Integrated Rotating Fibrous Bed Bioreactor-Ultrafiltration Process for Xanthan Gum Production from Whey Lactose DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ching-Suei Hsu Graduate Program in Chemical and Biomolecular Engineering The Ohio State University 2011 Dissertation Committee: Professor Shang-Tian Yang, Advisor Professor Jeffrey J. Chalmers Professor Kurt W. Koelling Copyright by Ching-Suei Hsu 2011 Abstract Biopolymer fermentation is an environmentally friendly process compared to petroleum-based polymer production. The goal of this study was to evaluate the feasibility of producing xanthan gum, an important biopolymer widely used in food and oil-recovery industries, from whey lactose, a low-value byproduct from cheese manufacturing in the dairy industry, in an integrated fermentation-ultrafiltration process. First, the fermentation kinetics of xanthan gum production from glucose, galactose, and their mixture, respectively, were studied with Xanthomonas campestris in stirred tank fermentors. In general, comparable fermentation performance in terms of productivity, product yield, and final product titer and quality (rheological properties) was obtained with these various carbon sources. Further batch fermentations with hydrolyzed whey permeate (lactose) showed the feasibility of xanthan gum production using whey permeate as an alternative low-cost feedstock. However, the high broth viscosity due to product accumulation can cause serious mixing and mass (especially oxygen) transfer problems in conventional stirred-tank bioreactors, resulting in low product yields and poor product quality. A rotating fibrous bed bioreactor (RFBB) operated under a high gravity field can increase mass transfer in viscous xanthan gum fermentation due to the shear-thinning property of xanthan gum broth, thus increasing reactor productivity and final product titer. Furthermore, cells immobilized in the RFBB would allow continuous production of xanthan gum in a low- ii cell or cell-free broth that can be readily concentrated by ultrafiltration (UF) before alcohol precipitation, thus reducing the amount of alcohol and energy used in the downstream processing by 10-fold. Ultrafiltration also allows the recycle of the fermentation spent medium in subsequent batch xanthan gum fermentations. The effects of recycling the ultrafiltration permeate on xanthan gum fermentation were thus studied, and the results showed no significant changes in productivity, yield, titer, and product quality when the fermentation medium consisted of 75% of recycled permeate, confirming the feasibility of recycling the fermentation medium through the integrated RFBB-UF process. To evaluate the scalability of the RFBB, xanthan gum fermentations in 20-liter RFBB operated in a repeated-batch mode were studied, and the results showed comparable performance to those obtained with 5-liter RFBB. A mathematical model for predicting the oxygen transfer rate, which affects the xanthan gum productivity, in the RFBB was also developed for process scale up. Finally, process and economic analyses were performed using SuperPro Designer, and the results confirmed that the integrated RFBB-UF process can reduce the xanthan gum production cost significantly, largely due to the improved reactor productivity and reduced raw materials and energy costs. Overall, the integrated RFBB-UF process is environmentally friendly and cost effective in producing xanthan gum from whey permeate. iii Dedication This document is dedicated to my family. iv Acknowledgments My grateful acknowledgment goes to my advisor, Dr. Shang-Tian Yang, for his invaluable guidance, patience, kindness and support throughout my Ph.D. study. I have benefited greatly from his expertise in science, deep insights in our research as well as his great personality. I wish to thank Dr. Robin Ng, Dr. Yunling Bai, and Dr. Liping Wang for their help in my early years of Ph.D. study, and to thank department staff members, Paul Green and Leigh Evrard, for their assistance in customizing and fabricating the bioreactor used in this study. I also want to thank all the members in my advisor’s research group for their friendship during these years. I would also like to thank Prof. Jeffrey Chalmers, Prof. Kurt Koelling, and Prof. Karen Mancl for taking time to serve on my dissertation committee, as well as their valuable suggestions and advices to my research project. Financial support from the United State Department of Agriculture Small Business Innovation Research (SBIR) Program via a subcontract from Bioprocessing Innovative Company is deeply appreciated. Finally, I wish to thank my family for their unconditionally love and support, and Dr. Xudong Zhang for his encouragements and company throughout my PhD study. v Vita 2002................................................................B.S. Chemical Engineering and Material Engineering, National Central University (Taiwan) 2004................................................................M.S. Chemical Engineering, National Tsing Hua University (Taiwan) 2005 to present ..............................................Graduate Research Associate, Chemical and Biomolecular Engineering, The Ohio State University Fields of Study Major Field: Chemical and Biomolecular Engineering vi Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita ..................................................................................................................................... vi Fields of Study ................................................................................................................... vi Table of Contents ............................................................................................................ vvii List of Tables ................................................................................................................... xiv List of Figures .................................................................................................................. xvi Chapter 1: Introduction ...................................................................................................... 1 1.1. Introduction ........................................................................................................ 1 1.2. Objectives ........................................................................................................... 3 1.3. References .......................................................................................................... 6 Chapter 2: Literature Review .............................................................................................. 9 2.1. Xanthan gum structure and rheology property ................................................... 9 2.2. Xanthan gum biosynthesis................................................................................ 13 vii 2.3. Xanthan gum production .................................................................................. 14 2.3.1. Xanthan gum fermentation using inexpensive substrates ................................ 14 2.3.2. Xanthan gum fermentation using whey related substrates ............................... 15 2.3.3. Effect of sugar mixtures on xanthan gum production ...................................... 17 2.4. Reactor design for xanthan gum production .................................................... 18 2.4.1. Rotating fibrous bed bioreactor (RFBB) .......................................................... 19 2.4.2. Xanthan gum fermentation using RFBB .......................................................... 20 2.4.3. Oxygen transfer in RFBB ................................................................................. 22 2.4.4. Mean residence time and liquid holdup in RFBB ............................................ 23 2.4.5. Cell adsorption in RFBB .................................................................................. 24 2.4.6. Other research on RFBB .................................................................................. 26 2.4.7. Other oxygen transfer study in xanthan gum fermentation .............................. 27 2.5. Ultrafiltration in biopolymer separation ........................................................... 27 2.6. Water reuse in fermentation ............................................................................. 28 2.7. References ........................................................................................................ 29 Chapter 3: Xanthan gum fermentation using hydrolyzed whey permeate (HWP) as an alternative carbon source .................................................................................................. 54 3.1. Summary .......................................................................................................... 54 3.2. Introduction ...................................................................................................... 54 viii 3.3. Materials and methods...................................................................................... 57 3.3.1. Culture and media ...........................................................................................