The University of Maine DigitalCommons@UMaine Electronic Theses and Dissertations Fogler Library 12-2009 Biological Conversion of Hemicellulose Extract into Value-Added Fuels and Chemicals Sara Lynn Walton Follow this and additional works at: http://digitalcommons.library.umaine.edu/etd Part of the Chemical Engineering Commons, and the Organic Chemistry Commons Recommended Citation Walton, Sara Lynn, "Biological Conversion of Hemicellulose Extract into Value-Added Fuels and Chemicals" (2009). Electronic Theses and Dissertations. 226. http://digitalcommons.library.umaine.edu/etd/226 This Open-Access Dissertation is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of DigitalCommons@UMaine. BIOLOGICAL CONVERSION OF HEMICELLULOSE EXTRACT INTO VALUE-ADDED FUELS AND CHEMICALS By Sara Lynn Walton B.S. University of Maine, 2005 A THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy (in Chemical Engineering) The Graduate School The University of Maine December, 2009 Advisory Committee: Adriaan R.P van Heiningen, Professor of Chemical Engineering, Co-Advisor G. Peter van Walsum, Associate Professor of Chemical Engineering, Co-Advisor Paul Millard, Associate Professor of Biological Engineering Jody Jellison, Professor of Biological Sciences Nancy Kravit, Adjunct Professor of Chemical Engineering DISSERTATION ACCEPTANCE STATEMENT On behalf of the Graduate Committee for Sara Lynn Walton, I affirm that this manuscript is the final and accepted dissertation. Signatures of all committee members are on file with the Graduate School at the University of Maine, 42 Stodder Hall, Orono, Maine. Signature December 2009 ii LIBRARY RIGHTS STATEMENT In presenting this thesis in partial fulfillment of the requirements for an advanced degree at The University of Maine, I agree that the Library shall make it freely available for inspection. I further agree that permission for ―fair use‖ copying this thesis for scholarly purposes may be granted by the Librarian. It is understood that any copying or publication of this thesis for financial gain shall not be allowed without my written permission. Signature: Date: BIOLOGICAL CONVERSION OF HEMICELLULOSE EXTRACT INTO VALUE-ADDED FUELS AND CHEMICALS By Sara Lynn Walton Thesis Co-Advisors: Dr. Adriaan R.P. van Heiningen and Dr. G. Peter van Walsum An Abstract of the Thesis Presented In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy (in Chemical Engineering) December, 2009 The research presented in this thesis describes the fermentation of hemicellulose extracts to produce ethanol and lactic acid. Hardwood hemicellulose extracts made at a variety of conditions using the green liquor and hot water extraction processes were characterized and then fermented by Escherichia coli K011 and Bacillus coagulans MXL-9. Hemicellulose extracts were found to contain a dilute amount of xylo- oligosaccharides and acetic acid as the major components, and many minor components including other organic acids, lignin-derived phenolics, and sugar degradation products. In order to generate ethanol economically, the fermentation must produce at least a 4% ethanol solution, which requires a starting sugar concentration of at least 8%. Since hemicellulose extracts did not contain high enough sugar titers, methods for concentrating the extracts prior to fermentation were investigated. Evaporation was found to be an ineffective method of concentrating sugars because it also concentrates such chemicals as acetic acid and sodium which inhibit fermentation. Ultrafiltration was then examined as a possible method of concentrating the oligomeric fraction of the extracts without effecting the concentrations of smaller molecules such as acetic acid and sodium. It does however concentrate the lignin fraction, which causes inhibition of fermentation organisms. Both E. coli and B. coagulans were found to consume all of the principal monosaccharides found in lignocellulosic biomass, including xylose and arabinose which cannot be utilized by traditional industrial yeast strains. These bacteria had higher tolerance to the inhibitors found in hemicellulose extracts than any other organism tested. The highest product titers achieved were 1% ethanol and 3% lactic acid. ACKNOWLEDGEMENTS I would like to express my sincere appreciation to my advisors, Dr. Adriaan van Heiningen and Dr. Peter van Walsum, for their guidance in this research. In addition, many thanks go to the members of my committee for their time and insight: Dr. Paul Millard, Dr. Jody Jellison and Dr. Nancy Kravit. Special thanks to fellow group members past and present for their help and input. I would like to also thank the technical support of Amos Cline, Keith Hodgins, and Nick Hill. For production of hemicellulose extracts, thanks go to Dwane Hutto and Nicholas Gramlich. I would also wish to extend my sincere gratitude to Martin Lawoko for the analytical support he provided. In particular, I am grateful for the research support and friendship provided by Justin Crouse. Thank you to my colleagues, friends and staff members in the Chemical and Biological Engineering Department, and in the Forest Bio-products Research Initiative. Many thanks also go to Ken Bischoff of the USDA ARS for providing Bacillus coagulans MXL-9 and insights on its culturing methods. This work was funded by the National Science Foundation (EPSCoR, Contract #: 0554545), by the US Department of Energy and by the Forest Products Laboratory, Forest Service, USDA (under Joint Venture Agreement No. 08-JV-11111122-036). Additional support was provided by the University of Maine Graduate School through a Doctoral Research Fellowship. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES ............................................................................................................. xi LIST OF FIGURES ......................................................................................................... xiii CHAPTER 1: INTRODUCTION .......................................................................................1 1.1 Literature Review ..................................................................................................2 1.1.1 Lignocellulose Composition ..........................................................................2 1.1.2 Hemicellulose Extraction ...............................................................................3 1.1.3 Hydrolysis of Hemicellulose into Monosaccharides .....................................4 1.1.4 Fermentation of Lignocellulose-derived Monosaccharides ...........................6 1.1.4.1 Metabolic Engineering of Bacteria ........................................................ 6 1.1.4.2 Escherichia coli K011 ........................................................................... 8 1.1.5 Inhibition of Fermentation ...........................................................................11 1.1.6 Concentration of Hemicellulose Oligomers.................................................13 1.1.7 Kraft Pulping ................................................................................................15 1.2 Objectives ............................................................................................................16 1.3 Outline of This Dissertation ................................................................................16 CHAPTER 2: EXPERIMENTAL METHODS .................................................................19 2.1 Raw Materials .....................................................................................................19 2.1.1 Wood chips ..................................................................................................19 2.1.1.1 Southern Hardwood Chips................................................................... 19 2.1.1.2 Northern Hardwood Chips................................................................... 20 iv 2.1.1.3 Aspen Strands ...................................................................................... 20 2.1.1.4 Siberian Larch ...................................................................................... 21 2.1.2 Green liquor and Sodium Carbonate............................................................21 2.1.3 Sugars and Other Chemicals ........................................................................22 2.2 Hemicellulose Extraction ....................................................................................22 2.2.1 Rotating Digester .........................................................................................22 2.2.2 Rocking Digester .........................................................................................23 2.2.3 Larch Extraction...........................................................................................24 2.3 Extract Concentration ..........................................................................................24 2.3.1 Evaporation ..................................................................................................24 2.3.2 Ultrafiltration ...............................................................................................25 2.4 Extract Hydrolysis ...............................................................................................26 2.4.1 Acid hydrolysis ............................................................................................26