exposure of EMS were streaked on MEA slants. These were named as PCEMS-1, PCEMS-2 and PCEMS-3 for Penicillium commune and PEMS-1 for Penicillium sp. IIB

36 respectively. These mutants were grown on M5 fermentation medium (before optimization) to find out the best mutated strain for the production of ergot alkaloids and also yield of ergot alkaloids was compared with the yield of wild strain. The composition of fermentation medium is given in the Table 3.8 as mentioned above.

Table 3.8: Composition of fermentation medium for the production of ergot alkaloids by mutated strains of Penicillium commune and Penicillium sp. IIB Medium g/100 ml Ingredients Sucrose 5 NH4Cl 0.2 Succinic Acid 0.5 Yeast Extract 0.5 Tryptophan 0.5 Asparagine 0.5 KH2PO4 0.5 MgSO4. 7H2O 0.03 FeSO4 0.01 ZnSO4 0.002 Fermentation conditions pH 5 Inoculum Size (ml) 5 Incubation 25 Temperature (°C) Incubation Time 21 (Days)

3.9.3. Fermentor studies for the production of ergot alkaloids by selected mutants

After screening step, PCUV-4, PUV-4 and PCEMS-3 strains of Penicillium commune and Penicillium sp. IIB were selected for the growth of mycelium and production of ergot alkaloids in one liter of fermentor. The composition of the fermentation medium is given in the Table 3.3 as mentioned above under the sub-section 3.5.

3.10. Determination of Ergot Alkaloids 3.10.1. Separation of flask culture material Each flask culture after 21 days of incubation was filtered under aseptic condition to separate the grown mycelial mass (intracellular) and culture liquid medium (extracellular). The collected mycelial mass and liquid culture medium was stored at 4°C for further analysis.

3.10.2. Standard Curves

The ergot alkaloids (extracellular and intracellular) synthesized by Penicillium commune and Penicillium sp. IIB were compared with two reference salts such as bromo-criptine mesylate (BCM) and dihydroergotamine methane sulfonate (DMS) salts to prepare the standard curves of these two standard salts as follows:

3.10.2.1. Standard curve of BCM and DMS salts

3.10.2.1.1. Preparation of stock solutions

Stock solution of bromocriptine mesylate was prepared by dissolving 0.875 g of BCM in 5 ml of the distilled water in a falcon tube. Dihydroergotamine methane sulfonate salt solution was prepared in a falcon tube by dissolving 0.01 g in 5 ml of the distilled water.

3.10.2.1.2. Preparation of Van Urk reagent

One gram of Dimethyl-aminobenzyldehyde was dissolved in 20 ml of concentrated HCl and 10 ml of the distilled water was added to make the final volume upto 30 ml.

3.10.2.1.3. Procedure for standard curve

This was done after the method of Ashour and Kattan (2013). One unit of ergot alkaloid yield was expressed as 1µ mol of ergot alkaloids released per minute in the given solution. Different concentrations of standard salts i.e. 40, 80, 120, 160 and 200 µg were prepared in sterilized double distilled water using the above mentioned stock solutions. 1ml of all these dilutions was taken separately in the test tubes and 2 ml of Van Urk reagent was added into each test tube. All test tubes were incubated at 37°C for 30

38 minutes and after incubation, optical density (OD) was measured at 590 nm by Spectrophotometer (Hitachi U2900/U2910 double beam) against blank (1 ml distilled water added in 2 ml of Van Urk reagent and incubated at 37°C). The optical density for all the dilutions of both standard salts is presented in Table 3.9. The graphical representation of the standard curves of BCM and DMS is given in Fig 3.1 and 3.2.

Table 3.9. Absorbance of the dilutions of bromocriptine mesylate (BCM) and dihydroergotamine methane sulfonate (DMS) salts Concentrations OD of BCM OD of DMS 40 0.28 1.06 80 0.60 1.65 120 0.90 2.43 160 1.15 2.91 200 1.397 3.12

Ergot alkaloids concentration was measured using the formula given below:

Concentration of standard SF = 1.6 Absorbance of standard

1.4

1.2

0.8

0.6

0.4

Optical at nm 590 density Optical 0.2

0 0 50 100 150 200 250 Concentrations (µg)

Fig. 3.1. Standard curve of BCM salt

3.5

2.5

1.5

Optical at 590 nmOptical density 0.5

0 0 50 100 150 200 250 Concentrations (µg)

Fig. 3.2. Standard curve of DMS salt

3.10.2.2. Assay for ergot alkaloids of culture liquid filtrate extracts (CLFE)

The culture liquid filtrates separated from mycelium were centrifuged at 5000 rpm/min for 5 min at 4°C. After centrifugation the supernatants were collected and named as culture liquid filtrates. The culture liquid filtrate (extracellular) of ergot alkaloids obtained from the two fungal species was purified using chloroform extraction method (Naude et al., 2005). In this procedure, ergot alkaloids were extracted three times in 50 ml of chloroform in a separating funnel. The supernatants were separately poured in a separate glass bottles. The remaining water in the extract was removed by rotary evaporator. After purification, 1 ml of culture liquid filtrate extract was taken in a test tube, and 2 ml of Van Urk reagent was added. The same process was adopted for all the samples. All the test tubes were incubated at 37oC for 30 min and OD was measured at 590 nm by Spectrophotometer (Hitachi U2900/U2910 double beam) against blank.

3.10.2.3. Assay for ergot alkaloids of mycelial filtrate extracts (MFE)

All the mycelial mass separated from the flask culture was analyzed for ergot alkaloids yield. Fresh weights of the fungal mycelial masses were recorded, and all mycelial samples were dried in oven at 40°C for 24 hours for dry weights. The dried mycelial samples were mixed with chloroform and after 3 hours the chloroform mixed mycelial samples were subjected to cell lysis by sonication process using Ultrasonic Generator at 200 rpm/ min for 15 min to release the contents of ergot alkaloids. All the sonicated material was homogenized in a homogenizer for 15 min for complete cell lysis so that all of the contents of ergot alkaloids may be released in the chloroform solution. Mycelial filtrate extracts were collected after passing from rotary evaporator. The concentrated filtrates were assayed with Van Urk reagent as mentioned above and OD was measured by Spectrophotometer (Hitachi U2900/U2910 double beam) at 590 nm against blank.

SECTION IV: ANALYTICAL STUDIES

3.11. Thin Layer Chromatography (TLC)

Thin Layer Chromatography was performed on 6 cm x 12 cm silica gel plates with 0.25 mm layers from Merck, Germany. For this purpose, dihydroergotamine methane sulfonate salt and bromocryptine mesylate (BCM) were used as the standard salts to compare the alkaloid contents of samples with the reference salts.

3.11.1. Preparation of standards

Dihydroergotamine methane sulfonate (DMS) and bromocriptine mesylate (BCM) salts 0.01g were separately dissolved in 5 ml of chloroform in separate falcon tubes and stored at 4°C for further studies. Solution of methergine tablet (0.125 mg each tablet) was prepared by dissolving two tablets in 5 ml of distilled water in a falcon tube. The contents were purified through rotary evaporator and dissolved in 2 ml of chloroform.

3.11.2. Preparation of samples

Samples were prepared by chloroform extraction method by adding 10 ml sample into 20 ml chloroform in a separating funnel. It was mixed well and after formation of two layers, the lower layer was separated into a funnel and the method was repeated. After this, a 3 µl drop of extracted sample was placed on silica gel 60F254 plates and left opened for air drying purpose.

3.11.3. Preparation of mobile phase

For this purpose, various mobile phases were screened in the first phase for the selection of final mobile phase for TLC purpose. The composition of the mobile phases used in phase I and phase II is mentioned below.

3.11.4. Screening of mobile phases for TLC (phase I)

Various mobile phases were composed and tested for the detection of ergot alkaloid compounds in the mycelial and culture liquid filtrate extracts produced by Penicillium

42 commune and Penicillium sp. IIB. Different mobile phases and their proportions are given in the Table 3.10.

Table 3.10: Screening of TLC mobile phases for the determination of ergot alkaloids Sr. No. Mobile Phase Proportion of Solvents Chloroform: Methanol: Ammonia Solution 1. A ( 80 : 20 : 0.5) 2. B Chloroform: Acetone: Acetic Acid ( 90 : 10 : 5) 3. C Chloroform: Ethanol ( 90 : 10) Chloroform: Acetone: Orthophosphoric Acid 4. D (90 : 10 : 5) 5. E Methanol: Ethyl-acetate ( 40 : 60) 6. F Chloroform: Ethanol (90 : 10) 7. G Methanol: Ethyl Methyl Ketone (40 : 60) 8. H Chloroform: Isopropanol: Water (120 : 30 : 20)

3.11.5. Selection of final mobile phase for TLC (phase II)

In the second phase of TLC analysis, TLC mobile phase “A and H” were selected from the above mentioned TLC mobile phases. The selected TLC mobile phases were modified to get the maximum separation of ergot alkaloid compounds. The composition and proportion of TLC phases is presented in the Table 3.11.

Table 3.11: Selection of TLC mobile phases for the determination of ergot alkaloids Sr. No. Mobile Phase Proportion of Solvents 1. A1 Chloroform: Methanol: Ammonia Solution ( 80 : 20 : 0.25) 2. A2 Chloroform: Methanol: Ammonia Solution (80 : 20 : 5) 3. A3 Chloroform: Methanol: Ammonia Solution ( 50 : 40 : 10) 4. A4 Chloroform: Methanol: Ammonia Solution (50 : 50 : 5) 5. A5 Chloroform: Methanol: Ammonia Solution ( 50 : 40 : 15) 6. H1 Chloroform: Isopropanol: Water (120 : 30 : 15) 7. H2 Chloroform: Isopropanol: Water (120 : 30 : 20)

All the TLC plates were developed in a horizontal chamber and a spot of 3 µl of the standard salt solutions “dihydroergotamine methane sulfonate salt (DMS)” and “bromocriptine mesylate (BCM)” were developed along with the 3 µl spot of the sample solution. The TLC spotted plates were placed in chromatographic chambers with respective mobile phases. After chromatography, Van Urk reagent was sprayed on the TLC plates and alkaloids were detected under UV light at 254 nm and Rf values were calculated. Two chromatograms were developed for each solute-solvent combination. The whole procedure was done after Polak and Rompala (2007). Rf values of culture liquid (extracellular) and mycelial (intracellular) filtrates were calculated using the formula given below:

Distance travelled by the substance 푅푓 = Distance travelled by the solvent

3.12. High Performance Liquid Chromatography (HPLC)

Extracts of culture filtrate and mycelia were subjected to HPLC analysis after Moussa (2003) and Safwan and Soulafa (2013) under following conditions:

1. HPLC equipment (HPLC Perkin Elmer (Perkin Elmer, Norwalk. CT, USA)

2. NH2 C-18 column 3. Mobile phase: Chloroform: Isopropanol (80:20) 4. Elution: isocratic 5. Flow rate: 1ml/min 6. Detection UV at 280 nm 7. Temperature: 25°C 8. Pressure: 1100 Psi 3.12.1. Preparation of standards

Two standards of ergot alkaloids were used in HPLC for confirmation of ergot compound in the samples as a comparison. These were Dihydroergometrine Methane Sulfonate (DMS) salt and Bromocriptine Mesylate (BCM). Solutions were prepared in falcon tubes by dissolving 0.01g in 5ml of chloroform and stored at -20°C. These were filtered through Nylon filter 0.20 µm when they were used for HPLC.

3.12.2. Preparation of samples

Already prepared samples in methanol were subjected to evaporation at 65°C for the evaporation of methanol from the samples. The 20 ml of sample was extracted in 20 ml of chloroform 50:50 (v/v) 3 times using chloroform extraction process. All of the samples and standard solution were filtered through 0.20 µm Nylon filter and poured into HPLC vials. Retention time of each sample was noted.

Concentration of various compounds of ergot alkaloids was measured by using the following formula:

ndard concentration x Dilution factor Area of the sample Concentration of compound = Area of the standard x Sta 3.12.3. Preparation of mobile phase

One liter of mobile phase was prepared by adding 800 ml of chloroform to 200 ml of isoprpoanol. This was then filtered through membrane filter and subjected to sonication for 10 minutes for degassing and de-foaming purpose.

3.12.4. HPLC of samples

Samples (20 µl) of culture liquid and mycelial filtrate extracts were taken in HPLC vials and subjected to HPLC analysis in UV detector at 280 nm at 25°C room temperature and at a flow rate of 1 ml/min. Retention time and the peak areas of all the samples were measured and compared with the retention times of standards. Concentration of compounds present in the samples was also measured by the formula mentioned above.

Chapter 4 RESULTS ______

For convenience this chapter has been divided into four separate sections, namely, Section-I, II, III and IV to handle it adequately.

SECTION-I: OPTIMIZATION OF CULTURE CONDITIONS BY OFAT METHOD 4.1. Screening of Fungal Organism and Fermentation Medium The test species of genus Penicillium such as Penicillium commune, Penicillium italicum, Penicillium oxalicum, Penicillium digitatum and Penicillium sp. IIB were cultured on five different fermentation media i.e. M1, M2, M3, M4 and M5 (Table 3.1). It was found that Penicillium commune and Penicillium sp. IIB produced maximum amount of ergot alkaloids in culture liquid (1.359 mg/ml and 1.154 mg/ml) and mycelial (0.958 mg/ml and 0.635 mg/ml) filtrate extracts, respectively. The M5 medium was proved to be the most suitable fermentation medium for the growth of mycelium and ergot alkaloid production as presented in the Table 4.1. The growth of mycelium in M5 fermentation medium of all the fungal species is presented in Fig. 4.1. The lowest amount of ergot alkaloids were produced by Penicillium oxalicum in all the fermentation media including M5.

Table 4.1: Screening of fungal organisms and fermentation medium for production of ergot alkaloids Fungal Different Fermentation Media organism M1 M2 M3 M4 M5 CLFE MFE CLFE MFE CLFE MFE CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) (mg/ml) (mg/ml) (mg/ml) (mg/ml) (mg/ml) (mg/ml) P. 0.012± 0.019± 0.096± 0.137± 0.308± 0.099± 0.796± 0.516± 1.359± 0.958± commune 0.001 0.001 0 0.002 0.001 0.001 0.006 0.002 0.002* 0.001* P. italicum 0.001± 0.002± 0.075± 0.013± 0.145± 0.095± 0.439± 0.096± 0.495± 0.135± 0 0 0.002 0.001 0.003 0.001 0.004 0.001 0.004 0.003 P. 0.001± 0.001± 0.087± 0.037± 0.252± 0.088± 0.054± 0.103± 0.097± 0.001± oxalicum 0 0 0.001 0.002 0.001 0.001 0.001 0.002 0.001 0

P. 0.010± 0.010± 0.097± 0.054± 0.954± 0.087± 0.096± 0.144± 0.144± 0.095± digitatum 0.005 0.005 0.001 0.001 0.001 0.001 0.002 0.003 0.003 0

Penicillium 0.015± 0.067± 0.075± 0.086± 0.956± 0.758± 0.981± 0.651± 1.154± 0.635± sp. Strain 0.001 0.001 0.002 0.002 0.001 0.001 0.001 0.002 0.001* 0.002* IIB Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates

3 y = 0.3486x2 - 2.1334x + 4.232 2.56 R² = 0.7026 2.5 2.37

2 1.58 P. commune 1.5 P. italicum 0.93 1 0.89 P. digitatum P. oxalicum 0.5 Growthof mycelium (g/100ml) Penicillium sp. IIB

0 P. commune P. italicum P. digitatum P. oxalicum Penicillium sp. IIB Fungal species

Fig. 4.1: Mycelial growth of all fungal species in M5 fermentation medium

4.2. Optimization Studies of Fermentation Medium

One factor at a time (OFAT) method was used to optimize different fermentation conditions to obtain the maximum yield of ergot alkaloids in culture liquid (extracellular) and mycelial (intracellular) filtrates of Penicillium commune and Penicillium sp. IIB. The results regarding the different parameters influencing the growth and the production of ergot alkaloids were as follows:

4.2.1. Optimization of substrate for ergot alkaloids production

4.2.1.1. Optimization of different carbon and nitrogen sources

Effect of different carbon and nitrogen sources was studied to enhance the yield of ergot alkaloids produced by Penicillium commune and Penicillium sp. IIB.

4.2.1.1.1. Effect of Carbon Sources

Many carbon sources, such as glucose, fructose, sucrose, maltose, mannose and mannitol were included in the culture medium to find out the significant carbon source to enhance the yield of ergot alkaloids by Penicillium commune and Penicillium sp. IIB. Culture liquid filtrate extracts of Penicillium commune and Penicillium sp. IIB produced considerable quantity of ergot alkaloids when sucrose was added in the fermentation medium i.e. 2.58 mg/ml and 2.78 mg/ml respectively (Table 4.2). The growth of mycelium of both species was recorded after the completion of incubation period which is described in Fig. 4.2 and presence of ergot alkaloids in mycelial filtrate (intracellular) extracts was determined and it was found that maximum alkaloid concentration was achieved by adding sucrose in the fermentation medium of Penicillium commune (1.88 mg/ml) and Penicillium sp. IIB (1.95 mg/ml) as presented in Table 4.2.

Lowest yield of ergot alkaloid concentration was obtained from culture liquid filtrate extract when maltose was added in the growth medium of Penicillium commune (1.71 mg/ml) and Penicillium sp. IIB (1.51 mg/ml). Lowest ergot alkaloids concentration was

49 also estimated in mycelial filtrate extract when glucose was added in fermentation medium of Penicillium commune (0.45 mg/ml) and Penicillium sp. IIB (0.35 mg/ml). Analysis of variance for the ergot alkaloids production by both fungal species was also done which is given in the Table 4.3.

Table 4.2. Effect of different carbon sources on the production of ergot alkaloids Carbon Source Penicillium commune Penicillium sp. IIB CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) Glucose 1.83±0.01 0.45±0.02 1.95±0.01 0.35±0.05 Fructose 1.79±0.04 0.56±0.02 1.65±0.06 0.96±0.01 Maltose 1.71±0.02 0.61±0.04 1.51±0.02 0.74±0.04 Sucrose 2.58±0.02* 1.88±0.01* 2.78±0.01* 1.95±0.02* Mannose 1.82±0.01 1.34±0.03 1.51±0.04 0.54±0.02 Mannitol 2.17±0.02 1.59±0.02 1.91±0.02 1.78±0.01 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Table 4.3. Analysis of variance for the effect of carbon sources

Mean

Sum of Squares Degree of freedom Square F-value Significance CLFE Between 3.457 1 3.457 22.14 0.001* Groups Within 1.718 11 0.156

Groups Total 5.175 12 MFE Between 1.051 1 1.051 2.829 0.121 Groups Within 4.085 11 0.371

Groups Total 5.136 12 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value in ANOVA is an average of three replicates and significant of factors are statistically analyzed at p <0.01.

