Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Production of Avermectins (avms) from Streptomyces avermitilis by Fermentation
BY
SAMIA SIDDIQUE
ROLL NO. 0 5 6 GCU PhD CHEM 2 0 1 0
Session 2010-2013
Department Of Chemistry Government College University Lahore
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Production of Avermectins (avms) from Streptomyces avermitilis by Fermentation
Submitted to GC University, Lahore in partial fulfillment of the requirements for the
Award of degree of
Ph.D In Chemistry
BY
SAMIA SIDDIQUE
ROLL NO. 0 5 6 GCU PhD CHEM 2 0 1 0
Session 2010-2013
Department Of Chemistry Government College University Lahore
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
RESEARCH COMPLETION CERTIFICATE
Certified that the research work contained in this thesis entitled “Production of Avermectins (avms) from Streptomyces avermitilis by Fermentation” has been carried out and completed by MS. SAMIA SIDDIQUE, Registration No.0056-PHD- CHEM-2010 under my supervision during her PhD (CHEMISTRY) in the Department of Chemistry, Government College University, Lahore /PCSIR Laboratories Lahore.
Dated: ______
Supervisor: Co-supervisor:
______
Dr. Fahim Ashraf Qureshi Dr. Quratulain Syed General Manager Director General Office of Research, Innovation and PCSIR, Laboratories, Lahore Commercialization, COMSATS Institute of Information and Technology
Submitted Through:
______
Prof. Dr Ahmad Adnan Controller of Examination Chairperson Government College University Department of Chemistry Lahore G. C. University, Lahore
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
D E C L A R A T I O N
I, Samia Siddique, Registration No. 0056-PHD-CHEM-2010, student of PhD in the subject of Chemistry session 2010-2013, hereby declared that the matter printed in the thesis entitled “Production of Avermectins (avms) from Streptomyces avermitilis by
Fermentation” is my own work and has not been printed, published and submitted as research work, thesis or publication in any form in any University, Research
Institution etc. in Pakistan or abroad.
Signature of Deponent Dated: ______SAMIA SIDDIQUE Department of Chemistry GC University, Lahore
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
A C K N O W L E D G E M E N T
All praises for the most merciful, omnipotent, omnipresent and omniscient Almighty ALLAH, Who enabled me with the blessing of His Prophet Hazrat Muhammad (P.B.U.H), Whose teachings inspired me to wider my thoughts and deliberate the things deeply and to complete this dissertation.
I am obliged to pay my heart rendering thanks and gratitude to my respected supervisor Dr. Fahim Ashraf Qureshi, General Manager, Office of Research, Innovation and Commercialization,COMSATS, Institute of Information and Technology, Islamabad. No words can suffice to express my indebtedness to Dr. Quratulain Syed, Director General, Food and BiotechnologyResearch Center, Lahore and Prof. Dr. Ahmad adnan, Department of Chemistry, GC University, Lahore, whose unremitting concentration, determination, hardworking, enthusiastic, highly courteous behavior, scholastic guidance, dedicated attitude, continuous help, congruent encouragement, valuable suggestions, inexhaustible inspiration and kindness in planning and execution of the present research projects and completion of the dissertation.
I express my heartiest gratitude and thanks to Prof. Dr. Islam Ullah Khan, Department of Chemistry, G.C. University Lahore, who provided me with all possible facilities of research work.
I can never forget the patience and graceful attitude offered to me by by the most compassionate Dr. Nadeem, Dr. Yasir Saleem and Dr. Rubina Nelofer, S.S.O. PCSIR Laboratories, Lahore. I feel pleasure in acknowledging their affectionate guidance, skilled advice and kind co-operation in Practical work. I also feel great pleasure in expressing my heartiest obligation and thanks to whole staff of Food and Biotechnology Research center, PCSIR Laboratories Lahore for their kind cooperation. I express my cordial thanks to Dr. Shahid Baig, Principal Scientists and Dr. Amir Ali,
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Jr. Scientist at National institute for Biotechnolgy and Genetic Engineering, Faislabad for their kind co-operation and personal interest towards completion of my research work.
I am delighted in feeling this occasion for making rightful acknowledgeent of vigorating suggestions blended with incredible patience of my brother Dr. Usman Siddique and my sisters. I owe them most of my life and my love for them will always be closed to my heart.
Finaly I would vemture to articulate my feelings of profound gratitude for my affectionate father, Dr. Muhammad Siddique and my husband Mr. Tehseen Aslam being a source of continuous encourgament and appreciation for me in completing my research work.
SAMIA SIDDIQUE
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
DEDICATED with Love To
My affectionate Father
Dr. Muhammad Siddique
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
LIST OF CONTENTS CHAPTER 1 Page # INTRODUCTION 01—33 1.1. Actinomycetes 1 1.1.1. Generic classification of Actinomycetes 2 1.1.1.1. Nocardioform Actinomycetes 2 1.1.1.2 Genera with multilocular sporangia 2 1.1.1.3. Actinoplanes 3 1.1.1.4. Streptomyces and other related genera 3 1.1.2. Characteristics of Streptomyces 3 1.1.3. Life cycle of Streptomyces 4 1.1.4. Streptomyces as secondary metabolites producing microorganisms 6 1.2. Streptomyces avermitilis 7 1.2.1. Glucose catabolism 7 1.2.2. Genome structure 8 1.3. Isolation and identification of Streptomyces avermitilis 9 1.4. Strian improvement and mutation 10 1.4.1. Screening of mutants 13 1.5. Fermentation 14 1.5.1. Range of fermentation process 15 1.5.2. Submerged fermentation 15 1.5.3. Components of fermentation 15 1.5.3.1. Medium composition 17 1.5.3.1.1. Carbon source 17 1.5.3.1.2. Nitrogen source 18 1.5.3.1.3. Inorganic phosphate 19 1.5.3.1.4. Inorganic salts 19 1.5.3.1.5 Trace metals 20 1.5.3.1.6. Precursors, co-factors and necleotides 20 1.5.3.1.7. Inhibitors 20
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
1.5.3.1.8. Inducers 21 1.5.3.2. Fermentation conditions 21 1.5.3.2.1. Ph 21 1.5.3.2.2. Temperature 22 1.6. Secondary Metabolites 22 1.6.1. Production 23 1.6.2 Functions 24 1.6.2. Resistance mechanisms and secondary metabolite secretion 24 1.7. Avermectins 25 1.7.1. Structure 25 1.7.2. Physical properties 27 1.7.3. Classification 28 1.7.4. Abamectin 29 1.7.5. Biosynthesis of avermectin from Streptomyces avermitilis 29 1.7.6. Spectra of activity 29 1.7.7. Toxicological effects 30 1.7.8. Environmental effects 31 1.8 Application of avermectin B1b 32 OBJECTIVES AND TARGETS 34 CHAPTER 2 LITERATURE REVIEW 35-54 CHAPTER 3 Materials and Methods Medium selection for the production of avermectin from 3.1 Streptomyces avermitilis 41445 55 3.1.1. Microorganism and maintenance 55 3.1,2. Inoculum development 55 3.1.3. Production of avermectin B1b 55 3.1.4. Effect of process parameters 56 3.2. Isolation and identification of microorganism 56
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
3.2.1. Isolation of Streptomyces 56 3.2.2. Isolation of Streptomyces avermitilis 57 3.2.2.1. Morphological characterization 57 3.2.2.2. Antimicrobial activities 57 3.2.2.3. Avermectin production 58 3.2.2.4. Agar colonies 58 3.2.2.5. Agar slants 58 3.2.3. Morphological characterization 58 3.2.4. Biochemical characterization 58 3.3. Mutagenesis of Streptomyces avermitilis 41445 59 3.3.1. Mutational analysis 59 3.3.2. Prepration of spore suspension 59 3.3.3. Stock solution of Ethidium bromide 59 3.3.4. Stock solution of ethylmethane sulfonate 59 Physical mutagenesis of Streptomyces avermitilis 41445 by ultra 3.3.5. violet irradiation 60 Physical mutagenesis of Streptomyces avermitilis 41445 by 3.3.6. ethylmethane sulfonate (EMS) 60 Chemical mutagenesis of Streptomyces avermitilis 41445 by 3.3.7. Ethidium bromide (EtBr) 60 Selection and screening of avermectin B1b hyper producing 3.3.8. mutants 61 Defined medium for the growth of mutant strain of Streptomyces 3.3.9. avermitilis 41445 61 3.4. Fermentation technique 61 3.4.1. Submerged fermentation 61 3.4.1.1. Inoculum development for Streptomyces avermitilis 41445 61 Shake flask study for avermectin B1b production from S. avermitilis 3.4.1.2. 41445 62 3.4.1.3. Inoculum development for mutant strain Streptomyces avermitilis 62
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
41445 UV 45(m)3 Shake flask study for avermectin B1b production from S.avermitilis 3.4.1.4. 41445 UV 45(m)3 mutant strain 62 3.5. Optimization 63 Optimization of cultural conditions for avermectin B1b production from Streptomyces avermitilis 41445 by Response surface 3.5.1. methodology 63 Single factor optimization methodology for the selection of key 3.5.1.1. factors for avermectin B1b production 63 3.5.2. Statistical experimental designs 63 3.5.2.1. Screening of cultivation variables by Plackett-Burman Design 63 Optimization by Central Composite Design (CDD) and statistical 3.5.2.2. analysis 64 3.5.2.3. Statistical model validation 65 3.6. Optimization 66 Optimization of cultural conditions for avermectin B1b production from Streptomyces avermitilis 41445 by Artificial Neural Network 3.6.1. (ANN) 66 3.6.1.1. Artificial neural network 66 3.6.1.2. Optimization capability of ANN 66 3.6.2. Statistical analysis 67 3.7. Optimization 67 Optimization of cultural conditions for avermectin B1b production from Streptomyces avermitilis 41445 UV 45(m) 3 by Response 3.7.1. Surface methodology (RSM) 67 Single factor optimization methodology for the selection of key 3.7.1.1. factors for avermectin B1b production 67 3.7.2. Statistical experimental design 68 3.7.2.1. Screening of cultivation variables by Plackett-Burman Design 68 3.7.2.2 Optimizaion by Central Composite Design (CDD) and statistical 69
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
analysis 3.7.3. Statistical model validation 70 3.8. Optimization 70 Optimization of cultural conditions for avermectin B1b production from S.avermitilis 41445 UV 45(m) 3 by Artificial neural network 3.8.1. (ANN) 70 3.8.1.1. Artificial neural network 70 3.8.1.2. Optimization capability of ANN 70 3.8.2. Statistical analysis 71 3.9. Fermenter scale production of avermectin B1b 71 Fermenter Scale Production of Avermectin B1b from Streptomyces 3.9.1. avermitilis 41445 72 3.9.1.1. Inoculum medium 72 3.9.1.2. Fermentation medium 72 Fermenter Scale Production of Avermectin B1b from Mutant strain 3.9.2. Streptomyces avermitilis 41445 UV 45(m) 3 72 3.9.3. Inoculum medium 72 3.9.4. Fermentation medium 72 3.1 Analysis 73 3.10.1. Avermectin Extraction 73 3.10.2. Thin layer chromatography 73 3.10.3. High performance liquid chromatography (HPLC) 73 3.10.4. Purification of avermectin B1b 73 3.10.5. Application of avermectin B1b 74 3.10.6. Bacterial cell count 74 3.10.7. Estimation of dry cell biomass 74 3.11. Kinetic parameters 74 3.12. Statistical analysis 75 3.13. Maintenance of culture 75 3.13.1. Short term storage 76
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
3.13.2. Medium term storage 76 3.13.3. Long term storage 76 CHAPTER 4 RESULTS AND DISCUSSION 77-159 4.1. Medium selection 77 4.1.1. Effect of different parameters on avermectin production 79 4.1.1.1. Effect of inoculum size on avermectin production 79 4.1.1.2. Effect of incubation time on avermectin production 80 4.2.3 Effect of temperature and pH on avermectin production 81 4.2. Isolation of microorganism from soil 82 Selection and screening of avermectin producing Streptomyces 4.2.1. avermitilis strains 83 4.3. Mutagenesis 91 Physical mutagenesis of Streptomyces avermitilis DSM 41445 by 4.3.1. UV light 91 Mutagenesis of Streptomyces avermitilis DSM 41445 by Ethidium 4.3.2. Bromide treatment 93 Susceptibility of Streptomyces avermitilis DSM 41445 against EtBr 4.3.2.1. treatment 93 4.3.3. Mutagenesis 94 Mutagenesis of Streptomyces avermitilis DSM 41445 by ethyl 4.3.4. methane sulfonate treatment 96 Susceptibility of Streptomyces avermitilis DSM 41445 against ethyl 4.3.4.1. methane sulfonate treatment 96 4.3.5. Mutagenesis 97 4.3.6. Heriditary stability 100 4.3.7. Confirmation of mutagenesis 101 4.3.7.1. Restriction enzyme analysis with Eco R1 101 4.3.7.2. Restriction enzyme analysis with Bam H1 102 4.4. Optimization 104
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Optimization of cultural conditions for avermectin B1b production from Streptomyces avermitilis DSM 41445 by Response Surface 4.4.1. methodology (RSM) 104 4.4.1.1. Selection of key factors for avermectin B1b production 104 4.4.2. Selection of key variables by Plackett-Burman design 105 4.4.3. Optimization by Central Composite Design and statistical analysis 109 4.5. Optimization 113 Optimization of cultural conditions for avermectin B1b production from Streptomyces avermitilis DSM 41445 by Artificial neural 4.5.1. networks 113 4.5.1.1. Optimization by Central Composite Design and statistical analysis 204 4.5.2. Comparison of different ANNS 116 4.5.3. The selected 38th ANN model 118 4.5.4. Optimization of fermentation variable using the selected ANN 119 4.5.5. Sensitivity analysis 123 4.6. Optimization 125 Comparative analysis of Response surface methodology and artificial neural network during medium optimization for the enhanced production of avermectin B1b from Streptomyces 4.6.1. avermitilis 41445 UV 45(m) 3 125 Single factor optimization for the selection of key factors for 4.6.2. avermectin B1b production 125 4.6.3. Statistical analysis 128 4.6.3.1. Screening of key variables by Plackett-Burman Design 128 4.6.3.2. Optimization by Central Composite Design and statistical analysis 129 4.6.4. Response Surface Methodology (RSM) 130 4.6.5. Artificial Neural network (ANN) 130 4.6.6. Optimizaion using RSM 132 4.6.7. Optimizaion using ANN 133 4.6.8. Sensitivity analysis by ANN 135
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
4.7. Application 143 4.7.1. Test substance 143 4.7.2. Contact filter paper toxicity of earthworms 143 4.7.3. Soil toxicity of avermectin of earthworms 144 4.7.4. Effect of avermectin B1b on cocoon formation 145 4.8. Fermenter 148 Laboratory Scale Production of Avermectin B1b from Streptomyces 4.8.1. avermitilis 41445 149 Laboratory Scale Production of Avermectin B1b from Streptomyces 4.8.2. avermitilis 41445 UV 45(m) 3 150 4.9. Kinetic parameters Study 151 Effect of different carbon sources on Streptomyces avermitilis 4.9.1. 41445 UV 45 (m) 3 growth and avermectin B1b production 152 Effect of pH on Streptomyces avermitilis 41445 UV 45 (m) 3 growth and avermectin B1b production in medium with potato 4.9.2. starch as carbon source 155 Effect of agitation speed on Streptomyces avermitilis 41445 UV 45 (m) 3 growth and avermectin B1b production in medium with 4.9.3. potato starch as carbon source at pH 7.5 156 4.9.4. Effect of various carbon sources on avermectin B1b production 158 Effect of pH on Streptomyces avermitilis 41445 UV 45 (m) 3 growth and avermectin B1b production in medium with potato 4.9.5. starch as carbon source 158 Effect of agitation speed on Streptomyces avermitilis 41445 UV 45 (m) 3 growth and avermectin B1b production in medium with 4.9.6. potato starch as carbon source at pH 7.5 159 CONCLUSION 160-163 REFERENCES 164-189 List of Publications Table # List of Tables Page #
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
1.1. Some antibiotics produced from Streptomyces species 6 1.2. Mutagen and their effects 11 1.3. Components of Fermentation 17 1.4. Microorganisms and antibiotics 18 1.5. Structural differentiation between avermectins 26 Nine variables screened by Plackett-Burman Design at lower and 3.1. higher vaues 64 Experimental variables, codes, units, range and levels of independent variables for response surface methodological 3.2. experiments 65 Nine variables screened by Plackett-Burman Design at lower and 3.3. higher levels 68 Experimental variables, codes, units, range and levels of independent variables for response surface methodological 3.4. experiments 69 Composition of different media used for the production of 4.1. avermectin B1b by S.avermitilis in submerged fermentation 77 Bacterial isolates with antimicrobial activities and avermectin 4.2. production 83 4.3. Antimicrobial activity of Streptomyces isolates 84 Morphological characteristics of active Streptomyces isolates on 4.4. Yeast extract malt extract agar (ISP-2) 86 4.5. Secondary Metabolite (avermectin) Production of Selected Isolates 87 4.6. Cultural characteristics of strain S1-C on different media 89 Morphological and biochemical characteristics of selected 4.7. Avermectin producing S1-C Streptomyces 90 4.8. Susceptibility of Streptomyces avermitilis DSM 41445 against EtBr 94 4.9. Susceptibility of Streptomyces avermitilis DSM 41445 against EMS 97 Comparative production of avermectin B1b from different mutant 4.10. strains 99
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
4.11. Analysis of Heriditary Stability of Strain UV 45(m) 3 100 PB Design for screening of nine variables with coded values and 4.12. observed avermectin B1b Production 106 (Analysis of Variance) ANOVA for experimental parameters of PB 4.13. design affecting the production of avermectin B1b 108 Variables with their coded values set at five different levels of 4.14. variations 110 Box-Wilson 23 factorial central composite design for the optimization of avermectin B1b production from S.avermitilis DSM 4.15 41445 for RSM 110 4.16. Model Coefficient 112 4.17. ANOVA (Analysis of Variance) 112 Variables with their coded values set at five different levels of 4.18. variations 114 Box-Wilson 23 factorial central composite design for the optimization of avermectin B1b production from Streptomyces 4.19. avermitilis 41445 by ANN 114 Comparison of prediction capabilities of different ANNs for the 4.20. avermectin B1b production from Streptomyces avermitilis 41445 117 Predicted optimal levels and avermectin B1b production obtained 4.21. through optimization using ANN 120 ANN Sensitivity analysis for optimization of avermectin B1b 4.22. production from Streptomyces avermitilis DSM 41445 124 PB Design for screening of nine variables with coded values and 4.23. observed avermectin B1b Production 128 Variable with their coded values set at five different levels of 4.24. variations 130 Box-Wilson 23 factorial central composite design for the optimization of avermectin B1b production from S.avermitilis 4.25. 41445 UV 45(m)3 for RSM 131
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Analysis of variance for optimization of avermectin B1b production from S.avermitilis UV 45(m) 3 using Box-Wilson Design calculated 4.26. by RSM data. 132 Sensitivity analysis by ANN for optimization of avermectin B1b 4.27. production from S.avermitilis UV 45(m) 3 135 The predicted optimum levels and avermectin B1b production obtained from optimization by S.avermitilis UV 45(m) 3 using 4.28. ANN and RSM 139 Comparison of optimization and prediction capability by ANN and RSM for avermectin B1b production obtained from optimization by 4.29. S.avermitilis 41445 UV 45(m) 3. 139 4.30. Physiochemical properties of avermectin B1b 143 Laboratory scale production of avermectin B1b from Streptomyces 4.31. avermitilis 41445 149 Laboratory scale production of avermectin B1b from Streptomyces 4.32. avermitilis 41445 UV 45(m) 3 150 Avermectin B1b fermentation by Streptomyces avermitilis 41445 4.33. UV 45(m)3 in SM2 medium 152 Kinetic parameter values of avermectin B1b fermentation by Streptomyces avermitilis 41445 UV 45(m)3 using different types of 4.34. carbon sources 153 Kinetic parameter values of avermectin B1b fermentation by Streptomyces avermitilis 41445 UV 45(m)3 using potato starch at 4.35. differnet initial culture pH 156 Kinetic parameter values of avermectin B1b fermentation by Streptomyces avermitilis 41445 UV 45(m)3 at medium pH 7.5 with 4.36. variable agitation speed 156 List Of Figures 1.1. Streptomyces life cycle 5 1.2 Linear physical map of the chromosome of S. avermitilis ATCC 9
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
31267 1.3. Structure of avermectin 26 4.1. Effect of different Media on Avermectin B1b Production 79 4.2. Effect of Inoculum Size on Avermectin B1b Production 80 4.3. Effect of fermentation period on Avermectin B1b Production 80 4.4. Effect of medium pH on Avermectin B1b Production 81 4.5. Effect of medium temperature on Avermectin B1b Production 81 Effect pf UV Exposure time on survival rate,lethality rate and 4.6. number of colony formation 92 Comparison between different UV exposure times on survival rate, 4.7. lethality rate and no, of colony formation 93 Effect of different concentrations of EtBr on survival rate, lethality 4.8. (A) rate and no, of colony formation at 10-20 minutes of exposure 95 Effect of different concentrations of EtBr on survival rate, lethality 4.8. (B) rate and no, of colony formation at 30 minutes of exposure 96 Effect of different concentrations of EtBr on survival rate, lethality 4.8. (C) rate and no, of colony formation at 50 minutes of exposure 96 Effect of different exposure intervals of EMS on survival rate, 4.9. lethality rate and no, of colony formation 98 Restriction enzyme analysis of S.avermitilis 41445, S.avermitilis UV 45 (3) mutant, S.avermitilis Ethidium Bromide mutant and 4.10. S.avermitilis EMS mutant with Eco R1 101 Restriction enzyme analysis of S.avermitilis 41445, S.avermitilis UV 45 (3) mutant, S.avermitilis Ethidium Bromide mutant and 4.11. S.avermitilis EMS mutant with Bam H1 103 4.12. Effect of different C-sources on Avermectin B1b production 104 Effect of different organic N-sources on Avermectin B1b 4.13. production 105 Effect of different inorganic N-sources on Avermectin B1b 4.14. production 105
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
4.15. Pareto Chart of t-values for coefficient 107 Topology of neural networks for avermectin B1b production during 4.16. ANN optimization 119 Surface plot obtained optimization using ANN for the combined
effect of yeast extract and MgSO4.7H2O on avermectin B1b 4.17. production by keeping all other variables constant 121 Surface plot obtained optimization using ANN for the combined effect of yeast extract and temperature on avermectin B1b 4.18. production by keeping all other variables constant 122 Surface plot obtained optimization using ANN for the combined
effect of MgSO4.7H2O and temperature on avermectin B1b 4.19. production by keeping all other variables constant 123 4.20. Effect of different C-sources on Avermectin B1b production 126 Effect of different organic N-sources on Avermectin B1b 4.21. production 127 Effect of different inorganic N-sources on Avermectin B1b 4.22. production 127 Topology of neural networks for avermectin B1b production during 4.23. ANN optimization 134 Surface plot obtained optimization using ANN for the combined effect of KCl and NaCl on avermectin B1b production by keeping 4.24. all other variables constant 136 Surface plot obtained optimization using ANN for the combined effect of KCl and pH on avermectin B1b production by keeping all 4.25. other variables constant 137 Surface plot obtained optimization using ANN for the combined effect of NaCl and pH on avermectin B1b production by keeping all 4.26. other variables constant 138 Difference in mortality after 48 and 72h during contact filter paper 4.27 test 144
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
4.28 Effect of concentration on earthworm mortality 144 4.29. Effect of concentration on earthworm cocoon formation 145 4.30. Kinetics of S.avermitilis 41445 and S.avermitilis UV 45(m)3 mutant 151 4.31. Rate of utilization of carbon substrates during fermentation 154 4.32. Effect of various carbon sources on cell biomass production 154 4.33. Effect of various carbon sources on avermectin B1b production 155
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Abstract
The main objective of this study was the optimization of medium for the maximum production of avermectin B1b from Streptomyces avermitilis DSM 41445. Different media were used for the production of avermectin B1b. However the maximum production of avermectin B1b (17 mg/l) was obtained by using SM2 growth medium containing soluble corn starch, yeast extract, KCl, CaCO3 and MgSO4 which was detected qualitatively by using TLC and quantitatively by HPLC. Maximum production was observed with initial medium pH of 7, 10% inoculum size with incubation temperature of 31oC for 10 days of fermentation period. In the next step the production of avermectin B1b from strain of Streptomyces avermitilis 41445 was enhanced by mutagenesis using Ultraviolet irradiation, ethidium bromide (EB) and ethyl methanesulfonate as mutagens. Selection of avermectin B1b hyper producing mutant produced from these three different methods was made on the basis of HPLC results. Mutants obtained after 45 minutes irradiation of ultraviolet rays on the spores of Streptomyces avermitilis 41445 was found to be the best mutant for the enhanced production of avermectin B1b component (254.14 mg/L). Other avermectin B1b hyper producing mutants obtained from EMS (1 µL/mL) and EB (30 µL/mL) treatment gave 202.63 mg/L and 199.30 mg/L of B1b respectively. The hereditary stability analysis of UV 45(m) 3 mutant showed that the production of avermectin B1b remained constant and there were no reverse mutation occurred after 15 generations. Streptomyces avermitilis belonging to Actinomycetes are specialized for the production of avermectin being used as anthelmintic and insecticidal agent. They are mostly present in the soil and their isolation from the soil is very crucial so s to obtain the medically important avermectin. In the present study 10 bacterial isolates lacking antimicrobial activities were isolated from soil samples collected from different areas of Lahore. Three distinctive localities of Lahore were opted for the assortment of soil to isolate Streptomyces avermitilis. About fifty isolates of Streptomyces species were attained through selective prescreening procedures. All of these isolates were studied for the production of secondary metabolite, the avermectin. Different test like soluble pigment colour and melanin formation were used for identification. Biochemical characterization of isolates closely resembling the
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation control was done. The 10 selected isolates were identified as avermectin producing strain by fermentation and were characterized on ISP2 medium for aerial and reverse side mycelia colour, soluble pigment colour and melanin formation in comparison with Streptomyces avermitilis DSM 41445. The best avermectin (10.15mg/L) producing isolate S1-C was when subjected for culture characteristics analysis in different media along with biochemical characterization showed similar result as were obtained for S.avermitilis DSM 41445. From the results it was concluded that agricultural lands around PCSIR Campus Lahore were the rich source of industrially important Streptomyces especially the S.avermitilis. Avermectin is an environment friendly bio- insecticide. Optimization of the culture conditions for avermectin B1b production was not carried out before using Artificial Neural Network (ANN) method. The present work is therefore conducted to optimize some important factors including yeast extract,
MgSO4.7H2O and temperature for the avermectin B1b production using ANN methodology from Streptomyces avermitilis DSM 41445. The optimum levels for the yeast extract, MgSO4.7H2O and temperature were 16.0 (g/L), 5.0 (g/L) and 32 °C respectively. Maximum effect was observed by yeast extract. Avermectin B1b yield was increased up to 150% after optimization. ANN was found a powerful technique for the optimization and prediction of avermectin B1b production from Streptomyces avermitilis DSM 41445. Present study was conducted to optimize avermectin B1b production from S.avermitilis 41445 UV45(m)3 using artificial neural network and Response surface methodology. Three variables NaCl, KCl and pH were used for optimization. Coefficient of determination and adjusted coefficient of determination have very poor values for RSM. Values predicted by RSM for experiments were also much different from the observed avermectin production. Comparatively predicted avermectin levels by ANN were very close to observed values with much higher R2 and adjusted R2. Optimum levels of NaCl, KCl and pH predicted by ANN were 1.0g/L, 0.5g/L and 7.46 respectively. Sensitivity analysis predicted highest effect being shown by pH followed by NaCl and KCl. About 37.89 folds increase in avermectin B1b production was observed at optimum levels of three variables envisage by ANN. Optimum levels, ranking order of variables and the predicted avermectin on the optimum levels by the RSM was much different from
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
ANN values. Results revealed that ANN is better optimization tool for given strain than RSM.
Use of avermectin B1b as anthelmintic and insecticidal agent has increased to protect the soil and for enhanced crop production. Enhanced production of avermectin B1b was obtained from mutant strain of Streptomyces avermitilis 41445. Modeling of mutant strain Streptomyces avermitilis 41445 UV 45(m) 3 growth and avermectin B1b production is therefore required for optimization during fermentation process. Kinetics of intracellular avermectin B1b production was studies in shake flask culture during submerged fermentation. Mathematical models based upon Logistic and Piret Equations have been used to investigate the kinetics of avermectin B1b production and substrate utilization from Streptomyces avermitilis 41445 UV 45(m)3. Effect of various carbon sources (glucose, maltose, lactose, potato starch, soluble corn starch, and wheat starch), pH (6.0, 6.5, 7.0, and 7.5), agitation speed (150, 200, and 250 rpm) on microbial growth and product formation were evaluated. Maximum avermectin B1b production (420.02 ±0.01 mg/L) and cell biomass (31.74 ±0.05 g/L) was obtained in media having potato starch as carbon substrate, medium pH of 7.5 with agitation speed of 250 rpm. Maximum specific growth rate (µmax), growth associated avermectin B1b production coefficient (α) and non-growth associated avermectin B1b production coefficient (β) obtained were 0.16h-1, 0 mg cell-1 h-1 and 3.5 mg cell-1 h-1 respectively. From the above results we can conclude that avermectin B1b production was non-growth associated process.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
INTRODUCTION
1.1. ACTINOMYCETES
Actinomycetes belonging to order Actinomycetales, are now known as Actinobacteria. They are spore forming gram positive bacteria and are usually characterized by formation of aerial mycelium. Also they have higher guanine and cytosine contents of DNA1 as compared to yeast and other microorganism. They possess circular genome of about 3300 or more genes encoding large number of coding sequences for morphological differentiation and secondary metabolite production2. Linear plasmids are also present in the Actinobacteria and have been implicated in the transfer of secondary metabolite biosynthetic genes and antibiotic resistant genes3.
Actinobacteria has the ability to produce bioactive secondary metabolites. About two third of the naturally occurring antibiotics are found to be produced by this group of bacteria4 and exhibit pharmacological and industrial application5,6. Application in the production of enzymes, removal of pollutants from the environment and synthesis of recombinant proteins reveal their versatile nature7,8,9. They have enzyme coding genes that are used in catalytic reactions to produce intramolecular C-C triple bonds. These triple bonds are the basic units of complex molecular structures of insecticidal compound. Other macrolide rarely exhibit this activity10.
The isolation and culturing of actinobacteria tailored to extreme conditions and symbiotic commensalism is very difficult11. Antibacterial diversity is exhibited under intense conditions. Cultivation of rare and novel genera thus can be done by suitable isolation procedures resulting in the enhanced microbial diversity12.
The actinomycetes being gram positive have very low growth rate13. Incubation of spores for 24 hours resulted in the colonies that were visible only under microscope. Colony formation is very slow process and usually took 3-4 days to be seen through naked eye14. Incubation period of 7-14 days or even 1 month are necessary for the colonies to form mature aerial mycelium15. They have been found to grow on media used in laboratory like nutrient agar, trypticase soya agar, blood agar and even brain-heart
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation fusion agar. Sporoactinomycetes require special medium for their growth, differentiation and development of characteristic spores and pigments16.
Culturing of Streptomyces on nutrient agar resulted in the configuration of hard, pale and shiny colonies which can simply be converted to yellow colonies if they are permitted to nurture on appropriate medium like oatmeal agar or inorganic salt starch agar16. A spore or a fragment of mycelium is required for the growth of actinomycetes and consequently for their development to hyphae. The resultant hyphae then pierce into the agar surface, get branched and form tough and leathery colony when lined mutually. Composition of growth agar medium determines the compactness and uniformity of the colonies14. Morphology of actinomycetes mounting on agar surface can be used as intimation for the recognition of species16.
1.1.1. Generic Classification of Actinomycetes
Prognostic and established system of classification for the Actinobacteria has amplified the competence and prospects of isolating novel compounds. Conventional and common means of categorization relied on morphological and physical depiction of the entire class. Based on the chemotaxonomic classification of Actinobacteria, they have been categorized into respective genera and species with varying degree of discrimination17,18.
1.1.1.1. Nocardioform Actinomycetes
This is the filamentous heterogeneous group of actinobacteria customarily fragmented into shorter elements. Some genera exhibit the aerial intensification and spore formation and are eminent by well chemotypes, existance or deficiency of mycolic acid and other chemical characters16.
1.1.1.2. Genera with multilocular sporangia
Genera whose filaments are alienated by longitudinal and transverse septa formation are included in this group. Normally motile (Dermatophilus, geodermatophilus) or non-motile (Frankia) coccoid-like elements are produced by them16.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
1.1.1.3. Actinoplanes
Filaments with little or even no aerial growth are formed by this actinobacteria group. Sporangia of Actinoplanes, Ampullariella, Dactylosporangium and Pilimelia produce motile spores as against that of Micromonospora and Catellatospora which form single or chain of spores respectively19. Cell hydrolysate contain the meso-DAP, glycine, arabinose and xylose16.
