University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 8-2013 Toward Direct Biosynthesis of Drop-in Ready Biofuels in Plants: Rapid Screening and Functional Genomic Characterization of Plant-derived Advanced Biofuels and Implications for Coproduction in Lignocellulosic Feedstocks Blake Lee Joyce [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Agronomy and Crop Sciences Commons, Biochemistry Commons, Biotechnology Commons, Molecular Biology Commons, Other Mechanical Engineering Commons, and the Plant Biology Commons Recommended Citation Joyce, Blake Lee, "Toward Direct Biosynthesis of Drop-in Ready Biofuels in Plants: Rapid Screening and Functional Genomic Characterization of Plant-derived Advanced Biofuels and Implications for Coproduction in Lignocellulosic Feedstocks. " PhD diss., University of Tennessee, 2013. https://trace.tennessee.edu/utk_graddiss/2440 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Blake Lee Joyce entitled "Toward Direct Biosynthesis of Drop-in Ready Biofuels in Plants: Rapid Screening and Functional Genomic Characterization of Plant-derived Advanced Biofuels and Implications for Coproduction in Lignocellulosic Feedstocks." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Plants, Soils, and Insects. Charles N. Stewart, Major Professor We have read this dissertation and recommend its acceptance: Feng Chen, Joseph Bozell, Bruce Bunting, Jonathan Mielenz Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Toward Direct Biosynthesis of Drop-in Ready Biofuels in Plants: Rapid Screening and Functional Genomic Characterization of Plant- derived Advanced Biofuels and Implications for Coproduction in Lignocellulosic Feedstocks A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Blake Lee Joyce August 2013 i Abstract Advanced biofuels that are “drop-in” ready, completely fungible with petroleum fuels, and require minimal infrastructure to process a finished fuel could provide transportation fuels in rural or developing areas. Five oils extracted from Pittosporum resiniferum, Copaifera reticulata, and surrogate oils for Cymbopogon flexuosus, C. martinii, and Dictamnus albus in B20 blends were sent for ASTM International biodiesel testing and run in homogenous charge combustion ignition engines to determine combustion properties and emissions. All oils tested lowered cloud point. Oils derived from Copaifera reticulata also lowered indicated specific fuel consumption and had emissions similar to the ultra-low sulfur diesel control. Characterization of the biosynthetic pathways responsible for the sesquiterpene-rich Copaifera-derived oils could lead to production of these oils in biofuel feedstocks. The Copaifera officinalis transcriptome sequencing, assembly, and annotation identified eight terpene synthase genes in C. officinalis and C. langsdorffii that produced mono- and sesquiterpene products in functional assays. The terpene synthases characterized produced the major fraction of sesquiterpenes identified in C. officinalis leaf, stem, and root tissues as well as the oils tested previously. This initial characterization will support future investigation of sesquiterpene biosynthesis in the Copaifera genus to understand how liters of sesquiterpene oils are produced for biotechnology applications and the mechanism responsible for the geographical biochemical variation seen in sesquiterpene-producing New World species compared to diterpene-producing African species. ii Lastly, Cymbopogon flexuosus and C. martinii biomass production in small field trials, as well as oil and ethanol yield from biomass were investigated to determine the feasibility of producing the advanced biofuels in lignocellulosic feedstocks. C. flexuosus and C. martinii ethanol yields from biomass were lower than Panicum virgatum, but had an average oil yield of 85.7 kg ha-1 [ha^-1] and 67.0 kg ha-1 [ha^-1], respectively. Combined ethanol and oil value for C. flexuosus and C. martinii were higher than P. virgatum ethanol value. This suggests that the oils from C. flexuosus and C. martinii are more suitable as high-value fermentation coproducts rather than as low-value advanced biofuels. Increasing yield of oil or alternative production schemes could lead to economically feasible advanced biofuel production. iii Preface Our growing world demands more and more from agriculture and natural resources. This reality is unavoidable. Natural population growth coupled with the fundamental search for a better quality of life has led to agricultural demands being levied on the world’s natural order. However, through technology and research, yields of food and agricultural products that seemed impossible to imagine 100 years ago are all too common today. In 1907, the average corn yield was 1 706 kg ha-1, but has since increased steadily to a reported 9 300 kg ha-1 in 2005 (Egli, 2008). Likewise, soybean yield increased from approximately 1000 kg ha-1 to 3 000 kg ha-1 in Illinois and Iowa from 1925 to 2006 (Egli, 2008). These documented continuous yield increases result from many interacting factors several of which are well known: fertilizer application, use of herbicides and pesticides, plant breeding and genetic improvement, and enhanced management practices to reference a few (Egli, 2008). Despite these and other past agricultural successes, a new generation of challenges has risen in the wake of global demands for the comforts of a modern lifestyle traditionally enjoyed primarily by citizens of developed economic powers. To meet these demands future generations will have to provide electricity, transportation, clean water, a stable high-quality food supply which includes protein, and sound housing for billions of people with existing arable land and natural resources. Additionally, modern agriculture will have to meet these demands with the added constraints of sustainable production as climate and land use change are inexorably linked to increasing agriculture production (Godfray et al., 2010; Lambin & Meyfroidt, 2011). Climate and land use change predictions portray reduction of food production iv and availability, stability of food supplies, access to food, and utilization of food with these impacts affecting poor developing countries disproportionately (Schmidhuber & Tubiello, 2007). The remaining amount of arable land that exists and the environmental impacts associated with bringing these lands into production are difficult to predict. In one estimate, there are 2.4 billion hectares of land suitable for cereal production of which 1.5 billion are already cultivated (Pingali et al., 2008). Pingali et al. note that the majority of the uncultivated land exists in South America and sub-Saharan Africa that require irrigation; however, availability of fresh water will be limited in the future and will likely be a major factor that ultimately determines future agricultural production (Strzepek & Boehlert, 2010). Therefore increasing agricultural production to feed our growing world is not as simple as opening more land for production. The intersection of agriculture and petroleum This leads to one seemingly simple conclusion: increasing production capacities on existing agricultural land will have to be the primary method to meet increased demand (Godfray et al., 2010). This has been traditionally accomplished through intensification of agriculture; which in turn leads to greater demands for agricultural inputs, e.g. liquid fuels for modern machinery, fertilizers, and pesticides (McMichael et al., 2007). This remains a complex issue in the face of increased global demand for liquid fuels and the impending predictions of global peak oil. A future peak oil production is now an accepted idea and debate has slowly shifted to timing peak oil production rather than its potential existence (de Almeida & Silva, 2009). A review of peak oil date predictions shows that independent analysts tend to predict peak oil between 2015- v 2020 while agencies like the U.S. Energy Information Administration (EIA), International Energy Agency, Shell, and the Cambridge Energy Research Associates (CERA) predict peak oil occurring after 2030 and as late as 2112 (de Almeida & Silva, 2009). Peak oil predictions, much like in global food production predictions of the past, have been confounded by technological development. Overall, declines in oil production from 2000 to 2008 suggest that about 1.8 million barrels day-1 needs to be replaced from countries that are not members of Organization of the Petroleum Exporting Countries (OPEC) (Allsopp & Fattouh, 2011). To meet this production decline, countries have turned to other sources of liquid fuels. Some of these new sources are hydraulic fracturing, or fracking, which breaks source rock to access sequestered gas and crude oil commonly referred