3.5 y = 0.0999x + 1.9644 R² = 0.2572 3

2.5 y = 0.0696x + 1.4477 R² = 0.1959 2

1.5 P. commune

1 Penicillium Growthof mycelium (g/100 ml) 0.5 sp. IIB

0 Glucose Fructose Maltose Sucrose Mannose Mannitol Carbon Sources (g/100 ml)

Fig. 4.2. Effect of different carbon sources on the mycelial growth of Penicillium commune and Penicillium sp. IIB

4.2.1.1.2. Effect of nitrogen sources

Effect of different nitrogen sources such as yeast extract, peptones, malt extract, meat extract, ammonium chloride and urea was evaluated on the growth of mycelium and ergot alkaloid production by Penicillium commune and Penicillium sp. IIB. Considerable quantity of ergot alkaloids was measured from culture liquid filtrate extract when yeast extract was added in the fermentation medium of Penicillium commune (2.85 mg/ml) and Penicillium sp. IIB (2.56 mg/ml). Lowest yield of ergot alkaloid in culture liquid filtrate extract was obtained when meat extract was added in the growth medium of Penicillium commune (0.61 mg/ml) and of ammonium chloride in the fermentation medium of Penicillium sp. IIB (0.04 mg/ml). Maximum ergot alkaloid yield was measured in mycelial filtrate extracts when yeast extract was added as a substrate in the fermentation medium i.e. 1.89 mg/ml and 1.97 mg/ml for Penicillium commune and Penicillium sp. IIB respectively. Least amount of ergot alkaloid concentration was obtained by the addition of ammonium chloride in the fermentation medium of Penicillium commune (0.34 mg/ml) and as well as addition of urea in the growth

51 medium of Penicillium sp. IIB (0.03 mg/ml) (Table 4.4). Measurement of mycelial growth after the completion of incubation time period is presented in Fig. 4.3 and highest growth of mycelium was observed by addition of yeast extract in the fermentation medium i.e. 2.57 g/100 ml and 2.17 g/100 ml of Penicillium commune and Penicillium sp. IIB respectively. Analysis of variance for the ergot alkaloids production by both fungal species is given in the Table 4.5.

Table 4.4. Effect of different nitrogen sources on the production of ergot alkaloids Nitrogen Source Penicillium commune Penicillium sp. IIB CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) Yeast Extract 2.85±0.02* 1.89±0.02* 2.56±0.01* 1.97±0.01* Peptones 2.31±0.03 1.48±0.01 2.13±0.04 1.32±0.03 Malt Extract 1.00±0.1 1.04±0.02 1.11±0.02 1.00±0.04 Meat Extract 0.61±0.03 1.05±0.03 0.09±0.01 1.01±0.01 Ammonium chloride 0.82±0.01 0.34±0.03 0.04±0.03 0.07±0.01 Urea 0.14±0.01 1.43±0.02 0.54±0.04 0.03±0.05 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates . Table 4.5. Analysis of variance for the effect of nitrogen sources

Sum of Degree of

Squares freedom Mean Square F-value Significance CLFE Between 1.293 1 1.293 1.257 0.286 Groups Within Groups 11.318 11 1.029 Total 12.612 12 MFE Between 1.032 1 1.032 2.547 0.139 Groups Within Groups 4.456 11 0.405 Total 5.488 12 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value in ANOVA is an average of three replicates and significant of factors are statistically analyzed at p <0.01.

2.5

1.5 P.commune 1 Penicillium sp.

0.5 Growthof mycelium (g/100 ml)

0 y = -0.3211x + 2.984 R² = 0.9382 Yeast Extract Peptones Malt Extract Meat Extract Ammonium Urea chloride y = -0.2631x + 2.5051 Nitrogen sources (g/100 ml) R² = 0.9592

Fig. 4.3. Effect of different nitrogen sources on the mycelial growth of Penicillium commune and Penicillium sp. IIB

4.2.2. Optimization of concentration levels of selected carbon and nitrogen sources for ergot alkaloids production

Sucrose and yeast extract were selected after screening of carbon and nitrogen sources respectively, for the production of ergot alkaloids. Effects of the concentration levels of sucrose and yeast extract on the yield of ergot alkaloids and growth of mycelium were assessed in the further experiments.

4.2.2.1. Effect of different concentration levels of sucrose

Table 4.6 shows the ergot alkaloids production in the culture liquid and mycelial filtrate extracts of Penicillium commune and Penicillium sp. IIB and it was found that, maximum alkaloid production was achieved in culture liquid filtrate extract at 35% of the sucrose level in the fermentation medium i.e. 2.67 mg/ml and 3.95 mg/ml by Penicillium commune and Penicillium sp. IIB respectively. Lowest ergot alkaloids concentration in culture liquid filtrate extract was observed at 10 % concentration (0.14 mg/ml) in growth media of both Penicillium species (Table 4.6).

Mycelial dry weights were recorded (Fig. 4.4) and ergot alkaloids production was quantified in mycelial filtrate extracts of Penicillium commune and Penicillium sp. IIB i.e. 1.34 mg/ml and 2.13 mg/ml respectively. Lowest ergot alkaloid concentration was measured in mycelial filtrate extracts at 10% addition of the sucrose in the growth medium (0.03 mg/ml and 0.07 mg/ml) of both species. Analysis of variance shows the significance of concentration levels of sucrose on the production of ergot alkaloids in culture liquid and mycelial filtrate extracts (Table 4.7). Table 4.6. Effect of concentration levels of sucrose on the production of ergot alkaloids Sucrose Penicillium commune Penicillium sp. IIB (g) CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) 10 0.15±0.02 0.03±0.03 0.14±0.04 0.07±0.02 15 0.35±0.01 0.31±0.01 0.22±0.01 0.17±0.03 20 0.69±0.02 0.67±0.01 1.23±0.02 0.98±0.02 25 0.95±0.01 0.95±0.04 2.96±0.01 1.53±0.04 30 1.66±0.01 1.23±0.02 2.97±0.01 1.97±0.01 35 2.67±0.01* 1.34±0.02* 3.95±0.01* 2.13±0.02* 40 2.33±0.01 1.67±0.03 3.16±0.01 2.01±0.01 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Table 4.7. Analysis of variance for the effect of concentration levels of sucrose

Degree Sum of of Mean Squares freedom Square F-value Significance CLFE Between Groups 18.194 6 3.032 5.082 0.025 Within Groups 4.176 7 0.597 Total 22.370 13 MFE Between Groups 6.260 6 1.043 8.492 0.006* Within Groups 0.860 7 0.123 Total 7.120 13 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value in ANOVA is an average of three replicates and significant of factors are statistically analyzed at p <0.01.

3.5

2.5 P. commune 2

1.5 y = 0.3657x + 1.0334 PenicilliumR² = 0.7701 sp. 1 Growthof mycelium (g/100 ml) 0.5 y = 0.1135x + 1.4033 0 R² = 0.4596 10 15 20 25 30 35 40 Sucrose conc. (g/100 ml)

Fig. 4.4. Effect of different concentrations of sucrose on the mycelial growth of Penicillium commune and Penicillium sp. IIB

4.2.2.2. Effect of different concentrations of yeast extract Table 4.8 presents that culture liquid filtrate extracts of Penicillium commune and Penicillium sp. IIB having 30% yeast extract level, produced the maximum ergot alkaloid yield i.e. 2.35 mg/ml and 2.23 mg/ml, respectively in the fermentation medium. Lowest ergot alkaloids concentration in culture liquid filtrate extract was observed at 5 % concentration level of fermentation media of Penicillium commune (0.43 mg/ml) and Penicillium sp. IIB (0.61 mg/ml). Ergot alkaloids production measured in mycelial filtrate extracts of Penicillium commune and Penicillium sp. IIB was found at the highest level i.e. 1.97 mg/ml and 1.53 mg/ml respectively for Penicillium commune and Penicillium sp. IIB in the presence of 30% yeast extract. Lowest ergot alkaloids yield was quantified in mycelial filtrate extracts at 5% of yeast extract in the growth medium (0.28 mg/ml and 0.21 mg/ml by Penicillium commune and Penicillium sp. IIB, respectively (Table 4.8). Mycelial dry weights were also measured after completing the incubation time as presented in Fig. 4.5. Analysis of variance shows that yeast extract addition was

55 highly significant for the production of ergot alkaloids extracted from culture liquid and mycelial filtrate extracts (Table 4.9).

Table 4.8. Effect of concentration levels of yeast extract on the production of ergot alkaloids Yeast Extract Penicillium commune Penicillium sp. IIB (g) CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) 5 0.43±0.04 0.28±0.03 0.61±0.02 0.21±0.02 10 0.65±0.02 0.49±0.02 0.71±0.05 0.22±0.02 15 0.79±0.02 0.61±0.02 0.93±0.03 0.61±0.03 20 0.81±0.01 0.91±0.01 1.35±0.01 0.78±0.04 25 1.91±0.01 1.24±0.01 1.95±0.02 1.21±0.01 30 2.35±0.01* 1.97±0.02* 2.23±0.01* 1.53±0.02* Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Table 4.9. Analysis of variance of the effect of concentration levels of yeast extract

Degree Sum of of Mean Squares Freedom Square F-value Significance CLFE Between 5.146 5 1.029 34.482 0.000* Groups Within Groups 0.179 6 0.030 Total 5.325 11 MFE Between 3.245 5 0.649 27.135 0.000* Groups Within Groups 0.143 6 0.024 Total 3.388 11 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value in ANOVA is an average of three replicates and significant of factors are statistically analyzed at p <0.01.

3 y = 0.2126x + 1.1527 2.5 R² = 0.9528

1.5 P. commune

1 Penicillium sp. 0.5 growthof mycelium (g/100 ml) 0 5 10 15 20 25 30 Yeast extract conc. (g/100 ml)

Fig. 4.5. Effect of different concentrations of yeast extract on the mycelial growth of Penicillium commune and Penicillium sp. IIB

4.2.3. Optimization of medium ingredients for ergot alkaloids production

Different concentration levels of KH2PO4, tryptophan, asparagine, succinic acid, ammonium chloride, MgSO4.7H2O, FeSO4.7H2O and ZnSO4 were optimized to evaluate their influence on the production of ergot alkaloids. The detail of the optimization of each ingredient is given in the following subsections.

4.2.3.1. Effect of different concentration levels of KH2PO4

Different concentrations of KH2PO4 ranging from 0.5 % to 3 % (w/v) were tested to achieve the maximum yield of ergot alkaloids from culture liquid and mycelial filtrate extracts of Penicillium commune and Penicillium sp. IIB. Presence of KH2PO4 in growth media supported the growth of mycelium and significantly increased the ergot alkaloids production. Table 4.10 shows the maximum ergot alkaloids yield in the culture liquid filtrate extract, observed at 2% concentration level i.e. 1.54 mg/ml and 1.90 mg/ml by Penicillium commune and Penicillium sp. IIB respectively. Least ergot alkaloids yield in culture liquid filtrate extracts was measured at 0.5% i.e. 0.26 mg/ml and 0.23 mg/ml for Penicillium commune and Penicillium sp. IIB, respectively. Mycelial growth of both of the species was greatly influenced by the varied concentration levels

57 of KH2PO4 as described in the Fig. 4.6. Ergot alkaloids concentration in mycelial filtrate extract was quantified and highest yield was observed at 0.98 mg/ml and 1.43 mg/ml by filtrates of Penicillium commune and Penicillium sp. IIB respectively as described in Table 4.10. Analysis of variance shows the significance effect of concentration levels of

KH2PO4 on the production of ergot alkaloids in culture liquid and mycelial filtrate extracts (Table 4.11).

Table 4.10. Effect of concentration levels of KH2PO4 on the production of ergot alkaloids

KH2PO4 Penicillium commune Penicillium sp. IIB (g) CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) 0.5 0.26±0.01 0.16±0.01 0.23±0.02 0.17±0.002 1 0.56±0.02 0.26±0.02 0.67±0.005 0.45±0.01 1.5 0.96±0.005 0.46±0.005 0.98±0.01 0.91±0.03 2 1.54±0.01* 0.98±0.01* 1.90±0.01* 1.43±0.01* 2.5 1.34±0.03 0.43±0.01 1.53±0.02 1.35±0.01 3 1.35±0.01 0.84±0.01 1.53±0.01 1.08±0.05 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Table 4.11. Analysis of variance for the effect of concentration levels of KH2PO4

Sum of Degree of Mean

Squares freedom Square F-value Significance CLFE Between 3.165 5 0.633 34.440 0.000* Groups Within Groups 0.110 6 0.018 Total 3.275 11 MFE Between 1.552 5 0.310 2.743 0.126 Groups Within Groups 0.679 6 0.113 Total 2.231 11 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value in ANOVA is an average of three replicates and significant of factors are statistically analyzed at p <0.01.

3.5 y = 0.0559x + 1.767 3 R² = 0.0423

2.5

P.commune 1.5 Pencillium sp 1

Growthofmycelium the (g/100 ml) 0.5

0 0.5 1 1.5 2 2.5 3 KH2PO4 conc (g/100 ml)

Fig. 4.6. Effect of different concentrations of KH2PO4 on the mycelial growth of Penicillium commune and Penicillium sp. IIB

4.2.3.2. Effect of concentration levels of tryptophan

Table 4.12 shows that, the presence of tryptophan in growth medium supported the mycelial growth and significantly increased the ergot alkaloids production. With the increase in the concentration of tryptophan, there was an increase in the production of ergot alkaloids. It was found that, maximum ergot alkaloids yield in the culture liquid filtrate extract was observed at 2% concentration level i.e. 2.58 mg/ml and 2.31 mg/ml from Penicillium commune and Penicillium sp. IIB respectively. Least ergot alkaloids yield in culture liquid filtrate extract was measured at 0.5% level i.e. 0.85 mg/ml and 0.95 mg/ml from Penicillium commune and Penicillium sp. IIB respectively. Impact of the concentration levels of tryptophan on mycelial growth of Penicillium commune and Penicillium sp. IIB was also monitored as described in the Fig. 4.7. Ergot alkaloids concentration in mycelial filtrate extract was assessed and the highest yield was observed at 2 % of the tryptophan concentration i.e. 1.67 mg/ml obtained from mycelial filtrate extract of Penicillium commune and Penicillium sp. IIB as described in Table 4.12.

Significance of the effect of different concentration levels of tryptophan was analyzed by ANOVA (Table 4.13). It was found that the effect of tryptophan concentrations greatly influenced the production of ergot alkaloids in culture liquid and mycelial filtrate extracts of both the fungal species in the fermentation medium.

Table 4.12. Effect of the different concentration levels of tryptophan on the production of ergot alkaloids Tryptophan Penicillium commune Penicillium sp. IIB (g) CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) 0.5 0.85±0.01 0.43±0.03 0.95±0.03 0.45±0.01 1.0 1.45±0.01 0.67±0.001 1.57±0.01 0.95±0.03 1.5 1.85±0.01 1.23±0.1 1.75±0.02 1.35±0.02 2.0 2.58±0.01* 1.67±0.02* 2.31±0.01* 1.67±0.001* 2.5 2.15±0.01 0.95±0.03 1.95±0.01 1.25±0.01 3.0 1.97±0.01 0.86±0.03 1.67±0.01 0.95±0.01 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Table 4.13. Analysis of variance of effect of concentration levels of tryptophan Sum of Degree of

Squares freedom Mean Square F-value Significance CLFE Between 2.721 5 0.544 27.518 0.000* Groups Within Groups 0.119 6 0.020 Total 2.839 11 MFE Between 1.788 5 0.358 22.432 0.001* Groups Within Groups 0.096 6 0.016 Total 1.884 11 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value in ANOVA is an average of three replicates and significant of factors are statistically analyzed at p <0.01.

4.5

4 y = -0.1063x + 3.4541 3.5 R² = 0.208 3

2.5

2 P. commune

1.5 Penicillium sp.

Myycelial Myycelial growth(g/100 ml) 1

0.5

0 0.5 1 1.5 2 2.5 3 Tryptophan Conc (g/100 ml)

Fig. 4.7. Effect of different concentrations of tryptophan on the mycelial growth of Penicillium commune and Penicillium sp. IIB

4.2.3.3. Effect of different concentration levels of asparagine

Presence of asparagine in growth medium supported the growth of mycelium and significantly increased the ergot alkaloids production during the present investigation. With the increase in the concentration of asparagines upto 2.5% in culture medium, an increase in the growth of mycelium and production of alkaloids was observed after which asparagine concentration retarded the growth of mycelium and production of ergot alkaloids. Table 4.14 shows the presence of maximum ergot alkaloids yield in the culture liquid filtrate extract of Penicillium commune at 2.5% concentration in the fermentation medium (2.35 mg/ml). 1.95 mg/ml of ergot alkaloids were obtained from culture liquid filtrate extract of Penicillium sp. IIB at 2% concentration of asparagine in the fermentation medium. Least ergot alkaloids concentration in culture liquid filtrate extract was estimated at 0.5% level i.e. 0.95 mg/ml and 1.00 mg/ml for Penicillium commune and Penicillium sp. IIB respectively. Mycelial growth of both of the fungi influenced by different concentration levels of asparagine as described in Fig. 4.8. Ergot alkaloids yield in mycelial filtrate extract of Penicillium commune and Penicillium sp. IIB

61 was found to be at the highest level i.e. at 2 and 2.5 % (Table 4.14). Significance of the effect of different concentration levels of asparagine was analyzed by ANOVA (Table 4.15). It was observed that the effect of asparagine concentrations partially influenced the production of ergot alkaloids in culture liquid and mycelial filtrate extract of both of the fungal species.

Table 4.14. Effect of different concentrations of asparagine on the production of ergot alkaloids Asparagine Penicillium commune Penicillium sp. IIB (g) CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) 0.5 0.95±0.02 0.75±0.02 1.00±0.02 0.97±0.01 1 1.23±0.03 0.95±0.03 1.09±0.01 1.21±0.002 1.5 1.75±0.01 1.00±0.04 1.35±0.02 1.85±0.01 2 1.95±0.01 1.32±0.02 1.95±0.01* 1.97±0.01* 2.5 2.35±0.01* 1.56±0.03* 1.53±0.02 1.73±0.03 3 2.00±0.001 0.95±0.01 1.01±0.01 1.32±0.03 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Table 4.15. Analysis of variance for the effect of concentration levels of asparagine Sum of Degree of Mean

Squares freedom Square F-value Significance CLFE Between 1.578 5 0.316 2.064 0.202 Groups Within Groups 0.917 6 0.153 Total 2.495 11 MFE Between 1.046 5 0.209 1.759 0.255 Groups Within Groups 0.713 6 0.119 Total 1.759 11 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value in ANOVA is an average of three replicates and significant of factors are statistically analyzed at p <0.01.

