Bioproduction of Riboflavin by Fungi using Spent Industrial Oils Submitted in fulfillment of the requirements of the degree of Master of Technology: Biotechnology in the Faculty of Applied Sciences at the Durban University of Technology Nazihah Khan B.Tech: Biotechnology August 2011 SUPERVISOR: Dr. F.M. Swalaha CO-SUPERVISOR: Prof. B. Odhav ii DECLARATION I declare that the work done in this dissertation was carried out in accordance with the Regulations of the Durban University of Technology. The work is original, except where indicated by special reference in the text, and part of the dissertation has been submitted for publication. Signed: ............................................. Date: ...................................................... Supervisor: ...................................... Date: ...................................................... Co-supervisor................................... Date: ...................................................... iii PREFACE Title of Publication: Spent Motor Oil as a Substrate for Riboflavin Production using a mutant of Eremothecium gossypii Submitted to Journal Title: Biotechnology Letters iv ABSTRACT Riboflavin (vitamin B2), an essential water-soluble vitamin is commercially produced because it cannot be synthesized by vertebrates. Although this vitamin is produced chemically, bioproduction is a better option since it is more economical, requires less energy, produces less waste and can use renewable sources. In this study we investigated spent oil from the food and motor industries as alternative cheap carbon sources for the bioproduction of this vitamin. Commercial fungal strains namely; Eremothecium gossypii ATCC 10895, Eremothecium gossypii CBS 109.51, Eremothecium ashbyi CBS 206.58 and the yeast, Candida famata ATCC 20850, as well as a laboratory mutated Eremothecium gossypii EMS 30/1 strain were used. Statistical experimental design using a series of fractional factorial experimental designs was used to optimize the effect of yeast extract, peptone, malt extract, K2HPO4 and MgSO4.7H2O to supplement the used oils for optimum riboflavin production. Response surface methodology based on central composite experimental designs was then applied and together with the point predictions made, production media for both substrates were further optimized. The optimized conditions were then tested with laboratory experiments. Results showed that mutant E. gossypii EMS 30/1 produced the most riboflavin in spent motor oil (20.45 mg.l-1) while Candida famata ATCC 20850 produced the highest concentration (16.99 mg.l-1) in spent vegetable oil. With these strains and using the experimental designs from the fractional factorial experiments, supplemented spent motor and spent vegetable oils produced 66.27 mg.l-1 and 72.50 mg.l-1 riboflavin, respectively. The central composite -1 -1 -1 optimization results showed that 0.18 g.l and 0.45 g.l K2HPO4 and 12.5 g.l malt extract increased the production to 91.88 mg.l-1 and 78.68 mg.l-1 in spent vegetable oil and motor oil respectively. A point prediction from the response surface methodology was used to validate these and it was found that 103.59 mg.l-1 riboflavin was produced by mutant E. gossypii EMS 30/1 using 2.5 g.l-1 yeast extract, 0.5 g.l-1 peptone, 12.5 g.l-1 -1 -1 malt extract, 0.18 g.l K2HPO4 and 0.3 g.l MgSO4.7H2O. After optimizing K2HPO4 in a one-factor-at-a-time experiment, 82.75 mg.l-1 riboflavin was produced by C. famataon v -1 -1 -1 -1 SVO using 6.5 g.l peptone, 12.5 g.l malt extract 0.15 g.l K2HPO4 and 1.75 g.l MgSO4.7H2O. This is a 5.08 and 4.87 fold increase respectively when compared to spent oil prior to optimization. This shows that spent motor oil and mutant E. gossypii produces 103.59 mg.l-1 riboflavin while spent vegetable oil and C. famata produces 82.75 mg.l-1 riboflavin. Hence, E. gossypii can be used to generate riboflavin using spent motor oil and C. famata, using spent vegetable oil. vi DEDICATION This thesis is dedicated to my late parents Sumdhara and Roopnarain Ramphal, my loving husband, Feiaz Mohamed Hassan Ally, and my children Farhana, Zaheer, Mohamed Zameer and Sumaya Hassan Ally. vii ACKNOWLEDGMENTS Firstly, I would like to thank my creator, the Almighty, for making everything possible and for giving me the wisdom and strength to persevere and finish this thesis under sometimes-difficult circumstances. My deepest gratitude to Dr. F.M.Swalaha, my supervisor, for his unselfishness, encouragement, guidance, patience and willingness to help in whatever way possible, throughout this study. I am indebted to my co-supervisor, Professor B. Odhav for her co-supervision of this project. Her guidance, support and corrections to this thesis contributed immensely towards finally completing the final