UNDERSTANDING METHANOTROPHIC POLYHYDROXYBUTYRATE (PHB) PRODUCTION ACROSS SCALE: LIFE CYCLE ASSESSMENT, PURE CULTURE EXPERIMENTATION, AND PATHWAY/GENOME DATABASE DEVELOPMENT A DISSERTATION SUBMITTED TO THE DEPARTMENT OF CIVIL AND ENVIRONMENTAL ENGINEERING AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Katherine Helen Rostkowski June 2012 © 2012 by Katherine Helen Rostkowski. All Rights Reserved. Re-distributed by Stanford University under license with the author. This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/ This dissertation is online at: http://purl.stanford.edu/mc120yq3299 ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Craig Criddle, Primary Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Michael Lepech I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Perry McCarty I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Peter Karp Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost Graduate Education This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives. iii ABSTRACT While the 140 million tons of plastics produced each year may contribute to quality of life, they also come at a significant cost. Their production requires large quantities of nonrenewable resources, contributing to climate change; they accumulate in landfills and natural environments; and they often contain harmful additives. One way to address the multiplicity of problems that arise from the widespread use of synthetic plastics–without compromising convenience and disposability—would be to replace them with functionally equivalent materials that are biobased, biodegradable, and biocompatible, such as polyhydroxyalkanoates—a class of bioplastics. Bacteria known as “methanotrophs” consume methane as feedstock, and some produce the PHA polymer poly-ß-hydroxybutyrate (PHB). PHB production from methane could take advantage of the abundant biogas methane that is currently flared or allowed to escape to the atmosphere by the waste sector (landfills and wastewater treatment plants) to produce a valuable product that biodegrades to methane at end-of-life, creating a closed-loop cycle. This research evaluates methanotrophic growth and PHB production across scale. (1) Life Cycle Assessment (LCA) is used to anticipate the environmental impacts of PHB production from waste biogas by extrapolation from laboratory scale studies. LCA is used as an early-stage design tool to identify opportunities for pollution prevention, reduce resource consumption, guide environmental performance improvements, and identify research needs. The LCA also enables comparison with published LCAs for PHB produced from other feedstocks. (2) Stoichiometry and kinetics are evaluated and modeled for PHB-producing methanotrophs to describe the effects of oxygen and nitrogen on growth and PHB production by two PHB-producing methanotrophs. Significant differences were observed, with major implications for the use of these species in biotechnology applications. Such analyses can better inform bioreactor design, scale-up models, and life cycle assessments (LCAs). (3) A pathway genome database is developed for Methylosinus trichosporium OB3b as a model iv organism using pathway reconstruction to predict the metabolic composition. The database provides a platform for the visualization of experimental data from omics experiments, facilitates comparative studies of pathways across species, and provides a resource for biotechnology applications of methanotrophs, such as through flux balance analysis. v ACKNOWLEDGMENTS I would like to thank my adviser, Craig Criddle, for his unending enthusiasm. I would also like to thank my committee members: Peter Karp for his patience and approachability, Perry McCarty for his thoughtful suggestions, and Michael Lepech for his attentiveness. I must also acknowledge my funding agencies; the research presented here was supported by the National Science Foundation Graduate Research Fellowship (NSF GRFP), the Stanford Graduate Fellowship (SGF), and by the California Environmental Protection Agency (CalEPA), Cal Recycle and the Department of Toxic Substances Control. I would like to express my gratitude to the other students in the Criddle group, but especially Andy Pfluger, Allison Pieja, Kurt Rhoads, and Eric Sundstrom who have been research collaborators, mentors, and friends. I also want to thank my family and friends for their support throughout my Ph.D. Most importantly, I’d like to thank my boyfriend, John, for providing love, encouragement, stability, and the occasional weather forecast. vi TABLE OF CONTENTS Abstract .............................................................................................................................. iv Acknowledgments.............................................................................................................. vi List of Tables ..................................................................................................................... ix List of Figures ......................................................................................................................x Introduction ..........................................................................................................................1 Problems caused by Petroleum-based Plastics ..............................................................1 Polyhydroxyalkanoates (PHAs): A Biodegradable and Biocompatible Plastic Alternative................................................................................................................2 Type I versus Type II Methanotrophs ............................................................................4 Industrial Ecology Principles in Bioplastic (PHB) Production......................................4 The Methane Opportunity: Waste Valorization and Industrial Symbiosis ..............5 Ecobiotechnology: Natural Selection for Plastic Production...................................6 Research Objectives .......................................................................................................8 Chapter 1: Cradle-to-Gate Life Cycle Assessment for a Cradle-to-Cradle Cycle: Biogas-to-Bioplastic (and Back) ..................................................................................10 Abstract ........................................................................................................................10 Introduction ..................................................................................................................10 Methodology ................................................................................................................16 Goal and Scope Definition .....................................................................................17 Inventory Analysis .................................................................................................17 Results ..........................................................................................................................23 Impact Assessment.................................................................................................23 Discussion ....................................................................................................................25 Chapter 2: Stoichiometry and Kinetics of the PHB-producing Type II Methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP .........................28 Abstract ........................................................................................................................28 Introduction ..................................................................................................................28 vii Requirements for Oxygen and Reducing Equivalents in Methanotrophic Proteobacteria ..................................................................................................30 Materials and Methods .................................................................................................31 Cultures ..................................................................................................................31 Culture Growth Conditions ....................................................................................31 Growth Monitoring and PHB production ..............................................................33 PHB Measurement .................................................................................................34 Modeling of Stoichiometry ..........................................................................................35 Modeling of Kinetics ...................................................................................................37 Results ..........................................................................................................................39