1.1.1.4. Streptomycetes and related genera
This is the group of actinobacteria in which cell hydrolysate is consisted of L- Diaminopimelic acid and glycine. In Streptomyces and Streptoverticillium, filaments with widespread aerial growth and elongated chains of spore are produced and these are the essential characteristics of this group20. Morphological, cultural, physiological and biochemical diversity in spore formation is found in other related genera like Intrasporangium, Kineospora and Sporichthya16,21.
1.1.2. Characteristics of Streptomycetes
Streptomyces are the largely subjugated actinomycetes allied to the production of antibiotics. Streptomycetes and other related species can also be used to degrade the cotton textiles, plastics, rubber and paper. They necessitate nutritionally, biologically and physically complex soil habitat for their growth and to execute broad range of metabolic processes14. Production of bioactive secondary metabolite is the main feature of this group22,23.
Members of Streptomycetes are omnipresent, however, the most appropriate habitat for their occurrence is the soil where they nurture and proliferate well. Many biotopes like fodder, grains and decaying wood get tainted by such soil. Bioactive Streptomyces are also found in the marine and fresh water habitats with marine Streptomyces being found extremely good for antibiotics production24,25.
Streptomycetes require different type of organic source (sugars, alcohols, amino acid, and aromatic acids), inorganic nitrogen source and other mineral salts for their proper growth. Production of extracellular hydrolytic enzymes and secondary metabolites
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation requires the presence of polysaccharides and hydrocarbons in the fermentation medium. Choice of carbon source utilization during macroscopic, microscopic and biochemical characterization of Streptomyces isolated from soil plays important role26. Other biochemical and physiological tests required for Streptomyces separation from other genera of actinomycetes are L-diamonipemelic acid, no featured precedent of sugars, a typical pattern of fatty acids, no mycolic acid formation, an emblematic pattern of menaquinones, melanin pigment formation and utilization of nine carbon27,28,29.
During life cycle, the aerial mycelia of Streptomyces are converted to conidia. Presence of extensively branching primary mycelium in addition to the less copious aerial mycelium is their characteristic feature30,31.
1.1.3. Life cycle of Streptomyces
The most characteristic aspect of Streptomyces is their intricate life cycle initiating with the formation of branching filamentous hyphae and ending with the formation of spores in these hyphae. These spores are the representation of semi-dormant stage of Streptomyces life cycle32,33. The dormant spores after germination form the germ tubes which after elongation stop the binary fission of cells14. The semi dormant spores will be forming the vegetative cell with time resulting in higher enzymatic activity as compared to the semi dormant stage of spore34.
Developmental life cycle on scrupulous medium consisted of following separate stages. Germination of spores to form vegetative mycelium encompassing filamentous multinucleotidal hyphae separated into larger sections with thin layered septa. Vegetative mycelium incessantly grows in and out of the medium as long as a concise growth apprehended and characterized by decreased macromolecular synthesis35,36 and resulting in the formation of reproductive mycelium. These mycelia crop up as vertical annex of the vegetative mycelium in the air37,38. The sections then instigate to recede due to the rapid mycelium expansion and hydrophobic rodlet layer then swathe the surface of aerial hyphae36. Extensive compartmentalization of aerial hyphae tips is now initiated and separated by double layered sporulation septa. This double layer is intended to be hydrophobic spore chains39,35,36. Wrapping fibrous sheath around spore chains begin to
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation rupture owing to the maturation of spores. The released spores are now geared up to germinate again as well as the life cycle. In short, the life cycle can be encapsulated as follow: vegetative hyphae, lysing of vegetative hyphae aerial hyphae, immature and mature spores, and germinating spores39.
Figure 1.1: Life Cycle of Streptomyces
Morphological differentiation of Streptomyces spp. is also interrelated with the formation of secondary metabolites. Time intervals related to the decline of exponential growth phase and initiation of stationary phase coincides strongly with the production of antibiotics from this genus. Some physiological and environmental factors in liquid cultures also contribute in the entire process40,41. In case of solid cultures, secondary metabolite production resulted immediately before or during the aerial hyphae development40. Nutritional constraints exceptionally the presence of carbon and nitrogen
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation sources in the production and growth media are essential for secondary metabolite production42,36.
1.1.4. Streptomyces as Secondary Metabolites Producing Microorganisms
The most salient property of Streptomyces is the degree to which they yield the antibiotics and other secondary metabolites during fermentation playing a significant role in medicine, agriculture and biotechnology. About 85% commercially and medically important and 60% agriculturally important antibiotics have been produced from this group of bacteria41,44.
More than 50 % of the isolated strains are capable of producing the secondary metabolites in the course of fermentation. Some microorganisms also produce more than one antibiotic and they are not structurally allied to each other. Antibiotics with miscellaneous chemical structures including aminoglycosides, ansamycins, anthracyclines, β-lactams, macrolides, peptides, polyenes, polyethers and tetracyclines and many others which do not fall in any of this category are produced from Streptomycete45.
Table 1.1: Some antibiotics produced from Streptomyces species
Structural class Antibiotic Producer Activity/Mode of action Aminoglycosides Streptomycin S. griseus Antibacterial Kanamycin A,B S. kanamyceticus agents, Hygromycin B S. hygroscopicus Some are broad Neomycin S. fradiae spectrum, Inhibit protein synthesis Tetracyclines Tetracycline S. aurofaciens Antibacterial, broad Chlorotetracycline S. aurofaciens spectrum, Inhibit protein synthesis
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Macrolides Avermectins S. avermitilis Antibacterial, Erythromycin S. erythrius inhibit Clindamycin S. lincolnesis protein synthesis β-Lactams Cephamycin A, B S. chartreusis Antibacterial, Clavulanic acid S. clavuligerus inhibit cell wall synthesis Polyenes Nystatin S. noursei Antifungal Amphotericin B S. nodosus Peptides/ Neocarzinostatin S. carzinostaticus Antitumor agents Glycopeptide Bleomycin A2, B2 S. verticillus Undefined Chloremphenicol S. venezuelae Antibacterial, broad Spectrum
Ordinary essentials of regulation are implicated in the physiological and morphological differentiation and also in the production of antibiotics46. Bald mutants which are not able to sporulate are flawed in the regulation process for the production of secondary metabolites47,48,49.
1.2. STREPTOMYCES AVERMITILIS
Streptomyces avermitilis belonging to mesophilic Actinomycetes50 are the aerobic gram positive bacteria having circular genome and form aerial spores51. The spores are spherical or oval in shape and usually occur in chains. For sporulation to occur, specialized media are required including the egg albumin, glycerol-asparagine, inorganic salt-starch and oatmeal agar medium. Grey colored aerial spores are formed on oatmeal agar giving colony reverse as dark brown to tan. The culture nurtures well at temperature between 27-37°C but not above 50°C 50. They are found to contain extensive amount of reiterated DNA. This group has a strong tendency to produce antibiotics having diverse chemical structure and different biological activities51.
1.2.1. Glucose catabolism
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
The most promising and distinctive feature of Streptomyces avermitilis is the production of secondary metabolites52 called ―Avermectins‖ showing characteristic anthelmintic and insecticidal activities53. Several parameters are involved in the regulation of secondary metabolite production chiefly the glucose metabolism. Avermectin production is suppressed when glucose is at earlier stages of fermentation due to the slow digestion of glucose by Streptomyces avermitilis. Two important pathways involved in the glucose metabolism are ―Embden-Meyerhof Pathway‖ and ―Pentose Phosphate Pathway‖ associated with certain enzymes including glucose-6- phosphogluconate dehydrogenase, phosphofructokinase and citrate synthase54.
The only enzyme that is affected by the higher concentrations of glucose is the glucose-6-phosphogluconate dehydrogenase thus acting as the rate limiting step in the pentose phosphate pathway. The production of this enzyme is significantly reduced by the addition of glucose at early stage of fermentation52.
1.2.2. Genome structure
Linear chromosome55 of Streptomyces avermitilis consisted of about 8.7 million base pairs56 encompassing the largest bacterial genome sequence and providing insight into the inherent assortment of the production of secondary metabolites during fermentation. Genome sequence contains genes for the production of 25 different secondary metabolites from S.avermitilis which are extensively positioned throughout the entire genome but they are mainly abundant near both ends of the sequence. About 6.4% of the S.avermitilis genome is subjugated by these gene clusters57. Four of the gene clusters are concerned with the biosyntheses of melanin pigments, two of them encode the tyrosinase and its cofactor and another two encode an ochronotic pigment and polyketide-derived melanin pigment. There are seven gene clusters that encode carotenoid biosyntheses along with five genes for siderophore construction58. Synthesis of type-I polyketide and type-II polyketide-derived compounds required eight and two gene clusters respectively. Meanwhile a polyketide synthase similar to phloroglucinol synthase was identified along with eight gene clusters for the biosyntheses of peptide compounds being synthesized by nonribosomal peptide synthetases56.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Figure 1.2: Linear physical map of the chromosome of S. avermitilis
1.3. ISOLATION AND IDENTIFICATION OF STREPTOMYCES FROM SOIL
Streptomyces belonging to the most profuse group in soil, the Actinomycetes are aerobic and gram positive bacteria59,60. Their distribution and presence in soil is highly effected by geographical conditions like soil temperature, type, pH, amount of organic matter and moisture contents. Those who are resistant to acidic environment are the most abundant of all Actinomycetes in the soil61. However they are less abundant in soils of alkaline pH62 and are known for their ability to produce industrially important enzymes and secondary metabolites during fermentation process63 and covering about 80% of the antibiotic products64. Screening and isolation of microorganisms producing secondary metabolites have been the main focus in recent many years65.
Components of media have pronounced effects on the isolation of Streptomycetes. Media if containing glycerol or starch as carbon source and arginine, casein or nitrate as nitrogen source will be giving the best isolation results. Different antifungal agents named nystatin; cycloheximide and pimaricin are usually employed during bacterial isolation to obtain the pure bacterial isolates. Identification of Streptomycetes is made on the basis of spore size, morphology, chains, pigmentation, physiological and biochemical characteristics, and antibiotic resistance66.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Standard microbiological methods, analysis of biochemical markers and DNA sequencing have also been employed for the selective identification of genus and species of the isolated microbes67. Members of Streptomycetes form stable filaments and also capable of producing long chains of spores with aerial growth. Direct and non direct methods of screening of antibiotic producing strains have usually been employed for the isolation of microbe of interest. Direct screening of strains involved the bioassay or some chemical methods while non direct screening involved co-relation of strain characteristic with antibiotic production68,69,70.
Bacteria belonging to genus Streptomyces are very important because their ability to produce several types of secondary metabolites71. Streptomyces avermitilis, gram positive bacteria is specialized for the formation of secondary metabolites, the avermectin being used as anthelmintic agents. The ivermectin which is the semisynthetic derivative of avermectin has been used widely in veterinary field for improved animal health and to eradicate the onchocerciasis72,73,74.
1.4. STRAIN IMPROVEMENT AND MUTATION
The process of altering one or more nucleotides, present at specific site of the DNA strand, using different sources is called Mutation. The resultant strain is called the Mutant Strain. The physical or the chemical source used for producing mutation is Mutagen. Nucleotide base pair present at DNA chromosomes can be rearranged, deleted or substituted in order to produce mutation. A mutation that occurs on a single nucleotide at a frequency of 10-5-10-10/generation is Point Mutation. Although certain mutations are harmful, yet they make the organism adapt to a particular environment, enhance its ability to produce secondary metabolites75 and improve Biocatalytic performance. The nucleotide sequence on chromosomal DNA regulates microbial metabolism to avoid any wasteful expenditure and enzymes which are required for the biosynthesis76.
Microbial strain improvement for greater metabolite production and better environmental adaptation is therefore a key factor. Gene sequence on chromosomal DNA must be altered and manipulated to shift and bypass regulatory controls and check points. Alteration and rearrangement in the genetic sequence of mutated cells result in the
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
population variability. The mutant strains are anticipated to evolve and are accomplished to confer their metabolic machinery towards key biosynthetic enzymes production. This will then enable them for over production of economical and industrially important metabolites77,78.
There are numerous genetic and molecular methods used for the mutagenesis of the strains and enhancement of secondary metabolite production including:
a. Classical random chemical mutagenesis b. Mutagenesis owing to irradiations c. Rational assortment of random and induced mutants d. Transposition mutagenesis e. Targeted deletion and duplication f. Genetic recombination‘s by protoplast fusion79
Table 1.2: Mutagen and their effects
Mutagen Mutation induced Impart on DNA Relative effect Radiation: UV radiation like X-rays, Single or double Deletions and High gamma rays stranded breakage of Structural changes DNA UV radiations Pyrimidines Transversions, deletion, frame Medium (Short wavelength) Dimerization shifts, transitions from GC-AT Cross links in DNA Chemicals: Base analogue 5-chlorouracil Results in faulty pairing GC-AT and AT-GC transitions Low 5-bromouracil - AT-GC and GC-AT transitions Low
2-aminopurine (Deaminating Errors in DNA Bi-directional translation, deletion Low agent) replication Hydroxylamine Deamination of AT-GC and GC-AT transitions Medium cytosine
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Nitrous acid Deamination of A,C & G
Alkylating agents MNNG Methylation, high pH GC-AT transitions High EMS Alkylation of C and A GC-AT transitions High Mustard-di-(2chloroethyl)- Alkylation of C and A GC-AT transitions High sulfide Intercalating agents Ethidium bromide, Intercalation between Frame shifts, Loss of plasmids and Low Acridinedyes two base pairs micro deletions
Biologicals: Phage, plasmids, DNA Base substitution and Deletion, duplication, insertion High transposing breakage
Random chemical mutagenesis and fermentation processes are the most valuable tools for the improved fermentation productivity79. Random mutagenesis is advantageous and valuable because of the high levels of mutations accomplished by chemicals with little or no information of constructive mutation being produced in the gene sequence. Manual or highly automated methods have been employed for the screening of mutants80,81.
Induction of limited spectrum of base pair substitutions is the main flaw of random chemical mutagenesis79. Mutagenic chemicals have been classified into three sets based on their genre of action:
a. Mutagen effecting non replicating DNA
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
b. Base analogs: incorporated into replicating DNA because of their resemblance to naturally occurring bases c. Frame shift mutagen: intercalating into DNA during replication causing inclusion or erasure of one or more nucleotide pairs82.
Induced mutagenesis prompts the alteration of gene sequence that normally resulted in the modified state of functioning. In secondary metabolite producing actinobacteria especially the Streptomyces, the mutagenesis abolishes the surplus intermediates and enhances the production of desired metabolites83,84.
Biosynthetic genes on chromosomal DNA can be identified by inducing mutation through UV irradiation, cold storage or chemical treatment. Most widely used chemical for mutagenesis are the Methyl methane sulfonate, Ethyl methane sulfonate, Hydroxyl amine, Ethidium bromide and N-methyl-N-nitro-N-nitrosoguanidine85,86,87,88,89. Use of MNNG as mutagen causing GC-AT transitions is not appropriate because of its carcinogenic effects although it is a very strong chemical mutagen82.
Mutations produced through different mutagens follow different mechanisms. Ultraviolet and ionization radiation both are also employed to induce mutation. Mutations caused by short wavelength UV irradiations lying in a range between 200-300nm are the most effective one90. They resulted in cross linking by formation of dimmers between Pyrimidines of either adjacent or complementary strands. GC-AT transversions, frame shift and deletions are also the consequences of Ultra Violet irradiation. Long wavelength UV irradiations although are less lethal, however, they are strong enough, and less survival and high death rate is resulted by introducing the interaction between DNA and different dyes. Ionization radiations resulted in the ionization of medium through which they travel resulting in the breakdown of single or double stranded DNA with higher probability. Translocations and inversions are resulted as a consequence of double stranded DNA cessation by ionization radiation exposure91.
1.4.1. Screening of mutants
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Selection of mutants produced as a result of treatment by various sources is very crucial step for the isolation and screening of hyper producing mutant. Two methods are generally employed for the screening of mutants including:
a. Random screening b. Selective screening
During random selection, the antibiotic producing capability of all the mutants is examined and the one with antibiotic hyper producing mutant is selected. This usually encompasses three important advantages. First: all the randomly selected mutants have single mutations strongly effecting the secondary metabolite production. Second: appropriate methods can be used for the screening and isolation of mutants in which rare mutation have been produced. Third: mutations consequential of unprompted mutations can be screened. During selective screening of mutants, high cell density of mutagenized population is shielded on the selected medium augmented with toxic substance preventing the growth of wild strain. Only the resistant mutant colonies are proficient to survive with capability to produce enhanced concentration of secondary metabolites91.
1.5. FERMENTATION
The word ―Fermentation‖ has been originated from Latin word ―Fervere‖ meaning ―to boil‖ and thus describes the outcome of yeast effect on fruit extracts and malted grains. The anaerobic catabolism of sugars causes the effervescence of carbon dioxide in the form of boiling. Reduction of pyridine nucleotides occur during sugar catabolism which is an oxidative process. These pyridine nucleotides must be re-oxidized so as to continue the entire process and for this purpose anaerobic conditions are provided. Electron transfer through cytochrome system causes the re-oxidation of reduced pyridine nucleotides and the oxygen provided act as terminal electron acceptor. The whole mechanism is accompanied by reduction of an organic compound obtained from catabolic pathway. Biochemical fermentation is the energy generating phenomenon in which organic compound acts as both the electron acceptor as well as the electron donor. Industrial microbiologists describe the fermentation as a process required for the
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation production of any product from microbes because of the microbial production of alcohol as metabolite on industrial scale for the very first time92.
The production of secondary metabolites from naturally occurring Streptomyces is very low and therefore this is not enough for the downstream chemical and biological activity categorization studies. In actinobacteria the production of secondary metabolites is initiated and improved by taking advantage of fermentation conditions93. Most important fermentations for secondary metabolites production are the submerged fermentation and solid state fermentation.
1.5.1. Range of fermentation processes
Fermentation processes have been classified into following five different groups:
a. Single cell protein producing fermentation b. Microbial enzyme producing fermentation c. Microbial metabolites producing fermentation d. Recombinant metabolite producing fermentation e. The process that caused the modification of a certain compound when added into the fermentation media92.
1.5.2. Submerged Fermentation
Submerged fermentation for secondary metabolite production employs the use of filamentous fungi and actinobacteria in fermentation vessels usually the angular baffles which help in the proficient mixing and amplified oxygen transfer94,95. The sufficient supply of oxygen is most recurrent problem coupled with shake flask96.
Different biochemical, physical and environmental factors affect the manifestation of secondary metabolism. Additives to fermentation media in submerged fermentation that bring forth enhanced secondary metabolite production is not really known. There is an assumption that there are certain compounds that resulted in enhanced secondary metabolite yield by trapping inhibitory substances. These embrace naturally stirring Zeolites like magnesium phosphate forming complexes with ammonium salts97,98.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Induction of various groups of secondary metabolites from different classes of actinobacteria is also possible using suitable nitrogen sources99,100.
1.5.3. Components of fermentation medium
Fermentation liquid media employed for secondary metabolite production from Actinobacteria might be either chemically defined synthetic or undefined naturally occurring complex media. To improve the yield and lower the cost of any process, inexpensive medium is used101. Complex undefined medium contained some distracted and vague components leading to variability and may customize the expression contour of the metabolites from actinobacteria102.
During fermentation, medium formulation plays important role. Cell biomass and secondary metabolite production from microorganism are related to constituents of medium. Microbial cell maintenance and its biosynthetic reaction require an appropriate supply of energy during any fermentation process. Stoichiometric relationship between medium components, cell biomass and metabolite formation during anaerobic fermentation is shown as:
Carbon+Nitrogen+Oxygen+other required components Biomass + CO2+H2O+Heat
Quantitative expression of the above equation is important for economical media designing. During the secondary metabolite production from microorganisms in any fermentation process, the medium formulation is required to meet maximum of the below mentioned criteria:
a. Give maximum cell biomass concentration per gram substrate b. Minimum or even no formation of by-products c. Consist of easily available and quality constituents d. Bear no or minimum problems during sterilization and medium formation e. Offer minimum resistance during aeration, agitation, extraction, purification and waste treatment f. Product formation should be free from any effects caused by foaming terminators
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Nutrient limitation and secondary metabolite formation are directly related to each other. During medium formulation following two factors are to be taken into consideration: a. Medium composition b. Fermentation conditions92
Table 1.3: Components of Fermentation
Medium composition Fermentation conditions
Carbon Source Ph Nitrogen Source Temperature Inorganic Phosphate Inorganic Salts Trace Metals Precursors Inhibitors Inducers
1.5.3.1. Medium Composition
1.5.3.1.1. Carbon Source
Carbon sources demonstrate species specific discrepancy in actinobacteria for cell growth and secondary metabolite production103,104. Glucose being one of the rapidly utilized sugars resulted in the ―Catabolic Repression‖105. It suppresses the synthesis of enzymes required for biosynthetic pathways. Glucose, lactose, maltose, sucrose, corn steep liqor, and molasses are the most widely used carbon sources that suppress the
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation production. Polyalcohols, polysaccharides, oligosaccharides, and oils are the favored non suppressing carbon sources due to the slow liberate of carbon during hydrolysis106,107.
Using glycerol as carbon source enhanced the production of metabolites due to their stabilizing effects on biosynthetic enzymes and production108,109. Alternatively sesame, groundnut and coconut oils have been used for anthracycline production110.
Cell biomass and secondary metabolite production is directly affected by the rate at which carbon source is utilized during fermentation process. Rapidly consumed sugars will reduce the production of metabolic products because of the fast growth of microorganism. During sterilization of the medium, the sugars should be sterilized separately to avoid the formation of black nitrogen containing compounds resulting from the reaction of sugars with amino acid and ammonium ion present in the medium. This will not inhibit the formation of secondary metabolite as well as the growth of the microorganism111.
1.5.3.1.2. Nitrogen Source Organic nitrogen sources when broken down into smaller units get incorporated + - into bacterial cells easily. Rapidly metabolized nitrogen sources e.g. NH4 , NO3 also inhibit the secondary metabolite production93. Reduction of antibiotic production however can be avoided by using appropriate nitrogen source112,107. Concentration of nitrogen needs to be in accordance to the carbon source concentration. Suitable physiological conditions favorable for secondary metabolite production in idiophase are created by selecting complex nitrogen source like soyabean meal in the medium. They maintain the balance of nutrient, low phosphorous contents and slow hydrolysis92,111.
Table 1.4: Microorganisms and antibiotics
Microorganism Antibiotic/Secondary metabolite Streptomyces antibioticus Oleandomycin Streptomyces erythreus Erythromycin
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Streptomyces kitasatoensis Leucomycin Streptomyces clavuligerus Cephamycin Streptomyces viridoflavum Candihexin Fusidium coccineum Fisidin
1.5.3.1.3. Inorganic Phosphate
Inorganic phosphate alterations strongly effect the secondary metabolites production from actinobacteria involving a mechanism consisting of six steps: a. Increased amount of inorganic phosphate in the fermentation medium causes the primary metabolism to accelerate due to increase in intracellular ATP concentration resulting in the active metabolic conversion113 b. Reallocate the carbohydrate metabolic pathway c. Constrain the synthesis of secondary metabolite pathway inducers114 d. Inhibits synthesis of secondary metabolite precursors115 e. Repress the production of phosphatases required for secondary metabolite production f. Divest the cell of with indispensible metals116
Consumption of carbon and nitrogen sources is greatly enhanced when Inorganic phosphate is used in large amounts. It also increased the respiration rate. As a result of which the growth of microorganism is accelerated but the production of secondary metabolites decreased consequently. During the fermentation of cephalosporin by Streptomyces clavuligerus, the production of antibiotic increased directly with concentration of inorganic phosphate until the concentration was 25mM. There was a corresponding decrease in the production of antibiotic with further addition of inorganic phosphate. Optimum concentration range of inorganic phosphate required for the growth of the microorganism is 0.3-300mM. However <10mM is required for the production of antibiotic117.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Biosynthetic enzymes production is also influenced by presence of inorganic phosphate at transcriptional level118. Surplus amount of glucose and phosphate proceed synergistically during fermentation process and represses the secondary metabolites production119.
1.5.3.1.4. Inorganic salts
Addition of inorganic salts like NaCl and KCl in the fermentation media enhances the production of secondary metabolites. However the concentration above optimum level has negative effects on antibiotic production111.
1.5.3.1.5. Trace metals
Fermentation processes require not only the enzymes but also the substrate and co-factors for their smooth completion and secondary metabolite production. Co-factors include the trace metals that are directly related to the production of antibiotics and show their active participation. Lower concentrations of trace metals are required for the secondary metabolite production as compared to that required for the growth of microorganism. Addition of metals above the optimal level will inhibit the production117.
1.5.3.1.6. Precursors, Co-factors and Nucleotides
The regulation of secondary metabolites production depends upon the accessibility of the precursors and co-factors120. Due to low substrate specificity of biosynthetic enzymes, precursors can be added as akin to give the hybrid products121,122. The production of efrotomycin by Nocardia lactamdurans, depends upon the availability of uracil affecting the rate limiting step during this metabolite production and is the precursor of pyridine moiety. Compounds that are commonly used as precursors for the secondary metabolite production are the short chain fatty acid123,124, purines and nucleotides. The precursors used for the regulation purpose are the adenosine and guanosine phosphate125.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
The precursors when added to fermentation medium are found to increase the production of antibiotics. Phenyl acetate when added as precursor during penicillin fermentation enchased the production of Penicillin-G from Penicillium chrysogenum126.
1.5.3.1.7. Inhibitors
Ethionine and sulfa drugs when added to the fermentation medium strongly inhibit the production of antibiotics. They effect the carbon transfer reactions to antibiotics and resulted in the demethylated antibiotic derivatives. However there is an assumption that there are few compounds that act as inhibitor for the production of one metabolite but not for the other metabolite111.
1.5.3.1.8. Inducers
Inducers and inhibitors not only effect the production of primary metabolites but also influence the production of secondary metabolites. Most often they work together and it is very difficult to identify the inducer in the medium formulation92.
1.5.3.2. Fermentation conditions
1.5.3.2.1. pH
The production of secondary metabolites as well as the growth of the microorganism is affected by the pH of the medium during fermentation in a similar way as affected by the medium constituents and temperature. Also the mycelial morphology is affected by this parameter127. Pronounced antimicrobial activity is shown by Microbispora spp. of actinobacteria by increasing the pH of medium from 5.5 to 7.5128.
When secondary metabolites production is decreased during fermentation it is not only due to the glucose or K2HPO4 but pH effects are also important. During
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation fermentation of helvolic acid and cerulenin from Cephalosporium caerulens, the pH of medium when adjusted to 6.0 with K2HPO4, CaCO3 or NaHCO3, the production of helvolic acid was increased while that of the cerulenin was decreased gradually. For optimum production of secondary metabolites during fermentation processes the pH has to be controlled either by adding any buffer to the medium or using a nutrient as part of medium constituent. Calcium carbonate (CaCO3) has been used most of the times to maintain the media pH at 7.0. Appropriate carbon to nitrogen ratio is also found to establish the constant pH of the medium. The pH is required to be constant after sterilization but the severe conditions of high pressure and temperature will change the medium pH so it needs to be maintain externally even after sterilization by the addition of ammonia or sodium hydroxide and sulfuric acid129.
Intracellular pH of microorganisms is kept neutral. An increased in the pH across the cytoplasm membrane triggers the cell to direct its resources towards maintaining the preferred intracellular pH130. Intracellular pH is thus the dynamic factor used for maintaining optimal conditions for extracting secondary metabolites131.
1.5.3.2.2. Temperature
The most important environmental factor that affects the production of secondary metabolite is the temperature. Secondary metabolite expression genes and decaying rate of Secondary metabolite are influenced by variations in the temperature132. Temperature range for excellent production of antibiotics should be very narrow i.e. 5 ~ 10 degrees111.
Production of secondary metabolites and antibiotics from thermophilic actinomycetes takes place at temperature greater than 40°C however for Streptomyces the temperature required for the production is near 27°C. In general practice the temperature has to be constant throughout the fermentation but temperature requirements for the microorganism growth and secondary metabolite production are different from each other as in case of penicillin producing strain which grow best at 30°C and the production is
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation good at 20°C. Thus temperature should be considered individually for growth and secondary metabolite production129.
1.6. SECONDARY METABOLITES
Complex and lengthy biosynthetic pathways of microorganism in submerged cultures resulted in the production of some complex intermediates structures known as secondary metabolites.
Precursors and energy produced during primary metabolic reactions resulted in the production of secondary metabolites133. They are obtained from only restricted groups of bacteria as a mixture of closely related compound of chemical nature134. The habitats usually influence the production of secondary metabolites. Terrestrial habitats are most suitable for their production as against the marine habitats which provide opportunity for producing the high yields of metabolites having novel structures135.
1.6.1. Production
The assumption about the production of similar secondary metabolites by different groups of microorganisms is that this is due to the direct transfer of the corresponding biosynthetic pathway genes which are responsible the production of secondary metabolites136. The β-lactams produced by actinobacteria and ascomycete fungi best illustrate the speculation as they follow the similar biosynthetic pathways for the production of these β-lactams137. The entire phenomenon is supported by the endosymbiont theory which states that during the evolution, whole microorganism with their complete genetic makeup and metabolism gets incorporated into eukaryotic cells and develops into mitochondria and chloroplast. Biosynthetic pathway once established for the production of secondary metabolites in one organism can then be transferred to other microorganism also138.
Secondary metabolites found in nature mostly contain polyketides, terpenes, steroids, shikimic acid and alkaloids groups133. Alteration and combination of various reactions during primary metabolic pathways resulted in diverse structure of secondary metabolites having low molecular weight about less than 1500 Daltons139.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Following four steps are involved in the biosynthesis of secondary metabolites in actinobacteria:
a. Nutrient uptake by the cells and their gradual conversion intermediate primary metabolites b. Production of secondary metabolite is then induced by the accumulation of primary metabolites and signaling molecules c. Breakdown of primary metabolites through certain pathways specific for the production of specific secondary metabolite and mostly include fatty acid metabolism and carbohydrate metabolism d. Specific genes are required for the production of secondary metabolites140
1.6.2. Functions
Production of secondary metabolites is beneficial most importantly to the microorganism producing it.
They destroy other microorganisms because of their chemical nature and increase their chances of fitness and survival in the nature141. Soluble metal ions are very important for the microorganism. Production of secondary metabolites helps in the providing these ions for the survival and nourishment of the microorganism142. They show their prompting action in the differentiation process for a no. of sporulating bacteria143.
1.6.3. Resistance mechanisms and secondary metabolite secretion
A number of resistance mechanisms are present in the actinobacteria that need to be elicited. These mechanisms will determine the degree of susceptibility to self inhibition from secondary metabolite144. These mechanisms which provide defense to the producing bacteria from these secondary metabolites are described as follow:
a. Antibiotic detoxification using enzymes b. Modification of target points in the cell
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
c. Restriction of antibiotic entry into the cell and altering the cell permeability for antibiotic to be out145 d. Sequestering of the secondary metabolite using the proteins present in the cytoplasm146,147 e. Reduction of antibiotic production during the growing phase of bacteria148 f. In addition to these mechanisms certain biosynthetic genes are also required that play very important role during this resistance regulation.
1.7. AVERMECTINS
During 1940‘s the production of anthelmintic products were only one every five years. The optimum dose (mg/kg) required to kill the gastrointestinal nematodes in different organisms especially the sheep had been reduced with increasing the production of these products and discovering the new of them149. The potency of anthelmintic entities was increased about 25 times more as compared to the other products that had been discovered at that time with the discovery of avermectins53. Also concentration required for the application was reduced from milligrams per kilograms to micrograms per kilogram. However avermectins were not active against the trematodes and cestodes150. They showed broad spectrum of activity against acarine and insect plant pathogens151. It has been found during veterinary development to show excellent microfilaricidal activity against Dirofilaria immitis infections present in dogs152 and Onchocerca cervicallis microfilaria found in horses153,154.
Ivermectin has been administrated in about 600,000 people against human onchocerciasis155 as most effective drug due to reduction in adverse reactions and skin microfilarial count156. They have been widely used in animal and human health and crop protection because of their safe mode of action. Large number of avermectin analogues showing endectocidal activity has been recognized157.
1.7.1. Structure
Avermectins are 16-membered pentacyclic compounds having polysaccharide of methylated deoxysugar L-oleandrose polyketide158. Avermectins are a combination of
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation eight components i.e. A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b159. The main difference in the structure of these components is at C5, C22-C23 and C26. The major component is B1a160,161. The ratio between ―a‖ and ―b‖ component is 80:20 or 90:10 showing that ―a‖ components are major and ―b‖ components are minor. So it is required to enhance the production of ―b‖ components which is achieved by mutagenesis162.