3.5 y = -0.1159x + 3.2481 3 R² = 0.4194

2.5

2 P. commune Penicillium sp. 1.5 Linear (Penicillium sp.) 1 Growthof mycelium (g/100 ml) 0.5

0 0.5 1 1.5 2 2.5 3 Asparagine Conc. (g/100 ml)

Fig. 4.8. Effect of different concentrations of asparagine on the mycelial growth of Penicillium commune and Penicillium sp. IIB

4.2.3.4. Effect of concentration levels of succinic acid Table 4.16 represents ergot alkaloids yield i.e. 1.35 mg/ml from Penicillium commune as well as for Penicillium sp. IIB obtained from culture liquid filtrate extract at 2% concentration level of succinic acid. Least ergot alkaloids concentration in culture liquid filtrate extract was measured at 0.5% level i.e. 0.35 mg/ml and 0.45 mg/ml from Penicillium commune and Penicillium sp. IIB respectively. Mycelial growth of both of the fungi influenced by the concentration levels of succinic acid was recorded as described in the Fig. 4.9. Ergot alkaloids yield produced by Penicillium commune and for Penicillium sp. IIB in mycelial filtrate extract was quantified and highest yield (0.98 mg/ml) was observed at 2 % of succinic acid (Table 4.16). It was also observed that with concentration of succinic acid beyond 2% in the fermentation medium, a decrease in the production of ergot alkaloids and mycelial growth occurred. Significance of the effect of different concentration levels of succinic acid was assessed by ANOVA (Table 4.17). It was observed that the effect of succinic acid concentrations significantly influenced the production of ergot alkaloids in culture liquid and mycelial filtrate extracts.

Table 4.16. Effect of concentration levels of succinic acid on the production of ergot alkaloids Succinic Acid Penicillium commune Penicillium sp. IIB (g) CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) 0.5 0.35±0.01 0.05±0.01 0.45±0.06 0.35±0.01 1 0.45±0.02 0.25±0.02 0.58±0.02 0.49±0.01 1.5 0.98±0.001 0.76±0.03 0.95±0.03 0.57±0.04 2 1.35±0.02* 0.98±0.01* 1.35±0.03* 0.98±0.02* 2.5 1.00±0.05 0.63±0.02 0.99±0.01 0.75±0.01 3 1.00±0.02 0.62±0.03 0.75±0.01 0.73±0.02 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Table 4.17. Analysis of variance for the effect of concentration levels of succinic acid Sum of Degree of Mean

Squares freedom Square F-value Significance CLFE Between 1.199 5 0.240 31.837 0.000* Groups Within Groups 0.045 6 0.008 Total 1.244 11 MFE Between 0.750 5 0.150 8.567 0.011* Groups Within Groups 0.105 6 0.018 Total 0.855 11 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value in ANOVA is an average of three replicates and significant of factors are statistically analyzed at p <0.01.

2.5 y = 0.6167ln(x) + 1.3068 R² = 0.6732 2

1.5 P. commune Penicillium sp. 1

Growthof mycelium (g/100 ml) 0.5

0 0.5 1 1.5 2 2.5 3 Succinic acid conc (g/100 ml)

Fig. 4.9. Effect of different concentrations of succinic acid on the mycelial growth of Penicillium commune and Penicillium sp. IIB

4.2.3.5. Effect of different concentrations of NH4Cl Highest yield of ergot alkaloids (1.25 mg/ml) in the culture liquid filtrate extract of

Penicillium commune was observed at 1.5% level of NH4Cl and at 2 % level for Penicillium sp. IIB (1.15 mg/ml). Least ergot alkaloids yield was measured at 0.5% level i.e. 0.35 mg/ml and 0.25 mg/ml by Penicillium commune and Penicillium sp. IIB respectively from their culture liquid filtrate extracts (Table 4.18). Mycelial growth of both of the fungi greatly influenced by the varied concentration levels of NH4Cl as presented in the Fig. 4.10. Assessment of production of ergot alkaloids from mycelial filtrate extracts indicated that highest yield was observed at 1.5% level of Penicillium commune (1.25 mg/ml) and 2 % of Penicillium sp. IIB (1.00 mg/ml) as shown in Table 4.18. Ammonium chloride in growth medium greatly influenced the growth of mycelium and increased the ergot alkaloids production but above 2% concentration hindered the production of alkaloids. Analysis of variance shows that NH4Cl concentrations significantly influenced the production of ergot alkaloids in culture liquid and mycelial filtrate extracts of both species of Penicillium.

Table 4.18. Effect of concentration levels of NH4Cl on the production of ergot alkaloids

NH4Cl Penicillium commune Penicillium sp. IIB (g) CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) 0.5 0.35±0.04 0.35±0.02 0.25±0.02 0.07±0.06 1 0.69±0.03 0.58±0.01 0.29±0.06 0.35±0.02 1.5 1.25±0.01* 1.25±0.001* 1.05±0.02 0.95±0.01 2 1.05±0.04 1.05±0.06 1.15±0.03* 1.00±0.003* 2.5 0.95±0.01 1.03±0.05 1.07±0.02 0.76±0.02 3 0.85±0.01 0.95±0.02 0.91±0.01 0.65±0.02 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Table 4.19. Analysis of variance of the effect of concentration levels of NH4Cl

Sum of Degree of

Squares freedom Mean Square F-value Significance CLFE Between 1.213 5 0.243 12.228 0.004* Groups Within Groups 0.119 6 0.020 Total 1.332 11 MFE Between 1.189 5 0.238 7.379 0.015* Groups Within Groups 0.193 6 0.032 Total 1.382 11 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value in ANOVA is an average of three replicates and significant of factors are statistically analyzed at p <0.01.

2.5

y = -0.0653x2 + 0.4618x + 0.9467 2 R² = 0.5348

1.5

P. commune 1 Penicillium sp.

0.5 Growthof mycelium (g/100 ml)

0 0.5 1 1.5 2 2.5 3 NH4Cl conc. (g/100 ml)

Fig. 4.10. Effect of different concentrations of NH4Cl on the mycelial growth of Penicillium commune and Penicillium sp. IIB

4.2.3.6. Effect of concentration levels of MgSO4.7H2O Table 4.20 shows that, the considerable yield of ergot alkaloids was obtained in culture liquid filtrate extract of Penicillium commune (1.53 mg/ml) and Penicillium sp. IIB (0.95 mg/ml) at 1.5% concentration level of MgSO4.7H2O in the fermentation medium.

Lowest yield of ergot alkaloids was quantified at 0.5% level of MgSO4.7H2O i.e. 0.59 mg/ml and 0.32 mg/ml from Penicillium commune and Penicillium sp. IIB respectively in their culture liquid filtrate extracts. Mycelial growth of the fungi was significantly influenced by the concentration levels of MgSO4.7H2O which is presented in the Fig. 4.11. Maximum ergot alkaloid yield was obtained from mycelial filtrate extracts of Penicillium commune (0.93 mg/ml) and Penicillium sp. IIB (0.33 mg/ml) at 1.5 % level of

MgSO4.7H2O (Table 4.20). Influence of concentration levels of MgSO4. 7H2O was analyzed by ANOVA (Table 4.21) and it was observed that MgSO4. 7H2O concentrations partially influenced the production of ergot alkaloids in culture liquid and mycelial filtrate extracts of Penicillium commune and Penicillium sp. IIB.

Table 4.20. Effect of the concentration levels of MgSO4.7H2O on the production of ergot alkaloids

MgSO4. 7H2O Penicillium commune Penicillium sp. IIB (g) CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) 0.5 0.59±0.01 0.35±0.03 0.32±0.02 0.03±0.05 1 1.35±0.01 0.74±0.01 0.56±0.01 0.23±0.01 1.5 1.53±0.005* 0.93±0.01* 0.95±0.01* 0.33±0.01* 2 0.95±0.01 0.65±0.02 0.43±0.005 0.21±0.02 2.5 0.75±0.01 0.54±0.01 0.33±0.02 0.13±0.01 3 0.32±0.01 0.34±0.04 0.13±0.006 0.03±0.003 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Table 4.21. Analysis of variance for the effect of concentration levels of MgSO4. 7H2O Sum of Degree of

Squares freedom Mean Square F-value Significance CLFE Between 1.333 5 0.267 2.110 0.195 Groups Within Groups 0.758 6 0.126 Total 2.091 11 MFE Between 0.304 5 0.061 0.618 0.693 Groups Within Groups 0.590 6 0.098 Total 0.894 11 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value in ANOVA is an average of three replicates and significant of factors are statistically analyzed at p <0.01.

3 y = -0.0268x2 + 0.1929x + 2.1507 R² = 0.5218 2.5

1.5 P. commune Penicillium sp. 1

Growthof mycelium (g/100 ml) 0.5

0 0.5 1 1.5 2 2.5 3 MgSO4.7H2O conc. (g/100 ml)

Fig. 4.11. Effect of different concentrations of MgSO4.7H2O on the mycelial growth of Penicillium commune and Penicillium sp. IIB

4.2.3.7. Effect of concentration levels of FeSO4.7H2O

Maximum ergot alkaloids yield in the culture liquid filtrate extract of Penicillium commune and Penicillium sp. IIB was obtained at 1.0% concentration level of FeSO4.7H2O i.e. 0.79 mg/ml and 0.93 mg/ml, respectively. Lowest yield of ergot alkaloids i.e. 0.25 mg/ml and 0.32 mg/ml in culture liquid filtrate extracts of Penicillium commune and Penicillium sp. IIB respectively was quantified at 3% concentration level in the fermentation medium (Table 4.22). Mycelial growth of both fungal species was significantly influenced by the different concentrations of FeSO4.7H2O (Fig. 4.12). Ergot alkaloid yield of Penicillium commune and Penicillium sp. IIB was found to be best at 1% level of FeSO4.7H2O i.e. 0.75 mg/ml and 0.83 mg/ml, respectively (Table 4.22). Analysis of variance (Table 4.23) showed that different concentrations of FeSO4.7H2O has significantly influenced the production of ergot alkaloids in culture liquid filtrate extract and slightly influenced the ergot alkaloids yield in mycelial filtrate extract of Penicillium commune and Penicillium sp. IIB.

Table 4.22. Effect of concentration levels of FeSO4.7H2O on the production of ergot alkaloids

FeSO4.7H2O Penicillium commune Penicillium sp. IIB (g) CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) 0.5 0.35±0.005 0.43±0.02 0.45±0.01 0.32±0.01 1 0.79±0.005* 0.75±0.02* 0.93±0.02* 0.83±0.01* 1.5 0.56±0.01 0.58±0.01 0.74±0.02 0.79±0.03 2 0.54±0.02 0.43±0.02 0.63±0.005 0.63±0.01 2.5 0.49±0.03 0.35±0.03 0.53±0.01 0.53±0.003 3 0.25±0.02 0.13±0.02 0.32±0.03 0.34±0.02 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Table 4.23. Analysis of variance for the effect of concentration levels of FeSO4. 7H2O Sum of Degree of Mean

Squares freedom Square F-value Significance CLFE Between 0.403 5 0.081 12.635 0.004* Groups Within Groups 0.038 6 0.006 Total 0.442 11 MFE Between 0.416 5 0.083 5.579 0.029 Groups Within Groups 0.090 6 0.015 Total 0.506 11 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value in ANOVA is an average of three replicates and significant of factors are statistically analyzed at p <0.01.

2 1.8 y = -0.117x + 1.8307 R² = 0.82 1.6 1.4 1.2 1 P. commune 0.8 Penicillium sp. 0.6 0.4 Growthof mycelium (g/100 ml) 0.2 0 0.5 1 1.5 2 2.5 3 FeSO4.7H2O Conc. (g/100 ml)

Fig. 4.12. Effect of different concentrations of FeSO4.7H2O on the mycelial growth of Penicillium commune and Penicillium sp. IIB

4.2.3.8. Effect of concentration levels of ZnSO4 Table 4.24 shows that the maximum ergot alkaloid yield was measured in the culture liquid filtrate extracts of Penicillium commune (1.00 mg/ml) at 1.5% addition of ZnSO4 in culture medium and of Penicillium sp. IIB (0.78 mg/ml) at 1% concentration level. Lowest yield of ergot alkaloids in culture liquid filtrate extract was obtained at 3%

ZnSO4 i.e. 0.43 mg/ml and 0.13 mg/ml from Penicillium commune and Penicillium sp. IIB respectively. Mycelial growth of both of the fungi has significantly influenced by the varied concentration of ZnSO4 (Fig. 4.13). Ergot alkaloid yield in mycelial filtrate extracts was found highest at 1% and 1.5% concentration level in culture medium of Penicillium sp. IIB (0.63 mg/ml) and Penicillium commune (0.85 mg/ml) (Table 4.24).

Influence of concentration levels of ZnSO4 was also estimated by the ANOVA (Table

4.25) showed that, ZnSO4 concentrations have significantly influenced the production of ergot alkaloids in mycelial filtrate extracts of Penicillium commune and Penicillium sp. IIB.

Table 4.24. Effect of concentration levels of ZnSO4 on the production of ergot alkaloids

ZnSO4 Penicillium commune Penicillium sp. IIB (g) CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) 0.5 0.53±0.02 0.43±0.01 0.59±0.01 0.43±0.01 1 0.79±0.01 0.63±0.02 0.78±0.01* 0.63±0.02* 1.5 1.00±0.03* 0.85±0.01* 0.65±0.02 0.59±0.03 2 0.95±0.01 0.42±0.01 0.53±0.03 0.43±0.02 2.5 0.79±0.02 0.32±0.03 0.32±0.01 0.33±0.02 3 0.43±0.03 0.10±0.03 0.13±0.03 0.03±0.06 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Table 4.25. Analysis of variance for the effect of concentration levels of ZnSO4 Sum of Degree of

Squares freedom Mean Square F-value Significance CLFE Between 0.414 5 0.083 1.619 0.286 Groups Within Groups 0.307 6 0.051 Total 0.721 11 MFE Between 0.537 5 0.107 17.717 0.002* Groups Within Groups 0.036 6 0.006 Total 0.573 11 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value in ANOVA is an average of three replicates and significant of factors are statistically analyzed at p <0.01.

2.5 y = -0.0796x + 1.9763 R² = 0.1532

1.5 P. commune Penicillium sp. 1

Growthof Mycelium (g/100 ml) 0.5

0 0.5 1 1.5 2 2.5 3 ZnSO4 conc (g/100 ml)

Fig. 4.13. Effect of different concentrations of ZnSO4 on the mycelial growth of Penicillium commune and Penicillium sp. IIB

4.2.4. Optimization of process parameters for the production of ergot alkaloids

Results of medium ingredients optimization revealed that Penicillium commune and Penicillium sp. IIB produced maximum ergot alkaloids in the medium containing sucrose (35%) as carbon source, yeast extract (30 %) as nitrogen source under surface culture fermentation process. Various ions in the form of salts i.e. KH2PO4 (2%), tryptophan (2 %), asparagine (2 %, 2.5%), succinic acid (2 %), ammonium chloride

(1.5%, 2%), MgSO4.7H2O (2%), FeSO4.7H2O (1%) and ZnSO4 (1%, 1.5%) supported the growth of mycelium of Penicillium commune and Penicillium sp. IIB respectively. Hence, the above formulation designated as M5 was used for further optimization of process parameters such as effect of pH, incubation temperature, incubation time and inoculum sizes was evaluated using surface culture technique. The effect of each parameter was evaluated individually in all experiments.

4.2.4.1. Effect of pH

Table 4.26 shows the best ergot alkaloids yield in the culture liquid filtrate extracts of Penicillium commune (2.14 mg/ml) and Penicillium sp. IIB (1.99 mg/ml) at pH 5 of the fermentation medium. Lowest yield of ergot alkaloids in culture liquid filtrate extracts was measured at pH 3 i.e. 0.82 mg/ml and 0.71 mg/ml of Penicillium commune and Penicillium sp. IIB respectively. Mycelial growth of both of the fungi greatly influenced by the varied pH levels as mentioned in the Fig. 4.14. Ergot alkaloid yield was estimated in mycelial filtrate extracts too and highest yield was measured at pH 5 of Penicillium commune (1.95 mg/ml) and Penicillium sp. IIB (1.89 mg/ml) and as presented in Table 4.26. Influence of different levels of pH was also estimated by the analysis of variance as described in the Table 4.27. It also shows that pH levels have significantly influenced the production of ergot alkaloids in mycelial filtrate extracts of Penicillium commune and Penicillium sp. IIB.

Table 4.26. Effect of pH on the production of ergot alkaloids on the production of ergot alkaloids pH Penicillium commune Penicillium sp. IIB CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) 3 0.82±0.01 0.65±0.02 0.71±0.02 0.54±0.01 4 1.71±0.02 0.96±0.01 1.35±0.01 0.98±0.01 5 2.14±0.001* 1.95±0.01* 1.99±0.01* 1.89±0.02* 6 1.74±0.01 1.57±0.02 1.54±0.02 1.05±0.01 7 1.85±0.02 0.96±0.01 1.01±0.03 0.95±0.02 8 1.34±0.01 0.44±0.01 1.00±0.04 0.65±0.03 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Table 4.27. Analysis of variance of the effect of various pH levels Sum of Degree of

Squares freedom Mean Square F-value Significance CLFE Between 1.934 5 0.387 4.527 0.047 Groups Within Groups 0.513 6 0.085 Total 2.447 11 MFE Between 2.604 5 0.521 18.898 0.001* Groups Within Groups 0.165 6 0.028 Total 2.769 11 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value in ANOVA is an average of three replicates and significant of factors are statistically analyzed at p <0.01.

