MICROBIAL PRODUCTION OF ERYTHRITOL AND MANNITOL: STRAIN IMPROVEMENT AND PROCESS OPTIMIZATION A THESIS SUBMITTED TO THE UNIVERSITY OF PUNE FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY BY LAXMAN S. SAVERGAVE UNDER THE GUIDANCE OF DR. RAMCHANDRA V. GADRE AT CHEMICAL ENGINEERING AND PROCESS DEVELOPMENT DIVISION NATIONAL CHEMICAL LABORATORY PUNE‐411008, INDIA AUGUST 2011 Dedicated to my teachers & parents... ii CERTIFICATE This is to certify that the work incorporated in the thesis entitled ‘MICROBIAL PRODUCTION OF ERYTHRITOL AND MANNITOL: STRAIN IMPROVEMENT AND PROCESS OPTIMIZATION’ submitted by Mr. Laxman S. Savergave was carried out by the candidate under my supervision at Chemical Engineering and Process Development Division, National Chemical Laboratory, Pune 411008 (India). Such material as has been obtained from other sources has been duly acknowledged in the thesis. Dr. Ramchandra V. Gadre Senior Principal Scientist Chemical Engineering & Process Development Division, National Chemical Laboratory, Pune – 411008 (India) August 2011 iii DECLARATION BY THE CANDIDATE I hereby declare that the work incorporated in the thesis entitled ‘MICROBIAL PRODUCTION OF ERYTHRITOL AND MANNITOL: STRAIN IMPROVEMENT AND PROCESS OPTIMIZATION’ is my own work conducted under the supervision of Dr. Ramchandra V. Gadre, at Chemical Engineering and Process Development Division, National Chemical Laboratory, Pune 411008 (India). I further declare that to the best of my knowledge, this thesis does not contain any part of work, which has been submitted for the award of any degree either of this University or any other University without proper citation. Mr. Laxman S. Savergave Dr. Ramchandra V. Gadre (Research student) (Research guide) iv ACKNOWLEDGEMENTS First and foremost, I would like to take this opportunity to express a deep sense of gratitude to my research supervisor, Dr. Ramchandra V Gadre, Scientist, National Chemical Laboratory (NCL), Pune, for his constant encouragement and sustained interest during my stay at NCL. Throughout my tenure at NCL we have had numerous fruitful discussions that I believe will contribute enormously towards my success in future career. I really consider myself honored for having him as my mentor. I am equally obliged to Dr. Sanjay N Nene, Head, Biochemical Engineering group, (NCL) for his encouragement and valuable suggestions in the project. I especially appreciate the freedom he has given me in the laboratory. His dynamic personality and energetic nature is an inspiration. I am extremely grateful to Mr. Vitthal V Jogdand, who gave me the opportunity to work in Biochemical Engineering group. I am grateful for his valuable suggestions, keen interest, constant encouragement and critical evaluation of my thesis. I thank all staff members of Biochemical Engineering Group, specially Dr H V Adikane, Dr Rajesh Singh, M D Jagtap, Dr D M Thakar and other members for their support in completing this work. I extend my warm gratitude to my dear friends Dr Bhalchandra Vaidya, Karthik Narayanan, Geetanjali Lale, Nayan Nayak, Amey Bhide, Asiya Khan, Vikas YSR and Sandeep Golegaonkar for helping me in my experimental work, for their valuable suggestions and technical support. I would like to thank Mukesh Patale, Santosh Dhule, Renuka Joshi, Abhijeet Karale, Amol Dive, Jahnvi Shah, Vaishali Sajjan, Supriya Patil, Hitesh Suthar, Prabhakar Shrivastav, Suresh Keshav, Dr Kumar Babu and my juniors Krunal, Sagar, Shailly, Samiksha, Sudipta, Gayatri and Jagdish for their co-operation and ensuring a friendly working ambiance. My special thanks to my close friends Prashant More, Dr Shailesh Pene, Ganesh Ingle, Prashant Nemmaniwar, Jyoti Patil, Sunil Nanwate, Sharad Borule, Ajit Belkone, Shivaji Kadam, Mahesh Pene, Ram Ambre, Amar Somvanshi, Pradeep Shrimangale, Santosh Deshmukh, Dr Shrikant Panchal, Dr Shivraj Nile, Dr Dnyanraj Choudhari, Dr Abhasheb, Dr Manmat, Satish, Nitesh, Satej, Digambar, Manoj, Yuraj, Anil, Narsingh, Praveen and Suraj for countless things that they have done for me and were always there whenever I needed them. I gratefully acknowledge the Council of Scientific and Industrial Research, New Delhi, for the award of Senior Research Fellowship and Director, NCL for allowing me to carry out my research in NCL. No words suffice the encouragement and moral support rendered by my parents, sisters, spouse Swati and brother in-laws’. This work would not have been possible without their support and persistent motivation. v CONTENTS CHAPTER 1 1.1 Erythritol 5 1.2 Physicochemical properties of erythritol 6 1.3 Functions 6 1.4 History 7 1.5 Biotechnological production of erythritol 8 1.6 Industrial production of erythritol 9 1.7 Erythritol biosynthesis pathway 10 1.8 