INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in ^ew riter fitce, while others may be from any type o f computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely afreet reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back o f the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Informaticn Company 300 North Zed) Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 A GAS-SOLID SPOUTED BED BIOREACTOR FOR SOLID STATE FERMENTATION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ellen M. Silva, M.S. ***** The Ohio State University 1997 Dissertation Committee: Approved by Professor Shang-Tian Yang, Adviser Professor Jeffrey Chalmers Adviser Professor David Tomasko Department of Chemical Engineering UMI Number: 9731715 Copyright 1997 by Silva, Ellen Mae All rights reserved. UMI Microform 9731715 Copyright 1997, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 Copyright by Ellen Mae Silva 1997 ABSTRACT Solid state fermentation (SSF) is the culture of microorganisms on moist solid substrate in the absence of free-flowing water. Composting and silage making are two frequently practiced examples of SSF, but other large scale examples are rare in Western countries, despite the many advantages SSF offers. These advantages include improved productivities and higher product concentrations than in many submerged fermentations and the ability to use simple plant biomass, including agri-industry waste stream material, as a substrate. The drawbacks that discourage industrial application include solids handling difficulties, difficulty in measuring and controlling process parameters, heat and mass transfer limitations in existing SSF bioreactors, and a lack of kinetic and design data. Spouted bed reactors are well known to have excellent heat and mass transfer characteristics and to easily handle solids of the types used for SSF substrates. For this reason, a novel spouted bed bioreactor for SSF was designed and tested to evaluate its ability to overcome some of the drawbacks previously mentioned. Several fermentations for the production of amylases by Aspergillus oryzae grown on rice were run using various operating strategies (continual, intermittent, and no spouting, the latter of which is equivalent to packed bed operation) and the results were compared to static SSF fermentations. 11 Static fermentation studies indicated that the optimal conditions, within those tested, for production of amylases from rice by Aspergillus oryzae were 29% initial moisture content, initial pH of 6.2 (no adjustment from that naturally occurring), temperature of 35 - 37° C, and nutrient supplementation of 0.5% (w/w) yeast extract. Inoculum size did not affect production within the ranges tested. Lentils gave superior fermentation results compared to rice and several other substrates. Fermentation in the gas-solid spouted bed bioreactor was found to be possible and to have several important advantages compared to the currently accepted best-performing reactor, the packed bed reactor with forced aeration. The best results, which were equivalent to the results achieved in the packed bed reactor, were achieved with intermittent spouting at 4 hr intervals. This was sufficiently frequent spouting to confer advantages of improved solids handling and uniformity compared to the packed bed fermentation. Overall protein productivity rates (which correlated to enzyme productivity) of about 6 mg/100 g reactor charge/h were achieved for both 4 hr intermittent spouting and packed bed operation; in comparison, deep static fermentation achieved less than 1 mg/100 g/h. Hydrodynamic studies showed that existing literature correlations could be used to approximate experimentally determined minimum spouting velocity, Ums. Linear annular speed and fountain height of the spout both increased in a linear manner with spouting velocity, though bed height and reactor geometry affected the exact correlation. Increasing the air inlet diameter from 1.27 cm to 2.54 cm decreased the stability of the spout and increased Ums; increasing the moisture content of rice from about 20% (w/w) to 45% had a similar effect. Ill to Wilson IV ACKNOWLEDGMENTS My grateful acknowledgment of many hours of consultation, advice, and encouragement goes to my adviser. Dr. Shang-Tian Yang. The relationship between an adviser and student can be critical to the success of a doctoral research project. I deeply appreciate the flexibility allowed me in scheduling my research by my adviser; without his understanding of my demands outside of the lab, my work here at the Ohio State University would not have been as fruitful or as pleasant. I have also had advice and guidance from the members of my dissertation committee. Dr. Jeff Chalmers and Dr. David Tomasko, and I appreciate the additions they have made to this research with their ideas. Even more, I appreciate their advice and their thoughts offered over the years on a variety of topics. For his assistance in running the static fermentation studies on pH and inoculum size, I thank Mr. Chad Laubenthal. Early in the development of this project. Dr. John Davidson generously shared his knowledge of spouted beds and helped generate the idea of intermittent spouting as a possible operating strategy. I thank him for this. Other early assistance came from Dr. Ping Cai, who directed me toward pertinent spouted bed literature, and Dr. Peijun Jiang, who helped me get a reactor set up and running. My thanks go to both of these individuals. Mr. Mike Kukla assisted with the design and construction of the reactor system; without him, I certainly would not have known where to start. Thank you, Mike. Mr. Carl Scott did a fine job building the second reactor, and I appreciate that, but I thank him most of all for the many times he graciously allowed me to interrupt his work in order to get some small question answered or part fixed. The staff of the Engineering Region 6 computer facilities, particularly Mr. Geoff Hulse, Mr. Mike Davis, and Mark Grzesiakowski assisted me many times with both critical and trivial computing problems, and for that I am grateful. I also appreciate the less direct, but equally important assistance of the administrative staff of the Department of Chemical Engineering, past and present, staff and work study students. Thanks for running the department so that we can run our research. Dr. Jaques Zakin and Dr. Liang-Shih Fan have helped me many times, mostly in the area of getting fellowships. This is, of course, deeply appreciated, as is their personal fellowship. My colleagues, both within my research group and in the Department of Chemical Engineering, have inspired and encouraged me. There were, of course, a special few who offered support when it was needed and a reality check when that was needed, too. Thanks, Paul, Rachel, Venkat, Suhas, Niki, Trish, and Yan. VI Financial assistance from the National Science Foundation, the Ohio State University Graduate Student Alumni Research Association, The Ohio State University, the Consortium for Plant Biotechnology Research, and the Association for Women in Science is gratefully acknowledged. Finally, those who are owed the greatest debt of gratitude are my family. Graduate school requires sacrifices of the student, but those are easy. The sacrifices of those for whom the student cares most are the hard ones. My parents, Mrs. Eloise Myers and Mr. Gib Myers, and my parents-in-law, Mrs. Helen Wilson Gibbins and Dr. Neil Gibbins, have given of themselves in too many ways to list. Perhaps the most important thing they have done has been to forgive me the lack of time spent with them while I pursued this degree. My daughters, Ms. Karen Gibbins and Ms. Jennifer Gibbins, have grown into fine, young women during this time and have kept my spirits high and my priorities straight. And, certainly, my heartfelt gratitude goes to my husband, Mr. Wilson Gibbins, for so many things; for computer advice, for coffee in the morning, for allowing me to clutter our lives with things like dogs and cats when it was already too cluttered with graduate school, for holding my hand during the tough spots, and for having faith in my ability to finish. I did not expect that one of the things I would learn in graduate school was what a wonderful husband I have. VII VTTA July 19, 1958 .......................................Bom - Medina, Ohio, USA 1980 .................................................... B.S. Chemical Engineering The Ohio State University Columbus, Ohio, USA 1980 - 1983 .........................................Engineer Diamond Shamrock Corporation Painesville, Ohio, USA 1985 - 1991 .........................................Technical Writer Virginia Polytechnic and State University Blacksburg, Virginia, USA 1991 - present .................................... Graduate Teaching and Research The Ohio State University Columbus, Ohio, USA 1993 .....................................................M.S.