3 y = 0.1117x + 1.5086 R² = 0.2049 2.5

2 P. commune 1.5

Penicillium sp. 1

0.5 Growthof mycelium (g/100 ml)

0 3 4 5 6 7 8 pH levels

Fig. 4.14. Effect of different pH levels on the mycelial growth of Penicillium commune and Penicillium sp. IIB

4.2.4.2. Effect of incubation temperature Table 4.28 describes that the maximum ergot alkaloids yield was obtained at 25°C in culture liquid filtrate extracts of Penicillium commune (2.28 mg/ml) and Penicillium sp. IIB (2.00 mg/ml). Lowest yield of ergot alkaloids in culture liquid filtrate extracts was quantified at 30°C i.e. 0.45 mg/ml and 1.00 mg/ml of Penicillium commune and Penicillium sp. IIB respectively. Mycelial growth of both of the fungi was significantly influenced by varying the incubation temperatures (Fig. 4.15). Ergot alkaloid yield obtained from mycelial filtrate extracts was highest at 25°C for Penicillium commune (1.13 mg/ml) and Penicillium sp. IIB (1.35 mg/ml) as presented in the Table 4.28. Influence of different incubation temperatures was analyzed by ANOVA (Table 4.29). It was found that incubation temperature influenced the production of ergot alkaloids significantly in culture liquid filtrate extracts of Penicillium commune and Penicillium sp. IIB and less significantly in mycelial filtrate extracts.

Table 4.28. Effect of incubation temperature on the production of ergot alkaloids Temperature Penicillium commune Penicillium sp. IIB (°C) CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) 23 1.35±0.01 1.03±0.02 1.43±0.01 0.83±0.01 24 1.59±0.02 1.04±0.003 1.59±0.01 0.95±0.02 25 2.28±0.01* 1.13±0.01* 2.00±0.01* 1.35±0.005* 26 2.15±0.001 1.10±0.02 1.96±0.03 1.15±0.02 27 2.10±0.005 1.09±0.02 1.75±0.02 1.00±0.003 28 1.95±0.01 1.00±0.01 1.63±0.01 0.09±0.06 29 1.05±0.01 1.00±0.02 1.10±0.03 0.04±0.04 30 0.45±0.02 0.75±0.02 1.00±0.01 0.09±0.03 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Table 4.29. Analysis of variance for the effect of different incubation temperatures Sum of Degree of Mean

Squares freedom Square F-value Significance CLFE Between 3.449 7 0.493 12.112 0.001* Groups Within Groups 0.325 8 0.041 Total 3.774 15 MFE Between 1.360 7 0.194 1.356 0.037 Groups Within Groups 1.146 8 0.143 Total 2.506 15 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value in ANOVA is an average of three replicates and significant of factors are statistically analyzed at p <0.01.

2.5 y = -0.0327x + 1.7836 R² = 0.0508 2

1.5 P. commune

Penicillium sp. 0.5 Growthof mycelium (g/100 ml)

0 23 24 25 26 27 28 29 30 Incubation temp. °C

Fig. 4.15. Effect of incubation temperature on mycelial growth of Penicillium commune and Penicillium sp. IIB

4.2.4.3. Effect of incubation time

Table 4.30 shows that the maximum ergot alkaloids yield was observed after 21 days of incubation period in the culture liquid filtrate extracts of Penicillium commune (2.96 mg/ml) and Penicillium sp. IIB (2.43 mg/ml). Lowest yield of ergot alkaloids in culture liquid filtrate extract was obtained after 30 days i.e. 1.23 mg/ml and 1.55 mg/ml of Penicillium commune and Penicillium sp. IIB respectively. Mycelial growth of fungi was influenced by different incubation time periods (Fig. 4.16). Ergot alkaloid yield was estimated in mycelial filtrate extracts and highest yield was observed after 21 days of incubation of Penicillium commune (2.35 mg/ml) and Penicillium sp. IIB (0.98 mg/ml) and as presented in the Table 4.30. Influence of different incubation times was also estimated by ANOVA (Table 4.31). It also showed that influence of incubation time periods was significant on the production of ergot alkaloids in culture liquid filtrate extracts of Penicillium commune and Penicillium sp. IIB.

Table 4.30. Effect of different incubation time periods on the production of ergot alkaloids Incubation time Penicillium commune Penicillium sp. IIB (Days) CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) 7 1.99±0.01 0.96±0.01 1.92±0.01 0.64±0.01 14 2.21±0.01 1.86±0.01 2.00±0.01 0.81±0.02 21 2.96±0.005* 2.35±0.01* 2.43±0.02* 0.98±0.002* 30 1.23±0.02 1.39±0.02 1.55±0.02 0.41±0.01 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Table 4.31. Analysis of variance for the effect of incubation times

Sum of Degree of

Squares freedom Mean Square F-value Significance CLFE Between 1.726 3 0.575 10.646 0.022 Groups Within Groups 0.216 4 0.054 Total 1.942 7 MFE Between 0.964 3 0.321 0.636 0.630 Groups Within Groups 2.021 4 0.505 Total 2.985 7 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value in ANOVA is an average of three replicates and significant of factors are statistically analyzed at p <0.01.

2.5

2 y = 0.182x + 1.18 R² = 0.1524 1.5 P. commune 1

Penicillium sp.

Growthof mycelium (g/100 ml) 0.5

0 7 14 21 30 Incubation time (Days)

Fig. 4.16. Effect of different incubation times on mycelial growth of Penicillium commune and Penicillium sp. IIB

4.2.4.4. Effect of size of inoculum Table 4.32 shows the maximum ergot alkaloids production in culture liquid filtrate extract of Penicillium commune was 3.34 mg/ml and of Penicillium sp. IIB, 2.53 mg/ml at 15 ml and 20 ml of inoculum addition in their fermentation media. Lowest yield of ergot alkaloids in culture liquid filtrate extract was observed with the addition of 5 ml inoculum i.e. 0.79 mg/ml and 0.58 mg/ml in the fermentation medium of Penicillium commune and Penicillium sp. IIB respectively. Mycelial growth of the fungi was significantly influenced by inoculating the fermentation medium with different sizes of inoculum as mentioned in the Fig. 4.17. Ergot alkaloid yield was measured in mycelial filtrate extracts of Penicillium commune and Penicillium sp. IIB. Highest yield was observed at 15 ml of inoculum in the mycelial filtrate extract of Penicillium commune i.e. 2.75 mg/ml and Penicillium sp. IIB 1.98 mg/ml (Table 4.32). Significance of the influence of different inoculum sizes was observed by ANOVA (Table 4.33). It showed that different inoculum sizes influenced the production of ergot alkaloids in culture liquid filtrate extracts of Penicillium commune and Penicillium sp. IIB, respectively.

Table 4.32. Effect of different size of inoculum on the production of ergot alkaloids Size of inoculum Penicillium commune Penicillium sp. IIB (ml) CLFE MFE CLFE MFE (mg/ml) (mg/ml) (mg/ml) (mg/ml) 5 0.79±0.02 0.56±0.02 0.58±0.02 0.35±0.02 10 2.48±0.01 0.98±0.03 1.95±0.01 0.97±0.01 15 3.34±0.005* 2.75±0.02* 2.13±0.01 1.35±0.02 20 1.96±0.02 1.38±0.01 2.53±0.002* 1.98±0.01* 25 1.97±0.01 2.59±0.01 1.97±0.03 1.63±0.06 30 1.38±0.01 1.78±0.02 1.87±0.02 0.13±0.04 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Table 4.33. Analysis of variance for the effect of various inoculum sizes Sum of Degree of Mean

Squares freedom Square F-value Significance CLFE Between 4.943 5 0.989 5.039 0.017* Groups Within Groups 1.177 6 0.196 Total 6.120 11 MFE Between 4.543 5 0.909 1.815 0.244 Groups Within Groups 3.004 6 0.501 Total 7.547 11 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value in ANOVA is an average of three replicates and significant of factors are statistically analyzed at p <0.01.

4.5

4 y = 0.089x + 2.4993 3.5 R² = 0.0255

2.5

2 P. commune

1.5 Penicillium sp.

1 Growthof mycelium (g/100 ml) 0.5

0 5 10 15 20 25 30 Inoculum size (ml)

Fig. 4.17. Effect of different inoculum size on mycelial growth of Penicillium commune and Penicillium sp. IIB

4.2.5. Production of ergot alkaloids in fermentor

Production of ergot alkaloids was carried out in one liter fermentor using optimum factors in the fermentation medium from through surface culture fermentation technique. The ergot alkaloids yield obtained from the culture liquid and mycelial filtrate extracts of both fungal organisms is presented in the Table 4.34 and growth of mycelium presented in Fig. 4.18.

Table 4.34. Production of ergot alkaloids in fermentor Penicillium commune Penicillium sp. IIB CLFE MFE CLFE MFE (mg/L) (mg/L) (mg/L) (mg/L) 4.38±0.01 2.93±0.01 5.51±0.02 2.68±0.01 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

12 y = 1.63x + 7.27 10.53 10 8.9

4 Growthof mycelium (g/l) 2

0 P. commune Penicillium sp. Fungus

Fig. 4.18. Mycelial growth of Penicillium commune and Penicillium sp. IIB in fermentor

SECTION-II: RESPONSE SURFACE METHODOLOGY

4.3. Response Surface Methodology Statistical designs such as Plackett-Burman Design (PBD) and Box-Behnken Design (BBD) were used for the screening and optimization of variables in fermentation medium influencing the yield of ergot alkaloids by Penicillium commune and Penicillium sp. IIB using surface culture fermentation technique.

4.3.1. Screening of variables using Plackett-Burman Design (PBD) for the production of ergot alkaloids by Penicillium commune

Plackett-Burman Design (PBD) was used for screening of individual variables/components (sucrose, yeast extract, succinic acid, asparagine, tryptophane,

KH2PO4, MgSO4, FeSO4, ZnSO4 and pH) of fermentation medium for the enhanced production of ergot alkaloids. The influence of individual variables on the yield of ergot alkaloids was observed in twelve runs/experiments. The variation in ergot alkaloids production ranged from 0.36 mg/100 ml to 14.76 mg/100 ml. Maximum production of ergot alkaloids (14.76 mg/100 ml) was obtained from culture liquid filtrate extract of run no. 2 (Sucrose, yeast extract and FeSO4) and the lowest yield was achieved from run no. 3 (Sucrose, yeast extract and FeSO4, 0.36 mg/100 ml). These variations reflected that the variables/components of fermentation medium greatly influenced the yield of ergot alkaloids (Table 4.35). The variations in the production of ergot alkaloids were calculated by first order polynomial model which was as follows:

Y = βo + ∑ βXi

Where Y is the yield of ergot alkaloids, βo is the intercept and βXi is the linear coefficient of independent variables.

Table 4.35: Screening of variables for ergot alkaloids production by Penicillium commune using PBD Run Yield of ergot alkaloids (mg/100 ml)

1. 10.98±0.01 2. 14.76±0.01* 3. 0.36±0.02 4. 5.50±0.03 5. 9.96±0.05 6. 12.99±0.01 7. 0.64±0.06 8. 5.63±0.03 9. 10.54±0.01 10. 5.95±0.02 11. 0.44±0.02 12. 12.38±0.01 Each value is an average of three replicates and “±” indicates the standard deviation among three replicates.

The fermentation medium variables/components with a p-value of < 0.05 at 90% confidence level were accepted as significant factors affecting the production of ergot alkaloids. The ANOVA for the model is described in the Table 4.36. The factors with p < 0.05 are considered significant for the production of ergot alkaloids. From the Table

4.36, it was clearly indicated that sucrose, yeast extract and FeSO4 significantly influenced the production of ergot alkaloids in the fermentation medium. So these three variables were selected for further analysis in the next step of RSM. These results also indicated the significance of PBD statistical design for the screening and identification of important variables influencing the production of ergot alkaloids in their culture liquid filtrate extracts. The screening of variables using PBD is also presented through Pareto chart to display the crucial factors that played a significant role of said factors for the production of ergot alkaloids. The values given in this chart indicates the significant role of sucrose, yeast extract and FeSO4 that for production of ergot alkaloids (Fig. 4.19).

Table: 4.36: Analysis of variance for ergot alkaloids yield by Penicillium commune using PBD Source Sum of Degree of Mean F-value p-value squares Freedom Square Intercept 0.46 1 0.46 8.26 0.21 Sucrose 147.78 1 147.78 2670.95 0.012 Yeast extract 22.54 1 22.54 4.7.44 0.032 Succinic acid 1.30 1 1.30 23.64 0.13 MgSO4 1.16 1 1.16 20.96 0.14 KH2PO4 0.16 1 0.16 2.86 0.34 FeSO4 12.87 1 12.87 232.57 0.042 ZnSO4 0.50 1 0.50 9.06 0.20 Asparagine 0.96 1 0.96 17.41 0.15 Tryptophan 0.36 1 0.36 6.47 0.24 pH 3.06 1 3.06 55.33 0.085 Error 0.06 1 0.06

(g /100 ml)

Fig. 4.19. Pareto chart showing the significant variables i.e. sucrose, yeast extract and FeSO4 influencing the production of ergot alkaloid yield by Penicillium commune

4.3.2. Optimization of suitable variables using Box-Behnken Design (BBD) for ergot alkaloids yield by Penicillium commune and Penicillium sp. IIB

Box-Behnken design (BBD) was employed to optimize the selected variables (Sucrose,

Yeast extract and FeSO4) and to find out the effect of their combined interactions on the production of ergot alkaloids. The BBD experimental design consisted of 13 runs and three independent variables such as sucrose, yeast extract and FeSO4 optimized at three different levels such as low (-1), medium (0) and high (+1) level. The yield of ergot alkaloids obtained from culture liquid filtrate extracts of Penicillium commune ranged from 5.42 mg/ 100 ml to 14.64 mg/100 ml and of Penicillium sp. IIB yield ranged from 13.50 mg/100 ml to 35.60 mg/100 ml, respectively. The observed values of ergot alkaloids produced by Penicillium commune and Penicillium sp. IIB were calculated using BBD and were compared with predicted values, as presented in the Table 4.37 and 4.38. Maximum ergot alkaloids yield was observed from culture liquid filtrate extract of run no. 6 (Sucrose, yeast extract and FeSO4, 14.64 mg/ml) of Penicillium commune, it was compared with the predicted value (14.99 mg/ml) that was considered as more significant than observed value. The lowest value of ergot alkaloids yield was observed at run 11 (Sucrose, yeast extract and FeSO4, 5.42 mg/100 ml) and it was more significant than the predicted value (5.15 mg/ml) (Table 4.37). These values were calculated from the polynomial equation as described under the subsection 3.6.3.2.

Table 4.37: Observed and predicted values of ergot alkaloids yield by Penicillium commune using BBD Runs Sucrose Yeast Extract FeSO4 Alkaloids Alkaloids Yield (g/100ml) (g/100ml) (g/100ml) Yield Predicted Observed 1. 41 5 0.06 7.86 7.52 2. 41 39 0.06 9.46 8.62 3. 41 22 0.01 7.38 7.96 4. 41 22 0.11 7.98 8.59 5. 5 5 0.06 7.42 8.27 6. 5 39 0.06 14.64* 14.99* 7. 5 22 0.01 11.48 10.88 8. 5 22 0.11 13.36 12.78 9. 23 5 0.01 7.83 7.61 10. 23 39 0.01 7.53 7.82 11. 23 5 0.11 5.42 5.15 12. 23 39 0.11 12.53 12.76 13. 23 22 0.06 6.39 6.39

Table 4.38: Observed and predicted values of ergot alkaloids yield by Penicillium sp. IIB using BBD Runs Sucrose Yeast Extract FeSO4 Alkaloids Alkaloids (g/100ml) (g/100ml) (g/100ml) Yield Yield Observed Predicted 1. 41 5 0.06 21.60 20.94 2. 41 39 0.06 15.00 16.51 3. 41 22 0.01 23.30 23.11 4. 41 22 0.11 26.60 26.94 5. 5 5 0.06 17.10 16.59 6. 5 39 0.06 13.50* 14.16* 7. 5 22 0.01 20.40 20.06 8. 5 22 0.11 24.10 24.29 9. 23 5 0.01 15.80 16.65 10. 23 39 0.01 17.90 17.56 11. 23 5 0.11 25.20 25.53 12. 23 39 0.11 17.60 16.75 13. 23 22 0.06 35.60 35.60

Maximum ergot alkaloids yield was observed from culture liquid filtrate extract of run 13 (35.60 mg/ml) of Penicillium sp. IIB, it was compared with the predicted value (35.60 mg/ml) and found as equal with the observed value. The lowest value of ergot alkaloids yield observed at run 6 was 13.50 mg/100 ml and it was less significant than

87 the predicted value (14.16 mg/ml), the values calculated from the polynomial equation described under the subsection 3.6.3.2.

4.3.2.1. Residual regression analysis for ergot alkaloids production

The residual regression analysis was accomplished by measuring the difference between the observed values of dependent variable (Y) and the predicted values (Y’) of the ergot alkaloids production and this was calculated by using the formula mentioned below: e = Y – Y’ Where e represents residues, Y is the observed response or yield and Y’ is the predicted response or yield of ergot alkaloids. The residual plots are drawn in Figs. 4.20 and 4.21 to describe the ergot alkaloid yield residual regression analysis for Penicillium commune and Penicillium sp. IIB respectively. Both plots are showing nearly linear relationship between the predicted and observed values for production of ergot alkaloids from culture liquid filtrate extracts.

(g /100 ml)

Fig. 4.20: Observed and predicted values of ergot alkaloid production by Penicillium commune using BBD

The Fig. 4.20 clearly indicates a non-random pattern among the predicted and observed values for the production of ergot alkaloids by Penicillium commune and it was observed that this model was the better fit model to describe the effect of optimized factors

(sucrose, yeast extract and FeSO4) as significant independent variables on the yield of ergot alkaloids which is considered as the dependent variable.

(g /100 ml)

Fig. 4.21: Observed and predicted values of ergot alkaloids production by Penicillium sp. IIB using BBD

The Fig. 4.21 clearly indicates a non-random pattern among the predicted and observed values for the production of ergot alkaloids by Penicillium sp. IIB and it was observed that this model was the better fit model to describe the effect of optimized factors

(sucrose, yeast extract and FeSO4) as significant independent variables on the yield of ergot alkaloids.

4.3.2.2. Desirability Analysis

Desirability analysis was conducted to study the influence of optimized factors on the Culture liquid filtrate extracts for the production of ergot alkaloids by Penicillium commune and Penicillium sp. IIB. In this approach, the measured properties of each predicted response is transferred to a dimensionless desirability value d. The scale of desirability functions ranged between d = 0, which suggests that response is completely unacceptable and d=1 suggests that response is exactly equal to the targeted value. The value of d increases as the desirability of the corresponding response increases. The approach was used to transform the response into a desirability value as described in the Figs. 4.22 and 4.23.

