Location: Sustainable Biofuels and Co-products Research2022 Annual Report
Objective 1: Develop fermentation technologies to synthesize and expand our collection of microbial biosurfactants (i.e., mannosylerythritol lipids, trehalose lipids, cellobiose lipids, sophorolipids, rhamnolipids etc.) and assess their commercial application potential through antimicrobial activity and surfactancy. Objective 2: Develop fermentation technologies to synthesize unique polyhydroxyalkanoates (PHA) biopolymers from low-value agro-industrial byproducts. Objective 3: Develop technologies that enable high performance products from agricultural fibers and biopolymers.
The aim of this project is to enable the development of new commercial uses for microbially-produced lipid-based molecules (i.e., glycolipid biosurfactants, biopolymers) and agricultural protein fibers (i.e., keratin, collagen) such that the newly formed materials are more cost-effective and commercially valuable. By using a multi-faceted synthetic approach, both microbial glycolipids (i.e., sophorolipids, rhamnolipids, mannosylerythritol lipids, trehalose lipids etc) and biopolymers (i.e., polyhydroxyalkanoates) will be synthesized using both lipid-based production (fermentation) strategies and modified using green chemistry synthesis techniques. Metabolic engineering and fermentation optimization of the bioprocesses for both glycolipid and biopolymer synthesis will be studied. Particular attention will be directed towards production economics and the use of inexpensive feedstocks for fermentation protocols as well as the creation of new functionalities to the bio-based products in an effort to improve their application potential. Structure-function analyses will be conducted on all newly-synthesized/produced materials such that their application potential will be fully understood for such areas as antimicrobial agents, lubricant additives, plastic substitutes, to be further derivatized to form new biobased precursors and products (i.e., polyurethanes, amphiphilic biopolymers) and be combined with protein fibers to form green composites with improved tensile strength and toughness. The effect of fiber length and diameter will be assessed for maximum potential and electrospinning will be employed to produce fibrous mats for application in green composite formation. Techno-economic analyses will be performed on all synthetic processes that result in favorable products.
Progress was made on all three objectives pertaining to the project associated with National Program 306. For Objective 1, scientists evaluated the genetic and molecular information from Pseudohyphozyma (P.) bogoriensis and Starmerella (S.) bombicola (sophorolipid producers) by investigating their genomic and transcriptomic sequences to identify the pertinent functional genes and enzymes and reconstruct the important metabolic pathways associated with sophorolipid biosynthesis from these two yeast strains. Petroleum-based surfactants are becoming problematic from the standpoint of disposal options while avoiding environmental pollution problems. DNA sequencing and genome assembly are the first steps towards understanding the molecular regulation and specific proteins and enzymes associated with sophorolipid (a bio-based naturally degradable surfactant) biosynthesis. Genomic sequencing of the high molecular weight (HMW) genomic DNA from Pseudohyphozyma bogoriensis (a known sophorolipid producing strain) utilizing genomic, transcriptomic and bioinformatic processes was performed. Construction of in silico models for sophorolipid metabolism, including the identification of genes, enzymes, and sugar transporters, as well as relevant carbohydrate metabolic pathways were identified and the contribution of different organic compounds to cell growth and sophorolipid accumulation was determined. Heterotrophic growth of P. bogoriensis using yeast and malt (YM) medium supplemented with organic compounds including C5 sugars and disaccharide substrates resulted in a positive contribution towards sophorolipid biosynthesis and a better understanding of the biosynthetic process. To that end, research included transcriptomics data analyses in P. bogoriensis which allowed in silico reconstruction of essential pathways for sophorolipid metabolism, as well as defined the capabilities of the yeast strains to use simple