Research Project: Commercial Products from Microbial Lipids

Location: Sustainable Biofuels and Co-products Research

2020 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
This is the final report for the Project 8072-41000-099-00D. The new NP306 approved project entitled “Commercial Products from Lipids and Fibers” has been reviewed and is currently being established. FY2020 (year 5): Under Subobjective 1A, efforts continued to assess the efficient utilization of recombinant Pseudomonas chlororaphis to produce rhamnolipids (RLs) from soy molasses and tofu whey. P. chlororaphis is an ARS-patented non-pathogenic bacterial strain that is capable of synthesizing both RLs and/or polyhydroxyalkanoate (PHA) biopolymers depending on the growth conditions. By genetically altering P. chlororaphis to express alpha-galactosidase activity, the economic potential of RL biosynthesis from P. chlororaphis was improved by utilizing inexpensive soy molasses and tofu whey. Throughout the past year, technical improvements were made in the synthetic process by utilizing a one factor at a time approach using the recombinant strain of P. chlororaphis. Using a two-stage method, it was determined that in the first stage the alpha-galactosidase activity could significantly reduce the amount of sucrose (disaccharide), raffinose and stachyose (two prevalent oligosaccharides) in the parental substrates by converting these into monosaccharides; then, in the second stage these monosaccharides could be metabolized for cellular growth and RL synthesis. Under Subobjective 1B, the technical aspects of scale-up and sophorolipid (SL) biosynthesis from Rhodotorula bogoriensis grown on soy molasses were determined through one factor at a time optimization protocols. R. bogoriensis produces a unique SL biosurfactant containing a hydrophobic fatty acid component that is 22 carbons in length. This molecule has been previously demonstrated in this project to possess antimicrobial activity against both Gram+ and Gram- bacteria. As with RLs, by utilizing soy molasses as the carbon source, it is anticipated that the economics of SL production may be improved. A crude economic comparison was performed between the SLs derived from soy molasses and other more conventional sugar molecules in terms of comparative feedstock costs and product yields. In addition, in collaboration with an industrial partner, further technical work was performed on the synthesis of SLs from other, less-conventional yeast strains at the shake flask scale. These research efforts were performed using additional cheap feedstocks such as centrate. Evaluation of product yields, structures and properties were performed. Under Subobjective 2 and in collaboration with ARS and university researchers we organized a collaborative effort to identify the gene(s) associated with SL biosynthesis in R. bogoriensis from the gene sequencing data obtained previously. In addition, efforts focused on the use of sugar-substitutes for SL and RL production have been concluded as well as the studies on the antimicrobial effects of SLs and RLs towards food pathogens such as Listeria monocytogenes, Escherichia coli, and various strains of Salmonella. In complementary work, collaborative work involving other ARS scientists focusing on the use of lignocellulosic biomass for the bacterial synthesis of PHA biopolymers has been concluded. The ongoing worldwide plastic pollution problem is generating increased focus on recycling, limited use, and plastic substitute methodologies. Polyhydroxyalkanoates (PHA) are bacterially derived natural polymeric materials whose physical characteristics can be controlled at the production level but which, in contrast to petroleum-based plastics, also demonstrate biorenewable, biodegradable, and biocompatible properties. Unfortunately, these environmentally benign polymers are normally much more costly to produce through fermentation than the currently used plastic materials. We successfully developed fermentation protocols designed to effectively utilize low-cost, high-volume plant biomass (i.e., corn stover) as a feedstock for PHA biosynthesis. By using known acid-hydrolysis procedures, the biomass was converted to monomeric sugar molecules that could be effectively metabolized by different bacterial strains. Then, by combining the processed plant biomass with small concentrations of levulinic acid, an inexpensive organic acid that can also be easily produced from plant biomass, PHA biopolymers were produced with favorable properties that could serve as plastic substitutes in many applications. The results of this study provide new possibilities for the cost-effective production of eco-friendly PHA biopolymers that can function as substitutes for the large-volume plastics in widespread use today. Previous work had shown that select bacterial strains can utilize sugars that are commonly found in cellulosic biomass. However, in order to improve PHA biosynthesis from additional bacterial strains, the inhibitory components (i.e. furfural, 5-hydroxymethylfurfural, organic acids etc.) of the acid-treated CSH were effectively removed/neutralized by a process known as overliming which broadened the array of bacteria that could use the CSH. This work lead to the synthesis of both poly(3-hydroxybutyrate-block-3-hydroxyvalerate) block copolymers and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxyvalerate) random terpolymers with tunable physical properties. Finally, in collaborative work with other ERRC scientists, phenolic fatty acid-based polymers were synthesized that exhibited antimicrobial properties towards both Gram+ and Gram- food pathogens. Life of the Project: Project 8072-41000-099-00D was primarily focused on improving the economics of the fermentative synthesis of select glycolipid biosurfactants and demonstrating new uses for these biobased products. These research goals were accomplished throughout the duration of the project. Sophorolipids (SLs) and rhamnolipids (RLs) are biobased surfactant molecules that are biodegradable and biorenewable. As with all surfactants SLs and RLs contain both a hydrophobic (water-hating; typically a hydroxy fatty acid) and hydrophilic (water-loving; typically a sugar) component which induce their accumulation at interfaces (i.e., oil:water, air:water) and produces surface tension variations. They are typically associated with detergent applications. Throughout this project, SLs and RLs were evaluated to improving production economics and for unique applications. RLs are naturally produced by members of the Pseudomonas family with P. aeruginosa being the most well-known source. Unfortunately, P. aeruginosa is a known opportunistic human pathogen which hinders the utilization of the RLs produced from that strain in certain applications. Previous work in our lab identified a non-pathogenic strain of Pseudomonas (P. chlororaphis) that produces RLs. Initially, this strain was identified as a RL producer using static growth conditions; however, genetic engineering conferred the ability to use agitation to improve RL yields. To help improve the economics of the RL production process using P. chlororaphis, alpha-galactosidase enzyme activity was conferred to the bacterium using genetic engineering protocols which allowed the enzymatic hydrolysis of the disaccharides (sucrose) and the oligosaccharides (raffinose, stachyose) in soy molasses and tofu whey into monomeric sugar which could be utilized for RL production. This discovery lead to the issuance of a patent on the developed technology. In addition, by transforming the glpF, glpK, and glpD genes from E. coli into P. chlororaphis, it was established that P. chlororaphis could better utilize glycerol (a large-volume coproduct from the transesterification reactions involved in biodiesel production) for RL biosynthesis. The ability to produce RLs from cheap, abundant substrates helps to enhance the production economics for these valuable biobased surfactants using a nonpathogenic bacterial strain. Most of the work performed within the project framework focused on SLs. SLs are well-known to be produced in high yields so the emphasis of the project plan with respect to SLs was in the discovery of new applications. Three primary application areas were explored including taste-sensory, antimicrobial, and plasticizer / lubrication. In what was perhaps the most interesting discovery pertaining to new applications of SLs, we developed an industrial collaboration to assess the taste-sensory properties of SLs. Interestingly, SLs block the bitter-taste response in mammals. This discovery lead to the issuance of a patent and opened additional possibilities for SL applications. With the continued concern about the overuse of antibiotics and the consequent bacterial resistance additional efforts focused on the application of SLs as antimicrobial agents. In collaboration with University, industrial and ARS scientists research was conducted on the antimicrobial effectiveness of SLs towards bacterial strains commonly associated with foodborne illness, dental caries, acne formation and hide degradation. In each case the efficacy of SLs towards these targeted bacterial strains was demonstrated. These results in combination with the taste-sensory qualities lead to the idea that SLs may have application potential in oral hygiene applications. In addition, a unique SL was produced using the yeast strain Rhodotorula bogoriensis that contained a 22-carbon hydroxy fatty acid which was also had antimicrobial character. In addition, genome sequencing was performed in collaboration with a university on the R. bogoriensis yeast and a method was developed to separate monoactylated, diacetylated and nonacetylated C22 SLs using organic solvents. This allowed the separate antimicrobial assessment among the three structures. In addition to the work performed on biobased surfactants, research was conducted to reduce the production economics of polyhydroxyalkanoate (PHA) biopolymers through the successful utilization of lignocellulosic feedstocks.