The desirability chart represents the predicted optimum levels and the predicted response of significant selected variables. The predicted optimum levels of sucrose, yeast extract and FeSO4 for ergot alkaloids yield from culture liquid filtrate extracts of Penicillium commune were 52, 50 and 0.14 (g/100 ml) (Fig. 4.22) and for Penicillium sp. IIB these were 23, 22 and 0.1 (g/100 ml) (Fig. 4.23) respectively. The predicted maximum ergot alkaloid yield at these levels for Penicillium commune and Penicillium sp. IIB were 16.95 mg/ml and 33.07 mg/100 ml, respectively. The observed ergot alkaloid yield of ergot alkaloids on these levels for Penicillium commune and Penicillium sp. IIB was 15.53 mg/ml and 36.76 mg/100 ml, respectively.

(mg /100 ml)

(g /100 ml)

Fig. 4.22: Desirability chart of the optimized factors i.e. sucrose, yeast extract and FeSO4 showing their predicted values for the production of ergot alkaloids by Penicillium commune

(mg /100 ml)

(g /100 ml)

Fig. 4.23. Desirability chart of the optimized factors i.e. sucrose, yeast extract and FeSO4 showing their predicted values for the production of ergot alkaloids by Penicillium sp. IIB

4.3.2.3. Analysis of Variance

The adequacy of the model was checked using ANOVA which was tested using Fisher’s statistical analysis as presented in Table 4.39 and 4.40. The effect of individually selected variables such as sucrose, yeast extract and FeSO4, and their interactions on the yield of ergot alkaloids were analyzed through ANOVA. It was investigated that sucrose remarkably influenced the production of ergot alkaloids in culture liquid filtrate extracts with a value of 15.34 mg/100 ml. Lowest amount of ergot alkaloids yield was obtained from the culture liquid filtrate extracts of Penicillium commune after the addition of yeast extract (1.72 mg/ml) in the fermentation medium. Among the

93 combined interaction effect of these three significant variables such as sucrose-yeast extract, sucrose- FeSO4 and yeast extract- FeSO4, the combination of yeast extract-FeSO4 interaction significantly influenced the production of ergot alkaloids in culture liquid filtrate extracts with a value of 13.74 mg/100 ml. Lowest yield of ergot alkaloids (0.40 mg/ml) was obtained from the combined interaction effect of “Sucrose-FeSO4” from culture liquid filtrate extracts of Penicillium commune.

Table 4.39: Analysis of variance for alkaloids production by Penicillium commune using BBD Variable Sum of Degree of Means F-Value p-Value t-Value Square Freedom Square Intercept 33.21 1 33.21 30.25 0.01 5.50 Sucrose 10.21 1 10.21 9.30 0.06 -3.05 Sucrose2 15.34* 1 15.34* 13.97* 0.03* 3.74* Yeast Extract 0.14 1 0.14 0.12 0.75 -0.35 Yeast Extract2 1.72 1 1.72 1.57 0.31 1.25 FeSO4 4.44 1 4.44 4.04 0.14 -2.01 FeSO4 2 2.61 1 2.61 2.38 0.22 1.54 Sucrose, Yeast 7.91 1 7.91 7.19 0.075 -2.68 Extract Sucrose, 0.40 1 0.40 0.37 0.59 -0.60 FeSO4 Yeast Extract, 13.74* 1 13.74* 12.51* 0.04* 3.54* FeSO4 Error 3.29 3 1.09761

Table 4.40: Analysis of variance for ergot alkaloid production by Penicillium sp. IIB using BBD Variable Sum of Degree of Means F-Value p-Value t-Value Square Freedom Square Intercept 176.25 1 176.25 157.48 0.001 -12.55 Sucrose 113.43 1 113.43 101.35 0.002 10.07 Sucrose2 117.26 1 117.26 104.77 0.002 -10.24 Yeast Extract 290.84 1 290.84 259.87 0.000 16.12 Yeast Extract2 309.56 1 309.56 276.59 0.000 -16.63 FeSO4 74.72 1 74.72 66.77 0.004 8.17 FeSO4 2 53.49 1 53.49 47.79 0.006 -6.91 Sucrose, Yeast 2.25 1 2.25 2.01 0.251 -1.42 Extract Sucrose, 0.04 1 0.04 0.04 0.862 -0.18 FeSO4 Yeast Extract, 23.52 1 23.52 21.01 0.019 -4.58 FeSO4 Error 3.35 3 3.35

The Table 4.40 indicates the effect of individually selected variables such as sucrose, yeast extract and FeSO4, and their combined interaction effect on the yield of ergot alkaloids were analyzed through analysis of variance. It was investigated that yeast extract remarkably influenced the production of ergot alkaloids in culture liquid filtrate extracts of Penicillium sp. IIB with a value of 290.80 mg/100 ml. Lowest amount of ergot alkaloids yield was obtained from the culture liquid filtrate extracts of Penicillium sp. IIB after the addition of FeSO4 (53.49 mg/ml) in the fermentation medium. Among the combined interaction effect of these three significant variables such as sucrose-yeast extract, sucrose- FeSO4 and yeast extract-FeSO4, the combination of yeast extract-FeSO4 interaction was significantly influencing the production of ergot alkaloids in culture liquid filtrate extracts with a value of 23.52 mg/100 ml. Lowest yield of ergot alkaloids

0.04 mg/ml was obtained from the combined interaction effect of “Sucrose-FeSO4” from Penicillium sp. IIB.

The R2 value (multiple correlation coefficients) was also calculated which was closer to 1 (0.97 for Penicillium commune and 0.99 for Penicillium sp. IIB respectively) described better correlation between the observed and predicted responses of ergot alkaloids production from Culture liquid filtrate extracts of both species. It was also indicated that the experimental design of BBD was highly reliable to optimize the significant variables. The p-values described the significance of the coefficients and also it helped in the understanding of the pattern of mutual/combined interaction effects between the variables.

The results obtained from the BBD model were put in a second order polynomial equation to explain the dependence of total ergot alkaloid production from the Culture liquid filtrate extracts of Penicillium commune and Penicillium sp. IIB on the fermentation medium components. The final shape of the equation achieved after putting the all values of ergot alkaloids yield for both of the species was described as follows:

Penicillium commune

Y=22.6674 -0.3443 x1 – 0.0425 x2 -78.5246 x3 + 0.008 x12 +0.003 x22 +427.75 x32 – 0.0046 x1x2 -0.3542 x1x3 + 2.1806 x2 x3

Penicillium sp. IIB

Y= -121.244 -19.656 x1 + 3.148 x2 + 55.350 x3 – 1.791 x12 - 0.02 x22 + 55.350 x32 – 0.019 x1x2 -0.100 x1x3 + 0.243 x2 x3

Where Y is the predicted yield of ergot alkaloids or response (total production), X1 , X2,

X3 are the coded values of sucrose, yeast extract and FeSO4 salt respectively.

The fit responses for the above regression model determined the yield of ergot alkaloids obtained from Penicillium commune and Penicillium sp. IIB using BBD experimental model. The combined interaction effects of three significant variables such as sucrose- yeast extract, sucrose- FeSO4 and yeast extract-FeSO4 are described in Figs. 4.24, 4.25, 4.26, 4.27, 4.28, 4.29. Three dimensional (3D) graphs were generated for the pair wise

96 combination of the three selected factors for total ergot alkaloids production. These graphs indicate the roles played by these factors in the final yield of total ergot alkaloids produced by both species. The dome shape curves shown by these graphs reflect the specified combination of interactive variables which enhanced the production of ergot alkaloids in culture liquid filtrate extracts.

(mg /100 ml)

(g /100 ml)

Fig. 4.24: Response surface graph showing combined interaction effect of sucrose and yeast extract on ergot alkaloid production by Penicillium commune

The interaction effect of sucrose and yeast extract on the production of ergot alkaloids is described in the Figure 4.24. It was observed that the interaction of these two factors had a less significant impact on the yield of ergot alkaloids. As the sucrose concentration increased in the fermentation medium, a positive impact on yield of ergot alkaloids was observed but with the increase in yeast extract concentration, a slight decrease in ergot alkaloids production was seen.

(mg /100 ml)

(g /100 ml)

Fig. 4.25: Response surface graph showing combined interaction effect of sucrose and FeSO4 on ergot alkaloids production by Penicillium commune

The combined interaction effect of sucrose and FeSO4 on the yield of ergot alkaloids is described in the Figure 4.25. It was observed that the interaction of these two factors had not been very significant impact on the yield of ergot alkaloids. As the sucrose concentration increased in the fermentation medium, a positive impact on yield of ergot

alkaloids was observed but with the increase in FeSO4 concentration, a sudden decrease in ergot alkaloids production was observed.

(mg /100 ml)

(g /100 ml)

Fig. 4.26: Response surface graph showing combined interaction effect of Yeast extract and FeSO4 on ergot alkaloids production by Penicillium commune

Figure 4.26 described the interaction effect of yeast extract and FeSO4 on the yield of ergot alkaloids. A cap pointed in upward direction was seen in the graph which

showed the significance of the presence of yeast extract and FeSO4 in the fermentation

medium. As the concentrations of yeast extract and FeSO4 were increased in the medium, their presence triggered the production of ergot alkaloids by Penicillium commune and a highest yield (15.64 mg/100 ml) was recorded in the fermentation medium.

100

(mg /100 ml)

(g /100 ml)

Fig. 4.27: Response surface graph showing combined interaction effect of sucrose and yeast extract on ergot alkaloid production by Penicillium sp. IIB

The interaction effect of sucrose and yeast extract on the yield of ergot alkaloids is described in the Figure 4.27. A dome shaped cap was seen in the graph which showed the significance of the presence of sucrose and yeast extract in the fermentation medium both at one time. As the concentration of sucrose and yeast extract was increased, they enhanced the production of ergot alkaloids by Penicillium sp. IIB and a highest yield of 31 mg/100 ml was estimated in the fermentation medium.

101

(mg /100 ml)

(g /100 ml)

Fig. 4.28: Response surface graph showing combined interaction effect of sucrose and FeSO4 on ergot alkaloid production by Penicillium sp. IIB

The interaction effect of sucrose and FeSO4 on the yield of ergot alkaloids is described in the Figure 4.28. Again a dome shaped structure was observed in the graph which showed that this combination was proved to be highly significant for the enhanced yield of ergot alkaloids by Penicillium sp. IIB.

102

(mg /100 ml)

(g /100 ml)

Fig. 4.29: Response surface graph showing combined interaction effect of Yeast extract and FeSO4 on ergot alkaloid production by Penicillium sp. IIB

The positive impact of the combination of yeast extract and FeSO4 in fermentation medium was also seen as described in the Figure 4.29. It was observed that the

interaction of yeast extract and FeSO4 was proved to be the best combination for the maximum production of ergot alkaloids. A significant increase in the yield upto 35

mg/100 ml of ergot alkaloids was estimated when 0.11 g/100 ml of FeSO4 and 39 g/100 ml of yeast extract was added in the fermentation medium.

103

SECTION-III: STRAIN IMPROVEMENT

4.4. Strain Improvement

One of the major issues in production of alkaloids at commercial level is the cost of the process. The cost can be reduced by improving the yield through optimization of culture conditions as well as by improving the wild strains. The wild strains of Penicillium commune and Penicillium sp. IIB were subjected to mutagenesis by physical and chemical mutagens such as UV irradiation and ethyl methane sulfonate (EMS) respectively, to improve the efficiency of these strains for the production of ergot alkaloids.

4.4.1. Mutagenesis by UV irradiations

Wild strains of Penicillium commune and Penicillium sp. IIB were exposed to UV irradiations for different time duration such as 15, 30, 45, 60, 75, 90, 105, 120, 135, and 150 min. After exposure to UV irradiations, survival rate (percentage) of colonies was calculated by comparing the developed colonies of mutated strains with colonies of wild strain as presented in the Table 4.41.

It was observed that the survival rate was decreased with the increase in exposure time. The minimal survival rate was observed as 5.7% and 2.04% of colonies of Penicillium commune and Penicillium sp. IIB, respectively, after 150 min of exposure under UV light. After exposure of 135 and 150 min, the mutated strains of Penicillium commune and Penicillium sp. IIB produced/developed five and four colonies respectively (Plate 1 and 2). These colonies were streaked on MEA medium slants and stored at 4°C after allotting them specified numbers.

104

Table 4.41. Survival rate of colonies of UV mutated strains of Penicillium commune and Penicillium sp. IIB UV exposure Penicillium commune Penicillium sp. IIB time (min) No. of colonies Survival rate No. of colonies Survival rate (%) (%) 0 35 100 49 100 15 31 89.5 44 89.7 30 29 82.8 41 83.6 45 28 80 37 75.5 60 25 71.4 31 63.2 75 18 51.4 28 57.1 90 10 28.5 20 40.8 105 6 17.1 14 28.5 120 5 14.2 7 14.2 135 3 8.5 3 6.12 150 2 5.7 1 2.04 Survival rate was calculated as: Survival rate (%) = 100 X (No. of mutated colonies/No. of wild colonies)

105

Plate 1: Effect of UV mutagen on the development of colonies of Penicillium commune (Magnification, 1X)

106

Plate 2: Effect of UV mutagen on the development of colonies of Penicillium sp. IIB

107

4.4.2. Mutagenesis by EMS Cell suspensions of wild strains of Penicillium commune and Penicillium sp. Strain IIB were treated with EMS for different time durations such as 10, 15, 20, 25 and 30 min. It was observed that the survival rate decreased readily and became 0% after exposure of both fungal strains in EMS for 30 min (Table 4.42). Minimum survival rate (11.1%) was achieved after 25 min of exposure in EMS of Penicillium commune that expressed 3 colonies. A survival rate of 3.2 % was observed for the colonies of Penicillium sp. IIB after 25 min of EMS treatment expressing only one colony (Plate 3 and 4).

Table 4.42. Survival rate of colonies of EMS mutated strains of Penicillium commune and Penicillium sp. IIB UV exposure Penicillium commune Penicillium sp. IIB time (min) No. of colonies Survival rate No. of colonies Survival rate (%) (%) 0 27 100 31 100 10 24 88.8 25 80.6 15 18 66.6 14 45.1 20 10 37.0 7 22.5 25 3 11.1 1 3.2 30 0 0 0 0 Survival rate was calculated as: Survival rate (%) = 100 X (No. of mutated colonies/No. of wild colonies)

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Plate 3: Effect of EMS mutagen on the development of colonies of Penicillium commune

109

Plate 4: Effect of EMS mutagen on the development of colonies of Penicillium sp. IIB

110

4.5. Production of Ergot Alkaloids by Mutated Strains 4.5.1. Screening of mutated strains of Penicillium commune and Penicillium sp. IIB for ergot alkaloids production

Mutated strains of Penicillium commune and Penicillium sp. IIB, treated with UV irradiation and EMS mutagens were screened for their ability to produce ergot alkaloids from culture liquid (extracellular) and mycelial (intracellular) filtrate extracts. The ergot alkaloids yield obtained from mutated strains was also compared with the yield of wild strains of both fungal species. 4.5.2. Screening of UV mutants Mutated strains of Penicillium commune (PCUV-1, PCUV-2, PCUV-3, PCUV-4, and PCUV-5) and Penicillium sp. IIB (PUV-1, PUV-2, PUV-3 and PUV-4) were screened for their ability to produce ergot alkaloids production. The ergot alkaloids yield obtained from culture liquid and Mycelial filtrate extracts of mutant strains was compared with the ergot alkaloids produced by wild strains of Penicillium commune and Penicillium sp. IIB as described in the Table 4.43. Table 4.43. Screening of UV mutant and comparison of ergot alkaloids production with wild strains UV treated CLFE MFE UV treated CLFE MFE strain of (mg/ml) (mg/ml) strain of (mg/ml) (mg/ml) Penicillium Penicillium commune sp. IIB PCUV-1 1.46±0.01 1.09±0.02 PUV-1 1.36±0.02 0.98±0.01 PCUV-2 1.58±0.02 1.73±0.03 PUV-2 1.67±0.01 1.32±0.02 PCUV-3 2.16±0.05 1.55±0.01 PUV-3 1.99±0.02 1.33±0.01 PCUV-4 4.36±0.01* 1.98±0.03* PUV-4 3.12±0.005* 1.42±0.01* PCUV-5 3.78±0.02 1.23±0.01 Wild 2 2.47±0.02 1.10±0.02 Wild 1 2.97±0.03 1.56±0.01 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Highest ergot alkaloids yield was achieved from culture liquid and mycelial filtrate extracts of PCUV-4 (4.36 mg/ml) and PUV-4 (3.12 mg/ml) mutants of Penicillium commune and Penicillium sp. IIB. Lowest yield was obtained from mutants PCUV-1 and PUV-1 (1.46 mg/ml and 1.36 mg/ml) from culture liquid filtrate extracts. Mycelial

111 growth was also monitored as presented in the Fig. 4.30. Similarly, maximum ergot alkaloids yield was obtained from mycelial filtrate extracts of Penicillium commune (1.98 mg/ml) and Penicillium sp. IIB (1.42 mg/ml). The ergot alkaloids yield of mutant strains was compared with the yield obtained from wild strains and it was determined that UV irradiation significantly influenced the production of ergot alkaloids both in culture liquid and mycelial filtrate extracts. The Fig. 4.30 and Table 4.43 show the maximum growth of mycelium and production of ergot alkaloids by PCUV-4 and PUV-4 strains of Penicillium commune and Penicillium sp. IIB respectively.

3.5 y = 0.0882x + 1.7255 3 R² = 0.3321

2.5

1.5

Mycelial dry 1 wts

Growthof mycelium (g/100 ml) 0.5

0 PCUV1 PCUV2 PCUV3 PCUV4 PCUV5 PUV1 PUV2 PUV3 PUV4 Wild 1 Wild 2 Mutated and Wild strains of Penicillium commune and Penicillium sp. IIB

Fig. 4.30. Comparison of mycelial growth of wild and UV mutated strains of Penicillium commune and Penicillium sp. IIB

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4.5.3. Screening of EMS mutants

The EMS mutant strains of Penicillium commune (PCEMS-1, PCEMS-2 and PCEMS-3) and Penicillium sp. IIB (PEMS-1) were screened for their ergot alkaloids productivity. The ergot alkaloids yield obtained from culture liquid and mycelial filtrate extracts of mutant strain was compared with the ergot alkaloids produced by wild strains of Penicillium commune and Penicillium sp. IIB (Table 4.44).

