Research Project: Commercial Products from Lipids and Fibers

Location: Sustainable Biofuels and Co-products Research

2024 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. For Objective 1, scientists continued to evaluate the antimicrobial characteristics of fermentation-based glycolipids. This year, emphasis was on sophorolipids, and mannosylerythritol lipids (MEL) which were synthesized under controlled fermentation conditions using plentiful, inexpensive feedstocks such as cellulose and cellulose-derived products (e.g., carboxymethyl cellulose, cellobiose, levoglucosan), as well as three types of waste grease to reduce production economics. Sophorolipids produced by the yeast Starmerella bombicola were provided to an international collaborator for assessment of their antimicrobial properties (determined by minimum inhibitory concentration; MIC) against various select halotolerant and halophilic bacteria, as well as extremely halophilic archaea typically found as colonizers in leather processing. In addition, sophorolipids were chemically deconstructed to obtain a variety of hydroxy fatty acids and amide derivatives which were supplied to an in-house collaborator for successful antimicrobial testing against various Gram-positive bacterial strains. These same hydroxy fatty acids and their methyl esters were also supplied to a separate ARS collaborator for use in the formation of oleogels (fat substitutes). Oleogels are commonly produced from ricinoleic acid because the location of the hydroxyl group within the fatty acid provides a favorable molecule for synthesis. Unfortunately, ricinoleic acid comes from castor beans which also contain ricin, a potent toxin. By utilizing the hydroxy fatty acids derived from sophorolipids, one can avoid the use of a potentially problematic substance and simultaneously provide additional value to the parent sophorolipid molecules. Total RNA samples were also extracted from the sophorolipid-producing yeast Pseudohyphozyma bogoriensis under varying conditions (e.g., nutrient deprivation) that drive synthesis and accumulation of sophorolipid biosurfactants. Transcriptomes were sequenced in collaboration with a sequencing facility at the University of Delaware. Bioinformatics analyses, performed in collaboration with an in-house collaborator, should identify relevant yeast genes in the glycolipid biosynthetic pathway as well as their expression level, a prerequisite to genetic modifications. MEL was synthesized using Moesziomyces aphidis from levoglucosan (a pyrolysis by-product), and waste grease, and the surface acting properties of the molecules determined. The antimicrobial properties, more specifically anti-algal and anti-fungal effects, are being tested by scientists on this project as well as an in-house collaborator. Additionally, scientists from this project participated in the characterization of biolubricants derived from oleic acid. The characterization techniques used in this study will be followed as additional fatty acid based biolubricants are developed from the hydroxy fatty acids derived from sophorolipids. 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 (e.g., catechol) and polyethylene glycol (PEG) derivatives to produce phenylated and amphiphilic PHA biopolymers. Phenylated PHA biopolymers were assessed for their antimicrobial properties against both Gram-positive (Listeria monocytogenes) and Gram-negative (Escherichia coli) bacteria while the PEGylated PHA biopolymers are currently being tested for surface-acting properties. In addition to the Project Plan, the described research supports the work defined in a funded grant (co-Principal Investigator) through the National Institute of Food and Agriculture (NIFA) which is focused on the preparation of antimicrobial thermosetting biopolymers from non-edible oils and their applications. This work resulted in an Invention Disclosure (Docket number 0047.24) that was approved by the chemical patent committee for patent application development. For Objective 3, scientists formulated green composites using PHA biopolymers (specifically poly-3-hydroxybutyrate; PHB) and biochar obtained from an in-house collaboration. The PHB/biochar composites were manually mixed in powdered form and pressed into dog bones that were amenable to tensile testing analyses. In addition, wool fibers were obtained from the American Wool Council in either their native or mercerized (scales removed) state for generation of PHA/wool composites with different component ratios using a compounding machine that produces homogeneous polymer fiber mixtures from longer scaled and descaled wool fibers. Different combinations of PHA and wool along with varying component ratios allow variation in the composite properties thus broadening application potential.

Accomplishments

Review Publications
Olanya, O.M., Yosief, H.O., Ashby, R.D., Niemira, B.A., Sarker, M.I., Ukuku, D.O., Mukhopadhyay, S., Msanne, J.N., Fan, X. 2023. Inactivation of foodborne and other pathogenic bacteria with pyrrolidine based fatty acid amide derivatives. Journal of Food Safety. https://doi.org/10.1111/jfs.13079.
Msanne, J.N., Ashby, R.D., Olanya, O.M. 2023. Heterologous expression of a yeast lipid transporter promotes accumulation of storage compounds in chlamydomonas. Biocatalysis and Agricultural Biotechnology. https://doi.org/10.1016/j.bcab.2023.102809.
Sarker, M.I., Mainali, K., Sharma, B.K., Yadav, M.P., Lew, H.N., Ashby, R.D. 2023. Synthesized biolubricants from naturally derived oleic acid: Oxidative stability and cold flow performance. Industrial Crops and Products. https://doi.org/10.1016/j.indcrop.2023.117315.
Kazem Rostami, M., Ryu, V.N., Wagner, K., Jones, K.C., Mullen, C.A., Wyatt, V.T., Wu, C., Ashby, R.D., Fan, X., Lew, H.N. 2023. Antibacterial agents from waste grease: Arylation of brown grease fatty acids with beechwood creosote and derivatization. ACS Sustainable Chemistry & Engineering. https://doi.org/10.1021/acssuschemeng.3c05767.
Qureshi, N., Ashby, R.D., Nichols, N.N., Hector, R.E. 2024. Novel technologies for butyric acid fermentation: use of cellulosic biomass, rapid bioreactor, and efficient product recovery. Fermentation. https://doi.org/10.3390/fermentation10030142.
Moser, J.K., Ashby, R.D., Yosief, H.O., Msanne, J.N., Peterson, S.C., Bantchev, G.B., Cermak, S.C., Felker, F.C. 2024. Properties of soybean oil oleogels produced from sophorolipid-derived hydroxy fatty acids, methyl esters and hydrogenated Lesquerella seed oil. Journal of the American Oil Chemists' Society. https://doi.org/10.1002/aocs.12843.
Ashby, R.D., Msanne, J.N., Munir, M., Inayat, A., Pastore, C., Mustafa, A. 2023. Overview of bioprocess engineering. In Abomohra, A., and Ende, S. (eds.) Value-added Products from Algae: Phycochemical Production and Applications. Switzerland: Springer, Cham. p. 123-155. https://doi.org/10.1007/978-3-031-42026-9_6.
Msanne, J.N., Ashby, R.D. 2023. Genetic and process engineering for select glycolipid biosynthesis from plant/algal oils or their derivatives. In: Liu, Z. and Kraus, K., editors. Green Chemistry and Green Materials from Plant Oils and Natural Acids: Green Chemistry Series. United Kingdom: Royal Society of Chemistry. p. 213-251. https://doi.org/10.1039/BK9781837671595-00213.