The difference between component ―A‖ and ―B‖ is that the component ―A‖ has methoxy group at C5 and component ―B‖ has hydroxyl group at C5. The ratio of ―A‖ and ―B‖ is 35:65163. Component ―A‖ has been originated from component ―B‖ by methylation at C5 of component "B‖164. Conversion of ―B‖ component to ―A‖ component is believed to catalyze by avermectin O-methyltransferase. It will shift methyl group S- adenosylmethionine (SAM) to C5 hydroxyl of avermectin ―B‖ component and produce avermectin ―A‖ component along with S-adenosylhomocysteine165.
Also group ―1‖ is supposed to be derived from group ―2‖ through dehydration at C22 and C23. In component ―a‖ 2 methylbutyryl group is present at C25 which is derived from Isoleucine. Component ―b‖ has isobutyl group at C25 obtained from Valine. There is no way to convert component ―A‖ to component ―B‖ via demethylation166.
Figure 1.3: Structure of Avermectin
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
The ―a‖ and ―b‖ components have been found to show almost the same biological properties and their separation is very difficult, that is why ivermectin is marketed as 167 mixture of > 80% 22,23 dihydro B1a and < 20% 22,23 dihydro B1b mixture . Doramectin, the avermectin with cyclohexyl group at C-25 is the mutational biosynthetic fermentation product of S.avermitilis168.
Table 1.5: Structural differentiation between Avermectins
Avermectin R1 R2 X-Y
A1a -CH3 -C2H5 CH=CH
A1b -CH3 -CH3 CH=CH
A2a -CH3 -C2H5 CH2-CH(OH)
A2b -CH3 -CH3 CH2-CH(OH)
B1a -H -C2H5 CH=CH
B1b -H -CH3 CH=CH
B2a -H -C2H5 CH2-CH(OH)
B2b -H -CH3 CH2-CH(OH)
1.7.2. Physical properties
Avermectins are lipophilic, therefore they are soluble in organic solvents and are insoluble in water167. They are photo degradable and result in the isomerization by cleavage of C8-C9 and C10-C11 bonds when exposed to ultra violet radiations. Acid sensitivity of avermectin resulted in the separation of C-13 sugars when treated with diluted hydrochloric acid169.
Abamectin is white to yellowish crystalline powder170. When exposed to heat of flame, it facades a slight fire vulnerability. There would be combustion and detonation hazards in the presence of oxidizers. When putrefied thermally it discharges toxic oxides of carbon171.
Chemical name: Avermectin
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Chemical family: Anthelmintic
Molecular formula: C48H72014 avermectin B1a
C47H70014 avermectin B1b
Molecular weight: 873.11 avermectin B1a
859.08 avermectin B1b
Physical form: colorless to pale yellow, crystalline
Melting point: 150-155°C
Vapour pressure: <200nPa
Specific gravity: 1.16g/cm
Stability: stable to hydrolysis in aqueous solutions at pH 7 and 9 at 25°C
Solubility: practically insoluble in water: 7.8ppb
Soluble in aecetone, methanol, acetonitrilie, isopropanol, toluene171
1.7.3. Classification
Avermectin insecticides include:
a. Abamectin
Avermectin B1a
Avermectin B1b
b. Ivermectin
22,23-dihydroxy-avermectin B1a
22,23-dihydroxy-avermectin B1b
c. Doramectin
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
25-cyclohexyl-5-O-demethyl-25-de(1-methylpropyl)-avermectin A1a
d. Emamectin
// // // (4 R)-4 -deoxy-4 -(methylamino)-avermectin B1a // // // (4 R)-4 -deoxy-4 -(methylamino)-avermectin B1b
e. Eprinomectin
// // 4 -deoxy-4 -epiacetylamino-avermectin B1a // // 4 -deoxy-4 -epiacetylamino-avermectin B1b
f. Selamectin
(5Z,25S)-25-Cyclohexyl-4/-O-de(2,6-dideoxy-3-O-methyl-alpha-L-rabino- hexopyranosyl)-5-demethoxy-25-de(1-methylpropyl)-22,23-dihydro-5- 172 (hydroxyimino)-avermectin A1a .
1.7.4. Abamectin (Mixture of B1a and B1b)
Abamectin, the animal endectocide173 has been obtained from the screening of avermectins and is found highly effective against phytophagous mites and insect pests and has been selected as pesticide for agriculture and horticulture crops. It is a mixture of B1b and B1a. About 5-27 grams per ha have been used as pesticide in the form of foliar spray to agricultural and horticultural crops, citrus fruits and cotton174.
For marketing purpose the recommended dose of injectable preparation was 200µg/kg175. Excellent anthelmintic and ectoparasiticidal activity was found for commercialized doramectin [25-cyclohexyl-5-O-demethyl-25-de(1-methylpropyl) avermectin B1a]168. Study of carcinogenicity in rat and mice exposed the abamectin to lack carcinogenic potential176.
1.7.5. Biosynthesis of avermectin from Streptomyces avermitilis
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
A novel method for the production of avermectin from Streptomyces avermitilis is mutation177. Substitution at C-25 position of avermectin resulted in a series of compound by addition of biosynthetic precursors to mutant strain of S.avermitilis that would have been otherwise impossible168.
1.7.6. Spectra of activity
The avermectin lack the significant activities against trematodes and cestodes being deficient to GABA-mediated neurotransmission properties in the producing microorganisms178. Nematocidal and insecticidal activities also depend upon the presence of glutamate-gated chloride channels172.
The endectocidal macrocyclic lactones show little effect on adult filarial parasites however they are extremely useful against microfilarial stages of filaroids. This stage demarcation is supposed to be found due to the lacking ability of the drug to reach the preferred site of action or intrinsic mechanistic insensitivity of the mature parasites. Avermectins are exceptionally valuable against the adult as well as the emergent larvae of gastrointestinal nematodes and the lungworm D.vivipurus when applied subcutaneously at a dose rate of 50pg/kg179.
Macrocyclic lactones particularly the avermectins show tremendous activities against the arthropods requiring the contact with or ingestion of host body tissues with the drug. Commercial applications are efficient against all parasitic stages of warble flies, burrowing mites, non-burrowing mites as well as the sucking lice. Efficiency of avermectins is however much less against the D. bovis feeding on outer layers of hair shafts and dermal scales. Parenteral application against mites will require a no. of days to be effective. Oral administration of ivermectin to cattle diminishes its efficacy due to lower or tainted tissue distributions. In dogs avermectins causes the prophylaxis of D.immitis. They show actions against canine nematodes including the Toxocara canis, Toxascaris leonine, Ancylostoma caninum, Ancylostoma brasiliensis and many other ectoparasites as well. Proficient activities are also shown against Dirofilaria immitis and Onchocerca cervicalis microfilaria in dogs and horses respectively157.
1.7.7. Toxicological effects
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Toxicological effects of avermectins were not found very high when compared with that of other therapeutic xenobiotics because of their low concentrations (tens of milligrams per kilograms) required as oral supplementation for the mammals. The key neurotransmitter in the central nervous system of mammals is gamma aminobutyric acid (GABA). The avermectins at toxic doses when given to the mammals caused the neurotoxicity176.
These anthelmintic compounds showed specific anti parasitic activity because very low concentrations are used for the stimulation of parasite specific glutamate gated -6 180 chloride ion channels whereas higher concentrations at about EC50 2 × 10 M are required to stimulate the release of GABA in the vertebrate brains. The macrocyclic lactones could not access the central nervous system GABA transmittance sites because of the presence of brain barriers which partially resist their distribution181.
In most of the mammalian species, the acute toxicity of avermectin in the form of ataxia, tremors and depression followed by death appears in the most similar way176,182. Identification of breed sensitivity in Murray Grey cattle in Australia was considered due to the presence of avermectin in the central nervous system183.
Qualitatively the toxicity of avermectins is considered to be similar to that of the ivermectin184, 182. Chronic effects of different doses of avermectin and milbemycins were studies and evaluated as 0.4 mg/kg/day for rats, dogs, 0.5 mg/kg/day for Rhesus monkey and > 1.2 mg/kg/day for neonatal Rhesus monkey. In human the estimated dose was 150µg/kg against onchocerciasis which was lesser than determined for Rhesus monkey185.
Ivermectin has been found to produce cleft palate and clubbed forepaw in rats and rabbits respectively when given in high dose. However, the administration of ivermectin at low level has revealed that it does not adversely affect the male reproductive system of animal species186,187 and therefore does not contribute towards the genotoxic activity177. In human the application of ivermectin against O-valvulus resulted in rash, fever, itching, headache, lymphadenopathy and tenderness in lymph node. The side effects of
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation ivermectin application are very low about 15% to 35% as compared to that of diethylcarbamazine application188,189.
1.7.8. Environmental effects
The fecal material of ivermectin treated animal degraded slowly as compared to the non ivermectin treated animals thus largely effecting the environment. Decaying rate of harmless degrades and the harmful effects of active excreta distress our ecosystem190. These excreta however sometimes show insecticidal properties to control the nuisance flies191,192,193. Half life of ivermectin in summer and winter is 7-14 and 91-217 days respectively and degradation in soil resulted in less toxic compounds which do not remain available for the soil organisms because of their binding to the soil194.
1.8. APPLICATION OF AVERMECTIN B1b
Avermectins mainly the abamectin are the secondary metabolites derived as fermentation product of S.avermitilis and have been used extensively to control both the endo- and ectoparasites195. Because of their broad spectrum effectiveness, easy handling and wide margin of safety to target animals, they have been used by the farmers all over the world. They are used as active components of many insecticidal and nematocidal products in agriculture196 and a part of veterinary medicines to control and prevent the parasitic diseases197,198,199. A very small dose is even very effective195,53.
The veterinary medicines can be degraded, transported and distributed among different soil compartments when released to the environment however very slow degradation of avermectin and their insolubility in water makes them to be less distributed in the soil200,201 and also allows them to be retained unaffected in the feces of effected animals and it is possible to recover more than 90% of the total drug used. Abamectin is easily photo degraded however it disappears from the soil slowly and has a half life of about 2-8 weeks202.
Different studies revealing the effect of abamectin on flies and beetles assaulting freshly deposit feaces in the field have been demonstrated however no study showing the dose-response relationship is available except for earthworms203.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Only a little research addressed the effect of avermectin mainly the ivermectin on soil dwelling organisms135 and the toxicity of abamectin to earthworm203.
Earthworms make up about 60-80% of the total animal volume in soil204,205. Their Presence in soil is very important in order to retain soil porosity, the fertility and improved soil structure, physical and chemical alteration of soil organic matter present in soil, configuration and stabilization of soil aggregates206,207,208. Being susceptible to soil chemicals because of their thick cuticle209 insecticidal bioaccumulation will not directly affect them however it will cause severe smash up to higher tropical levels210. Being apposite bioindicators of soil contamination earthworms can be used to impart safety sill for insecticidal treatments211.
Although beneficial to the soil, the presence of earthworms resulted in reduction of biomass and thickness of litter layer as is previously reported212. Forest decline, loss of native plant species, Soil erosion, increased humification and decomposition is resulted as litter layer depleted213,214.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
OBJECTIVES AND TARGETS:
Objectives:
The main objective of the present study is the enhanced production of avermectin B1b used as anthelmintic and insecticidal agent from Streptomyces avermitilis DSM 41445 and by locally isolated strains. The aim of the study was also to boost the Socio-economic status of Pakistan.
Targets
To design a medium suitable for the production of avermectin B1b from Streptomyces avermitilis DSM 41445 through submerged fermentation To isolate Streptomyces from soil samples To screen Streptomyces avermitilis strains capable of producing avermectin from the soil isolated Streptomyces To improve production of avermectin B1b through strain improvement strategies involving UV irradiation and chemical mutagenesis To manually optimize different carbon and nitrogen sources and their concentration to obtain maximum yield of avermectin B1b To statistically optimize medium components and fermentation conditions to obtain maximum yield of avermectin B1b To compare and select two statistical optimization approaches named artificial neural networks and response surface methodology To purify the avermectin B1b obtained from hyper producing Streptomyces avermitilis strain To check the anthelmintic and insecticidal properties of the purified avermectin B1b
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
LITERATURE REVIEW
1. Streptomyces avermitilis 2. Isolation, selection and screening of Actinomycetes 3. Secondary metabolite production 4. Effect of different factors on secondary metabolite production 5. Mutagenesis 6. Optimization 7. Avermectins
2.1. Streptomyces avermitilis
Avermectins lacking antimicrobial activities and oligomycin with characteristic antifungal properties have been reported to be produced as fermentation products and had been extracted using solvents like methanol215.
Certain species of Streptomyces avermitilis inspite of having enzymes necessary for the production of avermectin had been reported to lack the ability to produce avermectin during fermentation. The cell free extracts of S. avermitilis possessed the avermectin aglycon enzymes named dTDP-Oleandrose glycosyltransferase216.
In the linear chromosome of S.avermitilis about 8.7 million base pairs were reported. Out of which 6.4% represented the base for secondary metabolite production53. Gene clusters of 25 different kinds are responsible for production of melanin pigments, tyrosinase and ochronotic pigments formation. Type-1 and type-2 polyketide compounds also synthesis required different gene clusters.
Endospores formation is the key feature exhibited by Streptomycetes during their life cycles and also by Streptomyces avermitilis during submerged fermentation. It was observed that higher concentration of cells was obtained after the fermentation medium
CP1 was resuspended in phosphate buffer of pH 7.2 and 0.2% CaCl2 and had more glucose and lesser amount of phosphate. Endospores resulting during submerged
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation fermentation showed heat and lysozyme resistance and pertained other characteristic properties of microbes217.
Ara et.al isolated only six Streptomyces strains from soils of Al-Kharj (K) and Al- Madina (M), and characterized them on basis of morphological and biochemical characterization. All isolated Streptomyces strains howed strong antimicrobial activities when tested against different human pathogenic microorganisms. The compound with highest antiviral activity obtained after fermentation was extracted by solvent extraction and mixed with tobacco mosaic virus (TMV) obtained from tomato plants after homogenization in water and simple filtration. One week incubation of mixture with Datura metel plant resulted in reduced lesions across the leaves showing their positive effects218.
2.2. ISOLATION, SELECTION AND SCREENING OF ACTINOMYCETES
Various medium and antifungal agents used for Actinomycetes isolation, the Starch-casein medium supplemented with actidione and nystatin as antifungal agents proved the best medium for actinomycetes isolation from soil219.
The complications of random screening can be avoided by using a combination of efficient culture selection and cultivation method along with the detection method during isolation of antibiotic producing strains from the soil220.
Spreading of soil dilution on water agar supplemented with75g/ml Cephalosporin C have been described221 for selective isolation of Actinomadura melliaura.
A method for isolation of Streptomyces hygroscopicus producing the antitumor antibiotic, the phospholine, has been reported222 along with methods for physical and chemical characterization of the antibiotic and taxonomic classification of the isolated strain.
A method had also been described earlier223 for isolation of antitumor antibiotic producing Micromonospora echinospora strain from soil.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Highly selective isolation of Streptosporangium and Dactylosporangium from soil and removal of unwanted bacteria and actinomycetes contaminations were made possible by methods of drug heating and benzethonium chloride (BC) 224.
To avoid the problems to be faced during isolation of actinomycetes a method has been specified225 for extracted DNA analysis for identification of Streptomyces obtained from different locations, habitates and environments.
A three step method including baiting the species with Pinus pollen grains, desiccation in silica gel and finally the spore liberation on water immersion for simplified isolation of Actinoplanes spp. from soil has been reported226. This method performed using HV Actinoplanes spp.
Plate culture technique avoiding contamination caused by unicellular bacteria and unwanted actinomycetes employed treatment of water suspended soil with 1% phenol and subsequent culturing on humic acid-vitamin agar and has been described227.
DNA analysis and physiological characterization of the culture Streptomyces KS3-5 isolated from soil and strongly inhibiting the growth of gram positive and gram negative bacteria had shown it to closely resembling to Streptomyces toxytricinii228.
A method has been described229 to isolate selective genera including Herbidospora, Microbispora, Microtetraspora and Streptosporangium of Streptosporiaceae family from soil using HAV agar plates treated with chloramine-T.
Conventional methods of characterization along with genetic and phenotypic database identified S. avermitilis MA-4680 to be closely related to S. cinnabarinus, S. griseochromogenes, S. resistomyces and S. viridochromogenes. Taxonomic status based on Polyphasic methods represented the phyletic line 165 rDNA Streptomyces tree as is reported50.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
It is reported230 that 19 out of 47 isolates from Antartic soil showed strong activity against both gram positive and gram negative bacteria. Two isolate numbered 23 and 29 were supposed to be of same class and all other were very different from each other. Fermentation of these strains resulted in the production of anti bacterial compounds with non polar domains and no. of active components.
About 22% Actinomycetes isolated from soil samples using chitin agar medium by Rong-Yang et.al showed characteristic antifungal activities. Extracellular secondary metabolite produced in late log phase from these antifungal isolate. Conventional methods of characterization proved one of the isolate to be Streptomyces showing promising activities against yeasts and molds231.
Soil oriented Streptomyces are good sources of antibiotics with wide spectrum of activities against bacteria as is reported before71. The excellent antimicrobial activities were shown by Moula and Turkey soil isolates when tested against different microorganisms with maximum 30mm zone of inhibition.
Streptomyces NRC-35 has been isolated and identified on the basis of spore morphology, cell wall chemo-type and nucleotide sequence analysis found to show β- lactamase inhibition as a result of clavulanic acid production232.
Four genera of Actinomycetes namely Nocardia, Streptomyces, Actinomyces and Micromonospora were isolated from Sundarbans soils. Results reveled that Sundarbans soils were rich source of Actinomycetes with broad spectrum of antimicrobial activities and characteristics of pharmaceutically important antibiotics production during fermentation233.
Actinomycetes isolates obtained from soils of Ghaziabad were found to exhibit antibacterial activities and were characterized in different culture media using conventional methods. Zone of inhibition>20mm were observed by seven isolates234.
Soil samples of India were tested for the isolation of actinomycetes using crowded plate method and tested for the production of antibiotics in Soyabean casein agar medium and actinomycete isolation agar medium. Isolate A-4 with good aerial and cellular growth
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation was proved to be the member of Actinomycete genus after morphological and physiological characterization according to the International Streptomyces Project235.
Different pathogenic bacteria had been employed for the effective isolation of 42 isolates from the mangrove forest soil pretreated with Dry heat method in media KUA, AIA and SCA supplemented with nalidixic acid and cycloheximide at concentration of 75µg/mL and 80µg/mL respectively. In the mentioned media the actinomycete population density was 22 CFU-10-6/gm, 12 CFU-10-6/gm and 8 CFU-10-6/gm respectively. Out of 42 isolates, only 22 were able to produce secondary metabolites and showed antibacterial activities. However only one isolates designated as A107 possessed the characteristic features of Streptomyces spp236.
2.3. SECONDARY METABOLITE PRODUCTION
Antibiotic cephamycins closely resembling to cephalosporin C and having extraordinary activity against broad spectrum of bacteria had been obtained during submerged fermentation of one of the actinomycete isolated from the soil237.
Secondary metabolite fumaramidmycin had been reported to be produced from Streptomyces kurssanovii only on agar plates isolated from soil and showed activity against gram positive and gram negative bacteria238.
Component crisamicin A of complex crisamicins belonging to isochromanequinone group of antibiotic has been produced from Micromonospora purpurea is highly active against positive bacteria239.
Batch and fed batch fermentations of Streptomyces coelicolor resulted in the production of actinorhodin in phosphate and nitrate rich fermentation medium. About two fold production of actinorhodin has been obtained by following the method with glucose being fed into the bioreactor continuously240.
A strain of Streptomyces with gray colored aerial mycelium and spores of smooth surfaces producing brownish-orange crystalline antibiotics named A-60 had been isolated from soil as is reported241. Improved antibiotic production was obtained in medium
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation supplemented with MgSO4, MgCl2, MnCl2 FeCl2, FeSO4 and BaCl2 with enhanced activities against gram positive bacteria.
Antibiotics aristostatin A and aristostatin B are strongly effective against gram positive bacteria and show antitumor activity. It is reported242 that these antibiotics are produced from actinomycete TP-A0316 and have been extracted by solvent extraction.
Antibiotics simocyclonones D4 and D8 with potent activities against gram positive bacteria have been produced from mycelium extract of Streptomyces antibioticus as reported243. They were found to show cytostatic effects on tumor cell lines. Their presence in the culture broth was confirmed by HPLC having diode array detector and HPLC electrospary mass spectrometry screening.
The peptide sequence of antibiotics Stretocidin A, B, C and D produced from Streptomyces spp. Tii 6071 has been found to be closely related to secondary metabolites tyrocidine and gramicidine produced from B. brevis as is reported244.
The mutant strains of S. peucetius having the ability to produce daunorubicin during fermentation were selected and isolated on the basis of colour on agar plates as is reported245. The steps involved during mutation were catalyzed by dnrE, dnrS and doxA.
The strain Streptomyces Tii 6239 was characterized for the production of three secondary metabolites belonging to macrolactum group, the ripromycin and had been isolated from the culture broth and mycelium extract after fermentation as is reported246.
Antibiotic belonging to heptaene group of polyene antifungal antibiotics has been reported to be produced from Streptomyces purpeofuscus CM 1261 showing antagonistic behavior against the most common human pathogenic fungi as is reported247.
Romanian soil isolated from Micromonospora strain Tii 6368 has been reported248 to produce retamycin, galtamycin B and Saquayamycin Z along with lumichrome derivatives active against human tumor cell lines.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Avermectin production from Streptomyces avermitilis is associated with synthesis of certain volatile odorous compounds like geosmin and oxolones as is revealed249 when studying the effect of cultivation time. About 2g/L and 4g/L enhanced oxolones and geosmin respectively was observed with increasing the cultivation time along with avermectins. Optimal cultivation time for Oxolones production was 7 days after that its production began to decrease.
Production of oligomycin A, showing pronounced activity against human hepotoma, chronic myelogenous leukemia and carcinoma cell lines, has been reported from strain L003 being morphologically and physiologically similar to Streptomyces avermitilis. HPLC and NMR had been used for the purification and structural verification of oligomycin A250.
Streptomyces lividans 66 and Mutant strains relA of Streptomyces coelicolor A3(2) have been reported to produce actinorhodin (Act) and undecylprodigiosin respectively251. Relationship between activated actinorhodin and undecylprodigiosin production and expression of ActII-ORF4 and RedD along with regulators that is specific for Act and RedD biosynthetic pathways had been studied using Western blotting analysis.
The production of anti parasitic agent, the Doramectin, has been observed from mutant strain of Streptomyces avermitilis obtained by deletion of two genes named as branched-chain α-keto acid dehydrogenase encoding gene (bkdF) and oligomycin PKS encoding gene cluster (olmA) in the chromosome of Streptomyces avermitilis 76-05 as reported252. On industrial scale this genetically stable mutant can be used for higher productions of Doramectin.
Avermectin producing Streptomyces avermitilis strain has been reported to produce anti cancer component, the oligomycin A253. Oligomycin A has been reported to be produced from S.avermitilis L033. It is highly effective in the inhibition of in-vitro proliferation of human hepotoma, chronic myelogenous leukemia and colonic carcinoma cell lines. Purification and structure elucidation of the compound was done by crystallization and Nuclear magnetic resonance and mass spectrometry respectively.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
2.4. EFFECT OF DIFFERENT FACTORS ON SECONDARY METABOLITE PRODUCTION
The enzyme avermectin O-methyltransferase responsible for the conversion of avermectin B component to avermectin A component coordinately regulated the genes responsible for the SM production. This enzyme will not control the rate of the reaction for the conversion but it will increase the production of avermectin in direct manner as reported previously254.
Carbon mainly the glucose being the major component of fermentation medium is found to suppress not only avermectin production but also the activity of 6- phosphogluconate dehydrogenase during pentose phosphate pathway as is reported52. About 2-fold enhanced avermectin component production observed when glucose was added at late stage of fermentations.
L-proline and glycine were the optimized nitrogen source for the maximum production of macrolide polyene antibiotics PA-5 and PA-7 from Streptoverticilllium spp.43/16 as is reported255 along with manganese and magnesium being the best metallic ion. Increase in concentration of glucose after an Optimum of 40mM, production began to decrease. Negatively effecting nitrogen sources reported were L-cysteine and L-valine.
Qualitative and temporal relation between production of avermectin and lipid fraction from S. avermitilis during stationary phase of fermentation has been reported256 and was found to be effected by glucose. The results revealed that avermectins and fatty acids products had competed for the same precursors.
Effect of limited amount of glutamate, growth rate, temperature and pH on antibiotic production from Streptomyces thermoviolaceus has been reported129. The optimum levels for maximum production were 0.1-0.15h-1, 35oC and 6.5-7.5 for growth rate, temperature and pH respectively.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Production of spiramycin from Streptomyces ambofaciens is strongly affected by concentration of ammonium as investigated257. Replacement of ammonium ion with catabolic intermediate in medium will mask its negative effect if used in excess.
The behavior of Streptomyces coelicolor towards formation of extracellular actinorhodin during fermentation using two liquids system named perfluorocarbon (oxygen carrier) has been investigated258 and resulted in five folds more actinorhodin production. Drop size distribution and liquid-liquid interfacial area being the major factors were subjected to the effect of dispersed phase volume fraction known as PFC phase and the results revealed the direct relationship between liquid-liquid interfacial area and PFC concentration.
Medium salinity has pronounced effects of avermectin and oxolones production as reported259. The optimal NaCl concentration was 0.5% after that the production of both began to decrease. However the salinity has indirect effect on geosmin production.
About 12.8-13.8% more avermectin B1a from Streptomyces avermitilis has been observed in medium supplemented with 0.8% propionate after 24h of incubation as reported260. Feeding of 1.0% glucose at 4d, 5d and 6d of fermentation resulted in
780mg/L avermectin B1a. The components of medium used in the present study made it possible to obtain higher concentrations of avermectin even on industrial scale.
Pronounced effects of potato paste and fresh yeast present in fermentation medium have been observed on avermectin production from Streptomyces avermitilis ATCC 31272 and its mutants named Sa-76-9 obtained through high energy electronic current and nitroso guanidine treatment. 3500-4000 µg/mL avermectin production was obtained from mutant strain in medium containing starch, dry yeast, soy bean meal and inorganic salts as mentioned261.
Decreasing the concentration of glucose in the fermentation medium resulted in increased avermectin ―A‖ and ―a‖ ratio. In a previous study it was reported mutant
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation obtained from the given strain was resistant and O-methyl-L-threonine resulting in increased production of avermectin ―a‖ component and decreased concentration of avermectin ―1‖ component262.
Nonpolyenic macrolide antibiotic AK-111-81 produced at low growth rate from Streptomyces hygroscopicus 111-81 was enhanced when Lactose or glycerol, ammonium succinate and Mn+2, Cu+2 and Fe+2 were used as carbon, nitrogen sources and divalent ions in the production medium as reported117. A combination of all these components resulted in about 6-folds increase in AK-111-81 production.
In a study conducted previously263, they revealed that 1-20 mmol/L phosphate when added in the fermentation medium, production of avermectin was not effected showing that biosynthesis of secondary metabolite can tolerate higher concentrations of phosphate present in the fermentation medium.
Effect of nitrogen on production of streptolydigin and various other secondary metabolites has been investigated from Streptomyces lydicus AS 4.2501. LCMS and photodiode array analysis revealed that two streptolydigin analogues were obtained when a mixture of Peptone, asparamide and glutamic acid was used in fermentation medium as N-source. Use of Soyabean meal resulted in three analogue of streptolydigin after fermentation. Negative effects on streptolydigin were shown by ammonium sulfate264.
Effect of various parameters including nitrogen source, inoculum size and dissolved oxygen on avermectin production from Streptomyces avermitilis has been investigated265. Mixture of soyabean meal and yeast meal in fermentation medium with at 4.3% inoculum was optimized for pellet formation and maintenance of morphology at DO>20% studied using image analysis tools. Enhanced pellet formation obtained at higher concentrations of dissolved oxygen.
Oxygen uptake rate being an important indicator of cellular activity greatly enhanced avermectin B1a production and has been employed for determining the effect of
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation glucose feeding during fed-batch fermentation for the production of avermectin B1a. The results revealing enhanced avermectin B1a production using this strategy. It is concluded that enhanced production of avermectin B1a during this technique as compared to the fed batch fermentation was due to the availability of 5228U/Ml necessary organic acid precursor266.
2.5. MUTAGENESIS
It is reported267 that the mutant strain Streptomyces fradiae JS6 obtained after physical and chemical mutagenic treatment lost its ability to recover the damaging of DNA. Just like the parent strain, the JS6 being resistant to all the mutagenic agents has the ability to control the mutagenic DNA repair system revealing the existence of strong mechanism in Streptomyces to avoid any error as compared to the single celled eubacteria.
In Streptomyces plasmid DNA can be removed by ethidium bromide being used as DNA intercalating agent as is reported86. Mutations caused by other sources not only resulted in the chromosomal and phenotypic changes but also loss of resistance against antibiotics and enzyme activity along with their ability to produce spores.
The isolation and characterization of avermectin methylation deficient mutants CR-1, CR-2, CR-3 and CR-4 of S. avermitilis has been reported268. This deficiency might be due the absence of avermectin B2 O-methyltransferase (B2OMT) activity and mutants were capable to produce only avermectin B component. They could not methylate the / // oxygen atom of oleandrose moiety at C3 and C3 . Second mutation of CR-1 resulted in CR-6 having ability to produce demethyl avermectin B component. The mutant CR-5 of
S. avermitilis had avermectin B2 O-methyltransferase and was capable of producing avermectin A and B components although did not have methyl group in their molecule.
The UV irradiation of Streptomyces spp. 1254 resulted in two mutant strains. One of the isolated mutant strains named 113 produced mutactimycins with excellent activity against bacteriophage of B. subtilis and was mycolic acid deficient. The other isolated
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
UV mutant strain named 2-6 resulted in water soluble antimicrobial antibiotic and was found to belong to Streptomyces genus just like the parent Streptomyces spp. 1254 strain after morphological analysis. It was concluded from results of southern hybridization that UV irradiation resulted in activation of only silent genes and failed to express the genes for mutactimycin expression269.
A method has been reported270 for the selective isolation of mutants of Streptomyces coelicolor A3(2) with improper DNA replication allowing the isolation and identification of initiation and elongation stop dna mutants . These mutants had the ability to produce RNA and proteins at temperature 39°C with reversion in inhibition of DNA replication at 30°C. They also obtained the mutants deficient in macromolecule synthesis.
A method has been developed271 for the specific production of S. avermitilis mutant strain deficient in toxic oligomycin. In their technique they transposed S. avermitilis chromosome with Streptomyces Transposon TN4560 obtained from temperature sensitive plasmid and vice versa and resulted in 0.1% mutant strain that abolished the production of antibiotics.
The importance of C-source utilization has been reported272 for the development and secondary metabolite production during any fermentation process. ccrA1 mutation was characterized in S. coelicolor showing effects on catabolite controlled promoters. ccrA1 mutants altered the expression of galP1 but did not affect the expression of galP2. It was observed and concluded that genes responsible for the regulation of C-catabolite repression were identified by ccrA1 in S. coelicolor.
New methodologies were reported273 to enhance the production of secondary metabolites from Streptomyces sp. Potent chemical mutagenesis produced limited mutation in Streptomyces with GC to AT transitions dominating. Although the AT to GC transitions is complemented with GC to AT transitions however there is no specific mutagen to regulate this pathway. Cloning and insertion of natural genes with other
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation copies of genes will be resulted in enhanced secondary metabolite production and was used for the production of tylosin and pristinamycin. Transposon mutagenesis has been found to be very effective not only in the activation of gene transcription during SM production but also during tylosin production from S. fradiae. S. rimosus is highly specific for the production of oxytetracyclin during fermentation. The production of this secondary metabolite has been enhanced using genetic recombinant technique.
The overproduction of antibiotics about 5 to 50 fold more than the parent strain from streptomycin resistant mutants due to the point mutations rpsL genes encoding S12 ribosomal proteins has been reported274. Theses point mutations resulted in the conversion of Lys-56 to Asn, Arg, Thr or Gln.
Bald mutants obtained after mutagenic treatment of Streptomyces halstedii and Streptomyces violaceusniger with Ethidium bromide and NTG were capable of producing geosmin and MIB and resulted in loss of linear plasmid DNA as reported275. Reduced production of compound by bald and plasmid cured strains resulted.
A novel mutation method of low energy ion implantation for the high yield of antibiotics from Streptomyces erythreus has been reported276. They implanted the microbe with nitrogen ions using very low amount of energy about 40-60keV and fluence from 1 × 1011 to 5 × 1014 ions/cm2. There was a good linear relationship between survival rate and fluence value. Mutants obtained through this method resulted in high yield of erythromycin showing direct relation of production with fluence period. Mutation induced by this method resulted in increased production of erythromycin as compared to other conventional mutagenesis techniques. Also broad spectrum mutations are produces through this method. Rutherford Backscattering method was used for the distribution analysis of implanted ion.