Table 4.44: Screening of EMS mutants and comparison of ergot alkaloids production with wild strains EMS treated CLFE MFE EMS treated CLFE MFE strain of (mg/ml) (mg/ml) strain of (mg/ml) (mg/ml) Penicillium Penicillium commune sp. IIB PCEMS-1 1.76±0.02 1.76±0.01 PEMS-1 1.37±0.01 0.77±0.02 PCEMS-2 2.38±0.01 2.30±0.03 Wild 2 2.17±0.01* 1.65±0.03* PCEMS-3 2.98±0.005* 2.98±0.04* Wild 1 2.17±0.02 2.20±0.02 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

Considerable higher ergot alkaloids production was achieved from the culture liquid and mycelial filtrate extracts of PCEMS-3 mutated strain of Penicillium commune was more than the wild strain, while PEMS-1 mutated strain of Penicillium sp. IIB did not show a positive response towards the EMS treatment and produced a low quantity of ergot alkaloids (CLFE=1.37±0.01, MFE=0.77±0.02) as compared to wild strain (CLFE=2.17±0.01, MFE=1.65±0.03). Mycelial growth shown by mutated and wild strains of both species is presented in Fig. 4.31. Ergot alkaloids yield was also estimated in mycelial filtrate extracts of PCEMS-3 showed higher yield of ergot alkaloids as compared to the wild strain i.e. 2.98 mg/ml (Table 4.44).

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3.5 y = -0.0449x + 2.2087 3 R² = 0.0277

2.5

1.5 Mycelial Dry Wts. 1

0.5 Growthof mycelium (g/100 ml)

0 PCEMS1 PCEMS2 PCEMS3 PEMS1 Wild 1 Wild 2 Mutated and wild strains of Penicillium commune and Penicillium sp. IIB

Fig. 4.31. Comparison of mycelial growth of wild and EMS mutated strains of Penicillium commune and Penicillium sp. IIB

4.5.4. Production of ergot alkaloids in fermentor using mutated strains

Mutated strains i.e. PCUV-4 and PUV-4 and PCEMS-3 of Penicillium commune and Penicillium sp. IIB were subjected to fermentor studies using optimized M5 fermentation medium (Table 4.45). The production of ergot alkaloids was determined in culture liquid and mycelial filtrate extracts separately for the mutated strains. It was observed that mutant PCUV-4 strain of Penicillium commune showed the higher ergot alkaloids production in culture liquid (12.32 mg/L) and mycelial (intracellular 3.89 mg/ml) filtrate extracts than PUV-4 strain of Penicillium sp. IIB. Ergot alkaloids yield was also compared with the yield obtained from wild strains as described in the Table 4.45. Comparison of mycelial growth of mutated and wild strains is presented in Fig. 4.32.

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Table 4.45. Comparison of the ergot alkaloids yield of selected mutant and wild strains Strains CLFE (mg/ml) MFE (mg/ml) Penicillium commune wild 8.98±0.02 2.36±0.03 PCUV-4 mutant 12.32±0.02* 3.89±0.01* PCEMS-3 mutant 9.31±0.01 2.58±0.01 Penicillium sp. IIB wild 9.11±0.03 2.75±0.02 PUV-4 mutant 7.69±0.01 1.73±0.03 Where, CLFE=Culture liquid filtrate extract, MFE= Mycelial filtrate extract. Each value is an average of three replicates and ± indicates the standard deviation of these replicates.

18 16 y = -2.44x + 15.812 R² = 0.6209 14 12 10 8 6 Mycelial Dry Wts 4 Growthof mycelium (g/L) 2 0 P. commune PCUV-4 PCEMS-3 Penicillium sp. PUV-4 Wild IIB Selected mutant and wild strains

Fig. 4.32. Comparison of mycelial growth of wild and selected UV and EMS mutated strains of Penicillium commune and Penicillium sp. IIB

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SECTION-IV: ANALYTICAL STUDIES

4.6. Thin Layer Chromatography (TLC)

Ergot alkaloids of culture liquid and Mycelial filtrate extracts produced from the experimental runs of RSM were subjected to TLC analysis to determine the presence of expected ergot alkaloids. The thin layer chromatography (TLC) analysis of culture liquid and mycelial filtrate extracts of Penicillium commune and Penicillium sp. IIB was processed under UV irradiation (254 nm) that facilitated the identification of each isolated ergot alkaloid. The separated ergot alkaloid compounds of samples were compared with the reference ergot alkaloid salts such as dihydroergotamine methane sulfonate salt, bromocriptine mesylate, solutions of migril and methergene standard drugs.

4.6.1. Screening of mobile phase for TLC studies (Phase-I)

Table 4.46 showing the separation of compounds and the values of culture liquid and Mycelial filtrate extracts of twelve runs of Penicillium 푅푓 commune produced during RSM step. Various mobile phases were screened i.e. A, B, C, D, E, F, G and H. In this phase TLC plates revealed Van Urk reagent positive spots in mobile phase A (chloroform 80: methanol 20: ammonia soln. 0.5) and H (chloroform 120: isopropanol 30: water 20) (Table 3.10) that produced characteristic pinkish purple color spots on silica gel plates. These pinkish color spots were obtained from the culture liquid and mycelial filtrate extracts of PCCLFE1, PCMFE1 with value of 0.57 and 0.42 in mobile phase H and in PCCLFE12, PCMFE12 with value 푅푓of 0.60 and 0.56 in mobile phase A (Plate 5). The colored spots of ergot alkaloids푅푓 and their values of culture liquid and mycelial filtrate extracts of Penicillium commune and Penicillium푅푓 sp. IIB were measured with the formula given (subsection 3.10.5). The values of TLC of culture liquid and mycelial filtrate extracts of Penicillium commune푅푓 are presented in the Table 4.46. The pinkish

116 purple color indicated the presence of ergocriptine and ergotamine in the culture liquid and mycelial filtrate extracts of both strains.

Table 4.46: values of culture liquid and mycelial filtrate extracts of Penicillium commune 푹풇 Filtrate No. Mobile Phase and Value Expected Ergot A B C D E F G H Alkaloids 푹풇 PCCLFE1 0.54 0.1 0.2 0.12 0.1 0.18 0.21 0.57 Ergocriptine PCMFE1 0.4 0.3 0.1 0.15 0.05 0.12 0.15 0.42 PCCLFE2 0.1 - 0.14 - - 0.12 - - - PCMFE2 - 0.10 0.05 PCCLFE3 0.1 ------PCMFE3 0.02 PC4CLFE 0.42 0.15 0.11 0.1 0.07 0.07 0.12 0.5 Ergocriptine, PCMFE4 0.32 0.10 - 0.01 0.1 - - 0.35 Ergotamine PC5CLFE ------PCMFE5 PCCLFE6 0.56 0.12 0.1 0.1 0.13 0.19 0.25 0.56 Ergocriptine, PCMFE6 0.42 0.10 0.05 0.02 0.1 - 0.12 0.39 Ergotamine PCCLFE7 0.12 - - 0.23 - - - 0.43 - PCMFE7 0.03 0.15 0.21 PCCLFE8 0.56 0.42 0.28 0.15 0.19 0.33 0.23 0.46 Ergocriptine, PCMFE8 0.35 0.12 0.15 0.10 0.27 0.16 0.17 0.23 Ergotamine PCCLFE9 0.58 0.07 0.09 0.08 0.15 0.17 0.21 0.45 Ergocriptine PCMFE9 0.54 - 0.01 - 0.01 0.01 0.08 0.13 PCCLFE10 0.23 - 0.28 0.03 - - - 0.03 - PCMFE10 0.12 0.28 0.01 0.01 PCCLFE11 0.51 0.1 0.05 0.12 0.21 0.23 0.15 0.45 Ergocriptine PCMFE11 0.45 - - 0.01 0.04 0.21 0.13 0.32 PCCLFE12 0.60 0.24 0.13 0.15 0.21 0.21 0.1 0.50 Ergocriptine, PCMFE12 0.56 0.23 0.1 0.14 0.03 0.02 0.01 0.41 Ergotamine PC: Penicillium commune, CLFE: Culture liquid filtrate extract, MFE: Mycelial filtrate extract, No. 1, 2 3 are the sample no. of runs of RSM

Table 4.47 shows the separated compounds and values of culture liquid and mycelial filtrate extracts of twelve runs of Penicillium푅푓 sp. IIB produced during RSM studies. Various mobile phases were screened i.e. A, B, C, D, E, F, G and H. In this phase, TLC plates revealed Van Urk reagent positive spots in mobile phase A and H that produced characteristic pinkish purple color spots (ergocriptine) on silica gel plates. Maximum value was obtained from the culture liquid and mycelial filtrate extracts of PCLFE8푅푓 (0.63), PMFE8 (0.53), PCLFE12 (0.62) and PMFE12 (0.59) in mobile

117 phase A and from PCLFE9 (0.62), PMFE9 (0.56), PCLFE12 (0.64) and PMFE12 (0.61) in mobile phase H. The colored spots of ergot alkaloids and their values were measured with the formula given under subsection 3.10.5. The values푅푓 of TLC of culture liquid and mycelial filtrate extracts of Penicillium sp. IIB are푅푓 presented in Table 4.47.

Table 4.47: values of culture liquid and mycelial filtrate extracts of Penicillium sp. IIB 푹풇 Filtrate No. Mobile Phase and Value Expected Ergot A B C D E F G H Alkaloids 푹풇 PCLFE1 0.55 0.12 0.1 0.25 0.21 0.15 0.05 0.45 Ergocriptine PMFE1 0.45 0.01 0.02 0.2 0.01 0.01 0.02 0.35 PCLFE2 0.58 0.15 0.13 0.21 0.12 0.15 0.13 0.55 Ergocriptine PMFE2 0.49 0.23 0.15 0.01 0.08 0.09 0.11 0.42 PCLFE3 ------PMFE3 PCLFE4 ------PMFE4 PCLFE5 ------PMFE5 PCLFE6 0.65 0.13 0.18 0.22 0.16 0.19 0.23 0.54 Ergocriptine PMFE6 0.55 0.12 0.21 0.12 0.14 0.12 0.13 0.42 PCLFE7 ------PMFE7 PCLFE8 0.63 0.19 0.24 0.21 0.23 0.2 0.21 0.56 Ergotamine PMFE8 0.56 0.12 0.12 0.01 0.21 0.15 0.12 0.45 PCLFE9 0.51 0.15 0.2 0.26 0.24 0.25 0.29 0.62 Ergotamine PMFE9 0.49 0.10 0.12 0.13 0.23 0.14 0.12 0.56 PCLFE10 ------PMFE10 PCLFE11 ------PMFE11 PCLFE12 0.62 0.23 0.12 0.25 0.19 0.25 0.25 0.64 Ergotamine PMFE12 0.59 0.12 0.16 0.15 0.03 0.12 0.23 0.61 P: Penicillium sp. IIB, CLFE: Culture liquid filtrate extract, MFE: Mycelial filtrate extracts, No. 1, 2 3 are the sample no. of runs of RSM

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4.6.2. Selection of mobile phase for final step of TLC

Mobile phases A and H were selected from the 1st Phase of TLC analysis. These mobile phases were modified to get the proper separation of ergot alkaloid compounds present in culture liquid and mycelial filtrate extracts of Penicillium commune and Penicillium sp. IIB. Mobile phase A was modified into A1, A2, A3, A4 and A5 and mobile phase H was modified into H1 and H2 (Table 3.11). The values of Penicillium commune and

Penicillium sp. IIB are given in the Tables 4.48 and푅푓 4.49.

Table 4.48 showing the highest was obtained from culture liquid and mycelial filtrate extracts of PCCLFE9 and PCMFE9푅푓 with values of 0.85 and 0.66 in mobile phase A1 (chloroform 80: methanol 20: ammonia soln. 0.25). Maximum value was obtained in H2 (chloroform 125: isopropanol 25: water 15) mobile phase푅푓 with the culture liquid and mycelial filtrate extracts of PCCLFE9 (0.96) and PCMFE9 (0.85) samples of Penicillium commune (Plate 6).

Table 4.48: values of selected culture liquid and mycelial filtrate extracts of Penicillium commune 푹풇 Extract No. Mobile Phase and Value Ergot Alkaloid A1 A2 A3 A4 A5 H1 H2 Compound 푹풇 PCCLFE1 0.70 0.66 0.31 0.54 0.50 0.90 0.80 Ergocriptine PCMFE1 0.64 0.56 0.23 0.45 0.46 0.72 0.62 Agroclavine PCCLFE4 0.63 0.54 0.45 0.56 0.65 0.75 0.85 Ergocriptine PCMFE4 0.56 0.55 0.35 0.51 0.62 0.69 0.74 PCCLFE6 0.65 0.60 0.52 0.41 0.55 0.86 0.95 Ergotamine PCMFE6 0.53 0.56 0.56 0.33 0.52 0.72 0.63 PCCLFE9 0.85 0.65 0.70 0.45 0.60 0.75 0.96 Ergocriptine PCMFE9 0.66 0.66 0.69 0.55 0.55 0.63 0.85 PCCLFE11 0.61 0.59 0.60 0.55 0.50 0.65 0.66 Ergotamine PCMFE11 0.52 0.51 0.43 0.45 0.48 0.55 0.41 PCCLFE12 0.58 0.50 0.45 0.63 0.65 0.65 0.68 Ergocriptine PCMFE12 0.42 0.43 0.43 0.47 0.43 0.52 0.59 Agroclavine PC: Penicillium commune, S: Supernatant/Culture liquid filtrate extract, M: Mycelial filtrate extract/extract

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Table 4.49 showing the highest obtained from culture liquid and mycelial filtrate extracts of PCLFE8, PMFE8, PCLFE9푅푓 and PMFE9 with values of 0.80, 0.79, 0.90 and 0.75 in mobile phase A1 (chloroform 80: methanol 20: ammonia soln. 0.25). Maximum value was obtained in mobile phase H2 (chloroform 125: isopropanol 25: water 푅푓 15) mobile phase with the culture liquid and mycelial filtrate extracts of PCLFE8 (0.90), PMFE8 (0.89), PMFE9 (0.95) and PMFE9 (0.96) samples of Penicillium sp.IIB (Plate 7).

Table 4.49: values of selected culture liquid and mycelial filtrate extracts of Penicillium sp. IIB 푹풇 Extract No. Mobile Phase and Value Ergot Alkaloid A1 A2 A3 A4 A5 H1 H2 Compound 푹풇 PCLFE1 0.70 0.58 0.65 0.59 0.50 0.70 0.75 Ergotamine PMFE1 0.65 0.46 0.52 0.43 0.35 0.66 0.71 Agroclavine PCLFE2 0.78 0.56 0.54 0.45 0.56 0.65 0.85 Ergocriptine PMFE2 0.62 0.46 0.55 0.41 0.46 0.61 0.82 Agroclavine PCLFE6 0.70 0.65 0.60 0.55 0.56 0.60 0.80 Ergotamine PMFE6 0.62 0.59 0.56 0.43 0.52 0.61 0.75 PCLFE8 0.80 0.71 0.65 0.55 0.60 0.71 0.90 Ergocriptine PMFE8 0.79 0.62 0.42 0.32 0.45 0.42 0.89 PCLFE9 0.90 0.65 0.32 0.40 0.50 0.65 0.95 Ergotamine PMFE9 0.75 0.59 0.12 0.33 0.42 0.55 0.96 PCLFE12 0.75 0.72 0.65 0.55 0.56 0.65 0.85 Ergotamine PMFE12 0.71 0.78 0.69 0.51 0.42 0.56 0.75 P: Penicillium sp. IIB, S: Supernatant/Culture liquid filtrate extract, M: Mycelial filtrate extract/extract

TLC analysis of culture liquid and mycelial extracts of Penicillium commune and Penicillium sp. IIB revealed 3 Van Urk reagent’s spots produced a pinkish and purplish blue color on silica gel plate. The major spot was identified as ergotamine (Rf=0.80-0.90) and other spots were identified as ergocriptine (Rf=0.70) and agroclavine (Rf=0.40-50).

120

Plate 5: Screening of mobile phase for TLC analysis of Penicillium commune and Penicillium sp. IIB filtrates.

121

Plate 6: Selection of suitable mobile phase for TLC analysis of Penicillium commune culture liquid filtrate extracts in phase 2 using mobile phases A1 and H2 (S: Standard Salt spot, PC: Penicillium commune, 1, 4 and 8: Filtrate No., M: Mycelial filtrate extract).

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Plate 7: TLC analysis of Penicillium sp. IIB extracts in phase 2 mobile phases A1 and H2 (S: Standard Salt spot, P: Penicillium sp. IIB, 1, 4, 9 and 12: Filtrate No., M: Mycelial filtrate extract).

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4.7. High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography (HPLC) analysis involved the use of C18 column with mobile phase H containing chloroform and isopropanol in 4:1 ratio (v/v). The effect of the composition of mobile phase on the retention times of culture liquid and mycelial filtrate extracts of Penicillium commune and Penicillium sp. IIB was investigated for the determination of ergot alkaloid compounds. The peak areas and the concentration of ergot alkaloid compounds were calculated in the sample filtrates. The retention time, peak areas and concentration of ergot alkaloid compounds present in the sample filtrate extracts were compared with the retention time of standard salts such as dihydroergotamine methane sulfonate (DMS), bromocriptine mesylate (BCM) and ergotamine salt. The best response for all the substances was obtained with 20 µl (1 mg/ml) solution of each reference salt and filtrate at 280 nm. The samples were analyzed under the same conditions to identify the ergot alkaloids. Retention times of reference salts are given in the Table 4.50:

Table 4.50: Retention times of reference salts of ergot alkaloids

Reference Salt Retention Time (min) Ergotamine Salt 3.69 DMS Salt 4.01 BCM Salt 5.89 Mixture of DMS and BCM and Ergotamine 3.64,4.62,6.35 Salts

The retention times and the peak areas of reference standards were compared with the HPLC analysis of the samples of culture liquid and mycelial filtrate extracts of Penicillium commune and Penicillium sp. IIB as described in Table 4.51 and 4.52.