and complex organic compounds for biomass growth and increased sophorolipid productivity. The availability of genome sequences also allowed the manipulation and genetic modifications of S. bombicola and P. bogoriensis for the development of specialized glycolipids and fatty acids some of which were modified to form unique fatty acid amides and assessed for their antimicrobial properties against various food pathogens including Listeria monocytogenes, Salmonella enterica, and Escherichia coli. In addition, Moesziomyces aphidis (mannosylerythritol producer) was procured from a commercial culture collection and work was initiated and successfully conducted to produce mannosylerythritol lipids from the wildtype strain using conventional carbon feedstocks. These efforts will allow further studies on the surfactancy, and antimicrobial properties of the purified glycolipid biosurfactants but also will result in the formation of unique hydroxy fatty acids which can be chemically modified for enhanced antimicrobial properties and/or applied in such areas as oleogel formation and estolide development for possible lubricant applications. For Objective 2, scientists utilized known epoxidation chemistry (specifically m-chloroperoxybenzoic acid) on the polyhydroxyalkanoate (PHA) biopolymers derived from the 12-month milestones which contained terminal side-chain double bonds to create PHA biopolymer side chains with terminal epoxy groups in preparation to produce polymeric surfactants by attaching polyethylene glycol (or PEG derivatives) of different molecular weights, antimicrobial biopolymers by attaching various phenol derivatives, and unique polyurethanes by converting the epoxy groups to diols and then using different diisocyanates to produce the polyurethanes, all of which are within the context of the existing 5-year project plan. In a related collaborative study, work focused on the use of high-volume, low-value corn stover hydrolysate and levulinic acid (a plentiful organic acid) as substrates for PHA biopolymer synthesis was concluded and a manuscript detailing the study was submitted and accepted for publication. For Objective 3, scientists formulated green composites composed of PHA biopolymers (specifically poly-3-hydroxybutyrate; PHB) and ground wool fibers. Wool fibers were ground into fine powders and then manually mixed in different ratios ranging from 0.5% to 2% fiber before extrusion into composite filaments at 180 C and 190 C under constant pressure using a melt flow index tester. Data showed that ground wool did not appreciably affect the flow rate / extrusion process indicating that the fibers must be longer in length to provide structural support in the PHB biopolymer matrix. Ground fibers were used here to assure a homogeneous mixture within the PHB matrix. This work resulted in a submitted manuscript. Future studies will utilize a recently purchased compounding machine that will produce homogeneous polymer fiber mixtures from longer wool fibers which will improve the properties of the composite material.
Liu, J., Liu, Y., Brown, E.M., Ma, Z., Liu, C. 2021. Fabrication of composite films based on chitosan and vegetable-tanned collagen fibers crosslinked with genipin. Journal of American Leather Chemists Association. 116(10):345-358.
Ashby, R.D., Liu, C. 2021. Agro-based waste-/co-products as feedstocks for polyhydroxyalkanoate biosynthesis. In: Sarker, M., Liu, L.S., Yadav, M., Yosief, H., Hussain, S., editors. Conversion of Renewable Biomass and Bioproducts. Washington DC: ACS Press, ACS Symposium Series 1392. p.261-286.
Chen, N., Liu, C., Ashby, R.D. 2021. Modification of wool fibers via base/cationic detergent pretreatment and transglutaminase-mediated reaction of keratin. Journal of Natural Fibers. https://doi.org/10.1080/15440478.2021.2002780.
Msanne, J.N., Sy Vu, H., Cahoon, E. 2021. Acyl-acyl carrier protein pool dynamics with oil accumulation in nitrogen-deprived Chlamydomonas reinhardtii microalgal cells. Journal of the American Oil Chemists' Society. 98(11):1107-1112. https://doi.org/10.1002/aocs.12539.
Ashby, R.D., Qureshi, N., Strahan, G.D., Johnston, D., Msanne, J.N., Lin, X. 2022. Corn stover hydrolysate and levulinic acid: mixed substrates for short-chain polyhydroxyalkanoate production. Biocatalysis and Agricultural Biotechnology. 43:102391. https://doi.org/10.1016/j.bcab.2022.102391.