© 2013 ANTHONY COFFMAN ALL RIGHTS RESERVED PRODUCTION OF CARBOHYDRASES BY FUNGUS TRICHODERMA REESEI GROWN ON SOY-BASED MEDIA A Thesis Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Anthony Coffman December, 2013 PRODUCTION OF CARBOHYDRASES BY FUNGUS TRICHODERMA REESEI GROWN ON SOY-BASED MEDIA Anthony Coffman Thesis Approved: Accepted: ___________________________________ ___________________________________ Advisor Department Chair Dr. Lu-Kwang Ju Dr. Lu-Kwang Ju ___________________________________ ___________________________________ Committee Member Dean of The College Dr. Gang Cheng Dr. George K. Haritos ___________________________________ ___________________________________ Committee Member Dean of the Graduate School Dr. Chelsea N. Monty Dr. George R. Newkome ___________________________________ Date ii ABSTRACT Trichoderma reesei RUT-C30 was cultivated in shaker flasks and pH-controlled, agitated batch fermentations to study the effects of soy-based media on the production of cellulase, xylanase, and pectinase (polygalacturonase) for the purposes of soybean polysaccharide hydrolysis. Growth on defatted soybean flour as sole nitrogen source was compared to the standard combination of ammonium sulfate, proteose peptone, and urea. Carbon source effect was also examined for a variety of substrates, including lactose, microcrystalline cellulose (Avicel), citrus pectin, soy molasses, soy flour hydrolysate, and soybean hulls (both pretreated and natural). Flask study results indicated exceptional enzyme induction by Avicel and soybean hulls, while citrus pectin, soy molasses, and soy flour hydrolysate did not promote enzyme production. Batch fermentation experiments reflected the flask system results, showing the highest cellulase and xylanase activities for systems grown with Avicel and soybean hulls at near-neutral pH levels, and the highest polygalacturonase activity resulting from growth on lactose and soybean hulls at lower pH levels, 4.0 to 4.5. Soy polysaccharides were hydrolyzed with fermentation enzyme broths of varying enzyme composition and concentration. Enzyme mixtures with larger proportions of cellulase and polygalacturonase showed slightly higher sugar release. Hydrolysis was incomplete in all experiments, solubilizing roughly one-third of the polysaccharides to oligomeric products. Additionally, methods for estimation of cell concentration via cell DNA extraction and cell wall chitin hydrolysis and a modified iii intracellular protein analysis procedure were investigated, showing high levels of error and interference by soy protein from the growth media. Finally, a consistent and reliable assay for polygalacturonase activity was developed and verified. iv ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Lu-Kwang Ju, for his valuable support and guidance throughout the completion of the research for my Master’s degree. I would also like to thank Dr. Gang Cheng and Dr. Chelsea Monty for serving on my thesis defense committee and for their willingness to help with research questions and my thesis preparation. I additionally would like to thank my group members, especially Dr. Srujana Koganti, Mr. Nicholas Callow, and Ms. Qian Li, for their able and generous assistance and valuable suggestions. Finally, I thank the Department of Chemical and Biomolecular Engineering and the United Soybean Board for the financial support that allowed me to complete my graduate studies. v TABLE OF CONTENTS Page LIST OF TABLES ...............................................................................................................x LIST OF FIGURES ......................................................................................................... xiii CHAPTER I. INTRODUCTION .......................................................................................................1 1.1 Project Justification ...............................................................................................1 1.2 Research Objectives ..............................................................................................2 II. INTRODUCTION TO CARBOHYDRASES .............................................................4 2.1 Production by Trichoderma reesei........................................................................5 2.2 Cellulase ................................................................................................................6 2.3 Hemicellulase ........................................................................................................9 2.4 Pectinase .............................................................................................................13 2.5 Carbon Sources and Inducing Substrates ............................................................17 2.6 Effect of pH.........................................................................................................19 2.7 Effect of Dissolved Oxygen and Agitation .........................................................20 III. INTRODUCTION TO THE SOYBEAN ..................................................................22 3.1 Seed Structure and Components .........................................................................22 3.2 Lipids ..................................................................................................................23 3.3 Proteins ...............................................................................................................24 3.4 Carbohydrates .....................................................................................................25 3.5 Minerals ..............................................................................................................32 vi 3.6 Uses of Soybeans ................................................................................................33 3.7 Soy Protein Isolates.............................................................................................36 3.8 Use of Enzymes ..................................................................................................38 IV. EXPERIMENTAL APPARATUS AND METHODS ..............................................47 4.1 Maintenance and Cultivation of Trichoderma reesei .........................................47 4.2 Enzyme Hydrolysis .............................................................................................49 4.3 Analysis of Carbohydrate Composition ..............................................................50 4.4 Enzyme Activity Assays .....................................................................................53 4.5 Determination of Cell Concentration ..................................................................56 4.6 Determination of Protein Content .......................................................................59 4.7 Measurement of Triton X-100 ............................................................................61 4.8 Determination of Moisture Content and Ash in Biomass ...................................62 V. EFFECT OF SOY FLOUR ON PELLET FORMATION .........................................63 5.1 Introduction and Rationale ..................................................................................63 5.2 Materials and Methods ........................................................................................64 5.3 Results and Discussion .......................................................................................65 5.4 Conclusions and Recommendations ...................................................................68 VI. EFFECT OF MEDIUM ON ENZYME COMPOSITION—FLASK STUDIES ..................................................................................................................70 6.1 Introduction and Rationale ..................................................................................70 6.2 Materials and Methods ........................................................................................71 6.3 Results .................................................................................................................75 6.4 Discussion ...........................................................................................................84 6.5 Conclusions and Recommendations ...................................................................90 6.6 Acknowledgements .............................................................................................91 vii VII. PRELIMINARY FERMENTATION STUDIES ......................................................92 7.1 Introduction and Rationale ..................................................................................92 7.2 Materials and Methods ........................................................................................93 7.3 Results and Discussion .......................................................................................94 7.4 Conclusions and Recommendations .................................................................101 7.5 Acknowledgements ...........................................................................................102 VIII. EFFECT OF MEDIUM AND PH ON ENZYME PRODUCTION IN BATCH FERMENTATION ...................................................................................103 8.1 Introduction and Rationale ................................................................................103 8.2 Materials and Methods ......................................................................................104