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Table 4.51: Retention time of culture liquid and mycelial filtrate extracts of Penicillium commune

Sr. No. Penicillium commune Sample No. Retention Time Concentration Ergot Alkaloid (min) of Compound Compound (mg/ml) Culture liquid filtrate extract (CLFE) 1. PCCLFE1 3.71 15.92 Ergotamine 2. PCCLFE2 3.68, 4.05 14.92 Ergocriptine 3. PCCLFE4 3.69 14.91 Ergocriptine 4. PCCLFE8 3.63 5.296 Ergocriptine 5. PCCLFE9 3.73, 4.04 18* Ergotamine 6. PCCLFE12 3.61 12.97 Ergotamine Mycelial filtrate extract (MFE) 7. PCMFE1 3.69, 4.06 14.91 Ergocriptine 8. PCMFE2 3.71, 4.07 16.25 Ergotamine 9. PCMFE4 3.74, 4.89 16.49 Ergotamine 10. PCMFE8 3.69, 4.05 14.91 Ergocriptine 11. PCMFE9 3.71 16.25 Ergotamine 12. PCMFE12 3.83 19.48* Ergotamine

Where PCS stands for: Penicillium commune supernatant extract (Culture liquid filtrate extract) and PCM stands for Penicillium commune mycelial extract (Mycelial filtrate extract).

Table 4.51 indicates the appearance of a peak that was obtained after 3.73 min of elution of culture liquid filtrate extract (CLFE) no. PCCLFE9 of Penicillium commune with a considerable concentration of ergot alkaloids i.e. 18 mg/ml. The peak of this filtrate was alike with the reference salt that indicates the presence of ergotamine in the PC9S filtrate. Table 4.51 also describes the appearance of a blunt peak after 3.83 min of elution of mycelial filtrate extract (MFE) no. PCMFE12 with a significant concentration of ergot alkaloids i.e. 19.48 mg/ml also indicates the presence of ergotamine in the filtrate.

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Table 4.52: Retention time of culture liquid and mycelial filtrate extracts of Penicillium sp. IIB

Sr. No. Penicillium sp. IIB Sample No. Retention Time Concentration Ergot Alkaloid (min) of Compound Compound (mg/ml) Culture liquid filtrate extract (CLFE) 1. PCLFE1 3.69 14.91 Ergotamine 2. PCLFE2 3.60 19.53 Ergotamine 3. PCLFE6 3.70 14.92 Ergocriptine 4. PCLFE8 3.63 14.49 Ergotamine 5. PCLFE9 3.61 20.12* Ergotamine 6. PCLFE12 3.62 19.95 Ergotamine Mycelial filtrate extract (MFE) 7. PMFE1 3.62 14.52 Ergotamine 8. PMFE2 3.63 5.296 Ergotamine 9. PMFE6 3.59 19.97 Ergotamine 10. PMFE8 3.60 19.95 Ergotamine 11. PMFE9 3.60 20.11* Ergotamine 12. PMFE12 3.64 19.98 Ergotamine

Where PS stands for: Penicillium sp. IIB supernatant extract (Culture liquid filtrate extract) and PM stands for Penicillium sp. IIB mycelial extract (Mycelial filtrate extract).

Table 4.52 indicates processing of culture liquid filtrate no. PCCLFE9 of Penicillium commune through HPLC produced maximum peak after 3.61 min with ergot alkaloids yield of 20.12 mg/ml. The peak of this filtrate was alike with the reference salt that indicated the presence of ergocriptine and ergotamine in the PMFE9 filtrate. Table 4.52 also describes the appearance of a longitudinal peak after 3.60 min of elution of mycelial filtrate extract (MFE) no. PMFE9 with a significant concentration of ergot alkaloids i.e. 20.11 mg/ml also indicates the presence of ergotamine in the specific filtrate.

126

Retention times of the reference standards were compared with the retention times of the samples of culture liquid and mycelial filtrate extracts of Penicillium commune and Penicillium sp. IIB. Analysis through HPLC identified the specified ergot alkaloid compounds in the culture liquid and mycelial filtrate extracts and confirmed the presence of ergocriptine and ergotamine in the extracts of Penicillium commune and Penicillium sp. IIB. Most of the filtrates were eluted after 3.60 min that confirmed the presence of ergocriptine and ergotamine in the filtrates (Figs. 4.33-4.54).

127

Fig. 4.33: Chromatogram of ergotamine standard (1 mg/ml) with retention time of 3.69 min.

128

Fig. 4.34: Chromatogram of Dihydroergotamine Methane Sulfonate Salt (standard salt 2) (1 mg/ml) with retention time of 4.01 min.

129

Fig. 4.35: Chromatogram of Bromocriptine Mesylate Salt (standard 3) (1mg/ml) with retention times of 3.66 min and 5.89 min.

130

Fig. 4.36: Chromatogram of Mixture of Ergotamine (1 mg/ml), DMS Salt (1mg/ml) and BCM Salt (1mg/ml) with retention times of 3.64, 4.89 and 6.36 min.

131

Chromatograms of culture liquid filtrate extracts of Penicillium commune

Fig. 4.37: Chromatogram of Penicillium commune culture liquid filtrate extract No. 1 (1mg/ml) with retention time of 3.71 min.

132

Fig. 4.38: Chromatogram of Penicillium commune culture liquid filtrate extract No. 2 (1mg/ml) with retention time of 3.68 min.

133

Fig. 4.39: Chromatogram of Penicillium commune culture liquid filtrate extract No. 9 (1mg/ml) with retention times of 3.73 min.

134

Fig. 4.40: Chromatogram of Penicillium commune culture liquid filtrate extract No. 12 (1mg/ml) with retention time of 3.61 min.

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Chromatograms of mycelial filtrate extracts of Penicillium commune

Fig. 4.41: Chromatogram of Penicillium commune mycelial filtrate extract No. 1 (1mg/ml) with retention times of 3.69 min.

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Fig. 4.42: Chromatogram of Penicillium commune mycelial filtrate extract No. 2 (1mg/ml) with retention times of 3.71 min.

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Fig. 4.43: Chromatogram of Penicillium commune mycelial filtrate extract No. 4 (1mg/ml) with retention times of 3.74 min.

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Fig. 4.44: Chromatogram of Penicillium commune mycelial filtrate extract No. 8 (1mg/ml) with retention time of 3.60 min.

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Chromatograms of culture liquid filtrate extracts of Penicillium sp. IIB

Fig. 4.45: Chromatogram of Penicillium sp. IIB culture liquid filtrate extract No. 1 (1mg/ml) with retention time of 3.69 min.

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Fig. 4.46: Chromatogram of Penicillium sp. IIB culture liquid filtrate extract No. 2 (1mg/ml) with retention time of 3.60 min.

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Fig. 4.47: Chromatogram of Penicillium sp. IIB culture liquid filtrate extract No. 8 (1mg/ml) with retention time of 3.63 min.

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Fig. 4.48: Chromatogram of Penicillium sp. IIB culture liquid filtrate extract No. 9 (1mg/ml) with retention time of 3.61 min.

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Fig. 4.49: Chromatogram of Penicillium sp. IIB culture liquid filtrate extract No. 12 (1mg/ml) with retention time of 3.62 min.

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Chromatograms of mycelial filtrate extracts of Penicillium sp. IIB

Fig. 4.50: Chromatogram of Penicillium sp. IIB mycelial filtrate extract No. 1 (1mg/ml) with retention time of 3.62 min.

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Fig. 4.51: Chromatogram of Penicillium sp. IIB mycelial filtrate extract No. 2 (1mg/ml) with retention time of 3.63 min.

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Fig. 4.52: Chromatogram of Penicillium sp. IIB mycelial filtrate extract No. 6 (1mg/ml) with retention time of 3.59 min.

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Fig. 4.53: Chromatogram of Penicillium sp. IIB mycelial filtrate extract No. 8 (1mg/ml) with retention time of 3.60 min.

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Fig. 4.54: Chromatogram of Penicillium sp. IIB mycelial filtrate extract No. 9 (1mg/ml) with retention time of 3.60 min.

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Chapter 5 DISCUSSION ______

Fungal organisms have been recognized as a warehouse of novel secondary metabolites and many of them show significant biological activities. These fungal organisms are ubiquitous in nature and are a potential source to meet the demand of new drugs in pharmaceutical industry (Devi and Prabakaran, 2014). Typical alkaloids are derived from plants and they contain one or more nitrogen atom in their structures and are called as amino-alkaloids. These are also known as protoalkaloids. True alkaloids such as i.e. lysergic acid alkaloids and gliotoxins are rarely present in lower plants but can be found in various species of fungi (Roberts and Wink, 1998). Ergot alkaloids belong to complex family of mycotoxins which are derived from prenylated tryptophan in several species of fungi and these are playing a very important role to cure many human toxicoses. Ergot alkaloids produced by several fungi, such as Claviceps, Epichole, Neotyphodium, Aspergillus and Penicillium, mostly representing two different orders i.e. Hypocreales and Eurotiales. These species can produce a wide variety of significant ergot alkaloids (Clay and Shardl, 2002). Pharmacologically, ergot alkaloids are very toxic but in some cases they act as important drugs to cure various ailments because of their vasoconstrictive impacts on human being. Various ergot alkaloids such as ergotamine, ergocriptine, ergometrine and agroclavine are prescribed to control acute headaches, induce labor contractions, terminate early pregnancies, inhibit mammary tumors and can stop postpartum bleeding (Shelby and Kelley, 1990).

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Section-I: Optimization of Culture Conditions by OFAT Method Among many species, Penicillium commune and Penicillium sp. IIB were screened as the potential ergot alkaloid producers through surface culture fermentation technique (Table 4.1). It was investigated that genus Penicillium is the targeted organism now for microbiological research purposes and many compounds of ergot alkaloids with cytotoxic and antimicrobial effects have been isolated from the mycelium of this genus (Rukachaisirikul et al., 2007; Ge et al., 2008). Kozlovsky et al. (2013) screened many species of genus Penicillium for their ability to produce ergot alkaloids and found that Penicillium palitans and Penicillium citrinum have the ability to produce fumigaclavin and agroclavin alkaloids, respectively (Antipova et al., 2011). Abulhamd (2009) also reported that Penicillium expansum is the most common ascomycetous fungus which is involved in the postharvest decay of many fruits leading to drastic economic losses in the fruit industry but it also has the ability to produce some secondary metabolites such as patulin which is a mycotoxin and can cause severe diseases on animals. Different culture conditions were optimized during the present study to investigate the effect of various organic and inorganic compounds on the ergot alkaloids yield. Reshetilova and Kozlovsky (1990) described that production of ergot alkaloids depends on many factors in which, composition of culture medium, pH, temperature and other organic and inorganic compounds play significant role to enhance the yield of the product in artificial media. Deep knowledge about the regulation of ergot alkaloids synthesis and metabolism of the producer is a prerequisite for biotechnological productions of ergot alkaloids. Some products of primary metabolism serve in the secondary metabolism and act as a precursor for the growth of the organism in a biotechnological process. However, the organic and inorganic compounds exhibit some regulatory effects or behave as structural elements that influence the biosynthesis of secondary metabolites. In the present work, carbon sources significantly influenced the yield of ergot alkaloids in fermentation medium and optimization of sucrose concentration in the fermentation medium played a significant improvement in the yield of ergot alkaloids (Table 4.2) as

151 described by Kren et al. (1984), they identified sucrose as the best substrate for the production of ergot alkaloids. They used monosaccharides and oligosaccharides as carbon sources such as glucose, sucrose, maltose and polyols (mannitol and sorbitol) and investigated their impact on the yield of ergot alkaloids. They described that sucrose was readily metabolized in the fermentation medium in the beginning of cultivation of Claviceps purpurea for the production of ergot alkaloids but glucose as a sole was not suitable because it promoted the mycelial growth and inhibited the production of ergot alkaloids (Kren et al., 1987). The free addition of glucose in the growth medium of Claviceps fusiformis supported the extracellular release of polysaccharides which complicated the oxygen transport mechanism in the fermentation medium. Likewise, Aimici et al. (1967) cultivated Claviceps species under higher concentrations of cell sap. They determined that high concentrations of sucrose in the fermentation medium favor the yield of ergot alkaloids. They also investigated that 30-35% sucrose concentrations proved to be the best to get the maximum yield of ergot alkaloids. Penicillium commune and Penicillium sp. IIB were able to utilize a wide range of nitrogen sources but the most suitable nitrogen source for the maximum yield of ergot alkaloids (Table 4.4) was found to be yeast extract for both species. Moussa (2003) used various nitrogen sources in his experiments and described that maximum mycelial growth and a low yield of ergot alkaloid was achieved by the addition of potassium nitrate and sodium nitrate in the fermentation medium. Moussa (2003) also described that addition of ammonium chloride greatly influenced the mycelial growth and ergot alkaloids yield (1.20 mg/l). Similar results were reported by Taber and Vining (1958) by culturing Claviceps purpurea in fermentation medium. They investigated that nitrates partially influenced the production of ergot alkaloids in the experiments and suggested that presence of nitrogen in the fermentation medium was a significant factor for the mycelial growth and ergot alkaloids production. Mycelial growth and ergot alkaloids yield from Penicillium commune and Penicillium sp.

IIB was significantly influenced by the addition of KH2PO4 in the fermentation medium

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(Table 4.10). Moussa (2003) and Gram et al. (1980) have reported the similar results of the effect of phosphates on the growth of mycelium and biosynthesis of ergot alkaloids by Claviceps species. They described the merits and demerits of the addition of phosphates in the fermentation medium. They reported that high phosphate concentration in the fermentation medium triggers the vegetative growth of mycelium but inhibits the biosynthesis of ergot alkaloids. In the present investigation, maximum production of ergot alkaloids was observed at 2 g tryptophan concentration level in the fermentation medium. It was observed that high concentration of tryptophan inhibited the mycelial growth and ergot alkaloids yield in the fermentation medium (Table 4.12). Addition of amino acids such as tryptophan and asparagine in the fermentation medium can play a crucial role in the biosynthesis of ergot alkaloids. They can act as a precursor for the formation of ergoline ring structure. Floss and Mothes (1964) suggested that tryptophan can act as inducer for the enzymes involved in the synthesis of ergot alkaloids in nature. Krupinski et al. (1976) also worked on the induction of tryptophan at the enzymatic level for the production of ergot alkaloids and suggested that tryptophan triggered the biosynthesis of ergot alkaloids in the absence of inorganic phosphate. They reported that thitryptophan can overcome the blockage of ergot alkaloids synthesis by inorganic phosphate. They indicated that tryptophan or its derivatives can work as an inducer in the synthesis of ergot alkaloids by activating DMAT synthetase enzyme of Claviceps strain SD58 which is the first enzyme in the alkaloids synthesis pathway. These results are in the concordance with Kozlovskii et al. (2006) who described the use of various amino acids for the synthesis of ergot alkaloids and suggested that tryptophan triggered the mycelial growth and ergot alkaloids production by Penicillium citrinum strain Thom 1910. Among various amino acids, asparagine and aspartic acid have been reported to be the ample source of nitrogen for the growth of several fungi (Grover, 1964). Asparagine and aspartic acid, both are the prime contributors of transmitting mechanism in many organisms.

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It was also reported that aspartic acid such as α-keto acids acts as the intermediates in the synthesis of tricarboxylic cycle of secondary metabolite (Davis, 1955). Similar studies on the impact of tryptophan on ergot alkaloids yield have been reported by Christiane and Shu-Ming (2011) in their review in which they described that tryptophan and DMAPP are the important precursors for the formation of ergoline ring structure. The nitrogen level of the fermentation medium greatly influences the growth of mycelium and production of ergot alkaloids. Effect of different concentration levels of

NH4Cl on the mycelial growth and ergot alkaloids production was studied during the optimization studies (Table 4.18). The results are in consonance with Moussa (2003) who reported that the addition of high concentration of NH4Cl in the fermentation medium partially supported the synthesis of ergot alkaloids. Gaberc-Porekar et al. (1987) also reported that ammonia being rapidly utilized nitrogen source and it depletes from the growth medium readily until a high production of ergot alkaloids starts. The effect of different salts such as MgSO4.7H2O, FeSO4.7H2O and ZnSO4 was determined on the mycelial growth and production of ergot alkaloids during the present study. It was observed that in the presence of all these salts there was a remarkable increase in the production of ergot alkaloids as described in the Tables 4.20, 4.22 and 4.24. These ions help in the growth of fungal organism and act as cofactors to trigger and initiate metabolic processes of the organism as described by Fujiwara and Yammato (1987). The production of ergot alkaloids was strongly controlled by pH of the fermentation medium during the present study. Penicillium commune and Penicillium sp. IIB were active on different pH levels ranged from pH 3 to 8 in which pH 5 was found as optimum for the production of ergot alkaloids in culture liquid and mycelial filtrate extracts of Penicillium commune and Penicillium sp. IIB respectively (Table 4.26). Similar findings have been reported by Mizrahi and Miller (1970), Bogo et al. (2003) and Moussa (2003) for the production of ergot alkaloids from different fungal species. Incubation temperature and incubation time period significantly influenced the growth of mycelium and production of ergot alkaloids in in-vitro fungal cultures of test species. It was observed that incubation temperature of 25°C proved as the best suitable

154 temperature for mycelial growth and ergot alkaloids production (Table 4.28) as described by Socic and Garberc-Porker (1992), in which they described that 24°C to 27°C temperature range supported maximum ergot alkaloid production. Different incubation periods for the growth and production of mycelium was observed during the present study and maximum quantity of ergot alkaloids was produced after 21 days incubation of Penicillium commune and Penicillium sp. IIB (Table 4.30). It was also noted that with the increase in incubation temperature above 30°C and incubation time above 30 days slowed down the mycelial growth and ergot alkaloids yield. This may be due to the inhibition of mycelial development in the high temperature which was not suitable for the production of ergot alkaloids and growth inhibition in incubation period longer than 21 days. This may be also be due to the depletion of nutrients in fermentation medium as described by Zerdani (2004) in his experiments. Spore suspension density always plays a significant role in mycelial growth and consequently enhances the production of ergot alkaloids. Bhardwaj et al. (2012) reported in their study that inoculum size is an important factor for the growth of mycelium. High density of inoculum suspension supports the moisture contents to a significant level to obtain the maximum yield of the product. On the other hand, low inoculum density introduces a lower number of cells and hence a low yield of the product in the fermentation medium. In the present study, it was observed that with the increase in the percentage/density of the spore suspension in the fermentation medium, an increase in the growth of the mycelium and production of ergot alkaloids was observed (Table 4.32). These results are in concordance with Lee and Shuler (2000) who also reported the higher ajmalicine yield throughout the experiments with an increase in the inoculum size in the fermentation medium.

