BIOCONVERSION OF CELLULOSE INTO ELECTRICAL ENERGY IN MICROBIAL FUEL CELLS DISSERTATION Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Hamid Rismani-Yazdi, M.S. * * * * * The Ohio State University 2008 Dissertation Committee: Approved by Dr. Ann D. Christy, Advisor Dr. Burk A. Dehority Dr. Olli H. Tuovinen Dr. Alfred B. Soboyejo Advisor Food, Agricultural and Biological Dr. Zhongtang Yu Engineering Graduate Program ABSTRACT In microbial fuel cells (MFCs), bacteria generate electricity by mediating the oxidation of organic compounds and transferring the resulting electrons to an anode electrode. The first objective of this study was to test the possibility of generating electricity with rumen microorganisms as biocatalysts and cellulose as the electron donor in two-compartment MFCs. Maximum power density reached 55 mW/m2 (1.5 mA, 313 mV) with cellulose as the electron donor. Cellulose hydrolysis and electrode reduction were shown to support the production of current. The electrical current was sustained for over two months with periodic cellulose addition. Clarified rumen fluid and a soluble carbohydrate mixture, serving as the electron donors, could also sustain power output. The second objective was to analyze the composition of the bacterial communities enriched in the cellulose-fed MFCs. Denaturing gradient gel electrophoresis of PCR amplified 16S rRNA genes revealed that the microbial communities differed when different substrates were used in the MFCs. The anode-attached and the suspended consortia were shown to be different within the same MFC. Cloning and analysis of 16S rRNA gene sequences indicated that the most predominant bacteria in the anode-attached consortia were related to Clostridium spp., while Comamonas spp. was abundant in the suspended consortia. ii The external resistance affects the characteristic outputs of MFCs by controlling the flow of electrons from the anode to the cathode. The third objective of this study was to determine the effect of various external resistances on power output and coulombic efficiency of cellulose-fed MFCs. Four external resistances (20, 249, 480, and 1000 ohms) were tested with a systematic approach of operating parallel MFCs independently at constant circuit loads for three months. A maximum power density of 66 mWm-2 was achieved by MFCs with 20 ohms circuit load, while MFCs with 249, 480 and1000 ohms external resistances produced 57.5, 53 and 47 mWm-2, respectively. The anode potential varied under the different circuit loads employed. Higher coulombic efficiencies were achieved in MFCs with lower external resistance. The effect of different external resistances on the bacterial diversity and metabolism in cellulose-fed MFCs was investigated as the fourth objective. DGGE analysis of partial 16S rRNA genes showed clear differences between the planktonic and the anode-attached populations at various external resistances. Cellulose degradation was complete (< 0.1% residual), and there were no discernible differences among the MFCs. HPLC analysis of short chain fatty acids (SCFA) revealed that anaerobic degradation of cellulose was accompanied by production of acetic, propionic, butyric, isobutyric, valeric, isovaleric, and lactic acids, with acetic acid being predominant. The profile of metabolites was different among the MFCs. The concentrations of SCFA were higher in MFCs with larger external resistance. High levels of SCFA indicated that fermentative metabolism dominated over anaerobic respiration, resulting in relatively low coulombic efficiencies. The accumulation of SCFA at higher circuit resistances corresponded to lower power outputs. iii Methanogenesis shifts the flow of electrons available from the substrate away from electricity generation in MFCs. The fifth objective of this research was to assess the influence of methane formation on the performance of cellulose-fed MFCs under long- term operation. Two-compartment MFCs were inoculated with a ruminal microbial consortium and fed colloidal cellulose (0.5 g l-1 d-1) as the sole substrate. Replicate MFCs were operated under two different external resistances of 20 and 100 ohms, designated as R20Ω and R100Ω. During the first week of operation 0.31 ± 0.004 (± SD) and 0.44 ± 0.004 mmol of methane was produced in R20Ω and R100Ω MFCs, respectively. Methanogenesis was suppressed, however, to below the detection limit (< 0.5 ×10-3 mmol) after 90 days of operation. The decrease in methane production was accompanied with an increase in the performance of MFCs. The current output of the MFCs increased from 0.1 mA during the first week to 3.3 and 2.2 mA on day 90, resulting in 29 and 25% coulombic efficiency for the R20Ω and R100Ω MFCs, respectively. A maximum volumetric power density of 3.5 -3 W m was achieved in R20Ω MFCs, which was three times greater than that obtained with -3 R100Ω MFCs (1.03 W m ). The diversity of methanogens in cellulose-fed MFCs was also characterized. It was shown that the suppression of methanogenesis was accompanied by a decrease in the diversity