Location: Sustainable Biofuels and Co-products Research
2023 Annual Report
Objectives
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.
Approach
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 Report
Progress was made on all three objectives pertaining to the project associated with National Program 306. For Objective 1, scientists continued to evaluate the yeast Pseudohyphozyma (P.) bogoriensis for growth and sophorolipid (an eco-friendly biosurfactant) production when grown on plentiful, inexpensive mono/disaccharides (e.g., xylose, cellobiose), glycerine (sourced from the biodiesel industry), and cellulose and cellulose-derived products (e.g., carboxymethyl cellulose). The sequencing of the yeast genome allowed the identification of enzymes that facilitate the utilization of organic substrates, for instance putative cellulase and cellulose-binding enzymes, emphasizing its capability to metabolize cellulose. These results will contribute to the understanding of the effects of the feedstock on the amount, nature, and physico-chemical properties of the biosurfactants produced, and simultaneously increasing the value of agricultural by-products. One of the prospective downstream uses for glycolipids (including sophorolipids) and/or their derivatives is in antimicrobial applications. Extensive work has been done by our research group evaluating the antimicrobial properties of intact sophorolipids; however, molecules that can be chemically and/or biologically derived from sophorolipids have seen very little application-based research. Different structural variants of intact sophorolipids were chemically deconstructed to obtain a variety of hydroxy fatty acids which were supplied to an ARS collaborator for ongoing studies on oleogel formation and were used to prepare various fatty acid amides which were supplied to an in-house collaborator for antimicrobial testing against various Gram positive bacterial strains associated with food-borne illness and dental caries. This work led to a recently submitted manuscript and to a new Material Transfer Agreement (MTA) with the University of Connecticut for studies on potential anticancer applications. Furthermore, glycolipids have been produced using industrial glycerine as the carbon source, and these molecules were supplied to an in-house collaborator to test against various microbial pathogens. The completion of genome sequencing of P. bogoriensis also allowed the identification of relevant genes involved in fatty acid and sophorolipid biosynthesis. In the yeast fatty acid metabolic pathways, characteristic elongase enzymes that may be involved in the synthesis of very long chain fatty acids (22-carbon and 24-carbon chain lengths) associated with the sophorolipids were identified and transformed in Saccharomyces cerevisiae to confirm their metabolic functions. These new genes, which can be used for metabolic engineering in heterologous hosts, should provide additional tools and means for the synthesis of beneficial long-chain fatty acids in oleaginous and industrially relevant organisms. Moreover, a glycolipid transporter was identified and heterologously expressed in green alga, resulting in mutants with higher accumulation of lipids and starch. This information was described in a recently submitted manuscript. Sequencing of P. bogoriensis is also a prerequisite to genetic manipulations of this yeast since this allows development of vectors with promoter/terminator combinations, antibiotics, and reporter genes. A reliable transformation method using Agrobacterium rhizogenes-based gene stacking technology was recently developed and is currently being adapted for the transformation of the yeast with multiple genes/traits. In addition, other glycolipid production systems have been assessed to expand our collection of microbial biosurfactants. Moesziomyces aphidis (a mannosylerythritol producer; MEL) and Rhodococcus erythropolis (a trehalose lipid producer) were used successfully to produce glycolipid biosurfactants from conventional fatty acids, triacylglycerols, and n-alkanes with varying chemical structures and mix ratios. These efforts will allow further studies on the surfactancy, and antimicrobial properties of these purified molecules but also will result in the formation of unique hydroxy fatty acids which can be chemically modified for enhanced antimicrobial properties or other applications. For Objective 2, scientists utilized polyhydroxyalkanoate (PHA) biopolymers created by fermentation using 10-undecenoic acid as substrate to create biopolymers that were epoxidized by well-known chemical techniques and subsequently reacted with various phenol derivatives (through an in-house collaboration) to produce phenylated PHA biopolymers which will be assessed for their antimicrobial properties in future research. In addition to the Project Plan, this research supports the work described in a funded grant (co-Principal Investigator) through the National Institute of Food and Agriculture (NIFA; accession# - 1026090) focused on the preparation of antimicrobial thermosetting biopolymers from non-edible oils. In a related international collaboration, a specific PHA biopolymer (poly-3-hydroxybutyrate-co-3-hydroxyhexanoate) was utilized as an initiator to produce unique methacrylate polymers and a poly(vinyl chloride)-based food packaging material with antioxidative and anticancer properties was developed. Both studies resulted in submitted and accepted manuscripts. For Objective 3, scientists formulated green composites using 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 degrees Celsius and 190 degrees Celsius under constant pressure using a melt flow index tester. This work resulted in a submitted and accepted manuscript. In addition, work was performed to remove the scales that are naturally present in wool fibers. These scales may provide a foothold for the accompanying PHA biopolymer in composite formation. Scientists utilized dilute formic acid with heat to reduce/eliminate the scales present in parental wool fibers. It is anticipated that by removing the scales, the composites formed with identical polymer to fiber ratios will have altered physical and mechanical properties. Future studies will utilize a compounding machine that will produce homogeneous polymer fiber mixtures from longer scaled and descaled wool fibers which will improve and/or vary the properties of the composite material thus broadening application potential.
Accomplishments
Review Publications
Ashby, R.D., Muhammad Zulkifli, W., Yatim, A.M., Ren, K., Mustafa, A. 2022. Glycolipid biosurfactants: Biosynthesis, and related potential applications in food industry. In: Inamuddin and Adetunji, C.O., editors. Applications of Next Generational Biosurfactants in the Food Sector. London, UK: Academic Press and Elsevier. p. 307-334. https://doi.org/10.1016/B978-0-12-824283-4.00006-X.
Yosief, H.O., Liu, C., Ashby, R.D., Strahan, G.D., Latona, N.P., Chen, N. 2023. Extrusion plastometry processing of poly(3-hydroxybutyrate)/ground wool fiber blends. Green Materials. https://doi.org/10.1680/jgrma.22.00026.
Hazer, B., Tasci, F., Modjinou, T., Langlois, V., Ashby, R.D. 2023. Free radical polymerization of dimethyl amino ethyl methacrylate initiated by poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) macroazo initiator: Thermal and physicochemical characterization. Journal of Polymers and the Environment. https://doi.org/10.1007/s10924-023-02857-3.
Msanne, J.N., Shao, J.Y., Ashby, R.D., Campos, P., Liu, Y., Solaiman, D. 2022. Draft genome sequences of the sophorolipid-producing yeast pseudohyphozyma bogoriensis ATCC 18809. Microbiology Resource Announcements. https://doi.org/10.1128/mra.00566-22.
Hazer, B., Karahaliloglu, Z., Ashby, R.D. 2023. Poly(vinyl chloride) derived food packaging applications with antioxidative and anticancer properties. ACS Food Science and Technology. https://doi.org/10.1021/acsfoodscitech.3c00021.
