Location: Sustainable Biofuels and Co-products Research
2016 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
Glycerol byproduct stream from biodiesel production is an attractive low-cost renewable feedstock for production of microbial lipids. To improve glycerol utilization in fermentative production of microbial bioproducts, we had PCR-cloned the E. coli glycerol-utilization genes (glpF, glpK, and glpD), introduced them into Pseudomonas chlororaphis using plasmid vectors, and showed that P. chlororaphis containing the cloned glpF and glpK genes together (but not individually) can adapt quicker than the wild-type in glycerol medium. We had separately performed real-time reverse-transcription (RT-) quantitative PCR (qPCR) study on genetically engineered P. chlororaphis containing glpD gene (alone or in combination with F and K) to show that the cloned heterologous gene was actively expressed. Further fermentation study is ongoing to optimize the bioprocess for utilization of crude glycerol plus cellulosic hydrolysate by these genetically engineered strains to produce microbial bioproducts.
Sophorolipids (SLs) contain uncommon hydroxy fatty acids that are potentially valuable to the cosmetics and lubricants industries. To obtain these fatty acids, we have established basic reaction conditions to carry out acid- and base-catalyzed hydrolysis of SL produced by Rhodotorula bogoriensis in order to isolate the uncommon 13-hydroxydocosanoic acid fatty acid (13-OH-C22 FA). We had also initiated the testing of enzymes to hydrolyze SL from Candida bombicola to obtain 17-hydroxy oleic acid while retaining the wholeness of the sugar moiety. The results of these initial studies provide the basis for the ensuing RSM study to optimize the processes to harvest the value-added components of SLs.
Our collaborative effort with ARS-National Center for Agricultural Utilization research scientists has resulted in the ability to produce short-chain microbial polyesters (poly-hydroxyalkanoates: PHA) from corn stover hydrolysate (CSH). Ligno-cellulosic feedstocks are plentiful in nature and easily hydrolyzed into their respective monosaccharide components. Success in this research area will result in the potential decrease in production cost for short-chain PHA biopolymers through the use of an overabundant cheap feedstock which could lead to an even more expanded interest in short-chain PHA. We have discovered 2 different bacterial strains that have the ability to utilize CSH as a feedstock for PHA biosynthesis. Azohydromonas lata (formerly Alcaligenes latus) has the ability to utilize only the glucose portion of the CSH sugars to produce high yields of poly-3-hydroxybutyrate (PHB), while Burkholderia sacchari has the capacity to utilize all of the available sugars (both C-6 and C-5) sugars to achieve the same results. Having 2 separate bacterial strains that can produce short-chain PHA from the different sugars associated with CSH opens up a large production mechanism to utilize mixed culture fermentations to increase biopolymer yields. In addition, it has been determined that the addition of propionic acid or levulinic acid into the production media induces the synthesis of copolymers from these strains. Under the appropriate growth conditions, both bacterial strains can produce a copolyester composed of 3-hydroxybutyric acid (3-HB) and 3-hydroxyvaleric acid (3-HV) with varying monomeric ratios (this is not unique). However, using levulinic acid as a co-substrate with A. lata results in the formation of a terpolyester composed of 3-HB, 3-HV, and 4-hydroxyvaleric acid (4-HV) which further expands the production potential for mixed cultures of these two strains.
SLs have antimicrobial activity against numerous potentially harmful bacteria. We have focused on using these molecules as bacteriostatic and/or bacteriocidal agents against many clinical, food-pathogenic, and environmental microorganisms. SLs from Candida bombicola are synthesized with either C-16 or C-18 fatty acid side-chains. In collaboration with ARS and international scientists, we have determined that these SL molecules can be used to inhibit the growth of common bacterial strains associated with foodborne illness as well as strains associated with hide degradation in the leather industry. Our C-22 sophorolipids that are produced via fermentation using Rhodotorula bogoriensis have also been successfully tested against potentially harmful bacterial strains. One of the short-term goals of this research is to produce enough sophorolipid via ozonolysis that we can test these structural derivatives on potentially harmful bacteria. To date, we have been able to produce C-9 SLs along with some dihydroxy, epoxy, and hydroperoxy derivatives. We are currently working on controlling the reaction to the point of producing enough C-9 material to test its antimicrobial properties.
Lastly, we have initiated the study of sophorolipid optimization through the use of Response Surface Methodology (RSM) from Rhodotorula bogoriensis by cultivating the yeast strain under varying culture conditions and measuring sophorolipid yields at different time points through each fermentation.
Accomplishments
1. Novel sweet-tasting and bitter-masking property of biosurfactants. Sophorolipids (SLs) are biosurfactants produced by yeast and have been used in niche-market commercial products for dish washing and surface cleaning. There is a need to make SLs commercially more attractive and valuable by identifying any new value-added property associated with it. ARS researchers in Wyndmoor, Pennsylvania and others in the past had shown the valuable antimicrobial activity of SLs against many bacteria important to various industries. Recently in collaboration with an industrial partner, we have initiated new research to determine the taste-sensory properties of SLs. The results led to the novel discovery that SLs are an excellent stimulant to the receptors of the cultured sweet-taste cells (from human tongue) and can thus be used as a sweetener and/or bitter-masking agent. We have filed a provisional patent application on this novel discovery, and expect it to have a great impact for expanding the use of SLs into oral-hygienic cares and foods industries in which SLs’ surfactant/emulsifier, antimicrobial, and taste-sensory properties could all be simultaneously and fully employed.
None.
Review Publications
Ashby, R.D., Solaiman, D., Liu, C., Strahan, G.D., Latona, N.P. 2015. Sophorolipid-derived unsaturated and epoxy fatty acid estolides as plasticizers for poly(3-hydroxybutyrate). Journal of the American Oil Chemists' Society. 93:347-358. doi: 10.1007/s11746-015-2772-7.
Solaiman, D., Ashby, R.D., Crocker, N.V. 2016. Genetic construction of recombinant Pseudomonas chlororaphis for improved glycerol utilization. Biocatalysis and Agricultural Biotechnology. doi: 10.1016/j.bcab.2016.08.011.
