Location: Sustainable Biofuels and Co-products Research
2018 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
Under Subobjective 1A, after completing the study and publishing the results of our glp-transformants of Pseudomonas chlororaphis (which is an ARS-patented nonpathogenic bacterium capable of producing both a biosurfactant (rhamnolipid/RL) and a bioplastic (polyhydroxyalkanoate/PHA), we embarked on the next phase of carrying out strain improvement of the P. chlororaphis to obtain new strains that express a cloned alpha-galactosidase gene (a-gal) coding for an a-Gal enzyme capable of breaking apart complex soy sugars found in low-cost industrial byproducts such as soy molasses and soy tofu whey. Testing of the new strains in defined culture medium confirmed their ability to break down a soy sugar called raffinose but not another soy sugar called stachyose. We had also completed HPLC analysis of tofu whey to obtain concentrations of soy sugars (i.e., sucrose, raffinose, and stachyose) in preparation for testing the new strains expressing a-Gal activity in this byproduct. Future work is now in progress to test the new strains’ ability to break down soy sugars found in complex soy molasses and tofu whey. Under Subobjective 1B, sophorolipids were synthesized from 3 (three) new Candida strains (C. kuoi, C. apicola, and C. riodocensis) at the shake-flask scale and the chemical compositions and structural ratios were determined. We had performed a 10 L-scale fermentation of Rhodotorula bogoriensis yeast in medium containing selenium to evaluate cell-biomass and very-long-chain-(vlc-) SL (containing a 22- carbon hydroxy fatty acid (13-hydroxydocosanoic acid)) production to verify the finding of a previous small-scale preliminary Response-Surface-Methodology (RSM) experiment. We initiated full-genome sequence determination of R. bogoriensis yeast and obtained preliminary results that should benefit genetic engineering effort to improve very-long-chain-(vlc-) SL production. We had now completed the evaluation of the four (4) short-chain (9-carbon) and functionalized (dihydroxy, epoxy, and hydroperoxy) SLs for antimicrobial activity against tooth decay-causing bacterium, showing that these derivatives were equally active in comparison to the well-known 16- and 18-carbon SLs. Under subobjective 2, both native and short-chain/functionalized sophorolipids were tested as antimicrobial agents against 2 Gram-positive bacterial strains associated with dental caries formation and epoxy estolide molecules were synthesized from the hydroxy fatty acids derived from sophorolipids and applied as plasticizers in short-chain polyhydroxyalkanoate polymer films. Lastly, further studies involving the use of cheap feedstocks to produce unique polyhydroxyalkanoate biopolymers was continued. The use of lignocellulosic sugars (xylose, glucose) along with levulinic acid (cheap and easily produced through acid catalysis of both 5 and 6-carbon sugars) were shown to induce terpolymers comprised of 3-hydroxybutyrate, 3-hydroxyvalerate, and 4-hydroxyvalerate. These terpolymers are unique as few wildtype strains have been documented to be capable of their production. In addition, we showed that mixed culture fermentations involving the same feedstocks can be used to induce mixed polymer synthesis where the tensile properties of polymer films derived from the polymer mixtures are greatly improved over the films produced from single polymers. Finally, further progress was made in a collaborative research in developing new application frontier for SLs in the taste sensory field, in which the initial success in identifying the ability of SLs to stimulate taste receptors of human taste-bud cells had resulted in patent application for use of SL as a bitter-testant inhibitor in pharmaceutical and related areas, and continuing collaborative researches to test human use of SLs. In addition, in a new collaboration with a Center ARS scientist, we determined previously unknown surface-active (detergent) property and anti-oral bacterium activity of several mono- and poly-phenolic derivatives of soybean oil.