Mutagenic treatments usually resulted in industrial strain with reduced secondary metabolites production therefore suitable mutagens are to be opted in order on obtain the higher yields. About 8.2 times more avermectin B1 component was produced when
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation treated the wild Streptomyces avermitilis strain with MMS. Mutagenesis of Streptomyces avermitilis 31267 resulted in IPT-85 mutants giving 16 times more avermectin production than the parent strain. The results revealed that IPT-85 mutant when treated with MMS second time resulted in mutant quite stable towards avermectin production and hence isolated and selected277.
A method has been developed278 for the enhanced actinohordin production from Streptomyces coelicolor through combined drug resistant mutagenesis. On agar plates containing streptomycin (Str), Gentamycin (Genr) and Rifampin (Rifr) drugs, highest frequency of actinohordin producing mutants were isolated. Double and triple mutants‘ production will enhance the production of antibiotic about 2.5 and 48 times greater respectively than the wild type strain. Point mutations in Str mutant and Rip mutants occurred at rpsL gene and rpoB genes respectively. Western Blotting analysis of single, double and triple mutants revealed that the mutagenesis effected the production of Act-II- ORF4 in similar way as it would affect the production of actinohordin antibiotic. They concluded that this novel mutation method can be adopted for obtaining greater production of different antibiotics and secondary metabolite from the wild type of strain.
Genetic rpsL mutations within two regions of S12 proteins in Streptomyces lividans resulted in 16 mutants with enhanced antibiotic production as is reported279. They concluded that site directed mutagenesis were resulted in activation of silent genes and also the efficacy of drug resistance site directed mutagenesis was indicated through their observations.
Improved secondary metabolite production from Streptomyces during fermentation can be achieved through mutagenic strain improvement and medium optimization as reported280. About 5% greater spiramycin antibiotic was observed from oil resistant mutant XC-1-29 of Streptomyces ambofaciens XC-2-37. It their study 61.8% more spiramycin production observed using 2% soyabean oil and 0.4% propyl alcohol in fermentation medium from Streptomyces ambofaciens XC-2-37.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Gene replacement method has been developed previously281 for the alternative production of avermectin. Eight components of avermectin show structural variations at three positions. A family consisted of A1a, A2a, B1a, and B2a of which B1a shows most anthelmintic activity. 1 and 2 components are different on the basis of C22-23 dehydration due to defective dehydratase of aveA1 module 2. In the present work they used homologous recombination to replace this dehydratase with functional dehydratase of erythromycin eryAII module 4 which generate saturated chain at C6-7 in erythromycin. PCR products were made and replaced with the original resulted in precursors with alkene at C22-23. The resultant recombinant strain named JW3105 produced on unsaturated C22-23 avermectin parts. They concluded that desired component of avermectin can be produced using this technology.
The mutagenesis of tylosin producing strain of Streptomyces fradiae using UV irradiation and NTG has been reported282 to produce and regenerate protoplast. Mutants with enhanced production of tylosin about 0.5-28.3% more than the parent strain were obtained through combined exposure of UV and NTG. Instability of tylosin producing mutants was studied in detail.
A study has revealed283 that microorganisms usually produce the primary and secondary metabolite during fermentation only in amount that can fulfill their needs. In order to have this metabolite in excess quantities different strain improvement strategies were adopted. Genetic recombination and mutagenesis are the main techniques used for this purpose. Strain improvement for higher production will reduce the cost of industrial process and resulted in tremendous enhancement of fermentation productivity.
A previous research has reported284 the recombinant methodology using intraspecific protoplast fusion for enhancing and selective production of avermectin B component from Streptomyces avermitilis by fermentation. B components being highly effective against parasites and their low level of toxicity have extensively been used in agriculture and veterinary fields. Intraspecific protoplast fusion was done between two strains of S. avermitilis for enhancing the production of avermectin B specifically. The
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation strain giving higher avermectin production was S. avermitilis strain 76-05. The other was the one that was genetically engineered contacting the mutations aveD- and olmA-. Isolation and characterization of two recombinant strains F23 and F29 showed that they were the hyper producing strains giving 84.20% and 103.45% avermectin B respectively. They did not give the production of oligomycin during fermentation. F23 and F29 were genetically more stable as compared to the parent strain. Also F29 was more tolerant against different fermentation conditions as against the parent strain. The results showed that this method can also be used on industrial level for the production of only avermectin B component.
Tylosin, veterinary drug and growth promoter, is known to be produced in improved quantity from Streptomyces fradiae NRRL-2701 after UV and gamma irradiation. Morphologically different colonies produced after irradiations were screened using B.subtilis bioassay. Almost 2.7±0.22 folds more tylosin production observed from mutant colony as compared to the parent strain. The results revealed that UV irradiation generated less stable mutants as compared to the gamma irradiation285.
A new technique reported286 for enhanced avermectin production is the atmospheric pressure glow discharge plasma technique for mutational breeding Streptomyces avermitilis resulting in 12% positive mutation. The plasma jet at low temperature with strong mutagenic effect on S. avermitilis spores resulted in genetically stable mutants and proved to be a powerful mutation tool in fermentation and biotechnology research.
Stimulator of Streptomyces avermitilis, the aveR, when incorporated into the chromosome of S.hygroscopicus TYQ0915 resulted in mutants with 274.9% enhanced rapamycin production as compared to the parent strain. The resulted revealed the effect of aveR on growth and sporulation as reported287.
It is reported288 that production of avermectin from Streptomyces avermitilis can be enhanced about 2 folds from wild type strain if the concentration of avtAB mRNA is
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation increased from 30-500 folds. Avt AB is the ABC transporter encoded with avtAB operon in the avermectin biosynthetic gene cluster. Avermectins showing the anthelmintic and anti parasitic characteristics are the compounds having 16-membered macrocyclic ring and belonging to the polyketide group. Production of avermectin B1a was found to increase about 50% on industrial scale fermentation process using engineered high yield producer strain in YMG fermentation medium. RT-PCR analysis revealed that avermectin biosynthetic gene was not over expressed with that of avtAB genes. Reduction in ratio of intracellular and extracellular B1a revealed that AvtAB, the ABC transporter, exports the avermectin thus resulting in the enhanced production of other antibiotics along with avermectin as well.
2.6. OPTIMIZATION
Conventional single parameter medium optimization and glucose feeding for enhanced avermectin B1a production from S. avermitilis during fermentation was employed resulting in 48.6% and two folds increase in avermectin B1a production respectively as compared to the non optimized medium289.
Statistical optimization using various equations has been employed290 for medium as well as condition parameters during the growth phase. Various equations employed were Luedeking- Equation for estimation and determination of glucose and oxygen in the medium along with Logistic equation and Luedeking- Piret Equation for growth and actinorhodin production determination and calculation.
Statistical optimization using response surface methodology for improved avermectin B1a production during fermentation of Streptomyces avermitilis 14-12A has been reported291. Placket-Burman design and steepest ascent method have been used for screening of effective parameters and their effective concentrations. RSM resulted in 1.45 folds increase in avermectin B1a production at optimum 149.57g/L and 8.92g/L concentrations of most important variables corn starch and yeast extract respectively.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Optimum pH of protoplast buffers determined for protoplast transformation and intact cells for getting hyper producing avermectin mutant Streptomyces avermitilis 31780 strain was 6.5. The pH 6.5 and 6.3 of protoplast buffers were used for 4.5 × 106 transformations per µg of Pij702 and 1.6 × 105 for L-9 transformations respectively. Decreased avermectin production associated with L-9 transformations at lower pH value can be avoided by a process of electroporation of intact cells without lysozyme treatment292.
RSM has been employed for medium optimization during actinorhodin production from S. coelicolor A3(2) made use of 24 full factorial central composite designs for combined effect of various component. Sucrose, glucose, yeast extract and peptone were the main carbon and nitrogen sources screened using this design with optimum 339g/L, 1g/L, 1.95g/L and 2.72g/L concentration respectively. P-value<0.0001 had been found for linear, quadratic and cross-product effect of two carbon source showing the critical effect of carbon sources on actinorhodin production. About 32% more actinorhodin production observed in RSM optimized medium as compared to the non optimized medium293.
2.7. AVERMECTINS
Macrocyclic lactones named avermectins with potent anthelmintic and insecticidal properties have been produced from S. avermitilis NRRL 8165 as a result of submerged fermentation. The effectiveness of the extracted avermectins was chiefly against Nematospiroides dubius53.
Avermectins with potent nematocidal properties against broad spectrum of nematodes and produced as a result of fermentation product of soil isolated S. avermitilis were found to lack of antimicrobial activities. The avermectins initiated the opening of GABA chloride channels at the neuromuscular junctions and resulted in the paralysis of target nematodes294.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Synthesis of odorous compounds is associated with the production of avermectin components from S. avermitilis as a result of fermentation. Direct increase in geosmin and a relative decrease in oxolones homologous production with avermectin had been reported295.
Direct recombinant technique has been used for the production of most important veterinary drugs named avermectin B1a and B1b from S. avermitilis through fermentation. Ivermectin being used for treatment of onchocerciasis, eye disorder and all kinds of other skin diseases can be generated from polyketide macrolide avermectin B1a and B1b as a result of chemical hydrogenation at C22-23 double bond296.
It is reported that substrates of multi drug resistance proteins, taxol and vincristine can be produce from Hep-2 and P388 tumor cells respectively by the action of avermectins. The modification ability is reported to be higher for resistant strains without any effect on sensitivity of tumor cell to H2O2 and cisplatin. However the naturally occurring avermectins normally effected the calcein accumulation in cells and efflux of rhodamine 123. Inhibitory action of avermectins strongly corresponds to Cell type and nature of substrates for multi drug resistance proteins. They concluded that avermectins can be used successfully for cancer cells treatment297.
Biochemical and molecular basis for avermectin production and regulation has been reported298. The 16 membered pentacyclic polyketide avermectins produced from S. avermitilis during fermentation were reported to be highly effective antiparasitic and anthelmintic agents. Genome sequence of S. avermitilis, labeling studies, study of genes being blocked biosynthetically and identifications of gene clusters have been used scrutinize biosynthetic pathway involved in avermectin production. They conclude the genetic engineering of S. avermitilis as the best way for analogous avermectin components production along with the improvement of new avermectin biosynthetic ways.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Medium acidification and pH and selection of suitable solvent were reported to influence the extraction of avermectin from fermentation broth. About 4.833 million per ton of avermectin B1b/B1a ratio in 1-2% ratio had been extracted from mother liquor using xylene as solvent at medium pH adjusted at 1.5-2.0299.
Avermectin B1 fractions with effective antiparasitic and anthelmintic activities have been used efficiently in veterinary and agricultural fields and can be produced through intraspecific protoplast fusion technique developed. The mutants F23 and F29 obtained from fusion of avermectin hyper producing strain and genetically engineered strain of S.avermitilis were genetically stable giving 2.66-folds and 3.50-folds more avermectin B components respectively as compared to the parent strain. Industrially important with selective avermectin B component production mutants can be obtained through this technique300.
About 1.6 folds more avermectin hyper producing strains were identified and screened by using a high-throughput screening method described earlier. The method employing the use of micro titer plates and UV absorbance had an edge of lesser time requirement over conventional shake flask production method301.
It is reported that gene aveR belonging to LAL-family of regulatory genes is of utmost importance for avermectin production from S.avermitilis without which the strain will completely lose its ability to produce any of the avermectin component. Conversion of avermectin intermediate required for avermectin production will be lost. For every biosynthetic pathway there is a maximum threshold of genes to obtain desired component above which the production will be diminished302.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
MATERIALS AND METHODS
METHODOLOGY
Study 1:
3.1. MEDIUM SELECTION FOR THE PRODUCTION OF AVERMECTIN FROM STREPTOMYCES AVERMITILIS 41445
3.1.1. Microorganism and Maintenance
The strain DSM Streptomyces avermitilis 41445 provided by ―Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH‖ and has been used for the production of avermectin B1b throughout the study. The DSM Streptomyces avermitilis 41445 was maintained on medium 65 as specified by DSMZ. The medium 65 (YMG medium) consisted of (g/l in distilled water) glucose 4.0, yeast extract 4.0, malt extract
10.0 and CaCO3 2.0. The pH of the medium was adjusted to 6.5 before autoclave. The pH 0 was adjusted at 7.0 with CaCO3 after autoclaving the medium at 121 C for 15 min. The medium was then inoculated with Streptomyces avermitilis 41445 and incubated at 280C in water bath shaker at 150rpm until the brownish liquid was obtained. Culture was then streaked on nutrient agar slants and incubated at 280C for 24h for further studies.
3.1.2. Inoculum development
One loopful of 24h old culture of S. avermitilis 41445 was transferred into the sterilized inoculum medium. Thereafter the inoculum medium was placed at 31oC in the water bath shaker (Eyela, Japan) for 24h at 150rpm. The seed medium consisted of (g/L in distilled water) glucose 4.0, yeast extract 4.0, malt extract 10.0 and CaCO3 2.0.
3.1.3. Production of Avermectin B1b
The production of avermectin B1b by Streptomyces avermitilis 41445 was studied into eight different growth media named M1, M2, M3, M4, SM1, SM2, SM3 and SM4. SM stands for the synthetic media. Each growth medium was inoculated with 2 ml (4%
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation v/v) of inoculum medium separately. After transforming of seed medium, each growth medium was incubated at 31oC in water bath shaker for 15 days at 150rpm.
3.1.4. Effect of Process parameters
Effect of inoculum sizes (ranging from 4% to 16% v/v) and incubation time (ranging from 6 to 15 days) were studied on the production of avermectin B1b from Streptomyces avermitilis 41445 by fermentation. The effects of temperature (ranging from 26 to 38°C) and pH (ranging from 6 to 7.8) were also optimized for the maximum production of avermectin B1b from Streptomyces avermitilis 41445.
Study 2:
3.2. ISOLATION AND IDENTIFICATION OF MICROORGANISM
Soil samples from all around the Pakistan Council of Scientific and Industrial Research Laboratories (PCSIR) were collected for the isolation of Streptomyces avermitilis proficient of producing the avermectin.
The collected soil samples were desiccated at 37°C for 1 hour in hot air oven and then cooled at room temperature followed by preservation in the polythene bags until the pretreatment. 1 gram of each soil sample in 250ml conical flasks was dissolved in 100 ml of sterile water separately and few drops of Tween-80 solution were added into it. In orbital incubator shaker, all flasks were shaken for 30 minutes at 27°C and were considered as stock cultures303.
3.2.1. Isolation of Streptomyces
For each soil sample, 9 ml of sterile water was taken in 10 separate test tubes. From stock cultures, 1ml of the suspension was aseptically transferred into 1st tube to make 10-1 dilution. After thorough mixing, 1ml suspension from 10-1 dilution was transferred to the next test tube containing 9ml of sterile water to make 10-2 dilution. In this way serial dilutions up to 10-10 were made in sterile water in respective test tubes234.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
0.1ml of the each dilution was spread onto the surface of Actinomycetes isolation agar plates71, Starch casein agar medium plates236 and Kuster‘s agar medium plates304 separately and the plates were incubated at 28°C for 2-7 days until sporulation of Streptomyces colonies occurred.
Based upon shape, size, colour and surface structure, well grown Streptomyces colonies were picked from all plates and were transferred to Yeast extract-Malt extract agar medium plates for the isolation and purification of Streptomyces colonies. Pure cultures of Streptomyces were then obtained by reiterated sub culturing on Yeast extract- Malt extract agar medium slants71.
3.2.2. Isolation of Streptomyces avermitilis
Antibiotic named avermectins produced by Streptomyces avermitilis are specified for lacking characteristic antibacterial and antifungal activities277. Following tests were performed for the isolation of Streptomyces avermitilis strains.
3.2.2.1. Morphological characterization
Microscopic slides for all the isolates were prepared and scrutinized under microscope. Also the gram reactions were studied for Streptomyces isolates.
3.2.2.2. Antimicrobial activities
Antibacterial and antifungal activities of all the isolated were checked against five bacterial strains named Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterobacter aerogenes and Bacillus subtilis. Also the activity against two fungal strains named Aspergillus niger and Rhizopus oligosporus and one yeast strain named Candida albican was tested. All these strains were obtained from Food and Biotechnology Department of PCSIR and were cultured in Nutrient Broth for 24h at 37±0.1 oC71.
For the cultures of yeast and fungi, the incubation period was 5 days. Well diffusion method was used for studying the antimicrobial activities of the isolates. Wells were made in each of the plate using the sterilized borer already seeded with 300µL of
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation the test organism‘s separately305. Each well was then filled with 300µL of supernatant of each isolate dilution and kept for at least 1h to ensure the complete diffusion of dilution into the nutrient agar medium in each plate. Diameters of zones formed after incubation period of 24 and 48h at 37oC were measured232. Control plates without inoculating the isolated strains were also made so as to investigate the normal growth of Actinomycete.
3.2.2.3. Avermectin production
Different Streptomyces species produced different types of antibiotics. Streptomyces avermitilis is specified for the production of avermectin as secondary metabolite. All the isolates were tested for the production of the antibiotics through fermentation. The isolates producing the avermectin were selected and further identified.
3.2.2.4. Agar colonies
Selected Streptomyces avermitilis bacterial strains were streaked on nutrient agar medium and incubated at 28°C for 24h. The colony characteristics of the strains were observed.
3.2.2.5. Agar slants
Nutrient agar slants were prepared and inoculated with the Streptomyces avermitilis strains and incubated at 28°C for 24h for the investigation of growth characteristics on slants.
3.2.3. Morphological characterization
Based upon gram reaction, colony growth, aerial mycelia morphology, reverse side colour, soluble pigment formation and melanin pigment production, Streptomyces colonies were characterized morphologically.
3.2.4. Biochemical characterization
The tests performed for biochemical characterization of the soil isolates were nitrate reduction test, milk coagulation and peptonization, gelatin liquefication, H2S
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation production test, Nitrogen source utilization, carbon source utilization and growth temperature measurement.
Study 3:
3.3. MUTAGENESIS OF Streptomyces avermitilis 41445
3.3.1. Mutational analysis
For the mutational study, one physical (Ultra Violet Irradiation) and two chemicals mutagens (Ethidium Bromide and Ethylmethane Sulfonate) were employed using the spore suspensions. The screening and selection of mutants was based on the enhanced avermectin B1b production as compared to the parent strain Streptomyces avermitilis 41445. The survival rate and lethality rate were also considered while selecting the mutants from a cultivation plate.
3.3.2. Preparation of spore suspension
Spores of Streptomyces species usually occur as chains in the aerial mycelium and several methods have been employed for the isolation and separation of these spores. Individual spores were obtained by suspending them in water followed by vortex. Sterile saline was added in the plate containing the spores of given strain. Crude spore suspension was transferred to the sterile test tube. The optical density of spore suspension was measured by spectrophotometer at 600nm and adjusted the no. of spores/mL. At optical density 1, the number of spores in the given suspension was about 106 spores/mL. Adjustment of optical density at 1 was done by adding the sterile saline to the spore suspension306.
3.3.3. Stock solution of Ethidium Bromide
Stock solution (1mg/mL) of Ethidium Bromide was made by dissolving 10mg of Ethidium Bromide in 10mL of distilled water.
3.3.4. Stock solution of Ethylmethane Sulfonate (EMS)
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Stock solution of EMS (1mL/mL) was made by taking 10mL of liquid EMS and mixing it into 10mL of the distilled water.
3.3.5. Physical mutagenesis of S. avermitilis 41445 by Ultra Violet irradiation
UV lamp was kept on for at least 30minutes before use to let it to reach maximum emission. The 0.5mL of the spore suspension with 106spores/mL (Number of bacteria/ml = number of colonies / dilution × amount plated) was poured into Petri plates containing the nutrient agar. Short wavelength (320nm) ultra violet radiations were irradiated on spore suspensions for time interval of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 minutes with constant shaking to ensure the equal spreading of the radiations from the UV lamp kept under dark in close box throughout the plate. The treated plates were kept in dark straight away to evade any photo reaction. The plate with no irradiation was also made for comparison purpose307.
3.3.6. Chemical mutagenesis of S. avermitilis 41445 by Ethylmethane Sulfonate (EMS)
Clean and sterilized eppendorf tubes were taken and were oven dried at 180°C. A spore suspension of Streptomyces avermitilis 41445 of about 500µL was transferred aseptically into them. From the stock solution (1mL/mL) of EMS, suitable amount was poured in each of the eppendorf tubes to make different working concentrations (1µL/ml, 2µL/ml, 3µL/ml and 4µL/ml) of EMS. The spore suspensions were incubated at 28°C for 1h. Afterwards they were mixed exhaustively by vortex for 1 minute and centrifuged for 15 minutes at 8000rpms at 4°C. The cell pellet in each eppendorf tube was collected and washed thrice with 0.5M EDTA solution to get rid of all the traces of EMS. The treated spores were the resuspended into the sterile saline. The experimental spore suspensions were used for the further study along with the untreated set of spore suspension for the comparison purpose.
3.3.7. Chemical mutagenesis of S. avermitilis 41445 by Ethidium Bromide (EtBr)
A spore suspension of Streptomyces avermitilis 41445 of about 500µL was transferred aseptically into separate clean and sterilized eppendorf tubes From the stock
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation solution (1mg/mL) of Ethidium bromide, apposite amount was added in each of the eppendorf tube to make different working concentrations (10µL/ml, 20µL/ml, 30µL/ml and 40µL/ml) of Ethidium Bromide. After incubating the spore suspensions at 28°C for 1h, spore suspensions were agitated thoroughly by vortex for 1 minute and centrifuged for 15 minutes at 8000rpms at 4°C.
The cell pellet in each eppendorf tube was collected and washed thrice with 0.5M EDTA solution to eradicate the traces of Ethidium Bromide. The treated spores were then resuspended into the sterile saline. The experimental spore suspensions were used for the further study along with the untreated set of spore suspension for the comparison purpose.
3.3.8. Selection and screening of avermectin B1b hyper producing mutants
All the mutant strains were studied for the production of avermectin B1b through fermentation. The mutant strain with highest avermectin B1b production was selected and used for the further studies.
3.3.9. Defined medium for the growth of mutant strain of S.avermitilis 41445
All the mutant strains were maintained on nutrient agar slants and DSMZ specified medium 65 named GMY medium slants (glucose 4.0g/L, malt extract 10g/L, yeast extract 4.0g/L, CaCO3 2.0g/L and agar 12.0g/L). The pH of the medium 65 was maintained at 7.2 before addition of agar. The media for the production of avermectin B1b S. avermitilis 41445 UV 45(m) mutant strain was selected after optimization through one parameter optimization and response surface methodology (RSM) and artificial neural network (ANN) technique.
3.4. FERMENTATION TECHNIQUE
3.4.1. Submerged fermentation
3.4.1.1. Inoculum development for S. avermitilis 41445
The vegetative inoculum for S.avermitilis 41445 was developed by taking 18-24h old culture of S.avermitilis 41445 into 250mL shake flask containing 50mL of sterile
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
GYM medium. The composition of GMY medium was: glucose 4.0g/L, malt extract10g/L, yeast extract 4.0g/L and CaCO3 2g/L. The pH of the medium was maintained at 7.2±2 after the addition of CaCO3. The flask was incubated at 31±2°C for 24h at 150rpm in orbital shaker. After incubation, the inoculum medium was transferred to the fermentation medium at rate of 10% v/v.
3.4.1.2. Shake flask study for avermectin B1b production from S. avermitilis 41445
Streptomyces avermitilis 41445 was studied for the production of avermectin B1b in 250mL shake flask containing 50mL of the synthetically made SM2 fermentation medium: soluble corn starch 50.0g/L, α-amylase 0.1g/L, CaCO3 0.8g/L, KCl 0.1g/L,
NaCl 0.5g/L, yeast extract 2.0g/L, MgSO4.7H20 0.1g/L. The pH of the medium was maintained at 7.2±0.2 after sterilization. Then 10% of vegetative inoculum was transferred to the fermentation medium aseptically. After inoculation the fermentation medium was incubated at 31±2°C for 10days at 150rpm in orbital shaker.
3.4.1.3. Inoculum development for mutant strain S.avermitilis 41445 UV 45(m)3
The vegetative inoculum for S.avermitilis 41445 UV 45(m)3 was developed by taking 18-24h old mutant culture S.avermitilis 41445 UV 45(m)3 into 250mL shake flask containing 50mL of sterile GYM medium. The composition of GMY was as: glucose
4.0g/L, malt extract10g/L, yeast extract 4.0g/L and CaCO3 2g/L. The pH of the medium was maintained at 7.2±2 after the addition of CaCO3. The flask was incubated at 31±2°C for 24h at 150 rpm in orbital shaker. After incubation, the inoculum medium was transferred to the fermentation medium at rate of 10% v/v.
3.4.1.4. Shake flask study for avermectin production from Streptomyces avermitilis 41445 UV 45(m) 3 mutant strain
From one parameter carbon and nitrogen source optimization, potato starch and peptone were found to be the best carbon and nitrogen source respectively for the mutant strain to produce improved yield of avermectin B1b. Streptomyces avermitilis 41445 UV 45(m)3 mutant strain was studied for the production of avermectin B1b in 250mL shake flask containing 50mL of the synthetically made SM2 fermentation medium: potato
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation starch 50.0g/L, α-amylase 0.1g/L, CaCO3 0.8g/L, KCl 0.1g/L, NaCl 0.5g/L, peptone
2.0g/L, MgSO4.7H20 0.1g/L. The pH of the medium was maintained at 7.2±0.2 after sterilization. Then 10% of vegetative inoculum was transferred to the fermentation medium aseptically. After inoculation the fermentation medium was incubated at 31±2°C for 10days at 150rpm in orbital shaker.
Study 4:
3.5. OPTIMIZATION
3.5.1. Optimization of cultural conditions for avermectin B1b production from Streptomyces avermitilis 41445 by Response surface methodology
3.5.1.1. Single factor optimization methodology for the selection of key factors for avermectin B1b production
For the better production of avermectin B1b from Streptomyces avermitilis 41445, distinctive carbon sources were employed in the production medium. These include the glucose, maltose, lactose, soluble corn starch, wheat starch and potato starch. Also diverse nitrogen sources that have been employed for the strain to obtain the maximum avermectin B1b production include the yeast extract, corn steep liquor, casein, malt extract, peptone, lab lemco powder and urea. The salts used as inorganic nitrogen source in the production medium were (NH4)2SO4, NH4Cl, NaNO3, KNO3, (NH4)2SO4 and
(NH4NO3).
3.5.2. Statistical experimental design
3.5.2.1. Screening of cultivation variables by Plackett-Burman Design
Plackett-Burman Design was used for the screening of selected variables showing significant effects on the production of avermectin B1b from S.avermitilis 41445. Nine variables were screened and consisted of soluble corn starch, CaCO3, α-amylase, KCl,
NaCl, yeast extract, MgSO4.7H2O, pH and temperature. Each factor in the present design was scrutinized at two different levels, low (-) and high (+). About 12 Plackett-Burman runs with these selected levels and their responses were done. As this design does not
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation give interaction among different selected variables, the screening of factors consequently ensue the linear approach. Model variables can thus be explicated well by first order polynomial equation given as follow:
∑
Y=response = interception coefficient = linear coefficient
Response surface methodology which is the multiple regression analysis technique considered only those variables having P<0.1 obtained from ANOVA of PB design for optimization with significant effect on the production of avermectin B1b.
Table 3.1. Nine variables screened by Plackett-Burman Design at lower and higher levels
Variables Symbol code Units Experimental values (g/L)
Lower values (-) Higher values (+) Soluble corn starch X1 G 20 140
CaCO3 X2 G 0.8 6 α-amylase X3 g 0.1 0.5 KCl X4 g 1 4 NaCl X5 g 0.5 4 yeast extract X6 g 2 16
MgSO4.7H2O X7 G 0.1 0.5 pH X8 - 7.0 7.5 Temperature X9 °C 28 37
3.5.2.2. Optimization by Central Composite Design (CCD) and statistical analysis
After screening the significant factors, Central Composite Design (CCD) was used for the optimization of three variables viz. yeast extract (X6), MgSO4.7H2O (X7)
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation and temperature (X9). Each of the variables was studied at five coded levels (-2, -1, 0, +1, +2) and model was explained with twenty runs.
Table 3.2. Experimental variables, codes, units, range and levels of independent variables for response surface methodological experiments
Variables Symbol Code Units Experimental Levels (g/L) -2 -1 0 +1 +2 Yeast extract X6 g 2 7.6 10.4 13.2 16
MgSO4.7H2O X7 g 0.1 0.26 0.34 0.42 0.5 Temperature X9 °C 26.2 29.8 32.8 34.8 36.8
Second order polynomial equation has been used to depict the relationship between the response and the independent variables.
∑ ∑ ∑
Y = Predicted response = Interception coefficient = Linear coefficient
Quadratic coefficient Interaction coefficient
Multiple regression analysis of the given model and construction of the response surface graphs were done using Design-Expert, Version 7 of STATISTICA. Coefficient of determination R2 has been used to determine the quality of regression equation. F-test was employed for checking the significance of the model. Three dimensional response graphs showed the relationship between the response and the experimental variables. To optimize the maximum response of all the significant variables, point optimization methodology has been employed.
3.5.2.3. Statistical model validation
Validation of statistical method employed for the optimization purpose, an experiment was conducted at conditions optimized by the given response surface method in triplicate and their mean values were used to confirm the accuracy of the model.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Study 5:
3.6. OPTIMIZATION
3.6.1. Optimization of Cultural Conditions for Avermectin B1b Production From S.avermitilis 41445 by Artificial Neural Network (ANN)
3.6.1.1. Artificial neural network
Highest coefficient of correlation and lowest selection errors were determined from sixty different trained networks and had been used for the selection of multilayer perception network (MPN). 10 training, 5 selection and 5 testing experiments were conducted. Topology constructed consisted of three layers one of which is the hidden layer.
3.6.1.2. Optimization capability of ANN
Adjusted R2, AAD and RMSE were determined along with the coefficient of correlation determination (R2). The formulas for calculating R2, Adjusted R2, AAD and RMSE are given in following equation.
∑ ( )
∑ ( ̅ )
Here X is the ANN predicted avermectin B1b concentration, ̅ is the observed B1b concentration and Y is the average observed B1b concentration.
[( ) ]
Here N is the total no. of observations and K is the number of input variables.
*[∑ ( - ] +
Here yi,exp and yi,cal are the experimental and calculated responses, respectively, and P is the number of experiments.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
∑( ) √
Here yi,exp and yi,cal are the experimental and calculated responses, respectively, and n is the number of experiments.
3.6.2. Statistical analysis
The STATISTICA software version 7 was employed for the statistical analysis. Variable estimates, their t and p values were calculated from multiple regressions for PB design. For determining the significance of model parameters, F and t tests were performed. Performance of regression equation was evaluated through coefficient of correlation determination (R2). Desirability charts were used for the determination of optimum levels of variables. The neural networks were constructed using intelligent problem solver of STATISTICA. The best obtained network then selected for prediction and optimization. Input variables were then ranked using the sensitivity analysis of selected ANN network.
Study 6:
3.7. OPTIMIZATION
3.7.1. Optimization of Cultural Conditions of Avermectin B1b Production From S. avermitilis 41445 UV 45 (m) 3 by Response Surface Methodology (RSM)
3.7.1.1. Single factor optimization methodology for the selection of key factors for avermectin B1b production
For the improved yield of avermectin B1b from mutant strain Streptomyces avermitilis 41445 UV 45(m) 3, distinctive carbon sources were employed in the production medium. These include the glucose, maltose, lactose, soluble corn starch, wheat starch and potato starch. Also diverse nitrogen sources that have been employed for the strain to obtain the maximum avermectin B1b production include the yeast
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation extract, corn steep liquor, casein, malt extract, peptone, Lab Lemco powder and urea. The salts used as inorganic nitrogen source in the production medium were (NH4)2SO4,
NH4Cl, NaNO3, KNO3, (NH4)2SO4 and (NH4NO3).
3.7.2. Statistical experimental design
3.7.2.1. Screening of cultivation variables by Plackett-Burman Design
Plackett-Burman Design was used for the screening of selected variables showing significant effects on the production of avermectin B1b from mutant strain S.avermitilis
41445 UV 45(m) 3. Nine variables were screened and consisted of potato starch, CaCO3,
α-amylase, KCl, NaCl, peptone, MgSO4.7H2O, pH and temperature. Each factor in the present design was scrutinized at two different levels, low (-) and high (+). About 12 Plackett-Burman runs with these selected levels and their responses were done. As this design does not give interaction among different selected variables, the screening of factors consequently ensue the linear approach. Model variables can thus be explicated well by first order polynomial equation given as follow:
∑
Y=response = interception coefficient = linear coefficient
Response surface methodology which is the multiple regression analysis technique considered only those variables having P<0.1 obtained from ANOVA of PB design for optimization with significant effect on the production of avermectin B1b.