Accomplishments

Review Publications
Hazer, B., Ayyildiz, E., Eren, M., Canbay, H., Ashby, R.D. 2019. Autoxidized oleic acid bifunctional macro peroxide initiators for free radical and condensation polymerization. Synthesis and characterization of multiblock copolymers. Journal of Polymers and the Environment. 27(11):2562-2576. https://doi.org/10.1007/s10924-019-01536-6.
Solaiman, D., Ashby, R.D., Crocker, N.V. 2020. Bioprocess for hydrolysis of galacto-oligosaccharides in soy molasses and tofu whey by recombinant pseudomonas chlororaphis. Biocatalysis and Agricultural Biotechnology. 24:101529. https://doi.org/10.1016/j.bcab.2020.101529.
Solaiman, D., Ashby, R.D., Nunez, A., Cross, N.V. 2020. Low-temperature crystallization for separating monoacetylated long-chain sophorolipids: characterization of their surface-active and antimicrobial properties. Journal of Surfactants and Detergents. 23:553-563. https://doi.org/10.1002/jsde.12396.
Solaiman, D., Ashby, R.D., Biresaw, G. 2019. Microbial lipids for potential tribological applications – An overview. In: Biresaw G, and Mittal Kl, editors. Boca Raton, FL: Surfactants in Tribology, Volume 6. CRC Press. p. 57-72.