Location: Sustainable Biofuels and Co-products Research
2017 Annual Report
Objectives
Objective 1: Enable commercial processes for converting microbial lipids and the
byproducts of their fermentation into marketable products.
Sub-objective 1: Production of microbial glycolipids and variants to enhance
commercial viability.
1A: Genetic engineering of P. chlororaphis for production of RL from low-cost
bioglycerol and soy-sugar byproduct.
1B: Fermentative production of short-chain (C=12) and very-long-chain (C22)
sophorolipids.
Sub-objective 2: Synthesis and testing of value-added products from glycolipids
and components.
Enabling chemical and/or enzymatic production of glycolipid components and testing products as novel antimicrobial agents and novel sugar substitutes.
Approach
To enhance the commercial viability of microbial glycolipids (i.e., sophorolipids, SLs; and rhamnolipids, RLs), their high-value antimicrobial property will be extensively researched in this project for full exploitation in end-user industrial applications. Accordingly, the structure-function relationship of the antimicrobial activity of these glycolipids will be established by first biosynthesizing various structurally varied glycolipids through the use of new producing strains and uniquely synthesized oleochemicals from fats and oils as fermentative feedstocks. The resultant microbial SLs containing very-long-chain (C22) and short-chain (C
Progress Report
Progress was made on the objective and associated subobjectives of this project which addresses NP306, Component 2B, Enable Technologies. Under subobjective 1A, we genetic engineered (GE) a nonpathogenic patented Pseudomonas chlororaphis (which produces both biosurfactant (rhamnolipid/RL) and bioplastic (polyhydroxyalkanoate/PHA) to successfully express an alpha-galactosidase (a-gal) gene for breaking down soy sugars in low-cost industrial byproducts (soy molasses and soy tofu whey). Work is ongoing to construct additional GE organisms with higher a-Gal activity to more effectively use soy byproducts to lower production costs of RL and PHA. With subobjective 1B, we collaboratively developed a process to attach phenyl groups to the hydroxy fatty acids obtained from sophorolipids (SLs); these could subsequently be used to synthesize new phenylated estolides (fatty acid oligomers) for use as antimicrobial lubricant additives or to polymerize into new antimicrobial food packaging film. We also made substantial progress in developing assay methods and in identifying potential significant factors in the Response Surface Methodology (RSM) optimization study to achieve high-yield production of very-long-chain-(vlc-)SL (which uniquely contains a 22-carbon hydroxy fatty acid (13-hydroxydocosanoic acid)) by Rhodotorula bogoriensis yeast. Under subobjective 2, we have completed, published, and presented talks on the antimicrobial activity testing of common SLs against tooth-decay and animal hide-degrading bacteria using a microtiter-plate format we developed. We also had synthesized and begun the testing of four (4) short-chain (9-carbon) and functionalized (dihydroxy, epoxy, and hydroperoxy) SLs for antimicrobial and antibiofilm activity against tooth-decay bacteria. Collaborations with Center scientists are ongoing to test combined application of SLs with bacteriocin to inhibit biofilm formation. Separately, olefinic (double-bond) and epoxy (oxygen-containing 3-member ring) estolides were synthesized from the hydroxy fatty acids of SLs and then tested successfully as plasticizers (plastic softeners) in poly-3-hydroxybutyrate (PHB) bioplastic film applications; this work was published in 2016. On our effort to harvest the valuable uncommon hydroxy fatty acids along with the disaccharide sophorose from SLs for application in cosmetics, lubricants and biomass cellulosics industries, we have continued to make progress in developing methods and the subsequent assay protocols to implement enzymatic (cellulases and glucosidases) as well as acid- (H3PO4) and base (NaOH)-catalysed reactions to dissociate intact fatty acid and sugar moieties from SLs. In collaboration with extramural scientists, research was developed to utilize xylose and levulinic acid as feedstocks for PHA biosynthesis process, leading to the discovery of 2 (two) bacterial strains capable of synthesizing PHA bioplastics under the conditions used. Depending on the specific organism being employed in the bioprocess, the specific type of biopolymers could be produced. In one process, a terpolyester was produced consisting of 3-hydroxybutyrate-3-hydroxyvalerate-4-hydroxyvalerate (P3HB-co-3HV-co-4HV), and in the other process using xylose as the sugar source a block co-polymer was produced consisting of different ratios of 3-HB and 3HV. Because xylose is a common component of lignocellulosic material and levulinic acid is easily produced from the same plant material, this result will be of benefit to biopolymer researchers as efforts move forward in finding new outlets for plant biomass and reducing the cost of fermentative biopolymers. Finally, the most exciting progress is made in a collaborative research effort to open a new application frontier for SLs in the taste sensory field. Initial success had shown the ability of SLs to strongly stimulate the sweet- and bitter-taste T1R3 receptor of human taste-bud cells on culture dish; a patent application had been filed and continuing collaborative research is ongoing to test human use of SLs.
Accomplishments
1. Natural oral care surfactant. Surfactants are compounds that contain oil- and water-loving parts/components, and are used in the formulation of oral care products such as tooth paste and mouth wash. Commonly used surfactants are made either with petrochemicals or through harsh processes, and they lack bacterial inhibitor and often must be supplemented with antimicrobial agents such as triclosan. ARS researchers at Wyndmoor, Pennsylvania, in collaboration with an industrial partner had successfully tested and showed the antimicrobial activity of several forms of sophorolipids (a natural glycolipid surfactant produced by benign yeasts) against tooth-decay causing bacteria called streptococci and lactobacilli. The outcome is expected to heighten commercial interests to use sophorolipids in the formulation of oral care products with value-added antimicrobial property.
Review Publications
Solaiman, D., Ashby, R.D., Birbir, M., Caglayan, P. 2016. Antibacterial activity of sophorolipids produced by candida bombicola against gram-positive and gram-negative bacteria isolated from salted hides. Journal of American Leather Chemists Association. 111:358-364.
Solaiman, D., Ashby, R.D., Uknalis, J. 2017. Characterization of growth inhibition of oral bacteria by sophorolipid using a microplate-format assay. Journal of Microbiological Methods. 136:21-29. doi: 10.1016/j.mimet.2017.02.012.