Table 3.3. Nine variables screened by Plackett-Burman Design at lower and higher levels
Variables Symbol code Units Experimental values (g/L) Lower values (-) Higher values (+) Potato starch X1 G 20 140
CaCO3 X2 G 0.8 6 α-amylase X3 G 0.1 0.5
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
KCl X4 G 1 4 NaCl X5 G 0.5 4 Peptone X6 G 2 16
MgSO4.7H2O X7 G 0.1 0.5 Ph X8 - 7.0 7.5 Temperature X9 °C 28 37
3.7.2.2. Optimization by Central Composite Design (CCD) and statistical analysis
After screening the significant factors, Central Composite Design (CCD) was used for the optimization of three variables viz. KCl (X4), NaCl (X5) and pH (X8). Each of the variable was studied at five coded levels (-2, -1, 0, +1, +2) and model was explained with twenty runs.
Table 3.4. Experimental variables, codes, units, range and levels of independent variables for response surface methodological experiments
Variables Symbol Code Units Levels (g/L) +2 +1 0 -1 -2 KCl X4 G 4.0 3.4 2.8 2.2 1.0 NaCl X5 G 4.0 3.3 2.6 1.9 0.5 pH X8 - 7.5 7.4 7.3 7.2 7.0
Second order polynomial equation has been used to depict the relationship between the response and the independent variables.
∑ ∑ ∑
Y = Predicted response = Interception coefficient = Linear coefficient
Quadratic coefficient Interaction coefficient
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Multiple regression analysis of the given model and construction of the response surface graphs were done using Design-Expert, Version 7 of STATISTICA. Coefficient of determination R2 has been used to determine the quality of regression equation. F-test was employed for checking the significance of the model. Three dimensional response graphs showed the relationship between the response and the experimental variables. To optimize the maximum response of all the significant variables, point optimization methodology has been employed.
3.7.3. Statistical model validation
Validation of statistical method employed for the optimization purpose, an experiment was conducted at conditions optimized by the given response surface method in triplicate and their mean values were used to confirm the accuracy of the model.
Study 7:
3.8. OPTIMIZATION
3.8.1. Optimization of Cultural conditions for avermectin B1b Production From S. avermitilis 41445 UV 45 (m) 3 by Artificial Neural Network (ANN)
3.8.1.1. Artificial neural network
Highest coefficient of correlation and lowest selection errors were determined from sixty different trained networks and employed for the selection of multilayer perception network (MPN). In the present model, 10 training, 5 selection and 5 testing experiments were conducted. Topology constructed consisted of three layers one of which is the hidden layer.
3.8.1.2. Optimization capability of ANN
Adjusted R2, AAD and RMSE were determined along with the coefficient of correlation determination (R2). The formulas for calculating R2, Adjusted R2, AAD and RMSE are given in following equation.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
∑ ( )
∑ ( ̅ )
Here X is the ANN predicted avermectin B1b concentration, ̅ is the observed B1b concentration and Y is the average observed B1b concentration.
[( ) ]
Here N is the total no. of observations and K is the number of input variables.
*[∑ ( - ] +
Here yi,exp and yi,cal are the experimental and calculated responses, respectively, and P is the number of experiments.
∑( ) √
Here yi,exp and yi,cal are the experimental and calculated responses, respectively, and n is the number of experiments.
3.8.2. Statistical analysis
The STATISTICA software version 7 was employed for the statistical analysis. Variable estimates, their t and p values were calculated from multiple regressions for PB design. For determining the significance of model parameters, F and t tests were performed. Performance of regression equation was evaluated through coefficient of correlation determination (R2). Desirability charts were used for the determination of optimum levels of variables. The neural networks were constructed using intelligent problem solver of STATISTICA. The best obtained network then selected for prediction and optimization. Input variables were then ranked using the sensitivity analysis of selected ANN network.
Study 8:
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
3.9. FERMENTER SCALE PRODUCTION OF AVERMECTIN B1b
3.9.1. Fermenter Scale Production of Avermectin B1b from Streptomyces avermitilis 41445
3.9.1.1. Inoculum medium
12-18h old culture of mutant strain Streptomyces avermitilis 41445 was scrapped from the nutrient agar slant with sterilized loop and was shifted into the sterilized inoculum medium. After inoculation the inoculum medium was placed at 31oC in the water bath shaker (Eyela, Japan) for 24h at 150rpm. The composition of inoculum medium was (g/L in distilled water) glucose 4.0, yeast extract 4.0, malt extract 10.0 and
CaCO3 2.0. The pH of the medium was adjusted at 7.2±0.2 after sterilization.
3.9.1.2. Fermentation medium
The optimized SM2 medium with composition soluble corn starch 140.0g/L, α- amylase 0.5g/L, CaCO3 6.0g/L, KCl 4.0g/L, NaCl 4.0g/L, yeast extract 16g/L,
MgSO4.7H20 0.5g/L was used to study the production of avermectin B1b from parent strain Streptomyces avermitilis 41445. The optimized fermentation temperature for the parent strain was 32°C. The pH of the medium was maintained at 7.46±0.2 after sterilization.
3.9.2. Fermenter Scale Production of Avermectin B1b from Mutant strain Streptomyces avermitilis 41445 UV 45(m) 3 3.9.3. Inoculum medium
12-18h old culture of mutant strain Streptomyces avermitilis 41445 UV 45(m) 3 was scrapped from the nutrient agar slant with sterilized loop and was transferred into the sterilized inoculum medium. After inoculation the inoculum medium was placed at 31oC in the water bath shaker (Eyela, Japan) for 24h at 150rpm. The inoculum medium consisted of (g/L in distilled water) glucose 4.0, yeast extract 4.0, malt extract 10.0 and
CaCO3 2.0. The pH of the medium was adjusted at 7.2±0.2 after sterilization.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
3.9.4. Fermentation medium
The optimized SM2 medium with composition Potato starch 140.0g/L, α-amylase
0.5g/L, CaCO3 0.8g/L, KCl 1.0g/L, NaCl 0.5g/L, peptone 2.0g/L, MgSO4.7H20 0.5g/L was used to study the production of avermectin B1b from mutant strain Streptomyces avermitilis 41445 UV 45(m) 3. The optimized pH of the fermentation medium for the mutant strain was 7.46. The medium was adjusted at this pH after sterilization. The fermentation was carried out 28oC.
3.10. ANALYSIS
3.10.1. Avermectin Extraction
Fermentation broth of each flask was centrifuged at 40C for 20 minutes at 8000rpm. The cell biomass was taken and supernatant was discarded. The cell biomass in the form of pallet was mixed with appropriate amount of methanol to completely dissolve it. The mixture was centrifuged again and the supernatant was collected for the analysis of avermectin by TLC and HPLC.
3.10.2. Thin Layer Chromatography (TLC)
The qualitative analysis of avermectin B1b was performed by applying the supernatant on TLC plate. The plates were then kept in the TLC tank having mobile phase methanol:acetonitrile (98 : 2 v/v). Short wave ultra violet light (254 nm) was used to detect avermectin on plate as described by (20) after a slight modification in mobile phase.
3.10.3. High Performance Liquid Chromatography (HPLC)
The concentration of avermectin B1b components were determined quantitatively by reverse phase HPLC. In HPLC 20 µl of the sample was injected. The samples were separated on C18 column and were eluted by methanol : acetonitrile (98 : 2 v/v) at a flow rate of 0.5 ml/min with a UV absorbance at 246 nm (21).
3.10.4. Purification of avermectin B1b
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Different techniques were used for the purification purpose.
Lyophilization Column chromatography
3.10.5. Application of avermectin B1b
Earthworm Cockroaches
3.10.6. Bacterial cell count
Appropriate serial dilutions up to 10-5 of bacterial culture were made and plated into Petri plates containing nutrient agar. The plates were incubated at 28±1°C for 24 hours. Total no. of cells was determined using the formula:
( )
3.10.7. Estimation of dry cell biomass
For the estimation of dry cell biomass, 10ml of the fermentation broth was taken and centrifuged at 8000rpm for 20 minutes at 4°C. The cell biomass in the form pellet was washed twice with distilled water and weighed after drying for 24h at 80°C.
3.11. KINETIC PARAMETERS
Kinetic parameters such as pH, cell biomass formation, product formation and substrate consumption were determined for the parent Streptomyces avermitilis 41445 strain after every 12h using Logistic and Luedeking-Piret equations.
For the mutant strain Streptomyces avermitilis 41445 UV 45 (m) 3 the kinetic parameters such as pH, cell biomass formation, product formation and substrate consumption were also determined after every 12h using Logistic and Luedeking-Piret equations.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Cell growth
* ( )+
Substrate Consumption
( )
⁄
Avermectin B1b production
( )
Where
X = the concentration of cell biomass (g/L)
Xmax = the maximum concentration of cell biomass (g/L)
-1 µmax = maximum specific growth rate (h )
S = Substrate concentration (g/L)
YX/S = Yield coefficient of cells on carbon substrate (g/g) ms = maintenance coefficient (g/g/h)
α = growth associated avermectin B1b production coefficient ()
β = non growth associated avermectin B1b production coefficient () t = time of fermentation (h)
3.12. STATISTICAL ANALYSIS
All the experiments were conducted in triplicates and standard deviations were calculated.
3.13. MAINTENANCE OF CULTURE
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
For culture maintenance, subsequent techniques were adopted.
3.13.1. Short term storage
For short term storage of the cultures, nutrient agar slants and Yeast extract-Malt extract glucose agar slants were used. The slants were refreshed after every two weeks
3.13.2. Medium term storage
a. Enviro beads (Microbank, Prolab) stored in refrigerator at 4°C were used for the midterm storage of the cultures
b. Dipped sterile platinum wire into dense cultures of all the isolates separately followed by stabbing smoothly into suitable agar medium viz. nutrient agar slants and Yeast extract-Malt extract glucose agar. The stabbed cultures incubated at 30°C for 50h were tightly capped and enfolded in parafilm to preclude medium desiccation and ultimately stored at room temperature in dark.
3.13.3. Long term storage
Culture isolates were lyophilized/freeze dried for long term preservation. Sublimed the dense spore suspensions of cultures under vacuum and stored at 4°C after vial sealing.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
RESULTS AND DISCUSSION
STUDY 1:
4.1. MEDIUM SELECTION
The selection of a suitable medium plays a vital role to make any fermentation process cost effective. In present study the most apposite medium was selected for the production of avermectin B1b from S. avermitilis DSMZ 41445 by submerged fermentation. Eight media with different compositions as shown in Table 4.1 were used in the study. The synthetic SM2 medium was found to be the best production medium because it gave maximum avermectin B1b production 17.7798 mg/L being estimated by HPLC. Other media that showed the avermectin B1b production were M1 and M3. The production of avermectin B1b in these media was 12.8118 mg/l and 13.6936 mg/l respectively.
Lazim et.al (2009) reported that nitrogen source is very important for the secondary metabolite production308. The concentration and nature of nitrogen source significantly manipulate the production of antibiotics. Rapidly utilized nitrogen source in a medium inhibit the production of secondary metabolites. Therefore desired secondary metabolite yield can be achieved in a medium with appropriate nitrogen source in apposite concentration comparative to the carbon source309.
In the present study the media SM1, SM3, SM4, M2 and M4 did not produce any of the avermectin components. Absence of avermectin production in media SM1, SM3 and SM4 might be due absence of any nitrogen source. The production of avermectin from Streptomyces avermitilis 41445 in different media is shown in Figure 4.1.
Table 4.1: Composition of different media used for the production of avermectin B1b by S.avermitilis 41445in submerged fermentation
Components SM1 SM2 SM3 SM4 M1 M2 M3 M4 g/L (Self (Self (Self (Self (Hon (Rezank (Wang (DSMZ constructe constructe constructe constructe g a, et.al , et.al specified
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
d d d d Gao, 2007) 2010) ) medium) medium) medium) medium) et.al 2009) Glucose 10.0 - 10.0 - - 3.0 - 4.0 Soluble - 50.0 - 50.0 140.0 - 70.0 - starch α-amylase - 0.1 - 0.1 - - - -
K2HPO4 0.5 - 0.5 - - 0.5 0.5 -
CaCO3 - 0.8 - 0.8 0.8 5.0 2.0 2.0 KCl 0.1 0.1 0.1 0.1 - - 4.0 - Yeast - 2.0 - - 10.0 4.0 16.0 4.0 extract
MgSO4.7H2 0.1 0.1 0.1 0.1 - 0.1 0.5 - O NaCl 0.5 0.5 0.5 0.5 - 2.0 - -
(NH4)2SO4 - - 1.0 - 0.25 2.0 - - Malt extract ------10.0
CoCl2.6H2O ------10.0 - Soya flour - - - - 28.0 - - -
MnSO4.H2O - - - - 0.002 0.05 - -
FeSO4.7H2 - - - - - 0.1 - - O
ZnSO4.7H2 - - - - - 0.1 - - O pH of each medium was adjusted at 7.2±0.2. All the experiments were performed in the shake flasks containing 50 ml of fermentation medium separately.
Rate of carbon source metabolism by microorganism has greatly influenced the production of cell biomass and secondary metabolite117. Rapidly metabolized sugars like glucose lowered the production of secondary metabolites by inhibiting the formation of oleandrosyl diphosphoneucleotide enzyme required in the biosynthetic pathway126. Secondary metabolites are normally produced in stationary phase and S.avermitilis has used carbon source for its maintenance purpose268,310.
In the present study glucose is used as a carbon source in media SM1, SM3, M2 and M6 which metabolized at early stages and did not remain available till last stages of
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation fermentation thus resulting in the absence of any of the avermectin component. In selected SM2 medium soluble corn starch has been used as carbon source. It is slowly degraded to glucose by the action of α-amylase therefore in this medium glucose remained available for late stages for the production of avermectin B1b.
20
15
10 (mg/L)
Avermectin B1b 5
0 SM1 SM2 SM3 SM4 M1 M2 M3 M4 Medium
Figure 4.1: Effect of different Media on Avermectin B1b Production
4.1.1. Effect of Different Parameters on Avermectin Production
The selected medium SM2 was optimized at various process parameters including inoculum size, incubation time, incubation temperature and pH to obtain maximum production of avermectin B1b from S.avermitilis DSM 41445.
4.1.1.1. Effect of Inoculum Size on Avermectin Production
The results of present study revealed that production of B1b was increased with increasing the inoculum size. The maximum production was obtained at inoculum size of 10% v/v and after that it began to decrease as is shown in Figure 4.2. This might be due to the reason that by increasing the inoculum size more than the optimum the nutrient requirements of S.avermitilis cells increased rapidly which resultantly affects the avermectin B1b production.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
25
20
15
(mg/L) 10 Avermectin B1b 5
0 0 5 10 15 20 Inoculum Size (%)
Figure 4.2: Effect of Inoculum Size on Avermectin B1b Production
4.1.1.2. Effect of Incubation Time on Avermectin Production
The avermectin B1b production was also increased by increasing the time of incubation in growth medium. Maximum production was obtained at day 10th of fermentation. After that it began to decrease as shown in Figure 4.3. It may be due to the consumption of maximum quantity of soluble corn starch after the action of α- amylase at 10th day.
25
20
15
10 (mg/L)
5 Avermectin B1b
0 0 4 8 12 16 -5 Fermentation Time (Days)
Figure 4.3: Effect of Fermentation Period on Avermectin B1b Production
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
4.1.1.3. Effect of Temperature and pH on Avermectin Production
Production of avermectin B1b from the selected medium was studied at temperature and pH ranging from 25-370C and 6.0-7.5 respectively. The results revealed that maximum production was obtained at temperature 310C at pH 7.0. The effect of pH and temperature is shown in Figure 4.4 and Figure 4.5 respectively.
22 20 18 16
14
12
10 (mg/L) 8
Avermectin B1b 6 4 2 0 5.5 5.7 5.9 6.1 6.3 6.5 6.7 6.9 7.1 7.3 7.5 7.7 7.9 pH
Figure 4.4: Effect of medium pH on Avermectin B1b Production
25
20
15
(mg/L) 10 Avermectin B1b 5
0 20 25 30 35 40 Temperature (°)
Figure 4.5: Effect of Temperature on Avermectin B1b Production
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
In a study conducted by Zhinan and Peilin 1999, about 310 mg/L avermectin B1a has been obtained from S. avermitilis during 5 days of submerged fermentation at a temperature of 28oC with 10% inoculum size. The medium pH was adjusted at 7.0±0.2289. Young and Byeon 2005 reported a fermentation period of 5 days for enhanced avermectin B1a production from S.avermitilis by gene replacement281.
About 1.5 folds increase in production of avermectin B1a has been reported from S.avermitilis 14-12A through fermentation when carried out at a temperature 28oC and inoculum size 5%. Selective production of avermectin B components (505 µg/ml) was also obtained by studying the fermentation for 10 days at 28-30oC temperature and the inoculum size of 5%284,291.
Streptomyces avermitilis ATCC 31267 has been reported by Aikawa et.al 1999 to produce 28 mg/L avermectin B1 (B1a and B1b) at 9th day of fermentation with 10% inoculum. The temperature of incubation was adjusted at 300C277. Inoculum size of 0.03% has also been reported for the production of avermectin265. Streptomyces avermitilis NRRL 8165 had been found to produce about 17.5 mg/L of avermectin during 14 days of fermentation at 28oC with 2% inoculum. In this study the medium pH was maintained at 7.3±2288. Production of Avermectin B1a up to 2400±200 mg/L has been obtained at medium pH 7.5±1 and incubation temperature of 28-30oC286.
STUDY 2:
4.2. ISOLATION OF MICROORGANISM FROM SOIL
Three distinctive localities of Lahore were opted for the assortment of soil to isolate Streptomyces avermitilis. About fifty isolates of Streptomyces species were attained through selective prescreening procedures. All of these isolates were studied for the production of secondary metabolite, the avermectin. The aim of the study was to isolate the strains unambiguous for the production of avermectin and lacking certain antimicrobial activities.
Soil samples for isolating desired bacterial species were obtained from agriculture soil around PCSIR Campus Lahore, Botanical Garden of GCU, Lahore and Rose Garden
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation of Punjab University, Lahore and were assessed as is shown in Table 4.2. Among the isolated strains, only the gram positive bacterial species were considered for the further studies. From the results it was concluded that agricultural lands around PCSIR Campus Lahore were the rich source of industrially important Streptomyces.
Table 4.2: Bacterial isolates with antimicrobial activities and avermectin production
Source Bacterial Antimicrobial No Antimicrobial Avermectin Isolates Activities Activities Producing species PCSIR 30 24 6 6
Botanical 10 8 2 2 Garden GCU Rose Garden 10 8 2 2 Punjab University
4.2.1. Selection and Screening of Avermectin Producing Streptomyces avermitilis Strains
In a research work conducted by Burg et.al 1979 it is reported that Streptomyces avermitilis lack characteristic antimicrobial activities53. Antimicrobial activities of 50 soil isolates were studied against two gram positive (S.aureus, B.subtilis) and three gram negative (E.aerogenes, E.coli, P.aerogenosa) bacterial species. Antifungal (R.oligosporous, A.niger) and anti yeast (C.albican) activities were also assessed for the isolated strains. Among 50 soil isolated strains, only 10 isolates were found to lack antimicrobial activities and were deemed the Streptomyces avermitilis species as is shown in Table 4.3. Comparison and confirmation of the isolates was made with plates of Streptomyces avermitilis DSM 41445.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Table 4.3: Antimicrobial activity of Streptomyces isolates
Sr. Soil E. E.aerog S.aure P.aerogeno B.subtil A.niger R.oligosporo C.alb No. Isolate coli enes us sa is us ican 1 S1-A - - - - + - - - 2 S1-B + + + - - - + + 3 S1-C ------4 S1-D - - + + ++ - - - 5 S1-E - - ++ - + ++ + + 6 S1-F ++ + - +++ - - + + 7 S1-G - - + - + - - - 8 S1-H - - - + - ++ - - 9 S1-I ++ ++ + + - - + + 10 S1-J ------11 S2-A - - - + + - ++ - 12 S2-B - - ++ - - - - +++ 13 S2-C + - - + - ++ - - 14 S2-D - + - - + 15 S2-E ------16 S2-F - - + - - + - - 17 S2-G - + ++ + - - - + 18 S2-H + - + - - - ++ - 19 S2-I + - + - + - - ++ 20 S2-J ------21 S3-A - - + - + - + - 22 S3-B - - + - ++ - + - 23 S3-C ++ + - + - ++ - + 24 S3-D ++ + - + - ++ - - 25 S3-E ------26 S3-F - - + - + - + - 27 S3-G - + + + - - - -
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
28 S3-H - ++ - - - + + - 29 S3-I ------30 S3-J ++ + - - +++ - - + 31 S4-A + ++ ++ - + - - + 32 S4-B - - + ++ - + + - 33 S4-C + + ++ - - - - + 34 S4-D ++ + - + - ++ - + 35 S4-E + - - + - ++ - - 36 S4-F ------37 S4-G + - - + - ++ - - 38 S4-H + - ++ - + - - + 39 S4-I + ++ ++ - + - - + 40 S4-J ------41 S5-A + - - + - ++ - - 42 S5-B ++ + - + - ++ - + 43 S5-C ------44 S5-D + ++ ++ - + - - + 45 S5-E + - + ++ + - - + 46 S5-F ++ + - +++ - - + + 47 S5-G + - - + - ++ - - 48 S5-H ------49 S5-I + - ++ - + - - + 50 S5-J ++ + - + - ++ - + 51 Contr ------ol + = Fair, ++ = Potent, +++ = Highly potent, - = No effect
The ten isolates lacking antimicrobial activities and selected for the supplementary characterization on ISP-2 medium were S1-C, S1-J, S2-E, S2-J, S3-E, S3- I, S4-F, S4-J, S5-C and S5-H. The results of morphological characterization revealed the colour of aerial mycelia ranging between dark grey to light grey. The reverse side
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation morphology of these 10 isolates speckled from moderate grey to whitish grey along with colour of soluble pigment ranging from dark yellow to yellow with characteristic pale yellow and brownish yellow colour for some isolates. Positive results were given by all isolates for Melanin pigment formation. The results of the present morphological study of selected 10 isolates on ISP-2 medium confirmed them to be Streptomyces avermitilis when compared with the control Streptomyces avermitilis DSM 41445. Also the results of present study were in agreement with the results of research conducted by Shirling and Gottlieb 1966, Cross 1989, Lechevalier 1989; Locci 1989; Waksman, 1961; Williams et al., 1989; Goodfellow 198926,311,312,313,314,315,316.
In a research work conducted by Ceylan et.al 2008 the aerial mycelia colour, reverse side morphology and melanin pigment formation of Streptomyces isolates were studies. The results of their study illustrated 13.3% formation of melanin pigments by isolates. The colour of aerial mycelium was 80% for brown-yellow, 6.6% for yellow and 13.3% for violet. About 93.3% brownish-yellow and 6.6% violet colour were observed for reverse side morphology71.
The results of the present study revealed that the percentage of soluble pigment formation was 10%, 60%, 20% and 10% for dark yellow, yellow, pale yellow and brownish yellow colour respectively with 100% melanin pigment formation as is shown in Table 4.4. The aerial mycelium colour was dark grey (10%), grey (70%) and light grey (20%). Likewise the percentage of reverse side colour was 10%, 50%, 20% and 20% for moderate grey, grey, light grey and white to grey respectively.
Table 4.4: Morphological characteristics of active Streptomyces isolates on Yeast extract malt extract agar (ISP-2)
Sr. Soil Aerial Reverse side Soluble Melanin No. Isolates mycelium colour pigment colour pigment 1 S1-C Dark Grey Moderate Grey Dark yellow + 2 S1-J Grey Grey yellow + 3 S2-E Grey Grey yellow + 4 S2-J Grey Light Grey yellow +
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
5 S3-E Grey Grey Pale yellow + 6 S3-I Light Grey White to Grey yellow + 7 S4-F Grey Grey Brownish + yellow 8 S4-J Grey Grey yellow + 9 S5-C Grey Light Grey Pale yellow + 10 S5-H Light Grey White to Grey Yellow + 11 Control Dark Grey Moderate to Dark yellow + light grey
Avermectins are the secondary metabolites produced from Streptomyces avermitilis after fermentation. All the 10 isolates when tested for the production of secondary metabolites were found to produce avermectin in their stationary phases. Maximum avermectin production given by isolates S1-C was 10.15mg/L as is given in Table 4.5.
In previous studies carried out by Kim and Goodfellow 2002 and Egerton et.al 1979 mentioned that Streptomyces avermitilis are fastidious for their ability to produce secondary metabolites, the avermectin. They reported that latent anthelmintic and insecticidal activities are exhibited by avermectin although depriving the featured antimicrobial properties50,317.
Table 4.5: Secondary Metabolite (avermectin) Production of Selected Isolates
Sr. No. Soil isolate of Streptomyces Avermectin Production avermitilis (mg/L) 1 *S1-C 10.15±0.04 2 S1-J 5.0±0.05 3 S2-E 7.35±0.01 4 S2-J 6.29±0.09 5 S3-E 8.35±0.06
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
6 S3-I 6.0±0.011 7 S4-F 3.86±0.02 8 S4-J 5.78±0.01 9 S5-C 5.64±0.02 10 S5-H 8.65±0.07 11 Control 17.0±0.03 *The designation of the best avermectin producing strain Shake flask fermentation at pH 7.0, temperature 31°C. Each value is an average of three replicates. The symbol (±) shows the standard deviation among the replicates.
The Isolate S1-C found strictly resembled with the control strain and giving highest avermectin production therefore was used for the further studies. Culture characteristics of Isolate S1-C were studied on nutrient agar medium, Yeast extract malt extract agar medium (ISP-2), Inorganic salt-starch agar medium (ISP-4), PDA agar medium, Oatmeal agar medium (ISP-3), Bennett‘s agar medium, Casein enzymic hydrolysate-yeast extract (ISP-1) and DSMZ specified Medium 65.
The results of the present study revealed a comparable pattern of growth, aerial mycelium and reverse side colony characteristics of S1-C isolate and the control strain as is shown in Table 4.6. On media ISP-2 and ISP-3, the morphology of S1-C isolate closely resembled with the control strain. Comparable results were obtained on nutrient agar medium with a little difference in colour pattern of aerial mycelium and reverse side morphology. However, in other media the colour varied from grey to light grey. Growth of S1-C soil isolate found to be good as against the control where growth pattern was excellent and aerial mycelium and reverse side colony characteristics varied only slightly as compared to the control. In a research conducted to study morphological characteristics of Streptomyces avermitilis strain 173 on different media by Li-Xia et al 2005, they reported extraordinary diversity not only in the growth pattern but also a wide scope of colors in different media318.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Table 4.6: Cultural characteristics of strain S1-C on different media
Sr. Type of Growth Aerial mycelium Reverse side colour No. medium S1-C Control S1-C Control S1-C Control 1 Nutrient agar Good Good Dark Light Light White to yellow yellow yellow yellow 2 Yeast extract Good Good Dark Dark Moderate Moderate malt extract agar Grey Grey Grey to light (ISP-2) grey 3 Inorganic salt- Moderate Excellent Grey Dark Light Light starch agar grey cinnamon grey (ISP-4) 4 PDA agar Moderate Good White Dark light Pale to yellow yellow yellow yellow 5 Oatmeal agar Good Good Grey Grey White to White to (ISP-3) grey grey 6 Bennett‘s agar Good Very Grey Grey Light Light Good grey grey 7 Casein enzymic Good Good Light Light Light White to hydrolysate- grey grey grey grey yeast extract (ISP-1) 8 DSMZ Medium Good Excellent Yellow Light Light White to 65 yellow yellow yellow
The selective avermectin producing S1-C Streptomyces avermitilis species isolated from soil when subjected to different biochemical tests was found closely similar to control strain. The strain S1-C and control showed the same results of all biochemical test performed. The results of the present study were quite close to the results obtained by
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Goodfellow et.al 1992 and Awad et.al 2009 who examined the morphological and biochemical characteristics for the taxonomic classification and identification of different strain of Streptomyces319,66. Based on morphological and biochemical testing as is given in Table 4.7, the strain S1-C was Streptomyces avermitilis.
Table 4.7: Morphological and biochemical characteristics of selected
Avermectin producing S1-C Streptomyces
Sr. No. Properties S1-C Streptomyces Control spp. A Morphological Characteristics 1 Spore morphology Oval shaped, smooth Smooth 2 Colour of aerial mycelium Dark Grey Grey 3 Colour of substrate mycelium Grey Light grey 4 Gram‘s reaction Gram positive Gram positive B Biochemical Characteristics 1 Growth temperature range 28-37°C 28-37°C 2 Nitrate reduction test - - 3 Milk coagulation and peptonization + + 4 Gelatin liquefication + +
5 H2S production test - - 6 N-source utilization Yeast extract + ++ Malt extract + + Peptone + + Urea + + Lemco powder + + 7 C-source utilization Glucose + + Soluble corn starch ++ ++
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Maltose + + Lactose + + Mannitol + + Wheat powder + + Potato starch ++ ++
From the results obtained it was confirmed that the strain S1-C belonged to genus Streptomyces. The ultimate confirmation of the strain to be Streptomyces avermitilis was from secondary metabolite production in form of avermectin. Finally the food and biotechnology department of PCSIR Lahore evaluated the strain to be the desired strain and it has been used for the further study.
STUDY 3:
4.3. MUTAGENESIS
4.3.1. Physical Mutagenesis of Streptomyces avermitilis DSM 41445 by UV Light
Strain improvement strategies are very important in order to make a fermentation process cost effective. Mutagenesis is the most beneficial tool for strain improvement and results in the enhanced production of secondary metabolites and enzymes. Mutagenesis changes the bacterial behavior towards production and synthesis102. The genome sequence of Streptomyces avermitilis which is specialized for the formation of avermectin is known. However, the mechanisms that how the genes are involved in secondary metabolite production is not clear281. In previous studies the avermectin hyper producing mutants are obtained through Ultra Violet Irradiation and chemical mutagenesis320. Burg et.al 1979 reported about 56 times more avermectin production from Streptomyces avermitilis ATCC 31267 through mutagenesis53.
In the present research work the avermectin B1b hyper producing mutant strains of Streptomyces avermitilis DSM 41445 were obtained by both physical and chemical mutagenesis. UV irradiation being safe to handle was used as physical mutagen. Vegetative growing cells of Streptomyces avermitilis DSM 41445 were exposed to UV-
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation irradiation for 60min with a regular interval of 5min. About 163 mutant colonies obtained and only 16 were selected for screening experiments related to avermectin B1b production. Figure 4.6 revealed the effect of UV exposure on survival rate, lethality rate and number of colony formation of S.avermitilis 41445.
120 Survival Rate 0.1 0.09 100 No. of Colonies
0.08
lethaility rate 0.07 80 0.06 60 0.05 0.04
40
No.of colonies Survival Survival Rtae(%)
0.03 Lethality (%)rate 0.02 20 0.01 0 0 -10 0 10 20 30 40 50 60 Exposure Time (min.)
Figure 4.6: Effect of UV exposure time on survival rate, lethality rate
and number of colony formation
Wang et.al 2010 reported that lethality rate should be very high and survival rate should be low with increasing the mutagen exposure in order to induce powerful mutations and to screen effective mutants286. In the present research work the mutants obtained after 45, 55 and 60 min UV exposure resulted in the 20%, 16.66% and 13.33% survival rate respectively followed by an increase in lethality rate from 0.05 to 0.075 with UV exposure time increasing from 45 min to 60 min. In this study UV exposure resulted in about 80-88% death rate.
The optimal death rate for improved secondary metabolites production from microorganisms is 70-95% as reported previously321. Enhanced secondary metabolite production was obtained from Streptomyces fradiae NRRL-2702 with survival rate being decreased from 100% to 8% and 5% at 100 and 120 second UV exposure respectively285.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Survival rate decreased from 100% to 8% for Streptomyces venezuelae as UV exposure increased from 0-25min322. In present research work exponential survival curve made for Streptomyces avermitilis DSM 41445 with UV exposure at variable time and is also reported for Streptomyces flaneolus by Kelner 1948323. The effect of UV irradiation at exposure time of 45, 55 and 60 minutes is revealed in Figure 4.7.
20
15
10
5
0 45 55 60 No. of Colonies Exposure Time (min Survival Rate (%)
Lethality Rate (%)
Figure 4.7: Comparison between different UV exposure times on survival rate, lethality rate and number of colony formation
Findings of HPLC revealed that UV exposure of 45 minute resulted in a mutant giving 14.65±0.2 folds (254.1443mg/L) increase in avermectin B1b production as compared to the parent Streptomyces avermitilis DSM 41445 (17mg/L) strain with
Estimated (RM) and positive (RP) mutation rate calculated on the basis of product formation were 92.22% and 8.42% respectively. The enhanced production of avermectin B1b after UV exposure might be due to the reason that UV exposure caused the genetic code of Streptomyces avermitilis to be highly unstable resulting in the chromosomal mutation and thus the strain improvement300.
4.3.2. Mutagenesis of Streptomyces avermitilis DSM 41445 by Ethidium Bromide Treatment
4.3.2.1. Susceptibility of Streptomyces avermitilis DSM 41445 Against EtBr Treatment
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Growth of microorganism has been found to inhibit as the concentration of chemical mutagen is increased324.