Accomplishments
1. Biosurfactant- and biopolymer-producing bacteria genetically enhanced to use processing waste streams. ARS scientists previously discovered a nonpathogenic bacterium called Pseudomonas chlororaphis capable of producing biosurfactant (called rhamnolipid or RL) and biopolymer (called polyhydroxyalkanoate or PHA) by using glucose as starting material. Costs of production could be lowered if this bacterium could be made to use cheap industrial waste streams such as soy molasses or bio-glycerol as starting material. ARS scientists at Wyndmoor, Pennsylvania successfully incorporated genes needed to use soy molasses and glycerol into this bacterium. Tests showed that these genes were active to allow the bacteria to degrade complex sugar in soy molasses and to use glycerol more efficiently. Previous work over the years using the unmodified Pseudomonas chlororaphis had resulted in 1 issued- and 1 pending patents, 2 license applications, 2 MTAs, 5 peer-reviewed articles, and many invitations to present nationally and internationally. The new strains described in this accomplishment attracted commercial interests for use in producing lower-cost RL because of their ability to use cheaper starting materials. This new accomplishment had so far resulted in 3 peer-reviewed publications and numerous invited presentations in national and international meetings.
2. Biobased plasticizer for biodegradable polymers. Poly-3-hydroxybutyrate (PHB) is a plastic material that is environmentally friendly but also rigid and brittle. Sophorolipids (SL) are unique molecules synthesized by various strains of yeast; they contain uncommon fatty acids (components of natural fats and oils) which can be used in a range of applications. ARS researchers at Wyndmoor, Pennsylvania used the unique fatty acids from SL to chemically synthesize distinct estolides (ester derivatives of fatty acids that are generally composed of at least two fatty acids) that were used as plasticizers to improve the properties of PHB films. This research resulted in a peer-reviewed publication and an invited presentation at the Annual Meeting of the Society for Tribology and Lubrication Engineers (STLE) which generated considerable interest among industrial tribologists for the further use of these unique estolides in lubrication applications. The estolides synthesized and reported in this accomplishment have demonstrated uses as plasticizers (to make stiff, brittle polymers, such as PHB softer and more useful) but also have conceivable uses as additives for fine-tuning the qualities of lubricants. These results afford new uses for SL as well as provide a means of controlling the material properties of PHB polymers making both microbially-produced molecules more commercially attractive.
Review Publications
Ashby, R.D., Solaiman, D., Nunez, A., Strahan, G.D., Johnston, D. 2018. Burkholderia sacchari DSM 17165: A source of compositionally-tunable block-copolymeric short-chain poly(hydroxyalkanoates) from xylose and levulinic acid. Bioresource Technology. 253:333-342.
Olanya, O.M., Ukuku, D.O., Solaiman, D., Ashby, R.D., Niemira, B.A., Mukhopadhyay, S. 2018. Effects of temperature and storage time on inactivation of Listeria monocytogenes, Salmonella enterica, and Escherichia coli 0157:H7 populations by sophorolipid and sanitizer in-vitro and on tomato. International Journal of Food Science and Technology. 53:1303-1315. http://doi.org/10.1111/ijfs.13711.
Zhang, X., Ashby, R.D., Solaiman, D., Liu, Y., Fan, X. 2017. Antimicrobial activity and inactivation mechanism of lactonic and free acid sophorolipids against Escherichia coli O157:H7. Biocatalysis and Agricultural Biotechnology. 11(C):176-182. doi: 10.1016/j.bcab.2017.07.002.
Solaiman, D., Ashby, R.D., Biresaw, G. 2017. Biosynthesis and derivatization of microbial glycolipids and their potential application in tribology. In: Biresaw, G., Mittal, K.L., editors. Surfactants in Tribology, Volume 5. Boca Raton, FL: CRC Press. p. 263-288.
Solaiman, D., Ashby, R.D., Aneja, K., Crocker, N.V., Liu, Y. 2017. Galacto-oligosaccharide hydrolysis by genetically-engineered alpha-galactosidase-producing Pseudomonas chlororaphis strains. Biocatalysis and Agricultural Biotechnology. 12:213-218. https://doi.org/10.1016/j.bcab.2017.12.008.