of methanogens and changes in the concentration of SCFA, as revealed by DGGE analysis of PCR-amplified 16S rRNA genes and HPLC analysis, respectively. Analysis of partial 16S rRNA gene Sequences indicated that the most predominant methanogens were related to the family Methanobacteriaceae. The results demonstrate that electricity can be generated from cellulose by exploiting rumen microorganisms as biocatalysts. Results suggest that oxidation of iv metabolites with the anode as an electron sink was a rate limiting step in the conversion of cellulose to electricity in MFCs. This study also demonstrates that the size of external resistance significantly affects the bacterial diversity and characteristic output of MFCs. Thus the external resistance may be a useful tool to control microbial communities and consequently enhance performance of MFCs. Furthermore, this study shows that methanogenesis competes with electricity generation at the early stages of MFC operation but operating conditions suppress methanogenic activity over time. An improved understanding of the microbial communities, interspecies interactions and processes involved in electricity generation is essential to effectively design and control cellulose-fed MFCs for enhanced performance. In addition, technical and biological optimization is needed to maximize power output of these systems. v Dedicated to my family vi ACKNOWLEDGMENTS I would like to express my gratitude and deep appreciation to Dr. Ann D. Christy, advisor of my dissertation research, for her infinite support, encouragement, and patience as well as the magnificent mentorship she provided me with throughout the time it took me to complete this research and write the dissertation. It was a tremendous honor working with her. The members of my dissertation committee, Dr. Olli Tuovinen, Dr. Zhongtang Yu, Dr. Burk Dehority and Dr. Alfred Soboyejo, have generously given their time and expertise to improve my work. I thank them for their contribution and their good-natured support. Specifically, I wish to extend my sincere thanks and appreciation to Dr. Olli Tuovinen whose knowledge, wisdom, and advice have supported me over the course of my academic training at the Ohio State University. His technical and editorial guidance have been an inspiration to me and a critical key to the completion of my dissertation. He was always available, even during his sabbatical leave, to work on my writings and discuss new topics, ideas, and results. I am grateful to Dr. Zhongtang Yu for his valuable insights and ideas as well as countless advice that supported and expanded my work. His wide expertise in the field of molecular biology has played as essential role in this research project. I would like to sincerely thank Dr. Burk Dehority who triggered my vii academic journey in the US, and provided me with an incredible technical training and innumerable guidance before and throughout my dissertation research. I am also thankful to Dr. Alfred Soboyejo, for his continued encouragement and inspiring advice that supported and enlightened my research. I am grateful too for the support and advice from my faculty colleagues in Department of Food, Agricultural and Biological Engineering. I would like to thank many persons who helped me with my experiments, especially Chris Gecik for helping with electrical instrumentation, Carl Cooper and Kevin Duemmel for their assistance with fabrication of MFCs, and Don Irvine for providing computer support. Carol Moody, Kay Elliot, and Kevin Davison are also thanked for their administrative support. I must acknowledge as well the many friends, colleagues and students who assisted, advised, and supported my research and writing efforts over the years. Especially, I need to express my gratitude and deep appreciation to Sarah Carver, Mike Nelson, James Douglass, Kerry Hughes Zwierschke, Eun Kyoung Kim, Jill Stephen, Nikki Skrinak, Bethany Frew Corcoran, Brian Henslee, Peter Gehres, and Clayton Bettin. The Iranian Students and Scholars at the Ohio State University and their families made these years in Ohio, away from home, very special, very lively and very enriching. The enjoyable times spent with friends provided a well-needed balance to the work; thank you for your support and friendship. I am deeply indebted to my family for their care, support and encouragement. I am grateful to my parents without whom I would never have been able to achieve so much. I am also thankful to my mother and father in-law for their support, and encouragement. My brothers Saied and Ehsan, I am thankful for having you in my life. I viii have been blessed with an absolutely superb grandfather and honor the memory of the three of my grandparents whom I have lost, peace be upon their souls. Last and most importantly, I would like to thank the love of my life, my wife Najmeh, for her unyielding devotion and love, endless support, patience, and encouragement and all the helps she provided me with during the course of completing this research and writing the dissertation.