The susceptibility of Streptomyces avermitilis DSM 41445 against Ethidium Bromide mutagen was observed at varied concentrations ranging from 10µL/mL to 40µL/mL in chemically defined SM2 medium. A parallel decrease in the survival rate was observed with increased concentration as is shown in Table 4.8. Maximum reduction in survival rate was observed at 30µL/mL and 40µL/mL of Ethidium Bromide and these concentrations were further for mutagenesis of Streptomyces avermitilis DSM 41445.
Table 4.8: Susceptibility of Streptomyces avermitilis DSM 41445 against Ethidium Bromide
Ethidium Bromide Living Cells Survival Rate (µL/mL) % 0 7.0×109±0.63 100 10 1.1×109±0.51 15.71±1.43 20 5.0×108±0.49 7.14±0.14 30 4.0×107±0.28 0.57±0.01 40 1.1×106±0.32 0.015±0.001
4.3.3. Mutagenesis
Concentration and exposure time of mutagen affect the results therefore to acquire the positive mutations; it is required to use the optimum dose of mutagen and over mutagenesis should be avoided. Number of genes directly influences the avermectin production. Poisson Model of mutagenesis described 37% survival rate for improved secondary metabolite production85.
Four different concentrations i.e. 10µL/mL, 20µL/mL, 30µL/mL and 40µL/mL of Ethidium bromide were used for vegetative growing cells of Streptomyces avermitilis DSM 41445 and exposed for variable time interval of 10 to 60 min as shown in Figure 4.8 A,B and C. The results revealed that about 11 folds (199mg/L) increased mutant
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation avermectin B1b production was observed from mutant obtained after 30 min exposure of 30µL/mL Ethidium Bromide as compared to the parent strain (17mg/L avermectin B1b) as revealed in Figure 4.8 (B).
The mutants obtained at lowered concentrations (10µL/mL, 20µL/mL) of Ethidium Bromide were almost similar to the parent strain in their ability to produce avermectin B1b because of minor reduction on survival rate with time and very little lethality rate. Naveena et.al 2012 reported drastic effects of increasing the concentration of chemical mutagen up to a certain limit on survival capacity of mutants322. In our findings by increasing the concentration of Ethidium Bromide up to 40µL/mL, the mutants even lost their ability to produce avermectin B1b component. Optimum exposure time of 50 min at 30µL/mL Ethidium Bromide concentration resulted in 12.5% and 0.080% survival rate and lethality rate respectively. Secondary metabolite production and structural differentiation in case of Streptomyces are closely related to each other and over mutagenesis with Ethidium Bromide resulted in bald mutants with no ability to produce the bio products325.
10-20 min
140 No. of Colonies 120 Survival Rate (%) 100 Lethality rate (%) 80 60 40 20 0 10µl/min 20µl/min 30µl/min 40µl/min Concentartion of Ethidium Bromide
Figure 4.8 (A): Effect of different concentrations of Ethidium Bromide on survival rate, lethality rate and number of colony formation at 10-20 minutes of exposure
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
No. of Colonies 150 30 min Survival Rate (%) 125 Lethality rate (%) 100 75 50 25 0 10µl/min 20µl/min 30µl/min 40µl/min Concentration of Ethidium Bromide
Figure 4.8 (B): Effect of different concentrations of Ethidium Bromide on survival rate, lethality rate and number of colony formation at 30 minutes of exposure
50 min No. of Colonies Survival Rate (%) 150 Lethality rate (%0
100
50
0 10µl/min 20µl/min 30µl/min 40µl/min Concentration of Ethidium bromide
Figure 4.8 (C): Effect of different concentrations of Ethidium Bromide on survival rate, lethality rate and number of colony formation at 50 minutes of exposure
4.3.4. Mutagenesis of Streptomyces avermitilis DSM 41445 by Ethyl Methane Sulfonate Treatment
4.3.4.1. Susceptibility of Streptomyces avermitilis DSM 41445 Against EMS Treatment
Growth of microorganism has been inhibited as the concentration of chemical mutagen is increased324.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
The susceptibility of Streptomyces avermitilis DSM 41445 against EMS mutagen was observed at varied concentrations ranging from 0.1µL/mL to 1µL/mL in chemically defined SM2 medium. A parallel decrease in the survival rate was observed with increased concentration as is shown in Table 4.9. Maximum reduction in survival rate was observed at 1µL/mL of EMS and these concentrations were further used for mutagenesis of Streptomyces avermitilis DSM 41445.
Table 4.9: Susceptibility of Streptomyces avermitilis DSM 41445 against EMS
EMS Living Cells Survival Rate (µL/mL) % 0 1.0×1010±0.11 100 0.2 4.0×109±0.25 40.0±0.01 0.4 6.0×108±0.32 6±0.01 0.6 3.0×107±0.28 0.3±0.01 0.8 5.0×105±0.49 0.005±0.001 1.0 1.0×103±0.61 0.00001±0.001
4.3.5. Mutagenesis
Alkylating agents like EMS and MMS follow the error prone pathway while mutating the bacterial cells resulting directly in the mispairing of Alkylating bases and are powerful mutagens. About 4 times more avermectin B1 was obtained after mutagenesis of Streptomyces avermitilis50,51. Selection of appropriate exposure time is very crucial in case of chemical mutagenesis. In present research work no fruitful results obtained at exposure time exceeding the 50 minutes and might be due to the reason that delayed incubation of cells with EMS results in DNA damage and ultimate cells death322.
Vegetative growing cells of Streptomyces avermitilis DSM 41445 were exposed to a single concentration 1µL/mL of Ethyl Methane Sulfonate at variable times ranging from 10 to 80 minutes. Survival rate decreased from 0.769% to 0.384% as exposure time increased with corresponding 1.3003% to 2.6041% increase in the lethality rate. Avermectin B1b hyper producing EMS mutant was obtained at exposure time of 50 min
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation giving 11 times (202.63mg/L) more production as compared to the parent strain (17mg/L). About 5.2 and 10.5 folds avermectin B1b production was given by mutants at exposure time of 10 and 20 minutes respectively. Figure 4.9 shows the effect of EMS on survival rate and lethality rate at various exposures.
3.5
3
2.5
2 No. of Colonies 1.5 Survival Rate (%) 1 Lethality Rate (%)
0.5
0 10 20 50 Exposure Time (minute)
Figure 4.9: Effect of different exposure intervals of EMS on
survival rate, lethality rate and number of colony formation.
Our results revealed that mutants when obtained through different sources resulted in enhanced avermectin B1b production as competed to the parent Streptomyces avermitilis DSM 41445 strain. Choice of mutagen strongly effects the production of avermectin326. The selection of best mutant was based on the maximum production of secondary metabolite as was already done by many researchers286,323,271.
The mutant named Streptomyces avermitilis 41445 UV 45 (3) gave maximum avermectin B1b production and has been selected as avermectin B1b hyper producing mutant strain of Streptomyces avermitilis DSM 41445 and has been opted for further studies. UV irradiation mutagenesis being safe to handle has already been employed by Ikeda et.al 1987 to obtain high avermectin producers and avermectin aglycon mutants from Streptomyces avermitilis327. The production profile of avermectin B1b in 250mL shake flask from mutants obtained from various sources is shown in Table 4.10.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Table 4.10: Comparative production of avermectin B1b from different mutant strains
Type of Exposure No. of Colonies Avermectin B1b Cell Biomass mutagen Time colonies producing Production (g/L) (m) avermectin (mg/L) B1b UV 45 6 2 UV(45)1=43.51±0.28 3.82±0.07 * UV(45)3=254.14±0.08 8.04±0.01
55 5 3 UV(55)1=72.43±0.01 4.75±0.05 UV(55)2=200.27±0.01 7.01±0.01 UV(55)3=64.11±0.01 4.11±0.13 60 4 4 UV(60)1=64.70±0.17 4.12±0.14 UV(60)2=31.46±0.01 3.14±0.01 UV(60)3=49.34±0.01 3.99±0.02 UV(60)4=106.35±0.01 5.11±0.02
EMS 10 2 2 EMS (10)1 = 5.82±0.03
90.01±0.01 EMS (10)2 = 5.84±0.04 90.01±0.01 20 2 2 EMS (20)1 = 5.98±0.02 179.93±0.02 EMS (20)2 = 7.09±0.01 192.06±0.14 50 1 1 EMS (50)1 = 7.05±0.04 202.63±0.01 EB 10 5 2 EB (10)1 = 70.68±0.01 3.80±0.01 EB (10)2 = 5.56±0.01 119.48±0.12 30 4 3 EB (30)1 = 6.72±0.04
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
138.43±0.01 EB (30)2 = 7.95±0.03 199.30±0.05 EB (30)3 = 63.17±0.07 4.11±0.02 50 2 2 EB (50)1 = 52.03±0.12 4.01±0.01 EB (50)2 = 84.96±0.06 4.92±0.01 * The designation of avermectin B1b hyper producing mutant Strain. Shake flaks fermetaton carried out at pH 7.0, temperature 31°C. Each value is an average of three replicates. The symbol (±) shows the standard deviation among the replicates.
4.3.6. Hereditary Stability
The selected mutant strain obtained in the present study was further subjected to genetic stability studies by multiple streaking methods and analysis of avermectin B1b production and proved to be highly stable up to 15 generations as is shown in table 4.11.
Table 4.11: Analysis of Heriditary Stability of Strain UV 45(m) 3
Generation Cell Biomass Avermectin B1b (g/L) Production (mg/L)
1 8.04±0.05 254.14±0.03
3 9.08±0.01 260.18±0.04
5 7.01±0.04 230.56±0.02
7 7.95±0.02 245.36±0.03
9 8.01±0.01 251.67±0.02
11 9.05±0.03 258.61±0.02
13 7.20±0.02 235.81±0.02
15 7.35±0.01 240.98±0.01
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Shake flaks fermetaton carried out at pH 7.0, temperature 31°C. Each value is an average of three replicates. The symbol (±) shows the standard deviation among the replicates
4.3.7. Confirmation of Mutagenesis
4.3.7.1. Restriction Enzyme analysis with Eco R1
Total volume is 20 µl 1.0 µg DNA 0.5 µl OR (10 U/µL) Eco R1 4 µL 10X Tango Buffer Incubation at 37°C for 4h Electrophoresis at 1.5% Agarose gel in 1X TBE Buffer Staining with 1.5% Ethidium Bromide Photographed with UV Marker is high ranger 1Kb DNA Ladder
Figure 4.10: Restriction enzyme analysis of S.avermitilis 41445, S.avermitilis UV 45 (3) mutant, S.avermitilis Ethidium Bromide mutant and S.avermitilis EMS mutant with Eco R1
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Lane M: Marker Production of avermectin B1b
Lane 1: Uncut parent DNA 17mg/L avermectin B1b Lane 2: Parent cut with Eco R1 Lane 3: Uncut UV mutant DNA Lane 4: UV mutant DNA cut with Eco R1 254.14 mg/L avermectin B1b Lane 5: Uncut EtBr mutant DNA Lane 6: EtBr mutant DNA cut with Eco R1 199.30 mg/L avermectin B1b Lane 7: Uncut EMS mutant DNA Lane 8: EMS mutant DNA cut with Eco R1 202.63 mg/L avermectin B1b
4.3.7.2. Restriction Enzyme analysis with Bam H1
10 µL DNA 5 U Bam H1 2 µL 1X Digestion Buffer 0.2 µL 100 µg/ml Bovine serum albumin Incubation at 37°C for 4h Electrophoresis at 2% Agarose gel in 1X TBE Buffer Staining with Ethidium Bromide Photographed with UV Marker is high ranger 1Kb DNA Ladder
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Figure 4.11: Restriction enzyme analysis of S.avermitilis 41445, S.avermitilis UV 45 (3) mutant, S.avermitilis Ethidium Bromide mutant and S.avermitilis EMS mutant with Bam H1
Lane M: Marker Production of avermectin B1b Lane 1: Uncut parent DNA 17mg/L avermectin B1b Lane 2: Parent cut with Bam H1 Lane 3: Uncut UV mutant DNA Lane 4: UV mutant DNA cut with Bam H1 254.14 mg/L avermectin B1b Lane 5: Uncut EtBr mutant DNA Lane 6: EtBr mutant DNA cut with Bam H1 199.30 mg/L avermectin B1b Lane 7: Uncut EMS mutant DNA
Lane 8: EMS mutant DNA cut with Bam H1 202.63 mg/L avermectin B1b
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
STUDY 4:
4.4. OPTIMIZATION
4.4.1. Optimization of Cultural Conditions for Avermectin B1b Production From Streptomyces avermitilis DSM 41445 by Response Surface Methodology (RSM)
4.4.1.1. Selection of key factors for avermectin B1b production
Among various carbon sources optimized, maximum production of avermectin B1b of avermectin B1b from Streptomyces avermitilis DSM 41445 was obtained by soluble corn starch (31.5mg/L) followed by potato starch, wheat flour, lactose, maltose and glucose as is shown in Figure 4.12. The cell biomass produced using different sources also increased in the same order as was the production using different sources.
35 12
30 10
25
8
(mg/L) 20
6 g/L Avermectin B1b B1b Avermectin 15 4 10 Cell Biomass 5 2 0 0 Soluble potato Lactose wheat Maltose Glucose Corn starch Flour Avermectin B1b Starch Production Carbon Sources Cell Biomass
Figure 4.12: Effect of different C-sources on Avermectin B1b production
Similarly yeast extract and NH4Cl proved to be the best organic and inorganic N- sources among various sources used for the better production of avermectin B1b from the parent Streptomyces avermitilis DSM 41445 strain as is shown in Figures 4.13 and 4.14 respectively along with the cell biomass produced.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
14 40
12 35
10 30
25 8 20 g/L 6 15 (mg/L)
Cell Cell Biomass 4
10 Avermectin B1b 2 5 0 0 Yeast Peptone Malt Lemco Urea Corn Casein Extract Extract Powder steep liquor Cell Biomass
Organic N Sources Avermectin B1b Production
Figure 4.13: Effect of different organic N-sources on Avermectin B1b production
18 Avermectin B1b 9 Production 16 Cell Biomass 8
14 7
12 6
10 5
8 4 (g/L) (mg/L)
6 3 Cell Cell Biomass
Avermectin B1b 4 2 2 1 0 0 NH4Cl NH4NO3 KNO3 (NH4)2SO4 (NH4)2SO3 NaNO3
Inorganic N Sources
Figure 4.14: Effect of different Inorganic N-sources on Avermectin B1b production
4.4.2. Screening of key variables by Plackett-Burman Design
Plackett-Burman (PB) Design was used to screen nine variables. Each variable was set at two levels, the high (+) and the low (-) as is already used by Gao et.al 2009 for
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
the medium optimization for the production of avermectin B1a using RSM291. Twelve runs with avermectin B1b production is shown in Table 4.12.
Table 4.12: PB Design for screening of nine variables with coded values and observed avermectin B1b Production
Run Soluble CaCO3 α- KCl NaCl Yeast MgSO4.7H2O pH Temp. Avermectin No. Corn amylase extract B1b starch OC Production g/L g/L g/L g/L g/L g/L g/L X1 X2 X3 X4 X5 X6 X7 X8 X9 mg/L 1 140.0 0.8 0.1 4.0 0.5 2.0 0.1 7.5 35.0 46.37755 (+) (-) (-) (+) (-) (-) (-) (+) (+) 2 140.0 6.0 0.1 1.0 4.0 2.0 0.1 7.0 35.0 55.11105 (+) (+) (-) (-) (+) (-) (-) (-) (+) 3 20.0 6.0 0.5 1.0 0.5 16.0 0.1 7.0 28.0 54.973 (-) (+) (+) (-) (-) (+) (-) (-) (-) 4 140.0 0.8 0.5 4.0 0.5 2.0 0.5 7.0 28.0 52.15565 (+) (-) (+) (+) (-) (-) (+) (-) (-) 5 140.0 6.0 0.1 4.0 4.0 2.0 0.1 7.5 28.0 60.13 (+) (+) (-) (+) (+) (-) (-) (+) (-) 6 140.0 6.0 0.5 1.0 4.0 16.0 0.1 7.0 35.0 48.76865 (+) (+) (+) (-) (+) (+) (-) (-) (+) 7 20.0 6.0 0.5 4.0 0.5 16.0 0.5 7.0 28.0 45.9291 (-) (+) (+) (+) (-) (+) (+) (-) (-) 8 20.0 0.8 0.5 4.0 4.0 2.0 0.5 7.5 28.0 44.27725 (-) (-) (+) (+) (+) (-) (+) (+) (-) 9 20.0 0.8 0.1 4.0 4.0 16.0 0.1 7.5 35.0 41.948 (-) (-) (-) (+) (+) (+) (-) (+) (+) 10 140.0 0.8 0.1 1.0 4.0 16.0 0.5 7.0 35.0 41.2621 (+) (-) (-) (-) (+) (+) (+) (-) (+) 11 20.0 6.0 0.1 1.0 0.5 16.0 0.5 7.5 28.0 45.74485 (-) (+) (-) (-) (-) (+) (+) (+) (-) 12 20.0 0.8 0.5 1.0 0.5 2.0 0.5 7.5 35.0 43.16035 (-) (-) (+) (-) (-) (-) (+) (+) (+)
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Pareto Chart of t-Values for Coefficients; df=2 Variable: Var10 Sigma-restricted parameterization
"Var7" 2.531829
"Var9" 2.397865
"Var6" 2.126982
"Var4" .9182493
"Var8" .844837
"Var2" .7616067
"Var1" .6259527
"Var3" .2925251
"Var5" .0413385
p=.05
t-Value (for Coefficient;Absolute Value)
Figure 4.15: Pareto Chart of t-values for coefficient
Pareto chart in Figure 4.15 represented the variables with maximum effect being at upper most portion of the chart with minimal effects of the variables gradually progressing down in the chart291.
The three variables, Yeast Extract (X6), MgSO4.7H2O (X7) and temperature (X9) being at upper moist portion of the chart were screened as significant variables. Negative values of estimates for these three variables depict their negative effects on avermectin B1b production. The significant effect of screened variables on avermectin B1b production was in MgSO4.7H2O> temperature> Yeast Extract order according to their p- values. The other variables having high p-values and being present at lower side in Pareto chart were therefore neglected and were considered as non significant. The effect of three
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation selected variables was determined by ANOVA of the PB model as is shown in Table 4.13.
Table 4.13: (Analysis of Variance) ANOVA for experimental parameters of PB design affecting the production of avermectin B1b
Variables Estimates t-values p-values Soluble Corn 0.1640 0.1640 0.5 starch X1
CaCO3 0.0455 0.0455 0.5 X2 α-amylase -0.1917 -0.1917 0.7 X3 KCl -0.8024 -0.8024 0.4 X4 NaCl -0.0024 -0.0024 0.9 X5 Yeast Extract -0.3983 -0.3983 0.16* X6
MgSO4.7H2O -1.6592 -1.6592 0.12* X7 pH -5.2534 -5.2534 0.4 X8 Temperature -1.0770 -1.0770 0.13* X9 * Columns with significant variables and their calculated values. R2 = 0.975, Adjusted R2 = 0.730
Analysis of variance (ANOVA) showed that variables were attributed for 97.50% of sample variation and only 1% of total variation could not be explained by the PB design as is clear from value of coefficient of correlation, R2 = 0.975. A value of R2 > 0.9 represent a very good fit between observed and the predicted values showing that model is significant for avermectin B1b production. Value of adjusted R2 = 0.730 was also satisfactory to reveal the significance of the model291,328.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Yeast extract already used by Gao et.al 2009 had been found to show negative effect on avermectin B1a production from Streptomyces avermitilis using RSM291. According to Yu et.al 2008 and Yuan et.al 2008 secondary metabolite production by Streptomyces is greatly dependent and influenced by nutritional requirements especially the nitrogen and carbon source329,330. The main role of nitrogen source in fermentation is its direct influence on mycelium growth as is reported by Lou and Nakai 2001331.
Temperature was found to play a negative role in the production of avermectin B1b from Streptomyces avermitilis DSM 41445 in the present study. By increasing the temperature of incubation during fermentation after the optimum level, the production will be reduced accordingly. Secondary metabolite production is reported by de Silva et.al 2012 to be influenced by temperature, incubation period and culture medium332.
Production of clavulanic acid from Streptomyces strain No. 325 was greatly influenced by temperature in a study conducted by Viana et.al 2010333. The optimum temperature for the production of avermectin from Streptomyces spp was 30 ºC334.
Negative effect was revealed by MgSO4.7H2O on avermectin B1a production in the present study. Addition of metallic substances especially the Manganese, cobalt iron and zinc to the fermentation medium affect the production of secondary metabolites from the Streptomyces as is reported by Stanbury 2000335. Presence of Fe+2, Mg+2 and Zn2 in fermentation medium inhibited the production of avermectin B1a from S. avermitilis IP 842 during fermentation by Zhinan and Peilin 1999289.
Yeast extract (X6), MgSO4.7H2O (X7) and temperature (X9) were further selected for the optimization of maximum production of avermectin B1b from Streptomyces avermitilis DSM 41445.
4.4.3. Optimization by central Composite design and statistical analysis
The optimum levels of three variables [Yeast extract (X6), MgSO4.7H2O (X7) and temperature (X9)] affecting the production of avermectin B1b were determined using central Composite design (CCD) after screening experiments.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Box-Wilson (BW) 23 full factorial central composite design (CCD) was used to conduct twenty experiments by setting each variable at five different levels of variations as is shown in table. The three variables along with their coded are shown in Table 4.14.
Table 4.14: Variables with their coded values
Variables Symbol Code Units Levels (g/L) -2 -1 0 +1 +2 Yeast extract X6 g 2 7.6 10.4 13.2 16
MgSO4.7H2O X7 g 0.1 0.26 0.34 0.42 0.5 Temperature X9 °C 26.2 29.8 32.8 34.8 36.8
The first eight experiments (23=8, factorial CCD) at factorial points for estimation of main effects and two factor interactions, six were at axial points (α=2) to estimate the quadratic effects and six replicates were at central points to estimate the pure process variability reassess gross curvature were conducted. The results of all the experiments conducted along with observed and predicted values are shown in Table 4.15.
Table 4.15: Box-Wilson 23 factorial central composite design for the optimization of avermectin B1b production from S.avermitilis DSM 41445 for RSM
Exp. Yeast MgSO4.7H2O Temp. Observed Predicted by Residual O No. Extract C avermectin RSM (% Values
B1b differencea) g/L g/L g/L production X9 X6 X7 1 7.6 0.26 29.8 676.913 (-0.6) -4.546 672.3673 (-1) (-1) (-1) 2 7.6 0.26 34.8 423.141 (-2.9) -11.967 411.174 (-1) (-1) (+1) 3 7.6 0.42 29.8 650.221 (-12) -70.887 579.3333 (-1) (+1) (-1) 4 7.6 0.42 34.8 400.2587 474.033 (-18) -73.775
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
(-1) (+1) (+1) 5 13.2 0.26 29.8 819.507 (-7.1) -54.374 765.1336 (+1) (-1) (-1) 6 13.2 0.26 34.8 655.531 (-9.5) -57.261 598.26995 (+1) (-1) (+1) 7 13.2 0.42 29.8 849.034 (-15) -116.181 732.85255 (+1) (+1) (-1) 8 13.2 0.42 34.8 762.642 (-19) -123.602 639.04 (+1) (+1) (+1) 9 16.0 0.34 32.8 1016.379 (9.6) 108.332 1124.71165 (+2) (0) (0) 10 2.0 0.34 32.8 688.057 (1.2) 8.807 696.86375 (-2) (0) (0) 11 10.4 0.5 32.8 802.055 (-34) -205.503 596.5527 (0) (+2) (0) 12 10.4 0.5 32.8 802.055 (29) 333.651 1135.70615 (0) (-2) (0) 13 10.4 0.34 36.8 290.573 (17.7) 62.682 353.25425 (0) (0) (+2) 14 10.4 0.34 26.2 576.744 (6) 41.899 618.6425 (0) (0) (-2) 15 10.4 0.34 32.8 632.233 (1) 6.818 639.05065 (0) (0) (0) 16 10.4 0.34 32.8 632.233 (-1.2) -7.818 624.41515 (0) (0) (0) 17 10.4 0.34 32.8 632.233 (0.5) 3.631 635.86405 (0) (0) (0) 18 10.4 0.34 32.8 632.233 (-0.9) -6.319 625.914 (0) (0) (0) 19 10.4 0.34 32.8 632.233 (-1.9) -11.876 620.3575 (0) (0) (0) 20 10.4 0.34 32.8 632.233 (21) 178.289 810.5221 (0) (0) (0) Italic= select bold=training normal=testing a% difference was calculated as the % difference between observed value and the corresponding predicted value over the observed value
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
The values of all other variables were fixed at central points.
Table 4.16: Model Coefficient
Effects Coefficient Standard Student’s t- Significance estimates Error test level (p) Intercept -6004 8663 -0.69305 0.504055 Yeast extract -4538 6857 -0.66183 0.523037 Yeast extract2 2150 1025 2.09692 0.062399
MgSO4.7H2O -156891 253574 -0.61872 0.549934 2 MgSO4.7H2O 2025205 2494112 0.81199 0.435698 Temperature 582 438 1.32988 0.213094 Temperature2 -11 6 -1.71377 0.117343 Yeast extract × 25098 99581 0.25203 0.806119
MgSO4.7H2O Yeast extract × 80 199 0.40256 0.695743 temperature
MgSO4.7H2O × 2425 6971 0.34782 0.735187 temperature
Table 4.17: ANOVA (Analysis of Variance)
Effects SS Degree of MS F P freedom Intercept 11949.6 1 11949.6 0.480320 0.504055 Yeast extract 10897.2 1 10897.2 0.438021 0.523037 Yeast extract2 109392.4 1 109392.4 4.397093 0.062399
MgSO4.7H2O 9523.8 1 9523.8 0.382814 0.549934 2 MgSO4.7H2O 16403.1 1 16403.1 0.659335 0.435698 Temperature 43999.2 1 43999.2 1.768576 0.213094 Temperature2 73068.0 1 73068.0 2.937014 0.117343
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Yeast extract × 1580.3 1 1580.3 0.063521 0.806119
MgSO4.7H2O Yeast extract × 4031.6 1 4031.6 0.162055 0.695743 temperature
MgSO4.7H2O × 3009.7 1 3009.7 0.120977 0.735187 temperature Error 248783.4 10 248783.4
Multiple polynomial regression analysis revealed that the model used for the optimization is not significant as was apparent from the results of Fisher‘s F-test (F = 2.137799) with high value of probability (p = 0.126140). Model‘s non significance can also be seen by determination coefficient (R2). Value of determination coefficient (R2 = 0.658005) indicated that 35% of total variation could not be explained by the model. The adjusted determination coefficient (adj R2 = 0.350209) confirmed the Response Surface Model to be highly non significant for the optimization of avermectin B1b production from Streptomyces avermitilis DSM 41445.
STUDY 5:
4.5. OPTIMIZATION
4.5.1. Optimization of Cultural Conditions for Avermectin B1b Production from Streptomyces avermitilis DSM 41445 by Artificial Neural Networks (ANN)
Response surface methodology being failed for optimization of avermectin B1b production from Streptomyces avermitilis DSM 41445 therefore the ANNs were used as optimization tool for the avermectin B1b production.
Screening experiments are not required for the ANN to apply and it can b applied to both statistically designed and non designed data336. The variables (Yeast extract,
MgSO4.7H2O and temperature) were screened from Plackett-Burman Design and were
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation used for statistical optimization through ANN. The benefit of ANNs is that if one of its elements failed to perform, it began to work through another element337.
4.5.1.1. Optimization by central Composite design and statistical analysis
The optimum levels of three variables [Yeast extract (X6), MgSO4.7H2O (X7) and temperature (X9)] affecting the production of avermectin B1b were determined using central Composite design (CCD) after screening experiments.
Box-Wilson (BW) 23 full factorial central composite design (CCD) was used to conduct twenty experiments by setting each variable at five different levels of variations as is shown in table. The three variables along with their coded values are shown in Table 4.18.
Table 4.18: Variables with their coded values set at five different levels of variations
Variables Symbol Code Units Levels (g/L) -2 -1 0 +1 +2 Yeast extract X6 G 2 7.6 10.4 13.2 16
MgSO4.7H2O X7 G 0.1 0.26 0.34 0.42 0.5 Temperature X9 °C 26.2 29.8 32.8 34.8 36.8
The first eight experiments (23=8, factorial CCD) at factorial points for estimation of main effects and two factor interactions, six were at axial points (α=2) to estimate the quadratic effects and six replicates were at central points to estimate the pure process variability reassess gross curvature were conducted. The results of all the experiments conducted along with observed and predicted values are shown in Table 4.19.
Table 4.19: Box-Wilson 23 factorial central composite design for the optimization of avermectin B1b production from Streptomyces avermitilis 41445 by ANN
O Exp. Yeast MgSO4.7H2O Temp. C Observed Predicted by No. Extract avermectin ANN (%
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
B1b differencea) g/L g/L g/L production X6 X7 X9 mg/L 1 7.6 0.26 29.8 672.3673 666.1472 (0.9) (-1) (-1) (-1) 2 7.6 0.26 34.8 411.174 550.6528 (-33) (-1) (-1) (+1) 3 7.6 0.42 29.8 579.3333 642.5023 (-10) (-1) (+1) (-1) 4 7.6 0.42 34.8 400.2587 516.0763 (-28) (-1) (+1) (+1) 5 13.2 0.26 29.8 765.1336 765.4970 (-0.04) (+1) (-1) (-1) 6 13.2 0.26 34.8 598.26995 584.0858 (2.3) (+1) (-1) (+1) 7 13.2 0.42 29.8 732.85255 872.1135 (-19) (+1) (+1) (-1) 8 13.2 0.42 34.8 639.04 805.2171 (-26) (+1) (+1) (+1) 9 16.0 0.34 32.8 1124.71165 973.7062 (13.4) (+2) (0) (0) 10 2.0 0.34 32.8 696.86375 745.9921 (-7) (-2) (0) (0) 11 10.4 0.5 32.8 596.5527 783.8915 (-31) (0) (+2) (0) 12 10.4 0.5 32.8 1135.70615 783.8915 (30) (0) (-2) (0) 13 10.4 0.34 36.8 353.25425 436.1454 (-23) (0) (0) (+2) 14 10.4 0.34 26.2 618.6425 627.1866 (-1.3) (0) (0) (-2) 15 10.4 0.34 32.8 639.05065 640.2415 (-0.1) (0) (0) (0) 16 10.4 0.34 32.8 624.41515 640.2415 (-2.5) (0) (0) (0) 17 10.4 0.34 32.8 635.86405 640.2415 (-0.6)
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
(0) (0) (0) 18 10.4 0.34 32.8 625.914 640.2415 (-2.2) (0) (0) (0) 19 10.4 0.34 32.8 620.3575 640.2415 (-3.2) (0) (0) (0) 20 10.4 0.34 32.8 810.5221 640.2415 (21) (0) (0) (0) Italic= select bold=training normal=testing
a% difference was calculated as the % difference between observed value and the corresponding predicted value over the observed value
4.5.2. Comparison of Different ANNS
Out of 40 constructed ANNs, 38th ANN model was selected based on the best statistical values. Statistical comparison of all the 40 ANNs is given is Table 4.20. The model‘s goodness to fit and integrity can be best justified by values of determination coefficient (R2) and adjusted R2 .Chen et.al 2009 reported that high correlation between a regression model can be explicated by higher values of R2 328.
The higher values of determination coefficient R2 = 0.9752 showed that model is highly significant for the optimization of avermectin B1b production. It also reflected a good fit between the observed values and the predicted values. Higher value of adjusted R2 can also be used to elucidate the model‘s accuracy and significance as is reported by Elibol 2004293. R2 values higher than 0.9 were shown by 12, 13, 23, 31, 32 and 38 ANN; however the highest value was of 38th model. Although 12, 13, 23, 31, 32 and 38 ANNs have R2 > 0.9 but value of Adjusted R2 was greater than 0.9 only for the selected ANN and it was therefore selected as best ANN model. Values of averages absolute deviation (ADD) and root mean square error (RMSE) are of utmost importance for method validation.
For ANN model to be highly significant statistically, the value of averages absolute deviation must be as small as possible while root mean square error must be
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation close to zero. For selected 38th ANN model, these two conditions were fulfilled in contrast to all other ANN models having higher values than the selected ANN.
