Diversity within the genus Thermoanaerobacter and its potential implications in lignocellulosic biofuel production through consolidated bioprocessing by Tobin James Verbeke A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirements of the degree of DOCTOR OF PHILOSOPHY Department of Microbiology University of Manitoba Winnipeg, Manitoba Canada Copyright © 2013 by Tobin James Verbeke i Abstract A major obstacle to achieving commercially viable lignocellulosic biofuels through consolidated bioprocessing (CBP) is the lack of “industry-ready” microorganisms. Ideally, a CBP-relevant organism would achieve efficient and complete hydrolysis of lignocellulose, simultaneous utilization of the diverse hydrolysis products and high yields of the desired biofuel. To date, no single microbe has been identified that can perform all of these processes at industrially significant levels. As such, thermophilic decaying woodchip compost was investigated as a source of novel lignocellulolytic, biofuel producing bacteria. From a single sample, a collection of physiologically diverse strains were isolated, which displayed differences in substrate utilization and biofuel production capabilities. Molecular characterization of these isolates, and development of a genome relatedness prediction model based on the chaperonin-60 universal target sequence, identified these isolates as strains of Thermoanaerobacter thermohydrosulfuricus. Application of this model to other Thermoanaerobacter spp. further identified that these isolates belong to a divergent and lesser characterized lineage within the genus. Based on this, the CBP-potential of a single isolate, T. thermohydrosulfuricus WC1, was selected for further investigation through metabolic, genomic and proteomic analyses. Its ability to grow on polymeric xylan, potentially catalyzed by an endoxylanase found in only a few Thermoanaerobacter strains, distinguishes T. thermohydrosulfuricus WC1 from many other strains within the genus. The simultaneous consumption of two important lignocellulose constituent saccharides, cellobiose and ii xylose was also observed and represents a desirable phenotype in CBP-relevant organisms. However, at elevated sugar concentrations, T. thermohydrosulfuricus WC1 produces principally lactate, rather than the desired biofuel ethanol, as the major end- product. Proteomic analysis identified that all likely end-product forming proteins were expressed at high levels suggesting that the end-product distribution patterns in T. thermohydrosulfuricus WC1 are likely controlled via metabolite-based regulation or are constrained by metabolic bottlenecks. The xylanolytic and simultaneous substrate utilization capabilities of T. thermohydrosulfuricus WC1 identify it as a strain of interest for CBP. However, for its development into an “industry-ready” strain as a co-culture with a cellulolytic microorganism, improved biofuel producing capabilities are needed. The practical implications of CBP-relevant phenotypes in T. thermohydrosulfuricus WC1 in relation to other Thermoanaerobacter spp. will be discussed. iii Acknowledgements With deepest gratitude, I would like to first thank my advisor, Richard Sparling for granting me the opportunity to do this research and challenging me throughout. Your insights and abilities as a researcher have provided an example of who I aspire to become. Thanks also to David Levin, whose commitment to this work has been nothing short of admirable and whose insights have been incredibly valuable. I also extend my sincere appreciation to Vladimir Yurkov and Teresa De Kievit, whose advice and unfaltering encouragement has provided me strength throughout this degree. Thank you to Tim Dumonceaux, who saw potential where I was oblivious. Thanks to Vic Spicer and Oleg Krokhin who guided me through many struggles I encountered; even when those struggles defied all logic. Thank you to Gideon Wolfaardt and Martina Hausner whose generosity and support have been limitless. To Tom Rydzak, John Schellenberg, Carlo Carere and Scott Wushke; thank you for everything our numerous discussions/debates have taught me. Finally, thank you to all who have encouraged and supported me throughout, particularly Nazim Cicek, John Wilkins, Justin Zhang, Peter McQueen, Brian Fristensky, Marcel Taillefer, Umesh Ramachandran, Nathan Wrana, Bruce Ford, Matthew Links, Janet Hill, Andrea Wilkinson, Alexandru Dumitrache, Dmitry Shamshurin, Georg Hausner and Ivan Oresnik. This work would not have been possible without support provided by a Natural Sciences and Engineering Research Council grant (STPGP 365076), the University of Manitoba, the Manitoba Rural Adaptation Council (MRAC) – Advancing Canadian Agriculture and Agri-Food (ACAAF) program (309009) and by a Genome Canada grant titled “Microbial Genomics for Biofuels and Co-Products from Biorefining Processes.” iv Dedication To my wife, Lindsay, None of this matters without you. And to my parents, Dan and Karen, and my sister, Aynsley, Encouragement. Confidence. Effort. Inspiration. Love. Enough said. v Table of contents Abstract ............................................................................................................................... i Acknowledgements .......................................................................................................... iii Dedication ......................................................................................................................... iv Table of contents ............................................................................................................... v List of tables..................................................................................................................... xv List of figures ................................................................................................................... xx List of copyrighted material for which permission was obtained ........................... xxiv List of abbreviations used ............................................................................................ xxv List of genus abbreviations used ................................................................................. xxxi Chapter 1. Literature review .......................................................................................... 1 1.1 Introduction .............................................................................................................. 1 1.2 Microorganisms for second generation biofuels through CBP ................................ 2 1.2.1 Naturally occurring consortia ............................................................................ 3 1.2.2 Established platform organisms......................................................................... 4 1.2.3 Novel platform organisms ................................................................................. 7 1.2.3.1 Bioprospecting for novel strains ................................................................ 7 1.2.3.2 Continued development of novel organisms............................................ 11 1.3 Criteria to evaluate in the selection or development of novel platform strains...... 14 1.3.1 Advantages of thermophilic microorganisms .................................................. 14 vi 1.3.2 Lignocellulose hydrolysis potential ................................................................. 16 1.3.3 Fermentation of lignocellulose hydrolysis products ........................................ 24 1.3.4 Biofuel production ........................................................................................... 32 1.3.5 Genetically tractable microorganisms ............................................................. 41 1.4 Thesis objectives .................................................................................................... 45 1.5 Thesis outline ......................................................................................................... 46 Chapter 2. Isolates of Thermoanaerobacter thermohydrosulfuricus from decaying wood compost display genetic and phenotypic microdiversity ................................... 49 2.1 Abstract ................................................................................................................... 49 2.2 Introduction ............................................................................................................ 50 2.3 Materials and methods ........................................................................................... 52 2.3.1 Reference strains .............................................................................................. 52 2.3.2 Media and substrates ....................................................................................... 52 2.3.3 Enrichment and isolation ................................................................................. 53 2.3.4 Microscopy ...................................................................................................... 54 2.3.5 Substrate use and niche overlap ....................................................................... 54 2.3.6 16S rRNA and cpn60 UT amplification, sequencing and phylogenetic analysis ................................................................................................................................... 57 2.3.7 Genetic fingerprinting...................................................................................... 59 2.3.8 Growth and metabolic