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Section-II: Response Surface Methodology Response Surface Methodology (RSM) is an empirical statistical modeling technique used for multiple regression analysis of quantitative data obtained from multi-factorial experimental design (Rao et al., 2000; Venil and Lakshmanaperumalsamy, 2009b). It has been increasingly used for different phases of screening and optimization of process parameters of fermentation technique (Prapulla et al., 1992; Mayers and Montogomery, 2002; Mao et al., 2005). It’s a powerful tool for testing multiple process variables because in this methodology fewer experimental trails are needed as compared to study of one variable/factor at a time (OFAT). The conventional method of optimization of process parameters by changing one parameter/factor at one time is very time consuming and inefficient so statistical methods like RSM are very common now a days for statistical optimization of factors in fewer experiments (Nelofar et al., 2011). The interaction between the individual variables affecting the response can also be identified and quantified by such statistical techniques. In the present study two statistical designs related to RSM were used such as Plackett- Burman Design (PBD) (screening of fermentation factors/components) and Box- Behnken Design (BBD) (optimization of selected fermentation parameters) for mycelial growth and production of ergot alkaloids by Penicillium commune and Penicillium sp. IIB. Screening of the fermentation medium components was accomplished using PBD (Table 4.35). The variation in the yield of ergot alkaloids reflected the importance of medium ingredients (variables) used in the fermentation medium to achieve the maximum yield. Sucrose, yeast extract and FeSO4 were screened as the most significant variables using PBD and it was investigated that these factors greatly influenced the yield of ergot alkaloids in the fermentation medium (Fig. 4.19). Wu et al. (2011) employed the same Plackett-Burman Design (PBD) and screened important factors/ingredients of fermentation medium for the production of fumigaclavine C and helvolic acid from endophytic Aspergillus fumigatus CY018 strain. They investigated that

156 pH, phosphate concentration and inoculums size were the three significant independent factors/variables to influence the production of fumigaclavine C and helvolic acid in the culture liquid filtrates of Aspergillus fumigatus strain CY018. In the present study it was found that ergot alkaloids yield was significantly influenced by high concentration level (+1) of sucrose and yeast extract among employed levels (-1 and +1) as in the Tables 4.37 and 4.38. With the increase in the concentrations of sucrose and yeast extract, a remarkable increase in yield of ergot alkaloids was achieved from culture liquid filtrates of Penicillium commune. Guo et al. (2010) have presented the similar results by PBD indicating that carbon and nitrogen sources were screened as important variables for the production of nisin in the fermentation medium of Lactococcus lactis subsp. lactis. Desai et al. (2006) and Sing et al. (2008) also have used PBD statistical models to screen important ingredients to get the maximum increase in the yield of nisin. Our results are also in accordance with Wu et al. (2011) who worked on the production of two active secondary metabolites and screened three key cultivation factors of fermentation medium such as pH, phosphate concentration and inoculums size using PBD (Plackett-Burman Design) and CCD (Central-Composite Design) for the production of fumigaclavin and havolic acid. Similar PBD model have been in use by different researchers in different experiments. Venil and Lakshmanaperumalsamy (2009a) used Plackett-Burman Design (PBD) for the production of prodigiosin by Serratia marcescens SWML08, Kim et al. (2008) used the same PBD for production prodigiosin by Hahella chejuensis KCTC 2396 and Guo et al. (2010) for nisin production. Therefore, RSM had been used in the past, extensively for the optimization of medium composition, process parameters and in food manufacturing processes (Vazquez and Martin, 1997; Ramirez et al., 2001; Park et al., 2005).

Effect of combinations of sucrose, yeast extract and FeSO4 was studied such as sucrose- yeast extract, sucrose-FeSO4 and yeast extract- FeSO4 and it was found that yeast extract-

FeSO4 interaction was found to be more significant for enhanced yield of ergot alkaloids from culture liquid and mycelial filtrate extracts of Penicillium commune and Penicillium sp. IIB (Table 4.39 and 4.40). Same Box-Behnken Design (BBD) was used by Venil and

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Lakshmanaperumalsamy (2009a) and they investigated the interaction effects of three variables such as incubation temperature, (NH4)2PO4 and trace salts on the prodigiosin yield in the fermentation medium. Wang and Liu (2009) also used the same BBD model to analyze the influence of different combinations of glucose, peptones and KH2PO4 on the production of cell biomass in fermentation medium and this design was proved to be an efficient statistical approach for the optimization of culture parameters. Desirability charts were prepared using BBD statistical model and it was investigated that the optimum levels of sucrose, yeast extract and FeSO4 predicted for the two strains were different from each other. The optimum levels of selected variables for Penicillium commune were observed as higher than the Penicillium sp. IIB. It was also observed that the ergot alkaloids yield of Penicillium sp. IIB was higher than the Penicillium commune. Therefore it was concluded that Penicillium sp. IIB was a better ergot alkaloid producer than Penicillium commune. A similar BBD experimental model was also used by Krishnaa et al. (2013) for the optimization of pH and to evaluate the effect of pH on biomass production of Borasus flabellifer. They evaluated the influence of pH on the adsorption of chromium in culture liquid filtrates of 15 runs of Borasus flabellifer and proved that BBD wass the better fit model to investigate the large number of experiments in a single batch. Results of BBD model in this study are in harmony of BBD model used by Amara (2013), he optimized the culture conditions for the production of polyhydroxybutyrate and protease synthesis in the fermentation medium using mixed Bacillus culture and achieved a maximum yield of protease i.e. 2.8g/L. The combination of Plackett-Burman Design (PBD) and Box-Behnken Design (BBD) for screening and optimization of significant variables was proved to be an effective and reliable tool for the production of ergot alkaloids by Penicillium commune and Penicillium sp. IIB. The present work demonstrated the application of PBD and BBD for a quick determination of process conditions leading to the optimum yield of the dependent response (ergot alkaloids). This study also explains the effect of various factors in the production of ergot alkaloids by Penicillium species and it was concluded that sucrose, yeast extract and FeSO4 significantly influenced the yield. These statistical tools can

158 successfully be applied to any bioprocess, where optimization of variables and evaluation of interaction effects of factors are mandatory.

Section-III: Strain Improvement The major limiting factor in the production of important drugs on a commercial scale is the cost of the drugs. The cost can be reduced by optimizing culture conditions of specified organisms or these organisms can be genetically modified to produce desirable products. In the present study, wild strains of Penicillium commune and Penicillium sp. IIB were treated with physical and chemical mutagens such as UV irradiation and EMS to improve their ability to produce ergot alkaloids. Wild strain of Penicillium commune and Penicillium sp. IIB were exposed to UV irradiation (Table 4.41). It was investigated that death of the fungal organism is attributed to the lethal effect of UV irradiations on the microorganism. Onyegeme-Okerenta et al. (2013) reported that UV irradiation is a classical technique used for genetic modification and it had been used first time in 1950s to produce high penicillin yield from Penicillium chrysogenum Q- 176. Similarly they also achieved mutant strains of Penicillium chrysogenum (UVP1 and UVP2) after UV treatment for 20 and 25 min. Veerapagu et al. (2008) reported that wild strain of Penicillium chrysogenum was treated with UV irradiations and mutated strains had shown enhanced impact on the production of antibiotics. There are a number of reports available in the literature indicate that with the increase in the dose of irradiations, an increase in the yield of products can be observed (Paster et al., 1985; Moussa, 2003) or sometime a decrease in the production of fungal metabolites can be observed (Sharma et al., 1990) or sometimes, an unaffected response can also be observed (Paster and Bullerman, 1988). EL-Bondkly and Abeer (2007) working on the effect of different doses of UV irradiations on Penicillium roquefortii and a gradual increase in the lethality with the increase in the mutagen dosage. The data obtained in the present study was similar with Buzilova et al. (2000) who induced UV mutation in wild Penicillium roquefortii and obtained mutant strains as better producers of alkaloids such as Penicillium roquefortii VKMF-141 and Penicillium roquefortii VKMF-1073. They

159 classify mutants into three groups i.e. unable to produce alkaloids, producers of alkaloids and those who can produce ergot alkaloids in large quantities. Moreover, Vinokurova et al. (2001) isolated new isomers of clavinet alkaloids produced from mutant strains of Penicillium roquefortii Thom1906. It was demonstrated that mutant strains produced isomers of agroclavine, epoxyagroclavine, fumigaclavines A and B, festuclavine and chanoclavine. Ethyl methane-sulfonate mutagen proved to be a powerful agent in inducing a wide range of genetic variations in wild strains. For this purpose 300 µl/ml concentration of EMS was used to treat wild strains of Penicillium commune and Penicillium sp. IIB for different time intervals. It was observed that with the increase in time duration of the exposure, lethality was increased (Table 4.42). It was observed that, with the increase in the exposure time there was a decrease in the survival percentage. EL-Bondkly and Abeer (2007) also treated Penicillium roquefortii with many doses of EMS. They used 100 µl/ml concentrated solution of EMS to induce mutation in the fungal strains Their results showed the expected gradual increase of the lethality was associated with the increase of the mutagen dosage. The mutated surviving colonies of Penicillium commune (PCUV-1, PCUV-2, PCUV-3, PCUV-4, PCUV-5) and Penicillium sp. IIB (PUV-1, PUV-2, PUV-3 and PUV-4), were tested for their ability to produce ergot alkaloids. Mutated strains PCUV-4 and PUV-4 were found to be the best ergot alkaloids producers (Table 4.43). EMS treated strains of Penicillium commune (PCEMS-1, PCEMS-2 and PCEMS-3) and Penicillium sp. IIB (PEMS- 1) were checked for their ergot alkaloids productivity. It was observed mutated strain of Penicillium commune (PCEMS-3) produced a enhanced amount of ergot alkaloids after 21 days of incubation period (2.98 mg/ml) but the mutant PEMS-1 of Penicillium sp. IIB could not produce a considerable amount of ergot alkaloids (1.37 mg/ml) as compared to wild type (Table 4.44). The present study indicated that UV irradiations improved the ability of PCUV-4 mutant of Penicillium commune for the production of ergot alkaloids by altering its genetic makeup (Table 4.45). The data obtained from the physical and chemical treatment of wild strains of Penicillium commune and Penicillium sp. IIB

160 revealed that UV treatment proved to be more effective as compared to EMS in the present investigation. This might be due to the fact that UV is a strong mutagenic agent and can alter the DNA structure. Similar work has been done by Hamad et al. (2001), in which they found that chemical treatment was more efficient than the physical treatment to induce high level mutations in the DNA structure. Mohsin (2006) worked on strain improvement of thermophilic fungi Humicola insolens using both UV and EMS mutagens and described a considerable increase in the product by UV mutated strain. Our results were in concordance of Nadeem (2009) working on strain improvement using UV, NTG and MMS treatment for the production of alkaline proteases for industrial use, described that all physical and chemical mutagens can equally contribute in the alteration of DNA structure to get the desirable results.

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Section-IV: Analytical Studies Endophytes are ubiquitous in plants, with populations dependent on host and their habitats (Bills and Polishook, 1991; Tan and Zou, 2001). Many species of fungi are endophytic and associated with the medicinal plants and they are the good source of bioactive compounds. Species of genus Penicillium are a major target of endophytic microorganism research and many metabolites with cytotoxic and anti microbial secondary metabolites has been isolated from these fungi (Rukachaisirikul et al., 2007; Ge et al., 2008). Ergot alkaloids form a very diverse group of nitrogen containing compounds and they have tertiary or quaternary amino groups in their structures. Analytical problems connected with the alkaloids are mostly concerned with their physiochemical properties and they are commonly divided according to their type of chemical structure into tropane, indole, diterpene and others (Flieger, 2000). Izamailow and Schraiber are the pioneers in the thin layer chromatography (TLC) technique and they worked on the analysis of plant material containing alkaloids for the first time in 1938. The ergot alkaloids fraction produced by both species was determined through TLC and HPLC techniques. Ergot alkaloids extracted from culture liquid (extracellular) and mycelial (intracellular) filtrate extracts were identified by their specific colors obtained from filtrates and compared to the colors of their respective reference salts using thin layer chromatography plates. Various mobile phases were tested initially for the separation of ergot alkaloid compounds present in the culture liquid and mycelial filtrates of Penicillium commune and Penicillium sp. IIB and their Rf values were calculated. It was found that ergot alkaloids in culture liquid and mycelial filtrate extracts of Penicillium commune (Table 4.46) and Penicillium sp. IIB were clearly separated in mobile phase A (Table 4.47). Mobile phase H was also screened as best for its better Rf values for the PCCLFE1 and PCLFE12 filtrates of Penicillium commune and Penicillium sp. IIB. The mobile phases A and H were further modified and culture liquid and mycelial filtrates (PCLFEC9, PCMFE9) of Penicillium commune and Penicillium sp.

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IIB (PCLFE8, PMFE8, PCLFE9, PMFE9) showed maximum separation of alkaloid compounds in mobile phases A1 and H2 (Table 4.48 and 4.49). These filtrates revealed Van Urk’s reagent positive spots and produced characteristic pinkish blue and purple colors on TLC plates when observed under UV light. The major spot were identified as ergocriptine and ergotamine. These results are found to be similar with the findings of Moussa (2003) who reported the presence of ergocriptine in the extracts of Penicillium species with an Rf value of 0.70. The spots revealed a characteristic blue color under UV light. Spot with an Rf value 0.40 and 0.28 were identified as agroclavin and emyloclavin respectively. Polak and Rompala (2007) worked on the TLC behavior of ergot alkaloids using various mobile phases including various acids in the mobile phases in a very low amount. It was reported that ergot alkaloids are basic in nature and the surface of silica gel plate is always acidic, and a very strong interaction between silica gel and ergot alkaloids is always expected. They also suggested that this is not true for all alkaloids, some were strongly retained by the surface of silica gel but some weakly retained. This interaction depends upon the surface of silica gel and on the choice of the mobile phase for the separation of compounds. The components of mobile phase form complexes with the alkaloid molecules and these complexes have different binding capacities with the stationary phase. However, the non polar complexes could migrate with the mobile phase so their retention will be poor and their Rf values will be higher. They also described that addition of acids to the mobile phase slightly reduced binding of the alkaloid molecules to the silica gel surface and slightly increased the Rf values of the alkaloids. In the second phase of TLC mobile phase A1 and H2 were proved as the best to get the maximum Rf values. It was noted that, some of the ergot alkaloids had their Rf values in the range of 0.10-0.30, 0.30-0.60 and 0.60-0.90. (Polak and Rompala, 2007) suggested that pseudoalkaloids and phenylethylamine achieved their Rf value ranged from 0.60 to 0.80 in all kinds of mobile phases and for some true alkaloids such as ephedrine the Rf has been reported from 0.16-0.23 Adriana and Godoy (2001) studied the simple and relatively inexpensive TLC method for the detection and semi quantitative

163 measurement of ergovaline in the leaves of Festuca arundinacea. They use choloroform, acetone and acetic acid in ratio of 90:10:5 and chloroform:ethanol in 9:1 ratio as mobile phases for TLC studies. The TLC plates were visualized under UV after spraying p- dimethylaminobenzyldehyde and sulfuric acid and their Rf value were compared with the standard ergotamine reference salt. They observed the same Rf value of the extract as of ergoline. The Rf value for ergoline has been reported in the range of 0.4-0.6. The TLC method is now a days used as a pilot method for the HPLC analysis. In the present study, after the TLC analysis, the culture liquid and mycelial filtrates of Penicillium commune and Penicillium sp. IIB were subjected to HPLC analysis for the confirmation of the ergot alkaloid compounds. For this purpose, retention time, formation of peaks, peak areas and concentration of ergot alkaloid compounds in filtrates were compared with their respective reference salts. Ergotamine, dihydroergotamine methane sulfonate salt, bromocriptine mesylate and a mixture of all the reference standards were used as reference in the present study. From culture liquid and mycelial filtrate extracts such as PCCLFE9 and PCMFE12 of Penicillium commune (Table 4.51) showed the presence of ergotamine in the samples and culture liquid and mycelial filtrate extracts PCLFE12 and PMFE12 belonging to Penicillium sp. IIB were considered as the ergotamine containing filtrates (Table 4.52). Moussa (2003) has reported the presence of ergocriptine, agroclavine and elymoclavine alkaloids in the mycelial extracts of various Penicillium strains. Morlock (2004) and Aranda and Morlock (2007) described the use of UV absorption at 274 nm for caffeine and metamizol alkaloids by using the ethylacetate, methanol and ammonia as a mobile phase. In the present study, chloroform, isopropanol and water mobile phase (Table 3.11) was used for the detection of ergot alkaloids using UV detector at 280 nm. Ashour and Soulafa (2013) described HPLC as a sensitive analytical method for the determination of ergotamine in standard drugs and reported the presence of alkaloids with bromocriptime mesylate and egotamine tartrate as the reference salts.

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CONCLUSIONS

The following conclusions can be drawn from the present study:

1. It was known that the ergot alkaloids are not only produced in plants but in fungi too. However, the amount of ergot alkaloids produced by fungal species is comparatively less but it can be enhanced by focusing on identifying the new fungal species having the ability to produce ergot alkaloids. The literature survey indicated that this aspect has not yet been focused across the globe. Therefore, various species of Penicillium were evaluated for their ability to produce ergot alkaloids in their culture liquid (extracellular) and mycelial (intracellular) filtrates. By the present study it was found that Penicillium commune and Penicillium sp. IIB can be the potential candidates for the better production of ergot alkaloids. 2. Ergot alkaloids have been in-use since many decades due to their pharmaceutical properties. Generally, these are used for the treatment of many human ailments. The present study revealed that the surface culture fermentation technique is comparatively better for the production of ergot alkaloids from Penicillium commune and Penicillium sp. IIB. Optimization of fermentation conditions using OFAT and RSM techniques revealed that statistical procedures such as PBD and BBD are more reliable to enhance the yield of ergot alkaloids in a single step. 3. The yield of ergot alkaloids was improved by using physical and chemical mutagens, such as UV and EMS. It was found that the yield of ergot alkaloids was significantly improved in mutants obtained after UV irradiations as compared to EMS mutagen. The comparative studies of the mutated strains of Penicillium commune and Penicillium sp. IIB indicated that the mutant designated

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as PCUV-4 of Penicillium commune proved as the best mutant for the enhanced production of ergot alkaloids.

4. The TLC and HPLC analyses revealed both qualitatively and quantitatively the presence of ergotamine, ergocriptine and agroclavine in the culture liquid (extracellular) and mycelial (intracellular) filtrate extracts of Penicillium commune and Penicillium sp. IIB. This profile of ergot alkaloids has been the major part of drugs used to cure various health problems and is also prescribed to control acute headaches, induce labor contractions, terminate early pregnancies, inhibit mammary tumors and can stop postpartum bleeding.

5. It was also concluded that these fermentation studies can contribute as an alternative and cost effective methods for the biosynthesis of the important ergot alkaloids on commercial scales used to formulate drugs.

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