Location: Sustainable Biofuels and Co-products Research
2019 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, using previously constructed and tested genetically altered Pseudomonas chlororaphis (an ARS-patented non-pathogenic bacterial strain capable of synthesizing both rhamnolipid biosurfactants and polyhydroxyalkanoate biopolymers) bacterial strains expressing alpha-galactosidase activity, we developed a multi-stage fermentation approach to directly hydrolyze the soy sugars (i.e. sucrose, a disaccharide; raffinose and stachyose, both oligosaccharides) in soy molasses and tofu whey. This resulted in the formation of simple monosaccharides that could be metabolized for microbial growth and product synthesis from these inexpensive feedstocks. Work is currently underway to improve the ability of P. chlororaphis to produce rhamnolipid biosurfactants more economically through the use of the previously mentioned low-cost substrates. Under Subobjective 1B, the techno-economic assessment of the sophorolipid biosurfactants derived from new Candida yeast strains (C. kuoi, C. apicola, and C. riodocensis) as well as Rhodotorula bogoriensis is underway. Each synthetic fermentation process was scaled up from shake flask to at least the bench-top (5-L – 10-L) scale and all sophorolipid products were isolated, purified and analyzed for chemical content/structure. Product yields were determined for each system and the relative costs-to-produce were compared based on feedstock costs and product yields. Rhodotorula bogoriensis produces uniquely interesting sophorolipid biosurfactants having a very long hydrophobic fatty acid component (C22-SL). Under Subobjective 2 and in collaboration with university researchers we completed the first draft of the genomic DNA sequence of R. bogoriensis to study and identify the potential genetic system responsible for C22-SL biosynthesis. Following a previous study on the effects of the concentration of glucose, phosphate, nitrogen source, and a micro-element (selenium) on C22-SL production, we now performed experiments to study the cell growth and C22-SL production of R. bogoriensis using soy molasses as the sole carbon source. In addition, we developed a means to effectively separate monoactylated and diacetylated (structural analogues) C22-SL congeners using a selective crystallization method. The ability to separate these molecules provided the materials needed to test and compare the antimicrobial behavior of both monoacetylated and diacetylated C22-SL against the acne-causing bacterium Propionibacterium acnes. Lastly, further studies involving inexpensive substrates were undertaken to produce unique polyhydroxyalkanoate biopolymers using acid-treated corn stover hydrolysate (CSH) as the sole carbon source. Previous work had shown that select bacterial strains have the ability to utilize sugars that are commonly found in cellulosic biomass such as xylose. However, the presence of phenolic compounds (typically found in lignin) and other inhibitory molecules (i.e. furfural, 5-hydroxymethylfurfural) can impair bacterial metabolism and reduce cell growth and biopolymer production. By using CSH we demonstrated that certain bacterial strains can overcome the presence of small concentrations of inhibitory compounds to produce polyhydroxyalkanoate biopolymers, and when combined with low concentrations (typically less than 0.5%) of levulinic acid (a cheap, easily-produced material through acid catalysis of both C-5 and C-6 sugars), unique block copolymers could be produced which exhibited distinct material properties. Finally, in collaborative work with other ERRC scientists, the surfactant/surface-active properties of many phenolic fatty acids were measured and their antimicrobial activities against select dental caries-causing bacterial strains were determined.
Accomplishments
1. Very-long-chain (C22) sophorolipid (vlc-SL) biosurfactants have improved antimicrobial activity than the better-known medium-chain sophorolipid (mc-SL) variants. The expanding number of antibiotic resistant bacteria has resulted in an urgent need for the discovery and development of new antimicrobial compounds that are preferably eco-friendly and functional in multiple applications. Sophorolipids (SLs) are biologically-derived molecules that can be synthesized in large yields (reportedly as high as 400 g/L) via fermentation and possess good surface-active properties making them valuable as detergent- and surface-cleaning additives. Interestingly, SLs also possess good antimicrobial activity against select bacterial strains. The most common SL-producing yeast is Starmerella bombicola which typically produces medium-chain (C16-C18) SLs. However, Rhodotorula bogoriensis is another SL-producing yeast strain that synthesizes vlc-SLs as acetylated mixtures but to date, very little is known regarding their antimicrobial activities. In order to better-understand the antimicrobial characteristics of vlc-SLs we first developed a quick and simple process to separate the monoacetylated (Ac1) and diacetylated (Ac2) structural variants of vlc-SLs using selective crystallization in organic solvent. The respective antimicrobial activity of the separate Ac1 and Ac2 variants against the acne-causing bacterium (Propionibacterium acnes) and two bacteria involved in tooth decay (Lactobacillus acidophilus and Streptococcus mutans) were then evaluated and confirmed. Results showed that both the Ac1-vlc-SL and the Ac2-vlc-SL served as more effective antimicrobial agents than mc-SL against the targeted bacteria and resulted in the discovery of new, environmentally-friendly antimicrobial biosurfactants with the potential to replace existing antimicrobial agents that are already or in the process of being phased-out. Interest in this technology has been demonstrated through the development of a Material Transfer Research Agreement (MTRA) with a commercial entity to test Ac1-vlc-SL and Ac2-vlc-SL in their proprietary formulations.
Review Publications
Ashby, R.D., Solaiman, D. 2019. Sophorolipids: Unique microbial glycolipids with vast application potential. In: Banat, I.M. and Thavasi, R.( eds.). CRS Press, Taylor & Francis Group, Boca Raton, Florida, pp.27-54.
Ashby, R.D., Solaiman, D., Strahan, G.D. 2019. The use of Azohydromonas lata DSM 1122 to produce 4-hydroxyvalerate-containing polyhydroxyalkanoate terpolymers, and unique polymer blends from mixed-cultures with Burkholderia sacchari DSM 17165. Journal of Polymers and the Environment. 27:198-209.
Ozdener, M.H., Ashby, R.D., Jyotaki, M., Elkaddi, N., Spielman, A.I., Bachmanov, A.A., Solaiman, D. 2019. Sophorolipid biosurfactants activate T1R3-mediated taste responses and block responses to bitter taste in vitro and in vivo. Journal of Surfactants and Detergents. 22:441-449. https://doi.org/10.1002/jsde.12246.
Ashby, R.D., Solaiman, D., Fan, X., Olanya, O.M. 2018. Antimicrobial potential of sophorolipids for anti-acne, anti-dental caries, hide preservation and food safety applications. In: Fan, X., Ngo, H., and Wu, C. (eds). Natural and Bio-based Antimicrobials for Food Application. ACS Symposium Series. 1287:193-208. https://doi.org/10.1021/bk-2018-1287.ch010.
Ashby, R.D., Solaiman, D. 2018. Levulinic acid: A valuable platform chemical for the fermentative synthesis of poly(hydroxyalkanoate) biopolymers. In: Cheng, H.N., Gross, R.A., and Smith, P.B. (eds.). Green Polymer Chemistry: New Products, Processes, and Ap. Book Chapter. ACS Press, Washington DC, ACS Symposium Series 1310:339-354.
Roelants, S., Solaiman, D., Ashby, R.D., Lodens, S., Van Renterghem, L., Soetaert, W. 2019. Production and applications of sophorolipids. In: Hayes, D.G., Solaiman, D., and Ashby, R.D. (eds.). Biobased Surfactants: Synthesis, Properties, and Applications (Second Edition).. Book Chapter. AOCS Press and Elsevier, London, UK:65-119.