version. A special thanks to fellow colleagues Zethu, Thulisile, Prebashnee and Naudene who made the Fermentation Laboratory such a wonderful workplace during these years of study and to Rena, Bilkis and Sharon who assisted whenever I ran out of laboratory consumables. A special thanks to Kathija and Mohamed Hassen for contributing towards my laptop, making the major part of my study possible. It was most useful in recording data, conducting statistical analysis on which my topic was largely based, as well as the compilation of my final dissertation. Sincere thanks to my family who gave me the courage and continuous support that I required, to return to study, after a dormant period of twelve years. viii LIST OF ABBREVIATIONS ANOVA - Analysis of Variance ARS – Agricultural Research Service ATCC – American Type Culture Collection CBS – Centraalbureau voor Schimmelcultures DOE - Design of Experiments EMS – ethylmethane sulphonate FAD – flavin adenine dinucleotide FMN – flavin mononucleotide NRRL – Northern Regional Research Laboratory O and K – Ozbas and Kutsal OFAT - One-factor-at-a-time rpm - revolutions per minute SMO – spent motor oil SPC - statistical process control SVO – spent vegetable oil Td - doubling time λ - lambda - A lambda value of 1 indicates that no transformation is required ie. The model lies within the 95% confidence interval. μ - specific growth rates μmax - maximum specific growth rate ix TABLE OF CONTENTS DECLARATION .......................................................................................................... ii PREFACE .................................................................................................................. iii ABSTRACT ............................................................................................................... iv DEDICATION ............................................................................................................ vi ACKNOWLEDGMENTS ........................................................................................... vii LIST OF ABBREVIATIONS .................................................................................... viii TABLE OF CONTENTS ............................................................................................. ix LIST OF FIGURES ................................................................................................... xvi LIST OF TABLES ...................................................................................................... xx CHAPTER ONE 1.0 INTRODUCTION ................................................................................................. 1 1.2 AIM ........................................................................................................................ 3 1.3 OBJECTIVES ......................................................................................................... 3 CHAPTER TWO 2.0 LITERATURE REVIEW ..................................................................................... 4 2.1 VITAMINS ............................................................................................................. 4 2.1.1 Production of Vitamins ........................................................................................ 4 2.2 RIBOFLAVIN ........................................................................................................ 5 2.2.1 Sources of Riboflavin ........................................................................................... 5 2.2.2 Structure and Properties of Riboflavin .................................................................. 6 2.3 SYNTHESIS OF RIBOFLAVIN ............................................................................. 8 2.3.1 Chemical Synthesis .............................................................................................. 9 2.3.2 Biological Synthesis ............................................................................................. 9 x 2.3.3 Biosynthesis of Riboflavin ................................................................................. 11 2.4 RIBOFLAVIN-PRODUCING MICROORGANISMS .......................................... 14 2.4.1 Bacteria .............................................................................................................. 14 2.4.2 Filamentous Fungi .............................................................................................. 16 2.4.2.1 Eremothecium ashbyi. ..................................................................................... 17 2.4.2.2 Eremothecium gossypii .................................................................................... 19 2.4.2.3 Other fungal riboflavin producers .................................................................... 23 2.4.3 Yeasts