Strategies for enhancement in erythritol production 18 1.8.1 Strain improvement by mutagenesis 18 1.8.2 Optimization of culture conditions 20 1.8.3 Supplementation with vitamins and trace metals 21 1.8.4 Removal of inhibitors and byproducts 22 1.9 Erythrose reductase (EC 1.1.21) 24 1.10 Uses of erythritol 27 1.11 Mannitol 29 1.12 Chemical process for mannitol production 30 1.13 Mannitol production by lactic acid bacteria (LABs) 31 1.13.1 Mannitol production by homofermentative LABs 31 1.13.2 Mannitol production by heterofermentative LABs 32 1.14 Mannitol production by yeast and filamentous fungi 38 1.15 Mannitol production by recombinant microorganisms 39 1.16 Mannitol dehydrogenase (EC 1.1.67) 40 1.17 Enzymatic production of mannitol 42 1.18 Uses of mannitol 43 1.19 REFERENCES 46 1 CHAPTER 2 2.1 INTRODUCTION 60 2.2 MATERIALS AND METHODS 63 2.2.1 Microorganisms 63 2.2.2 Culture conditions 64 vi 2.2.3 Analysis 64 2.2.4 Screening of cultures for production of erythritol 65 2.2.5 Screening of nitrogen sources for selected erythritol producing cultures 65 2.2.6 Choice of carbon source 65 2.2.7 Evaluation of combinations of yeast extract and inorganic nitrogen sources for 65 erythritol production by Y. lipolytica and C. magnoliae 2.2.8 Effect of medium volume in shake flask for erythritol production 66 2.2.9 Effect of pH and on erythritol production by Y. lipolytica and C. magnoliae 66 2.2.10 Evaluation of growth and erythritol production by C. magnoliae in medium 66 with different yeast extract concentrations 2.2.11 Effect of initial sucrose concentration on erythritol production by C. 67 magnoliae 2.2.12 Requirement of phosphate for erythritol production by C. magnoliae 67 2.2.13 Time course of erythritol production by C. magnoliae using sucrose as carbon 67 source 2.2.14 Effect of sucrose and glucose feeding on erythritol production by C. magnoliae 67 2.2.15 Production of polyols by resting cells of C. magnoliae 67 2.3 RESULTS AND DISCUSSION 68 2.3.1 Screening of cultures for erythritol production 68 2.3.2 Screening of nitrogen sources for selected erythritol producing cultures 71 2.3.3 Choice of carbon sources 73 2.3.4 Evaluation of combinations of yeast extract and inorganic nitrogen sources for 75 erythritol production by Y. lipolytica and C. magnoliae 2.3.5 Effect of medium volume in shake flask for erythritol production 78 2.3.6 Effect of pH on erythritol production by Y. lipolytica and C. magnoliae 80 2.3.7 Evaluation of growth and erythritol production by C. magnoliae in a medium 81 with different yeast extract concentrations 2.3.8 Effect of initial sucrose concentration on erythritol production by C. magnoliae 81 2.3.9 Requirement of phosphate for erythritol production by C. magnoliae 82 2.3.10 Time course analysis of erythritol production by C. magnoliae using sucrose 83 as carbon source 2.3.11 Effect of sucrose and glucose feeding on erythritol production by C. magnoliae 84 2.3.12 Production of polyols by resting cells of C. magnoliae 85 2.4 CONCLUSIONS 86 2.5 REFERENCES 87 1 vii CHAPTER 3 3.1 INTRODUCTION 92 3.2 MATERIALS AND METHODS 94 3.2.1 Cultures 94 3.2.2 Mutagenesis 94 3.2.3 Mutant selection 96 3.2.4 Optimization of erythritol production by C. magnoliae mutant M572 96 3.2.4.1 Choice of carbon sources for erythritol production 96 3.2.4.2 Effect of initial glucose concentration on erythritol production 97 3.2.4.3 Effect of yeast extract concentration on erythritol production 97 3.2.4.4 Effect of medium volume on erythritol production 97 3.2.4.5 Effect of metal ions on polyol production 97 3.2.4.6 Nutritional requirements of mutant M572 98 3.2.4.7 Effect of glucose feeding on erythritol production 98 3.2.4.8 Comparison of C. magnoliae parent strain and its mutant M572 99 3.2.4.9 Comparison of erythritol producing mutants M572 and R23 of C. magnoliae 99 3.2.4.10 Comparison of mannitol producing mutants R1 and R9 of C. magnoliae 99 3.2.5 Media optimization for maximum erythritol and minimum mannitol and 100 glycerol formation from mutant R23 using Response Surface Methodology (RSM) 3.2.6 Characterization of R23 mutant of C. magnoliae 101 3.2.7 Studies on intracellular enzymes of mutant R23 102 3.2.7.1 Preparation of cell extracts 102 3.2.7.2 Erythrose reductase assay 103 3.2.7.3 Glucose‐6‐P dehydrogenase assay 103 3.2.7.4 Partial characterization of ER from mutant R23 103 3.2.7.5 Effect of product and by‐products on ER activity of mutant R23 104 3.3 RESULT AND DISCUSSION 104 3.3.1 Strain improvement for erythritol production by classical mutagenesis 104 3.3.2 Optimization of erythritol production by the C. magnoliae mutant M572 112 3.3.2.1 Effect of carbon sources on erythritol production 112 3.3.2.2 Effect of initial glucose concentration on erythritol production by C.