Table 4.20: Comparison of prediction capabilities of different ANNs for the avermectin B1b production from Streptomyces avermitilis 41445
Sr. No. R2 Adjusted R2 AAD RMSE 1 0.741 0.5079 -16.30 4.45 2 0.657 0.3426 -15.10 4.80 3 0.525 0.0975 -12.30 5.50 4 0.712 0.4528 -15.10 3.69 5 0.765 0.5535 -18.58 4.30 6 0.562 0.1678 -12.35 5.40 7 0.867 0.7473 -20.78 2.75 8 0.763 0.5497 -18.60 4.25 9 0.825 0.6675 -18.35 2.68 10 0.651 0.3369 -15.30 3.80 11 0.824 0.6656 -18.30 2.69 12 0.900 0.8100 -20.22 2.01 13 0.915 0.8385 -20.38 2.05 14 0.781 0.5839 -18.30 4.40 15 0.849 0.7131 -19.45 2.99 16 0.682 0.3958 -16.84 3.98 17 0.799 0. 6181 -17.99 4.96 18 0.763 0.5397 -18.60 4.25 19 0.852 0.7188 -19.44 3.01 20 0.863 0.7397 -19.40 3.98 21 0.841 0.6979 -19.35 2.47 22 0.876 0.7644 -18.55 3.98 23 0.911 0.8309 -20.57 2.045 24 0.769 0.5611 -18.40 4.28 25 0.872 0.7568 -18.50 3.75 26 0.736 0.4984 -15.90 3.65
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
27 0.791 0.6029 -18.66 4.50 28 0.842 0.6998 -19.33 3.46 29 0.798 0.6162 -18.69 4.45 30 0.832 0.6808 -19.12 3.58 31 0.939 0.8845 -20.76 2.26 32 0.936 0.8784 -20.98 2.33 33 0.845 0.7055 -19.35 2.01 34 0.793 0.6067 -17.91 4.80 35 0.775 0.5725 -16.65 4.00 36 0.842 0.6998 -19.30 2.01 37 0.693 0.4167 -15.60 3.35 38 0.9752 0.95288 -29.85 1.076 39 0.753 0.5307 -15.60 3.30 40 0.735 0.4965 -15.98 3.35
4.5.3. The selected 38th ANN Model
Values of determination coefficient (R2=0.989) enlightened that selected 38th ANN model was unable to explain only 1.1% of total variation. The precision and goodness to fit of model was well exhibited from R2 and adjusted R2 values. Also the observed production of avermectin B1b was very close to the ANN predicted values showing the model‘s accuracy.
The main stride for applying ANN as statistical optimization tool is to design the appropriate network topology which involves choice of activation function, training algorithms, training parameters number of hidden layers, number of neurons in each hidden layer, initial weights and training durations. All of these factors affect the optimization process appreciably. Topology of network normally consisted of a single hidden encompassing large no. of hidden neurons as mention by Hornik et.al 1989338. Network topology for selected 38th ANN model consisted of three layers (3:7:1) as is shown in Figure 4.16.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Figure 4.16: Topology of neural networks for avermectin B1b production during ANN optimization. Triangles represent the input (neurons added for ANN processing):
yeast extract, MgSO4.7H2O and temperature. The squares represent the hidden and the output layer (neurons generated during ANN processing). Small open circles represent the input and output layers (the neurons that can be observed in the form of numerical values)
4.5.4. Optimization of fermentation variable using the selected ANN
The ANN predicted optimal levels for Yeast extract; MgSO4.7H2O and temperature were 16.0 g/L, 0.5 g/L and 32οC respectively with 1095.556mg/L avermectin B1b production from Streptomyces avermitilis DSM 41445. The production of avermectin B1b at these optimum values of three variables keeping the other variables at their central points was 1050.756±0.789mg/L which was in good agreement with the predicted value and hence showing the prediction capability of the selected ANN model as is shown in Table 4.21.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Table 4.21: Showing the predicted optimal levels and avermectin B1b production obtained through optimization using ANN
Sr. Method Yeast MgSO4.7H20 Temp. Predicted Experimental no. extract by ANN data X1 X2 X3 (% diff.a) g/L g/L οC 1 ANN 16.0 0.5 32 1095.55 1050.75±0.78 predicted optima 2 Optima 2.0 0.34 33 745.9921 (- 696.86±0.75 before 7) optimizati on
During fermentations, production can be enhanced through optimization289,263,291.
Fermentation medium with ANN optimized levels of Yeast extract, MgSO4.7H2O and temperature resulted in about 1.5 fold increase (150%) in avermectin B1b production as compared to the non optimized SM2 medium. Medium optimization for avermectin B1a production from Streptomyces avermitilis IP 842 was enhanced up to 22.6% in optimized medium as compared to the non optimized CSYC medium as reported by Zhinan and Peilin 1999289. In RSM optimized medium production of avermectin B1a was improved up to 1.45 folds as compared to the non optimized medium291.
The 3D response surface interaction plots for avermectin B1b are represented in Figures 4.17, 4.18 and 4.19. The 3D response surface plot for yeast extract and
MgSO4.7H2O showed that both have a positive interaction effect on avermectin B1b production. Profound effect was seen by yeast extract. Production enhanced linearly as the concentration of the two variables increased and for these two variables the optima is located at near their maximum concentration as is shown in Figure 4.17.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Figure 4.17: Surface plot obtained optimization using ANN for the combined effect
of yeast extract and MgSO4.7H2O on avermectin B1b production by keeping all other variables constant
3D response surface plot for yeast extract and temperature revealed that the effect of yeast extract on avermectin B1b is positive however temperature began to effect the production negatively as the optima reached. For yeast extract the optima is located near the maximum concentration while for temperature it is at the middle point as is shown in Figure 4.18.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
4.18: Surface plot obtained optimization using ANN for the combined effect of yeast extract and temperature on avermectin B1b production by keeping all other variables constant
The 3D response surface plot for temperature and MgSO4.7H2O revealed that the effect of MgSO4.7H2O on avermectin B1b is positive however temperature began to effect the production negatively as the optima reached. For MgSO4.7H2O the optima is located near the maximum concentration while for temperature it is at the middle point as is shown in Figure 4.19.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Figure 4.19: Surface plot obtained optimization using ANN for the combined effect
of MgSO4.7H2O and temperature on avermectin B1b production by keeping all other variables constant
4.5.5 Sensitivity Analysis
ANN Sensitivity analysis illustrates the sensitivity of network towards a particular variable and was used to calculate the ratio and rank of each variable. The ratio equal to or less than one for a particular variable revealed the variable to be non significant for output in contrast to the variable with ratio more than one as is reported by Lou and Nakai 2001331.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Predictive error ratio for each input variable is calculated according to their degree of validity representing the contributions of different variables in predicting the outcome. ANN sensitivity analysis for three variables revealed that maximum effect on avermectin B1b production can be seen from yeast extract with Ratio = 1.322824 followed by temperature having Ratio = 1.280772. The least significant factor for avermectin B1b production from Streptomyces avermitilis DSM 41445 was
MgSO4.7H2O with Ratio = 1.072251 as is shown in the Table 4.22.
Table 4.22: ANN Sensitivity analysis for optimization of avermectin B1b production from Streptomyces avermitilis DSM 41445
Parameter Yeast extract (X1) MgSO4.7H2O (X2) Temperature (X3) g/100mL g/100mL ο C Ratio 1.32 1.07 1.28 Rank 1.00 3.00 2.00
Ratios are values given by ANN as a result of sensitivity analysis to input variables. Rank is the order according to the ratios.
Enhanced avermectin B1a production from Streptomyces avermitilis 14-12A through RSM proved yeast extract to have significant effect on product formation391. Yeast extract used as nitrogen source in the fermentation medium was found to be the most important variable affecting the production of avermectin B1b from Streptomyces avermitilis DSM 41445. The optimal level of yeast extract for maximum avermectin B1b production obtained through ANN was 16g/L. Secondary metabolite production and growth of microorganism from Streptomyces is highly associated with nitrogen source present in the fermentation medium because it affects the mycelium growth directly308, 339, 340.
According to ANN sensitivity analysis, the temperature was ranked as second most important parameter affecting the production of avermectin B1b. the optimal level
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation of temperature depicted by ANN was 32°C. A temperature of 30 ºC was optimized for enhanced avermectin production from Streptomyces spp. by Oskay 2009334.
Various factors effecting the production of secondary metabolites from Streptomyces include temperature, incubation period and culture medium332. The most significant variable during clavulanic acid production from Streptomyces strain No. 325 was temperature333.
Addition of metals in the fermentation medium strongly effects the production of secondary metabolites. The most important effect can be seen by Manganese, cobalt iron 335 and zinc . The optimum concentration of MgSO4.7H2O obtained from ANN optimization was 0.5g/L and has been characterized as the least effective variable from ANN sensitivity analysis. The production of avermectin B1a from S. avermitilis IP 842 inhibited when Fe+2, Mg+2 and Zn2 were added in the production medium289.
STUDY 6:
4.6. OPTIMIZATION
4.6.1. Comparative Analysis of Response Surface Methodology and Artificial Neural Network During Medium Optimization For the Enhanced Production of Avermectin B1b from Streptomyces avermitilis 41445 UV 45(m) 3
4.6.2. Single factor optimization for Selection of key factors for avermectin B1b production
Among various carbon sources optimized, maximum production of avermectin B1b from Streptomyces avermitilis 41445 UV 45(m)3 was obtained by potato starch (473.27 mg/L) followed by maltose, wheat flour, soluble corn starch, glucose and lactose as shown in Figure 4.20. The cell biomass produced using different sources also increased in the same order as was the production using different sources.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
30 500 450 25 400 350
20
300
15 250
g/L mg/L
200 Cell Cell Biomass
10 Avermectin B1b 150 100 5 50 0 0 Potato Maltose Wheat Soluble Glucose Lactose starch flour Corn Starch Avermectin B1b
Cell Biomass Carbon Sources
Figure 4.20: Effect of different C-sources on Avermectin B1b production
Similarly peptone and NH4Cl proved to be the best organic and inorganic N- sources among various sources used for the better production of avermectin B1b from the parent Streptomyces avermitilis DSM 41445 strain as is shown in Figures 4.21 and 4.22 respectively along with the cell biomass produced.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
700 25
600 20
500
400 15 (g/L)
(mg/L) 300 10 Cell Cell Biomass 200 5
Avermectin ProductionB1b 100
0 0 Peptone Yeast Malt Lemco Urea Corn Casein Extract Extract Powder steep liquor Avermectin B1b
Organic N Sources Cell Biomass
Figure 4.21: Effect of different organic N-sources on Avermectin B1b production
18 600 16 500 14
12 400
10
300 (mg/L) 8 (mg/L)
Cell Cell Biomass 6 200 4 100 Avermectin ProductionB1b 2 0 0
Cell Biomass
Inorganic N Sources Avermectin B1b
Figure 4.22: Effect of different inorganic N-sources on Avermectin B1b production
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
4.6.3. Statistical Optimization
The present study was conducted for the comparison of two methods for the production of avermectin B1b from Streptomyces avermitilis 41445 UV 45(m) 3 obtained from ultra violet irradiation of Streptomyces avermitilis 41445. Statistical optimization of medium and process parameters using two methods ANN and RSM was performed using KCl (X1), NaCl (X2) and pH (X3) as significant variables. In routine optimization processes, one parameter at one time optimization has been employed most of the time.
Single parameter at a time optimization technique is unable to predict the optimal conditions and correlation between different parameters for a bioprocess. RSM is advantageous over conventional single parameter optimization. ANN is better method expresses the non linearities in much simple and better way as compared to the RSM and has been successfully used as most suitable technique for medium as well as process parameter optimization for many bioproducts341,342,343.
4.6.3.1. Screening of key variables by Plackett-Burman Design
Plackett-Burman (PB) Design was used to screen nine variables. Each variable was set at two levels, the high (+) and the low (-) as is already used by Gao et.al 2009 for the medium optimization for the production of avermectin B1a using RSM291. Twelve runs with avermectin B1b production is shown in Table 4.23.
Table 4.23: PB Design for screening of nine variables with coded values and observed avermectin B1b Production
Run Potato CaCO3 α- KCl NaCl Peptone MgSO4.7H2O pH Temp. Avermectin No. starch amylase B1b g/L g/L g/L g/L g/L g/L g/L OC Production
X1 X2 X3 X4 X5 X6 X7 X8 X9 mg/L 1 140.0 0.8 0.1 4.0 0.5 2.0 0.1 7.5 35.0 143.58±2.12 (+) (-) (-) (+) (-) (-) (-) (+) (+)
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
2 140.0 6.0 0.1 1.0 4.0 2.0 0.1 7.0 35.0 161.454±0.77 (+) (+) (-) (-) (+) (-) (-) (-) (+) 3 20.0 6.0 0.5 1.0 0.5 16.0 0.1 7.0 28.0 251.819±0.79 (-) (+) (+) (-) (-) (+) (-) (-) (-) 4 140.0 0.8 0.5 4.0 0.5 2.0 0.5 7.0 28.0 491.289±1.21 (+) (-) (+) (+) (-) (-) (+) (-) (-) 5 140.0 6.0 0.1 4.0 4.0 2.0 0.1 7.5 28.0 231.484±1.31 (+) (+) (-) (+) (+) (-) (-) (+) (-) 6 140.0 6.0 0.5 1.0 4.0 16.0 0.1 7.0 35.0 151.78±0.72 (+) (+) (+) (-) (+) (+) (-) (-) (+) 7 20.0 6.0 0.5 4.0 0.5 16.0 0.5 7.0 28.0 444.959±1.73 (-) (+) (+) (+) (-) (+) (+) (-) (-) 8 20.0 0.8 0.5 4.0 4.0 2.0 0.5 7.5 28.0 128.65±1.17 (-) (-) (+) (+) (+) (-) (+) (+) (-) 9 20.0 0.8 0.1 4.0 4.0 16.0 0.1 7.5 35.0 95.2331±0.43 (-) (-) (-) (+) (+) (+) (-) (+) (+) 10 140.0 0.8 0.1 1.0 4.0 16.0 0.5 7.0 35.0 188.547±0.75 (+) (-) (-) (-) (+) (+) (+) (-) (+) 11 20.0 6.0 0.1 1.0 0.5 16.0 0.5 7.5 28.0 131.494±0.67 (-) (+) (-) (-) (-) (+) (+) (+) (-) 12 20.0 0.8 0.5 1.0 0.5 2.0 0.5 7.5 35.0 156.226±1.08 (-) (-) (+) (-) (-) (-) (+) (+) (+)
The three variables selected for statistical optimization by RSM and ANN were KCl (X4), NaCl (X5) and pH (X8).
4.6.3.2. Optimization by central Composite design and statistical analysis
The optimum levels of three variables [KCl (X4), NaCl (X5) and pH (X8)] affecting the production of avermectin B1b were determined using central Composite design (CCD) after screening experiments. A total of 20 experiments using KCl, NaCl and pH variables were conducted according to the Box-Wilson (BW) 23 full factorial central composite design (CCD). Each variable was set at five different levels of variations as is shown in Table 4.24.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Table 4.24: Variable set at five different levels of variations
Variables Symbol Code Units Levels (g/L) +2 +1 0 -1 -2 KCl X4 G 4.0 3.4 2.8 2.2 1.0 NaCl X5 G 4.0 3.3 2.6 1.9 0.5 Ph X8 - 7.5 7.4 7.3 7.2 7.0
4.6.4. Response Surface Methodology (RSM)
Second order model (Eq. 1) used for the calculation of predicted response and the optimum values. 2 2 2 Y = βο + β4X4 + β5X5 + β8X8 + β44X4 + β55X5 + β88X8 + Β45X4× X5 + β48X4× X8 + β58X5 ×
X8 (Equation 1)
Where Y is the response variable and (βο) is the interception coefficient. Β1, β2 and β3 are the coefficients of linear effects. Β44, β55 and β88 are the coefficients of quadratic effects. Β45, β48 and β58 are coefficients of interaction effects for three independent variables (X4= KCl, X5= NaCl and X8= pH).
4.6.5. Artificial Neural Network (ANN)
The data that has been used for the optimization of avermectin B1b production from S. avermitilis UV 45(m) 3 by RSM was also used to be optimized by ANN for comparison of two techniques. Regression based network, 30 in number constructed by STATISTICA were studied for the best, depending on the highest correlation coefficient determination (R2) and lowest selection error. From these networks, the best one was a multilayer perception network that was then selected and used for the optimization and prediction. The experiments used for testing (5), training (10) and selection (5) are shown in Table 4.25.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
The first eight experiments (23=8, factorial CCD) at factorial points for estimation of main effects and two factor interactions, six were at axial points (α=2) to estimate the quadratic effects and six replicates were at central points to estimate the pure process variability reassess gross curvature were conducted. The results of all the experiments conducted along with observed and predicted values are shown in Table 4.25.
Table 4.25: Box-Wilson 23 factorial central composite design for the optimization of avermectin B1b production from S.avermitilis 41445 UV 45(m)3 for RSM
Exp. KCl NaCl pH Observed Predicted Residuals Predicted Residuals No. (g/L) (g/L) (X3) by RSM by ANN (X1) (X2) 1 2.2 1.9 7.2 59.293 -204.234 263.527 75.885 -16.6 (-1) (-1) (-1) 2 2.2 1.9 7.4 2037.1109 1223.188 813.923 1999.68 37.43 (-1) (-1) (+1) 3 2.2 3.3 7.2 884.255 393.520 490.735 907.051 -22.8 (-1) (+1) (-1) 4 2.2 3.3 7.4 784.2876 604.063 180.255 809.126 -24.83 (-1) (+1) (+1) 5 3.4 1.9 7.2 694.5715 125.870 568.702 715.000 -20.42 (+1) (-1) (-1) 6 3.4 1.9 7.4 961.8703 703.679 258.192 1002.85 -40.98 (+1) (-1) (+1) 7 3.4 3.3 7.2 832.3824 897.379 -64.996 797.007 35.38 (+1) (+1) (-1) 8 3.4 3.3 7.4 743.7067 258.308 485.399 761.712 -18.0 (+1) (+!) (+1) 9 4 2.6 7.3 258.8651 408.102 -149.237 263.948 -5.08 (+2) (0) (0) 10 1 2.6 7.3 107.5976 374.126 -266.529 117.531 -9.93 (-2) (0) (0) 11 2.8 4 7.3 501.1463 556.823 -55.676 487.669 13.48 (0) (+2) (0)
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
12 2.8 0.5 7.3 85.5713 393.682 -308.111 65.977 15.6 (0) (-2) (0) 13 2.8 2.6 7.5 596.2421 1039.784 -443.502 608.892 -12.64 (0) (0) (+2) 14 2.8 2.6 7.0 152.0718 287.816 -135.744 179.242 -27.17 (0) (0) (-2) 15 2.8 2.6 7.3 144.7085 458.759 -314.051 159.817 -15.10 (0) (0) (0) 16 2.8 2.6 7.3 141.1498 458.759 -317.610 159.817 -18.67 (0) (0) (0) 17 2.8 2.6 7.3 249.9209 458.759 -208.838 259.817 -9.9 (0) (0) (0) 18 2.8 2.6 7.3 170.8978 458.759 -287.862 159.817 11.08 (0) (0) (0) 19 2.8 2.6 7.3 156.5448 458.759 -302.215 169.817 -13.3 (0) (0) (0) 20 2.8 2.6 7.3 252.4276 458.759 -206.332 259.817 -7.4 (0) (0) (0) Italic= select bold=training normal=testing
4.6.6. Optimization Using RSM
Values of R2 (0.4214) and adjusted R2 (0.338) are very small as calculated by the RSM regression analysis. The calculated p values were greater than 0.05 for all parameters, interaction effects and the model. Thus the RSM model is not significant according to the above mentioned statistical values (Table 4.26). The non significant RSM model means that it is not explaining the data correctly.
Table 4.26: Analysis of variance for optimization of avermectin B1b production from S.avermitilis UV 45(m) 3 using Box-Wilson Design calculated by RSM data.
Variables SS Degree of MS F t-value P- C.L C.L
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
freedom value -95% +95% Intercept 13729 1 13729.1 0.054 0.234 0.819 -- -- KCl (X1) 35631 1 356315.8 1.424 1.193 0.260 -29.463 97.434 6 KCl2 (X12) 7104 1 7103.6 0.028 -0.168 0.869 -2.905 2.496 NaCl (X2) 72300 1 723008.5 2.890 1.700 0.119 -15.043 111.905 9 NaCl2 (X22) 1852 1 1852.2 0.007 0.086 0.933 -2.041 2.2051 pH (X3) 58764 1 58764.5 0.234 -0.484 0.638 -55.443 35.631 pH2 (X32) 16512 1 165126.2 0.540 0.734 0.479 -30.315 60.157 6 KCl (X1) * NaCl 15095 1 15095.3 0.060 0.245 0.810 -4.622 5.767 (X2) KCl (X1) * pH 36092 1 360921.7 1.442 -1.201 0.257 -97.626 29.235 (X3) 2 NaCl (X2) * pH 74039 1 740398.3 2.959 -1.720 0.116 - 14.443 (X3) 8 8 112.328 Error 25014 10 250148.9 89
This can also be seen from the big differences of observed values and values predicted by RSM (Table 3). No factor and their pair wise interaction effects showed the p-values less than 0.5. Maximum effect was due to the NaCl followed by KCl and pH sequentially. The optimum levels of KCl, NaCl and pH given by RSM insignificant model were 2.14 g/L, 2.56 g/L and 7.5 respectively.
In the ANOVA test, the F and P values determined the significance of the input variables. Higher values for the calculated F and p values smaller than 0.05 indicated the significance of a model291,344,345. In the present work the values of F and p also indicates the non significance of RSM model. Therefore it is concluded that RSM method could not be used for optimization and prediction of the present process.
4.6.7. Optimization Using ANN
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
The ANN predicted values are much closer to the observed vales as compared to the RSM predicted values. Topology of network consisted of three layers (3:7:1), an input layer consisted of three fermentation variables, a middle hidden layer of seven neurons and one output layer for avermectin B1b production (Figure 4.23). Different colors represented the activation levels of neurons in ANN processing.
Figure 4.23: Topology of neural networks for avermectin B1b production during ANN optimization. Triangles represent the input (neurons added for ANN processing): KCl, NaCl and pH. The squares represent the hidden and the output layer (neurons generated during ANN processing). Small open circles represent the input and output layers (the neurons that can be observed in the form of numerical values)
During the application of ANN modeling the main step is the designing of network topology346,347. The statistical optimization procedures are highly affected by different designing parameters including the choice of activation function, training algorithms, training parameters and no. of hidden layers, no. of neurons in each hidden
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation layer, initial weights and training durations. Customarily one hidden layer with large number of hidden neurons depicts the precise estimations to any continuous nonlinear function. In the present study during ANN modeling the developed topology network consisted of three layers: (1) an input layer consisting of three neurons (2) a hidden layer with seven neurons and (3) output layer of one neuron.
4.6.8. Sensitivity Analysis by ANN
The optimum levels of KCl, NaCl and pH given by ANN were 1.0g/L, 0.5g/L and 7.46 respectively. The highest effect was from pH followed by NaCl and KCl as is shown by sensitivity analysis in Table 4.27.
Table 4.27: Sensitivity analysis by ANN for optimization of avermectin B1b production from S.avermitilis UV 45(m) 3
Parameter KCl (X1) NaCl (X2) pH (X3) g/L g/L Ratio 1.44 1.51 2.47 Rank 3.00 2.00 1.00
The RSM calculate the effects from factors alone and interaction effects of different factors in the form of regression analysis. ANN method gives the sensitivity analysis, which tells about the ranking order of factors and the significance of factors. The ratio and ranking of each variable from ANN sensitivity analysis represents the network sensitivity for a fastidious variable331.
The ratio equal to one or less than one represent the variable to be less significant for output as compared to the variables with ratio more than one. Chiu et al., 2006 predicted from their research work that predictive error ratio for each input variable can be calculated according to their degree of validity which will demonstrate the
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation contributions of distinctive variables in predicting the outcome. Ratios are ranked in order of descending importance348.
In the present study conducted for the production of avermectin B1b from Streptomyces avermitilis 41445 UV 45(m) 3, the sensitivity analysis revealed maximum effect on production was from pH followed by NaCl and KCl respectively.
This order of effects was not in accordance to that obtained from RSM. However ANN model accuracy is best supported by better values of R2 (0.9353) and adjusted R2 (0.8706). The interaction effects of these parameters are shown by 3D surface plots as shown in Fig. 4.24, 4.25 and 4.26. It was observed from curvature of the surfaces that no interaction has strong effect on avermectin B1b production.
Figure 4.24: Surface plot obtained optimization using ANN for the combined effect of KCl and NaCl on avermectin B1b production by keeping all other variables constant
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Figure 4.25: Surface plot obtained optimization using ANN for the combined effect of KCl and pH on avermectin B1b production by keeping all other variables constant
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Figure 4.26: Surface plot obtained optimization using ANN for the combined effect of NaCl and pH on avermectin B1b production by keeping all other variables constant
Comparative analysis of RSM and ANN
The predicted avermectin B1b values obtained from selected neural network of ANN and selected model RSM for experimental runs are shown in Table 4.25. Table revealed that predicted ANN values were much closer to the experimental values as compared to the RSM predicted values. The ANN predicted levels for KCl, NaCl and pH were 1.0g/L, 0.5g/L and 7.46 respectively with predicted 4105.76 mg/L of avermectin B1b production. The RSM predicted levels for KCl, NaCl and pH were 2.1 g/L, 2.56 g/L and 7.5 respectively with predicted 4105.76 mg/L of avermectin B1b production. The experiments performed at optimum levels of three variables predicted by ANN and RSM (4077.40 and 3080.556 for ANN and RSM respectively) revealed that the observed
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation production of avermectin B1b was much closer to the value predicted by ANN as is shown in Table 4.28. Table 4.28: The predicted optimum levels and avermectin B1b production obtained from optimization by S.avermitilis UV 45(m) 3 using ANN and RSM
Sr. Method KCl NaCl pH Predicted by Predicted Experiment no. X1 X2 X3 ANN by RSM al data g/L g/L 1 ANN predicted 1.0 0.5 7.4 4105.76 -- 4077.40 optima 6 2 RSM predicted 2.1 2.56 7.5 -- 4105.76 3080.556 optima 3 Optima before 1 2.6 7.3 117.531 374.126 107.5976 optimization
According to the RSM analysis NaCl has maximum effect; KCl and pH are following respectively. While In ANN method maximum effect was from pH. The second most effect is from NaCl and the least effect is from KCl.
For comparison of ANN and RSM, four evaluation parameters named R2, Adjusted R2, AAD and RMSE were used as shown in Table 4.29. The values of R2 and Adjusted R2 calculated from selected ANN model are much higher as compared to those calculated from RSM. The values of AAD and RSME are sufficiently lower for ANN as compared to the RSM. These results revealed that ANN is much better technique for the optimization of avermectin B1b production from mutant strain Streptomyces avermitilis 41445 UV 45(m) 3 of Streptomyces avermitilis 41445.
Table 4.29: Comparison of optimization and prediction capability by ANN and RSM for avermectin B1b production obtained from optimization by S.avermitilis 41445 UV 45(m) 3
Sr. No. Statistics ANN RSM 1 R2 0.998 0.4214 2 Adjusted R2 0.997 0.338
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
3 AAD 7.586 127.68 4 RMSE 0.105 1.73 Value of coefficient of determination (R2) higher than 0.9 for a regression model characterized the high correlation328. The integrity and accuracy of any statistical model can be well explained from the values of R2 and adjusted R2 293. In the presently conducted research for medium and process parameter optimization for avermectin B1b production from Streptomyces avermitilis 41445 UV 45 (m)3 through RSM, multiple correlation coefficients (R) and determination of coefficient (R2) have been used for the verification of the required model. Value of multiple correlation coefficients (R) in the present study is very small; hence the selected RSM model is not significant for the production of avermectin B1b from Streptomyces avermitilis 41445 UV 45(m) 3. Also the values of determination of coefficient (R2) and adjusted R2 were very small and are not significant for the model. Good values of R2 and adjusted R2 were obtained291. For the medium optimization of avermectin B1a production from Streptomyces avermitilis 14- 12A using Response surface methodology. But the medium and conditions used were different that might be possible reason for the different results from the present work.
During statistical process parameter and medium optimization for avermectin B1b production from Streptomyces avermitilis 41445 UV 45 (m)3 through ANN, values of R2 obtained for ANN was 0.9353 represented the goodness of fit of the model for the optimization. The value of adjusted R2 (0.8706) also advocates the high significance of the model. For a model to be significant the difference between coefficient of determination (R2) and adjusted R2 should be very small. In the present case this difference is the 0.0647 thus proving the model to be highly significant for the process parameter and medium optimization for avermectin B1b production from Streptomyces avermitilis 41445 UV 45 (m) 3.
Values of ADD and RMSE are also important for a statistical method to be significant for the given optimization. For a method to be good the value of ADD should be as small as possible and the value of RSME should be close to zero. In the present study the calculated values of ADD and RSME for RSM were higher than the ANN
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation values. The lower value of RSME for ANN as compared to the RSM revealed the model accuracy and proved the model to be more significant. From all the above parameters it can be concluded that ANN model showed much better performance for the optimization of avermectin B1b production from Streptomyces avermitilis 41445 UV 45 (m)3, in contrast to the RSM.
In case of Streptomycetes, the production of secondary metabolites is restricted to stationary phase cell biomass. Various factors and process parameters including carbon source, nitrogen source, pH, temperature, aeration and incubation time are therefore responsible for the suppression of antibiotic production129.
Selection of suitable medium, pH, incubation period and temperature effect the growth of microorganism and the production of secondary metabolites. Maximum antibiotic production was obtained at optimum pH with higher growth rate of microorganism. With increasing the pH of the medium the antibiotic production and growth began to decrease349. The pH of medium strongly affects the yield of cell biomass. Cell biomass was decreased from 0.8g/L to 0.6 g/L when pH of medium was raised up to 8.0332. The pH found suitable for the enhanced production of granaticine from Streptomyces thermoviolaceus was between 7.3 and 7.5129.
The prominent effect on enhanced production of secondary metabolites is attributed to the pH of fermentation medium. About 1.6 folds increase in the production of avermectin was observed from Streptomyces avermitilis using high-throughput screening method291. This enhanced production is attributed due to the pH of medium being maintained at 7.5 in 360 m3 fermentation batch study. In another study conducted by Gao et.al 2009 it was observed that the production of avermectin B1a in optimized medium was 5128 ± 144 mg/L at pH 7.0291.
Enhanced production of oligomycin was obtained in production medium adjusted at pH 7.0-7.2250. Avermectin B1a production of 5228 U/ml was obtained using OUR
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation monitoring of glucose feeding rate as reported by Liang et.al 2010 in a medium with pH adjusted at 7.5 on industrial scale266.
Impact of pH was testified by de Silva et.al 2012 on the production of antibiotics from Streptomyces spp. isolated from soil332. They revealed that production is best observed at pH 7-8.5 through statistical optimization using RSM. In the present study the pH was optimized at 7.5 and 7.46 for the maximum production of avermectin B1b through RSM and ANN network respectively, these values are within the range of pH used by the other researchers.
Production of secondary metabolites is directly influenced by medium composition332. Salinity is the promising environmental factor effecting the production of metabolites from the microorganism. High salt stress prompts the overall quantity of secondary metabolites produced and the metabolite profile and hence regulation is required in very controlled manner. Good avermectin production was observed with a transformed strain of Streptomyces avermitilis in a medium containing 0.1g NaCl349,350.
Production of avermectin from Streptomyces avermitilis studied by a research group, at varied concentrations of NaCl and the production remained constant up to 0.5% salt concentration in the cultivation medium259. A decrease in production reaching to zero occurred above 0.5-2.5% NaCl concentration. From all the above studies it is observed that a higher concentration of salts inhibits the growth. NaCl was required in small amount for an optimal growth of S. avermitilis. In the present study the optimum levels of NaCl depicted by ANN was very low as compared to RSM. The lower concentration predicted by ANN is found therefore more productive than the RSM predicted NaCl concentration.
In the present study the least significant effect on avermectin production was shown by KCl as predicted by the ANN method. The optimum levels of this variable in the cultivation medium was almost half from the RSM predicted level. Medium salinity effects the growth of microorganism351. The production and composition of secondary
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation metabolites also decreased in medium containing higher KCl concentrations. A research was conducted by Lin et.al 2009 for the enhanced production of oligomycin A from Streptomyces avermitilis and maximum production of oligomycin was obtained in a medium containing 4g/L KCl250. In the present studies the product is different, which may be the possible reason for the lower optimal level of KCl.
STUDY 7:
4.7. APPLICATION
4.7.1. Test Substance
For all the experiments conducted, the avermectin B1b was obtained as fermentation product of S.avermitilis 41445 in SM2 medium by a method as described earlier352 and used in the present study after purification by lyophilization. It is the minor component of commercially available abamectin. It is macrocyclic lactone containing an isopropyl reside in the 25-position showing broad spectrum of anthelmintic activity. The physiochemical properties of avermectin B1b are given in Table 4.30353.
Table 4.30: Physiochemical properties of avermectin B1b
Molecular mass 859.1 Physical appearance White
Molecular formula C47H70O14 Solubility Ethanol, Methanol, DMF, DMSO Form Solid
4.7.2. Contact Filter Paper Toxicity of Earthworms
The results of contact filter paper for earthworms revealed that the effectiveness of avermectin B1b varied with time of exposure. The LC50 values determined from Probit‘s analysis after 48 and 72h were 500µg/cm2 and 300µg/cm2 respectively. Difference in mortality recorded after 48 and 72h is shown is Figure 4.27. Mortality increased as the concentration of avermectin B1b increased.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
100 90 % mortality after 48h 80 % mortality after 72h
70 60 50
40 Mortality% 30 20 10 0 0 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 Concent. (g/cm2)
Figure 4.27: Difference in mortality after 48 and 72h during contact filter paper test
4.7.3. Soil toxicity Of Avermectin of Earthworms
Soil toxicity test of avermectin B1b showed a clear concentration dependent relationship. The mortality increased with concentration and exposure time as is shown in Figure 4.28.
