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

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. 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