MICROBIAL BIOPROCESSING: FROM NATURE TO INDUSTRY By Jenna M. Young A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Microbiology and Molecular Genetics 2012 ABSTRACT MICROBIAL BIOPROCESSING: FROM NATURE TO INDUSTRY By Jenna M. Young Microorganisms play an essential role in the processing of plant-derived matter in nature and in industrial settings. Decomposition of plant matter in nature is a key process in the feedback between soils and the atmosphere and an important parameter to incorporate into climate models to increase their predictive value. The mechanisms that enable decomposers to process plant matter in nature are not known. To gain insights, I investigated the response of Cellulomonas decomposers to nitrogen disturbances such as those caused by the addition of fertilizers or atmospheric deposition of greenhouse gases. These decomposers adapt to low nitrogen by specifically colonizing plant-derived substrates as biofilms and sequestering significant amounts of carbon in the biofilm matrix. The biofilm strategy enabled cells to be closer to the substrate and to degrade it more efficiently despite the low availability of nitrogen sources. The process is reversible and potentially manageable, thus showing promise for its use as a carbon remediation technology to mitigate the accumulation of greenhouse gases. Knowledge gained from studying Cellulomonas uda can be utilized in industry as interest in cellulolytic microorganisms has increased in recent years due to their role in bioethanol production. To minimize the cost of bioethanol, agricultural residues and dedicated bioenergy crops are preferable as substrates instead of starch and sugar. These lignocellulosic substrates are more recalcitrant and require a chemical pretreatment step plus an additional step of enzymatic hydrolysis to make them fermentable. Consolidated bioprocessing (CBP), i.e. a combined platform that catalyzes the breakdown and fermentation of cellulose in one single step, is a cost-effective approach to producing ethanol from lignocellulosic substrates. C. uda is an attractive candidate for industrial consolidated bioprocessing of lignocellulose. The activities of this organism is limited, however, by the accumulation of ethanol and fermentation byproducts, which in nature are rapidly removed by other organisms. Our lab has developed a platform for the bioprocessing of chemically-pretreated agricultural residues such as corn stover by C. uda and Geobacter sulfurreducens into ethanol and biohydrogen using a microbial electrochemical cell (MEC). As predicted, nitrogen supplementation prevented the accumulation of carbon as a curdlan biofilm matrix and resulted in 2-fold increases in the energy recoveries from the fermentation of Ammonia Fiber Expansion pretreated corn stover (AFEX-CS). Improving culture conditions and developing a faster fermenting strain led to a 12-fold increase in ethanol productivity. Another bioprocessing scheme of industrial significance is the generation of value-added co-products from glycerol, the major waste product in the production of biodiesel. Harnessing the glycerin waste stream to produce value-added products will diminish the cost and waste of biodiesel production. We identified a glycerol-fermenting bacterium (Clostridium cellobioparum) that converts glycerol into ethanol at high rates and generates waste fermentation byproducts that are converted into hydrogen in Geobacter-driven MECs. This scheme shows promise as a wastewater treatment method as optimization of the platform resulted in glycerol consumption of 50g/L. DEDICATION This dissertation is dedicated to “family” in every sense of the word. In particular to my family back home in Georgia. They are an amazing support system and have listened tirelessly to both the good and the bad over the years. I would also like to dedicate this to my Michigan family, the friends and labmates who helped me through the tough times and laughed with me through the good times. Lastly, to Tucker, who makes me laugh every day. iv ACKNOWLEDGEMENTS The work described in this dissertation is the result of the help, support, and work of many good people. First and foremost, my mentors Dr. Gemma Reguera and Dr. Bruce Dale for the good fortune of an ever interesting project that excited and challenged me throughout the process. I also want to thank my committee members who guided me in addition to my mentors, Dr. James Tiedje, Dr. Claire Vieille, and Dr. Kurt Thelen. I want to thank Susan Leschine for making my projects possible as she provided our cellulolytic strains. A special shout-out to the Vieille lab members for all their assistance with HPLC analysis and troubleshooting, in particular thanks to Dr. Bryan Schindler and Nik McPherson. Thanks to Dr. Melinda Frame and Dr. Per Askeland for microscopy guidance resulting in some very pretty pictures of bacteria. I want to thank my generous funding sources, especially the College of Natural Science at Michigan State University for the Marvin Hensley Endowed, continuation, and dissertation completion fellowships as well as the Rackham Fund Foundation. To the past and present members of the Reguera lab, thank you to those who helped keep the lab running, edited papers and fellowships, helped with the many many growth curves over the years, and became my surrogate family. In particular, I want to acknowledge our old lab techs for their help in the early days. Blair Bullard trained me on day one in the lab and was always around to lend a helping hand. Kwi Kim helped with the original cellulolytic screening before I ever started my project and managed to sneak in advice about the important things in life into everyday conversation. A special thanks to Dr. Sanela Lampa-Pastirk for being an amazing source of knowledge, even v though Microbiology isn’t her thing. She taught us the ways of the academic world and how science learning should be approached. Tucker and I also want to thank her for her cheese pie. Special recognition goes to Dena for being the rock of the lab. To round out the bunch, Allison Speers. As I am the only member of the Reguera lab not working with Geobacter, an extra extra special thanks to Allison, who was my scientific connection to the rest of the lab. With a parallel project as well as being co-contributer to another project, Allison was my major resource for discussions, experimental planning, and trouble shooting in the lab. Many of my experiments have a fingerprint or two of hers within them. Lastly, I want to recognize the MMG grad students both past and present. We are lucky as a department to have such a close knit group. I encourage that tradition to continue as I know that I could not have gotten through this without them. vi TABLE OF CONTENTS LIST OF TABLES .......................................................................................................... x LIST OF FIGURES ....................................................................................................... xi LIST OF SYMBOLS AND ABBREVIATIONS ............................................................. xiii Chapter 1: A history anD applications of inDustrial microbiology .................................. 1 1.1 InDustrial Microbiology: An UnDerstanDing ................................................... 2 1.2 InDustrial Microbiology: A History ................................................................. 3 1.3 Biofuels ......................................................................................................... 7 1.3.1 Overview ......................................................................................... 7 1.3.2 Ethanol ............................................................................................ 8 1.3.3 BioDiesel ......................................................................................... 9 1.4 Pretreatment technologies as the first step in biomass conversion to ethanol ................................................................................................................. 10 1.5 Native Microbial Bioprocessors .................................................................. 12 1.5.1 Microbial Bioprocessing in Nature................................................. 12 1.5.2 Microbial Bioprocessing in InDustry: Bioconversion to Ethanol ..... 13 1.5.3 CellulomonaDs .............................................................................. 14 1.5.4 Microbial Bioprocessing in InDustry: Wastewater treatment .......... 16 1.5.5 ClostriDia ....................................................................................... 16 1.6 Summary .................................................................................................... 17 REFERENCES ................................................................................................. 18 Chapter 2: Reversible control of biofilm formation by Cellulomonas spp. in response to nitrogen availability ...................................................................................................... 24 2.1 SUMMARY ............................................................................................... 25 2.2 INTRODUCTION ...................................................................................... 25 2.3 RESULTS ................................................................................................. 29 2.3.1 Effect of N availability on growth anD biofilm formation in Cellulomonas spp. ......................................................................