% mortality at day 7 100 % mortality at day 14 80 % mortality at day 28
60
40
Mortality% 20
0 0.1 1 10 50 100 500 1000 -20 Conc. (mg/Kg)
Figure 4.28 Effect of concentration on earthworm mortality
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
The LC50 values calculated after 7, 14 and 28 days from Probit‘s analysis were 712.5248 mg/Kg, 382.6 mg/Kg and 74.6 mg/Kg respectively. No observed effective concentration (NOEC) and lowest observed effective concentration (LOEC) for mortality calculated on the basis of student t-test were 0.1 mg/Kg and 1.0 mg/Kg respectively. The concentration-response relationship was also demonstrated in the form of cocoon formation. No. of cocoon formed after 7, 14 and 28 days were calculated. The results revealed that with increasing the concentration, no. of cocoon formation decreased. Also the exposure time effected the formation of cocoon in a linear manner as is shown in the Figure 4.29.
25 7th day
20 14th day
28th day 15
10 Cocconformation 5
0 control 0.1 1 10 50 100 500 1000 Concent. (mg/kg)
Figure 4.29: Effect of concentration on earthworm cocoon formation
4.7.4. Effect of Avermectin B1b on cocoon formation
Maximum cocoon formation occurred at lowest test concentration of 0.1 mg/kg of avermectin B1b. No cocoon formation was observed at concentration of 1000mg/kg. Also formation of cocoon was found to be the maximum after 28 days. The effect of concentration and exposure time on biomass of earthworm resulted in the maximum reduction in weight loss at highest concentration of test chemical at 14th and 28th day of
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation exposure when compared to the control. Very little different in biomass between treated and non-treated test organisms was observed after 7th day of exposure (Data not shown).
Abamectin which is a mixture of avermectin B1a and B1b is very efficient against parasites and potentially perilous for invertebrates in the soil354. Wang et al, (2012) reported that toxicity inference of an insecticide deceptively fluctuate when different test methods are adopted for the testing248. Avermectins resulted in the paralysis of muscles and cause unspecific effects on the metabolism of test animals355.
The paralysis of muscles occurs due to the suppression of electrical activity caused by the activation of irreversible chlorine permeability356 resulting in Behavioral changes, Mortality, reduction in biomass and cocoon formation effects357. In the present study different test method implemented to check the toxicity gave different results.
During contact filter paper test insecticide is absorbed trough skin therefore it is employed for relative toxicity determination of insecticides to earthworms and is one of the preliminary screening techniques. However in case of soil ecosystem this technique does not give fruitful results358,359. In the present research work the contact filter paper test when employed for the earthworms proved the avermectin B1b to be moderately toxic at a concentration of 500µg/cm2 and 300µg/cm2 after 48 and 72h respectively. From the LC50 values calculated from Probit‘s analysis it was estimated that avermectin B1b was most effective on earthworms after 72h of exposure.
Roberts and Dorough, (1984) classified the chemical as super toxic at LC50 <1.0 2 2 2 µg/cm , extremely toxic at LC50 = 1-10 µg/cm , very toxic at LC50 = 10-100 µg/cm , 2 moderately toxic at LC50 = 100-1000 µg/cm and relatively non toxic at LC50 >1000 µg/cm2 360. In a study conducted by Wang et al, (2011), they reported the intermediate toxicity response of antibiotics, carbamates and organophosphate against E. fetida in 248 contact filter paper assay . The LC50 value for ivermectin in their study was 23.08µg/cm2 owing it to be very toxic. However in present study the avermectin B1b showed moderate toxicity against the tested organism.
It is reported that toxicity of insecticides against earthworms can be accessed through artificial soil toxicity due to the absorbance of chemical through gut210,361,362. In a
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation study conducted by Wang et al, (2011), the LC50 value for ivermectin after 7 and 14days of exposure were 31.05mg/kg and 27.86mg/kg showing that with increasing the exposure 248 time the LC50 values decreased . Mortality increased with time and concentration and intermediate toxicity was exhibited by antibiotics.
In the present study soil test was when performed to evaluate the toxicity of avermectin B1b against earthworms, represented the same decrease in the LC50 values with increasing the exposure time. The estimated LC50 values for avermectin B1b found in the present research were 712.5248 mg/Kg, 382.6 mg/Kg and 74.6 mg/Kg after 7, 14 and 28 days of exposure respectively. The higher concentrations of avermectin B1b observed in this study might be due to the reason that only one abamectin component is effecting the target animals.
Diao et al, (2007) mentioned in their study the values of NOEC and LOEC to be 5.00mg/Kg and >5.00mg/Kg for earthworm survival against abamectin195. NOEC and LOEC for mortality of target species in the present study were 0.1mg/Kg and 1.0mg/Kg respectively. The EC50 value estimated after 48h of exposure of abamectin in a research conducted by Nunes and Espindola, (2012) against Eisenia andei in soil test was 3.92mg/kg with NOEC and LOEC values 0.85mg/kg and 1.75mg/kg respectively363.
Diao et al, (2007) reported the potential risks of abamectin against earthworms confirming the abamectin to be toxic against them231. Gunn and Sadd, (1994) performed a research work showing the effect of ivermectin on earthworm survival rate and reporting that in OECD artificial soil there was no survival observed at above 20mg/kg 364.
The LC50 value reported by Halley et al, (1989) for ivermectin in OECD artificial soil against E. fetida was 314mg/kg 194. In another study conducted by Wislocki et al,
(1989) the LC50 value for abamectin against earthworms after 28 days of exposure was 365 th 28mg/kg of soil dry weight . At 14 day of exposure the calculated value of LC50 for abamectin against earthworm was 17.1mg/kg in OECD artificial soil representing acute 203 toxicity of the abamectin . In a study conducted by Kolar et al, (2010), the LC50
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation determined for abamectin against isopod survival was 71mg/kg of dry soil366 In their study the values for NOEC and LOEC were 3mg/kg and 10mg/kg respectively.
Exposure time and concentration of test chemical also affects the biomass significantly. In a study conducted by Diao et al, (2007) it was observed that there was a significant reduction of the biomass of earthworms after 14 and 28 days at 5.0mg/kg abamectin. However the difference between the control and the experimental species after 14 days of 2.5mg/kg abamectin exposure was less significant195.
In the present study the maximum reduction in the biomass of the exposed species was after 14 and 28 days of exposure at highest concentration (1000.0 mg/Kg) of the avermectin B1b. In a study conducted by Kolar et al, (2008) it is reported that earthworms showed a dose related biomass reduction. In their study the values of NOEC and LOEC calculated for biomass reduction of earthworms in OECD artificial soil were 9.8mg/kg and 29mg/kg respectively196.
In another experiments performed by Koller et al, (2010), it is reported that different in biomass between treated and non-treated isopods was less significant when fed with 10, 20, 100 and 300mg abamectin per kg of dry soil366. Jensen et.al 2007 reported EC50 value for abamectin to be 0.46mg/kg against biomass of earthworms with 0.25mg/kg NOEC367. Diao et al, (2007) reported that no. of cocoon formation is directly influenced by concentration of abamectin. Significant reduction in cocoon formation was observed at 0.25mg/kg of abamectin195.
In the present study the similar dose response relationship was observed for avermectin B1b against earthworms. Gunn and Sadd, (1994) reported that at a concentration of 4mg/kg of ivermectin there was a momentous reduction in the cocoon formation364.
STUDY 8:
4.8. FERMENTER
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
4.8.1. Laboratory Scale Production of Avermectin B1b from Streptomyces avermitilis 41445
The table showed the production of avermectin B1b from Streptomyces avermitilis 41445 on laboratory scale. Glass stainless steel fermenters of 1-liter, 2-liter, 30-liter and 50-liter capacity were used for the fermentations. The working volume of SM2 medium was maintained at 60% of the total Fermenter capacity in each case. In fermentation medium, soluble corn starch 140.0g/L, α-amylase 0.5g/L, CaCO3 6.0g/L,
KCl 4.0g/L, NaCl 4.0g/L, yeast extract 16g/L, MgSO4.7H20 0.5g/L were used to study the production of avermectin B1b from parent strain Streptomyces avermitilis 41445. The optimized fermentation temperature for the parent strain was 32°C. The pH of the medium was maintained at 7.46±0.2 after sterilization.
To ensure the homogeneous mixing of fermentation medium, the agitation speed was maintained at rate of 150 rpm. After 10days of incubation period, the soluble corn starch consumption and avermectin B1b production were noted. The results obtained related that rate of sugar consumption and avermectin B1b production remained the same from 1-liter Fermenter to 30-L Fermenter. A decrease in avermectin B1b production and soluble corn starch consumption occurred in 50-Liter Fermenter and might be due to the improper handling as shown in Table 4.31.
Table 4.31: Laboratory scale production of avermectin B1b from
Streptomyces avermitilis 41445
Fermenter Working Run NO. Sugar Avermectin Volume volume Consumption B1b production (Liter) (g/L) (mg/L) 1-Liter 1.0 3 11 1040.756±0.7 2Liter 1.2 3 11 1025.45±0.04 30-Liter 18.0 3 11 1010.32±0.02 50-Liter 30.0 3 12 1009.24±0.01
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
4.8.2. Laboratory Scale Production of Avermectin B1b from Streptomyces avermitilis 41445 UV 45(m) 3
The table showed the production of avermectin B1b from Streptomyces avermitilis 41445 UV 45(m) 3 on laboratory scale. Glass stainless steel fermenters of 1- liter, 2-liter, 30-liter and 50-liter capacity were used for the fermentations. The working volume of SM2 medium was maintained at 60% of the total Fermenter capacity in each case. In fermentation medium, Potato starch 140.0g/L, α-amylase 0.5g/L, CaCO3 0.8g/L,
KCl 1.0g/L, NaCl 0.5g/L, peptone 2.0g/L, MgSO4.7H20 0.5g/L was used to study the production of avermectin B1b from mutant strain Streptomyces avermitilis 41445 UV 45(m) 3. The optimized pH of the fermentation medium for the mutant strain was 7.46. The medium was adjusted at this pH after sterilization.
The fermentation was carried out 28 °C. To ensure the homogeneous mixing of fermentation medium, the agitation speed was maintained at rate of 150 rpm. After 10days of incubation period, the potato starch consumption and avermectin B1b production were noted. The results obtained related that rate of sugar consumption and avermectin B1b production remained the same from 1-liter Fermenter to 30-L Fermenter. A decrease in avermectin B1b production and potato starch consumption occurred in 50- Liter Fermenter and might be due to the improper handling a is revealed in Table 4.23.
Table 4.32: Laboratory scale production of avermectin B1b from
Streptomyces avermitilis 41445 UV 45(m) 3
Fermenter Working Run NO. Sugar Avermectin Volume volume Consumption B1b production (Liter) (g/L) (mg/L) 1-Liter 1.0 3 11 3080.556±0.01 2Liter 1.2 3 11 3079.45±0.04 30-Liter 18.0 3 11 3075.32±0.02 50-Liter 30.0 3 12 3035.24±0.02
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
4.9. KINETIC PARAMETER STUDY
Comparison of Streptomyces avermitilis 41445 UV 45(m) 3 growth, avermectin B1b production and substrate consumption evaluated in SM2 media during submerged fermentation is shown in Figure 4.30. From Figure 4.30 it is clear that the growth arrived at stationary phase after 72h in SM2 medium with 10.30±0.02 g/L cell biomass. The avermectin B1b production was not observed up to 72 h of fermentation an started in stationary phase revealing the process to be non growth associated. After the growth entered the stationary phase, the production of avermectin B1b started and increased with increasing the cell biomass.
The production of avermectin B1b was maximum (258.6±0.01) at 10th day of fermentation after that it began to decrease. Substrate which is soluble corn starch in the present case was consumed for growth and avermectin B1b production and completely utilized at the end of fermentation.
50 300 280 40
260
240 30
220
20 200 (g/L) 180
10 Avermectin B1b Cell Cell Biomass(g/L) 160
Substrate Utilization SubstrateUtilization (g/L) 140 0 0 48 96 144 192 240 120 -10 100 Time (h)
Figure 4.30: Kinetics of S.avermitilis 41445 and S.avermitilis UV 45 (3) Mutant
The results revealed that the proposed models based upon the Logistic and Piret Equations were significantly describing the relation between growth of Streptomyces
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation avermitilis 41445 UV 45(m) 3, avermectin B1b production and substrate consumption. The kinetic parameter values for present fermentation in SM2 medium are represented in -1 Table 4.33. The maximum specific growth rate (µmax) was 0.15h in SM2 medium with 3.824 non growth associated avermectin B1b production coefficients (β). The higher value of non growth associated avermectin B1b production coefficient (β) than growth associated avermectin B1b production coefficient (α) revealed the process to be non growth associated.
Table 4.33: Avermectin B1b fermentation by Streptomyces avermitilis 41445 UV 45(m) 3 in SM2 medium
SOLUBLE CORN PARAMETERS STARCH
Maximum cell conc Xmax (g/L) 25.6
Initial cell conc. X0 (g/L) 0.5
-1 Maximum specific growth rate µ max (h ) 0.1541
Initial avermectin B1b Production P0 (g/L) 0
Maxi. avermectin B1b Production Pmax (g/L) 0.2586106
Maintenance Coefficient ms 0.003
YP/S 0.737 Growth associated avermectin B1b production coefficient, α 0.001 Non-Growth associated avermectin B1b production coefficient, β 3.824
Cell yield YX/S 0.644
4.9.1. Effect of different carbon sources on Streptomyces avermitilis 41445 UV 45(m)3 growth and avermectin B1b production
Effect of different carbon sources on Streptomyces avermitilis 41445 UV 45(m) 3 growth and avermectin B1b production has been shown in Table 4.34. The kinetic parameter values were calculated from the data obtained during the fermentation process in SM2 media with changed carbon source. Results revealed that production and growth
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation was effected significantly by type of carbon source used. Maximum specific growth rate -1 (µmax) (1.29 h ) was obtained in medium having potato starch as carbon source followed by glucose (0.8 h-1), maltose (0.34 h-1), wheat starch (0.24 h-1), lactose (0.16 h-1) and soluble corn starch (0.15 h-1). The production of secondary metabolites is related to cell biomass.
In the present work Maximum avermectin B1b production (420.02 mg/L) was observed in medium with potato starch as carbon source having highest value of 31.74 g/L for Xmax (g/L) followed by wheat starch, soluble corn starch, maltose, lactose and glucose. Rate of utilization of different carbon substrate and their effect on cell biomass production formation and production formation are shown in Figures 4.31, 4.32 and 4.33 respectively
Table 4.34: Kinetic parameter values of avermectin B1b fermentation by Streptomyces avermitilis 41445 UV 45(m) 3 using different types of carbon sources
Soluble corn Wheat Potato Parameters Glucose Maltose Lactose Starch Starch Starch
Maximum cell conc. Xmax (g/L) 14.74 20.74 16.7 25.6 26.74 31.74
Initial cell conc. X0 (g/L) 0.1 0.1 0.2 0.5 0.3 0.5
-1 Maximum specific growth rate µ max (h ) 0.875 0.3458 0.16 0.1541 0.2416 1.291
Initial avermectin B1b Production P0 (g/L) 0 0 0 0 0 0
Maxi. avermectin B1b Production Pmax (mg/L) 130.01 143.11 138.01 182.14 258.61 420.02
Maintenance Coefficient ms 0.027 0.006 0 0.003 0.001 0.005
Avermectin yield (YP/S) 1.055 0.778 0.29 0.737 0.779 0.662 Non-Growth associated avermectin B1b production coefficient, β 11.36 3.124 3.72 3.824 56.66 22.29 Growth associated avermectin B1b production coefficient, α 0 0 0 0 0 0
Cell yield YX/S 0.375 0.529 0.42 0.644 0.7344 1.077
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
45
40 35 30 25 20 15 10 SubstrateConcent.(g) 5 0 0 24 48 72 96 120 144 168 192 216 240 264 Time (h)
Glucose Maltose Lactose Soluble corn Starch Wheat Starch Potato Starch
Figure 4.31: Rate of Utilization of carbon substrates during fermentation
35 30
25 20
15 Biomass (g) 10 5 0 0 24 48 72 96 120 144 168 192 216 240 264 288 Time (h)
Glucose Maltose Lactose Soluble corn Starch Wheat Starch Potato Starch
Figure 4.32: Effect of various carbon sources on cell biomass production
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
450 400 350 300 250 200 150
Production(mg/L) 100 50 0 0 24 48 72 96 120 144 168 192 216 240 264 Time (h)
Glucose Maltose Lactose Soluble corn Starch Wheat Starch Potato Starch
Figure 4.33: Effect of various carbon sources on avermectin B1b production
4.9.2. Effect of pH on Streptomyces avermitilis 41445 UV 45(m) 3 growth and avermectin B1b production in medium with potato starch as carbon source
Effect of different initial pH on Streptomyces avermitilis 41445 UV 45(m) 3 growth and avermectin B1b production has been shown in Table 4.35. The highest cell concentration (Xmax) (21.3±0.04g/L) was obtained at pH 7.5 with 180.04±0.03mg/L avermectin B1b production. Slight reduction in growth and avermectin B1b production observed as the pH decreased from 7.5 to 7.0 with great inhibition at pH 6.5 followed by 6.0.The final cell concentration was about three times lowered at pH 6.0 as compared to the pH 7.5. However two time reduction in avermectin B1b production was observed at pH 6.0 as compared to the pH 7.5. As for as the cell yield (YX/S) and avermectin B1b yield (YP/S) is concerned, the cell yield vary significantly as the pH was reduced from 7.5 to 6.0. The results revealed that for higher avermectin B1b production and growth of Streptomyces avermitilis 41445 UV 45(m) 3, the pH of 7.5 is very suitable.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Table 4.35: Kinetic parameter values of avermectin B1b fermentation by Streptomyces avermitilis 41445 UV 45(m) 3 using Potato starch at different initial culture pH.
Parameters 6.0 6.5 7.0 7.5
Maximum cell conc. Xmax (g/L) 7.01 10.12 18.4 21.3
Initial cell conc. X0 (g/L) 0.11 0.13 0.21 0.4
-1 Maximum specific growth rate µ max (h ) 0.11 0.12 0.10 0.16
Initial avermectin B1b Production P0 (g/L) 0 0 0 0
Maxi. avermectin B1b Production Pmax (mg/L) 90.1 125.10 160.2 180.04
Maintenance Coefficient ms 0.001 0.003 0.003 0.004
YX/S 0.412 0.47 0.50 0.889 Non-Growth associated avermectin B1b production coefficient, β 3.36 3.124 2.70 3.5 Growth associated avermectin B1b production coefficient, α 0 0 0 0
4.9.3. Effect of agitation speed on Streptomyces avermitilis 41445 UV 45(m) 3 growth and avermectin B1b production in medium with potato starch as carbon source at pH 7.5
During shake flask fermentation, the effect of agitation speed is shown in Table 4.36 on Streptomyces avermitilis 41445 UV 45(m) 3 growth and avermectin B1b production. The concentration of cell biomass did not vary considerably with changing the agitation speed, however, a slight increase in avermectin B1b production was observed with increasing the speed. The resulted revealed that highest avermectin B1b was obtained at agitation speed of 250rpm. The other kinetic parameters such as (YX/S),
(YP/S) and µmax did not vary with speed of agitation.
Table 4.36: Kinetic parameter values of avermectin B1b fermentation by Streptomyces avermitilis 41445 UV 45(m) 3 at medium pH 7.5 with variable agitation speed
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
PARAMETERS 150.0 200.0 250.0
Maximum cell conc. Xmax (g/L) 21.32 21.31 21.30
Initial cell conc. X0 (g/L) 0.11 0.13 0.4
-1 Maximum specific growth rate µ max (h ) 0.14 0.15 0.16
Initial avermectin B1b Production P0 (g/L) 0 0 0
Maxi. avermectin B1b Production Pmax (mg/L) 175.02 178.03 180.04
Maintenance Coefficient ms 0.004 0.004 0.004
YX/S 0.888 0.888 0.889 Non-Growth associated avermectin B1b production coefficient, β 3.4 3.3 3.5 Growth associated avermectin B1b production coefficient, α 0 0 0
Fermentation is very complex process and it is not possible to depict all the process going on in a fermentation process. Production of secondary metabolites from microorganisms usually occurs after the cells entered the exponential growth phase from lag phase. The logistic rate equations have been used as alternative empirical equations to examine large no. of data obtained during fermentations368.
In the present study, the avermectin B1b production also followed the typical trophophase-idiophase fermentation. Production of intracellular compounds is effected strongly by medium composition and the culture conditions369. In the present study effect of various carbon sources and cultural condition was seen on avermectin B1b production and growth of Streptomyces avermitilis 41445 UV 45(m) 3 during submerged fermentation. Using the chemically defined media helped in better understanding of nutrient requirement for growth and secondary metabolite production369 as compared to the other complex medium.
The results of the present study revealed that carbon source used as substrate along with initial culture pH and agitation speed played a key role for growth of
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Streptomyces avermitilis 41445 UV 45(m) 3 and in avermectin B1b production. The modeling study revealed that the avermectin B1b production is non growth associated and occurred in stationary phase. During stationary phase the production increased with time and the cell biomass gradually. Specific growth rate of 1.29 h-1 gave higher cell yield (1.07g.g-1) and avermectin B1b yield (0.66 mg.g-1) in SM2 medium having potato starch as carbon source. In a previous study it is reported that at specific growth rate of 0.36 h-1, the maximum cell yield obtained was 0.37g.g-1 with xylanase yield being 154.09 U mg-1 from E.coli DH5α revealing that cultivation of cells at lower growth rate allowed the allocation of more cellular resources for product expression genes369.
4.9.4. Effect of various Carbon sources on avermectin B1b Production:
The medium and culture condition optimization for enhanced secondary metabolite production can be made more fruitful if the positive or negative effects of the component of medium and fermentation condition are known as is explained during the xylanase production from E.coli370,371,372.
In the present study various carbon source employed were glucose, lactose, maltose, soluble corn starch, wheat starch and potato starch. Maximum specific growth rate obtained was 1.2 h-1 when the carbon source was the potato starch with maximum cell biomass 31.74±0.01g/L. A specific growth rate 0.06±0.007 h-1 has been reported during the tylosin production from Streptomyces fradiae NRRL-2702373. Nitrogen source -1 NH4Cl is associated with specific growth rate 0.69 h during xylanase production from E.coli DH5α369.
4.9.5. Effect of pH on Streptomyces avermitilis 41445 UV 45(m) 3 growth and avermectin B1b production in medium with potato starch as carbon source
Production of enzymes and secondary metabolites from microorganisms at variable initial pH make them very selective towards a specific bioprocess as is reported previously374. High specific growth rate and specific production rate were obtained at pH 7.0 from both the wild type and the mutant strain of Streptomyces venezuelae.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
In the present research work the highest specific growth rate obtained at pH 7.5 was 0.16 h-1 from Streptomyces avermitilis 41445 UV 45(m) 3 with maximum specific production rate 180.04 mg/L. This pH is associated with highest cell biomass 21.3±0.01g/L. It is reported by James et.al 1991 that antibiotic production from S. therrnoviolaceus is highly dependent on medium pH and temperature. Maximum cell biomass was obtained at pH 5.5-6.5 at maximum growth rate of 0.15 h-1 after which it began to decrease and so the Granaticin production375.
Farliahati Mohd Rusli et al. 2009 reported 2122.5 U mL-1 and 4.59 g L-1 maximum xylanase production and maximum cell concentration respectively from E.coli DH5α at initial medium pH of 7.4. At this initial medium pH the maximum specific growth rate (μmax), growth associated xylanase production coefficient (α) and non-growth associated xylanase production coefficient (β) were 0.41 h-1, 474.26 U mg cell-1and 0 U mg cell-1 h-1 respectively. Reduction of medium pH resulted in lowered xylanase production369.
4.9.6. Effect of agitation speed on Streptomyces avermitilis 41445 UV 45(m) 3 growth and avermectin B1b production in medium with potato starch as carbon source at pH 7.5
In previous research it is reported that agitation speed does not contribute greatly towards the growth of microorganism as well as the enzyme production. Production of cell biomass varied a little with agitation speed. The optimal agitation speed reported by them is the 200rpm with 2122.5 U/mL xylanase productions369.
In the present research work the results are in close agreement with the results obtained previously. Maximum cell biomass of 21.32 g/L was obtained at agitation speed of 150rpm. However specific rate and avermectin B1b production were maximum at agitation speed of 250 rpm although not very different from that obtained at 150rpm.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Summary
The present study was aimed for the selection of suitable avermectin B1b producing medium and further the optimization of selected medium for the maximum production of avermectin B1b from Streptomyces avermitilis DSM 41445. Different media were used for the production of avermectin B1b. However, the maximum production of avermectin B1b (17 mg/l) was obtained by using SM2 growth medium composed of (g/L) soluble corn starch 50.0g, KCl 0.1g, NaCl 0.5g, Yeast extract 2.0g,
MgSO4.7H2O 0.1g, CaCO3 0.8g and α-amylase 0.1g at pH 7.2±0.2. The qualitative and quantitative detection of avermectin B1b was performed by using TLC and HPLC respectively. Maximum production was observed with initial medium pH of 7.0±0.2, 10% inoculum size with incubation temperature of 31°C for 10 days of fermentation period.
Streptomyces avermitilis belonging to Actinomycetes are specialized for the production of avermectin being used as anthelmintic and insecticidal agent. They are mostly present in the soil and their isolation from the soil is very crucial so as to obtain the medically important avermectin. Three distinctive localities of Lahore were opted for the assortment of soil to isolate Streptomyces avermitilis. About fifty isolates of Streptomyces species were attained through selective prescreening procedures. All of these isolates were studied for the production of secondary metabolite, the avermectin.
Different test like soluble pigment color and melanin formation were used for identification. Biochemical characterization of isolates closely resembling the control was done. The 10 selected isolates were identified as avermectin producing strain by fermentation and were characterized on ISP2 medium for aerial and reverse side mycelia color, soluble pigment color and melanin formation in comparison with Streptomyces avermitilis DSM 41445. The best avermectin B1b (10.15mg/L) producing isolate S1-C was when subjected for culture characteristics analysis in different media along with biochemical characterization, showed similar result as were obtained for S.avermitilis DSM 41445. From the results it was concluded that agricultural lands around PCSIR
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
Campus Lahore were the rich source of industrially important Streptomyces especially the S.avermitilis.
Avermectin B1b producing Streptomyces avermitilis strains isolated and screened from soil samples were compared with S.avermitilis DSM 41445 for avermectin B1b production. S.avermitilis DSM 41445 being hyper producing avermectin B1b strain was further subjected for strain improvement by mutagenesis to obtain the strain with highest avermectin B1b production. Streptomyces avermitilis DSM 41445 was mutagenized using Ultraviolet irradiation, Ethidium Bromide and ethyl methane sulfonate (EMS).
Selection of avermectin B1b hyper producing mutant produced from these three different methods was made on the basis of HPLC results. Mutants obtained after 45 minutes irradiation of ultraviolet rays on the spores of Streptomyces avermitilis 41445 was found to be the best mutant for the enhanced production of avermectin B1b component (254.14 mg/L). Other avermectin B1b hyper producing mutants obtained from EMS (1 µL/mL) and EB (30 µL/mL) treatment gave 202.63 mg/L and 199.30 mg/L of B1b respectively. The hereditary stability analysis of UV 45(m) 3 mutant showed that the production of avermectin B1b remained constant and there were no reverse mutation occurred after 15 generations.
Single factor optimization being failed to describe interactive effects amongst various fermentation variables had been replaced by statistical optimization in the present research work. Two statistical tools used were Response surface methodology (RSM) and artificial neural network (ANN).
During RSM, screening experiments using Plackett-Burman design for fermentation variables screened nine parameters as significantly effecting the avermectin B1b production from given strains followed by optimization using Central composite design (CCD). In case of ANN, no screening experiments were required and the statistical approached was applies on data used for RSM. Improved avermectin B1b production was obtained through statistical process and medium parameter optimization from both mutated and non mutated S.avermitilis DSM 41445 and S.avermitilis DSM 41445 [UV 45(m)3] strains.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
A comparative study of Response surface methodology (RSM) and artificial neural network (ANN) for UV mutated S.avermitilis DSM 41445 [UV 45(m) 3] proved the ANN to be the best statistical optimization process. The optimum levels of KCl, NaCl and pH given by ANN were 1.0g/L, 0.5g/L and 7.46 respectively with optimum 4105.76 mg/L avermectin B1b production. Similarly for RSM the predicted levels for KCl, NaCl and pH were 2.1 g/L, 2.56 g/L and 7.5 respectively with predicted 4105.76 mg/L of avermectin B1b production. The observed production of avermectin B1b at optimum levels of three variables at ANN and RSM optimum levels was 4077.40mg/L and 3080.556mg/L respectively) and results revealed that the observed production of avermectin B1b was much closer to the value predicted by ANN. Therefore ANN proved to be the best statistical optimization tool for avermectin B1b production from UV mutated S.avermitilis DSM 41445 [UV 45(m)3] strain.
Similarly in case of non mutated parent strain, RSM was not successful as an optimization tool for avermectin B1b production from Streptomyces avermitilis DSM 41445 therefore the various ANNs were used as optimization tool for the avermectin B1b production. As ANN can be applied to both statistically designed and non designed data, therefore the variables (Yeast extract, MgSO4.7H2O and temperature) were screened from Plackett-Burman Design and were used for statistical optimization through ANN. A total of 40 ANNs were used for the optimization and 38th ANN model proved to be the best model based upon R2, adjusted R2, ADD and RMSE values. The ANN predicted optimal ο levels for Yeast extract; MgSO4.7H2O and temperature were 16.0 g/L, 0.5 g/L and 32 C respectively with 1095.556mg/L avermectin B1b production from Streptomyces avermitilis DSM 41445. The observed avermectin B1b production at ANN predicted optimum levels of Yeast extract; MgSO4.7H2O and temperature keeping the other variables at their central points was 1050.756±0.789mg/L and being in agreement with the predicted value proved the prediction capability of the selected ANN model for Streptomyces avermitilis DSM 41445.
Avermectin B1b possesses strong anthelmintic and insecticidal properties being highly toxic against earthworms. Contact filter paper test and soil toxicity tests when performed for avermectin B1b against earthworms proved it to be highly toxic against
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation earthworms as is clear from LC50 values, NOEC and LOEC values obtained using
Probit‘s analysis as statistical tool. The results revealed that LC50 values determined for contact filter paper test after 48 and 72 h were 500µg/cm2 and 300µg/cm2 respectively.
The mortality increased as the concentration of the applied substance increased. The LC50 values calculated after 7, 14 and 28 days from Probit‘s analysis were 712.5248 mg/Kg, 382.6 mg/Kg and 74.6 mg/Kg respectively showing a clear concentration and mortality relationship. NOEC and LOEC values determined for mortality calculated on the basis of student t-test were 0.1 mg/Kg and 1.0 mg/Kg respectively. At lower concentrations the cocoon formation was observed with subsequent elimination at 1000mg/kg of avermectin B1b.
Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
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Production of Avermectins (AVMs) from Streptomyces avermitilis by Fermentation
List of Publications
1) Samia Siddique et.al. (2013) Production of avermectin B1b from Streptomyces avermitilis 41445 by Batch Submerged Fermentation. Jundishapur Journal of Microbiology. 6(8): e7198
2) Samia Siddique et.al. (2014). Production and Screening of High Yield Avermectin B1b mutant of Streptomyces avermitilis 41445 through Mutagenesis. Jundishapur Journal of Microbiology. 7(2): e8626
3) Samia Siddique et.al. (2014). Isolation, Characterization and Selection of Avermectin Producing Streptomyces avermitilis Strains from Soil Samples. Jundishapur Journal of Microbiology. 7(6). e10366.
4) Samia Siddique et.al. (2014). Avermectin B1b production optimization from Streptomyces avermitilis 41445 UV 45(m)3 using response surface methodology and artificial neural network‖. Journal of the Korean Society of Applied Biological Chemistry. 57(3): 371-378.
5) Samia Siddique et.al. (2014). Medium optimization for the enhanced production of avermectinB1b from Streptomyces avermitilis 41445 using Artificial Neural Network. Journal of the Korean Society of Applied Biological Chemistry. 57(5): 677-683.
6) Samia Siddique et.al. (2015) Toxicity of avermectin B1b to Earthworm and Cockroaches. The Journal of Animal and Plant Science. 25(3): 844-850.
7) Samia Siddique et.al. Kinetics of Avermectin B1b Production from Mutant Strain of Streptomyces avermitilis DSM 41445 in Shake Flask Culture. Paper submitted.