1a. Objectives (from AD-416):
The long term goal of the project is to develop commercially viable integrative bioprocesses encompassing biological and bio/chemical technologies to produce environmentally friendly biobased materials and chemicals using agricultural fats (i.e., tallow, lard, chicken fats, etc.), oils (i.e., soybean oil, cuphea oil, sunflower oil, etc.) and coproducts as feedstocks. 1: Develop biocatalysts and bioprocesses that enable the commercial production of new biobased products from agricultural lipids and related coproducts such as bioglycerol. 1A: Improve the efficiency of feedstocks utilization and enable the production of new varieties of bioproducts in high yields through metabolic engineering of microorganisms. 1B: Systematic optimization of bioprocesses to improve yields and economics of biobased products. 2: Develop new commercially viable applications for sophorolipids, rhamnolipids, cyanophycin and gamma-polyglutamic acid. 2A: Increase the values of product components. 2B: Develop biomaterials for end-use products.
1b. Approach (from AD-416):
Microorganisms capable of producing new variants of rhamnolipids (RLs) and sophorolipids (SLs) will be created by introducing mutated genes that originally encode the synthesis of native RL and SL. Increasing the RL yields in non-pathogenic P. chlororaphis by metabolic engineering will be attempted by increasing the copy-number and transcription level of the responsible genes (i.e., rhlAB), or by ‘knocking out’ the gene(s) responsible for diverting precursors away from RL biosynthesis. We will also enhance the value of P. chlororaphis by introducing another gene, rhlC, to allow the biosynthesis of dirhamnose lipids. The efficiency of RL- and SL-producing microorganisms to utilize soy molasses (SM) will be improved by introducing genes encoding enzymes that breakdown the complex sugars in SM. We will also express in Candida bombicola (an SL-producing yeast) and P. chlororaphis the genes that are used for glycerol metabolism to improve the organisms’ efficiency of using bioglycerol. To enhance colonization and biofilm formation for higher RL yields from P. chlororaphis, we will experiment with attaching a sponge-like adapter to the impellers of a fermenter and then use minimal aeration (slow impeller rotation, reduced air introduction, or both). Separately, process optimization will be carried out for the fermentative synthesis of SL, gamma-polyglutamic acid (PGA) and cyanophycin (Cp) using such low-cost feedstocks as crude glycerine from biodiesel production, soy molasses and meat and bone meal hydrolysates. Fermentation parameters such as carbon source content and concentration, feed rate, aeration, pH etc. will be varied and optimized using Response Surface Methodology (RSM). To increase the values of bio-product subcomponents or for application in end-use products, biological and chemical syntheses will be performed to obtain the followings for subsequent characterization: 1) novel epoxy and polyhydroxy fatty acids by using functionalized fatty acids (i.e., vernolic acid, ricinoleic acid, lesquerellic acid) as fermentative substrates, 2) cyanophycin-derived biosurfactants by linking fatty acids to the polar dipeptide unit of Cp, 3) terminally unsaturated fatty acids by elimination reactions on hydroxy fatty acids, 4) biopolymer/biosurfactants composites by solution-casting of PHA and SL or RL, 5) oligoamides by converting the hydroxy fatty acids to amino fatty acids and self-coupling them, and 6) water-soluble SLs by linking charged amino acids to the sugar headgroups.
3. Progress Report:
To meet the objectives of developing commercially viable enzymes, bioprocesses, and biobased products through genetic engineering, fermentation optimization, and chemical modification, we tested fermentation conditions to produce glucose lipids and phytosterol glucosides using a recombinant yeast we previously constructed; evaluated various culture conditions to produce large amount of rhamnolipids using a recombinant non-pathogenic pseudomonad we previously constructed; investigated methods to overcome cloning roadblock encountered in the continuing effort to isolate a new glycosyltransferase from a unique sophorolipid-producing yeast; completed sugar-composition analysis of tofu whey in preparation for testing as cheap culture medium to make biochemicals; attached a highly reactive adapter to fatty acids to link to various chemicals to make surfactants, polymer building blocks, and antioxidant for lubricant; established collaborations to investigate the use of sophorolipids and rhamnolipids as antimicrobial biosurfactants in food-safety and hides/leather research; investigated various conditions of stirred-tank fermentation at 10-liter scale to increase production yields of rhamnolipids; and successfully synthesized and tested an unsaturated estolide and an epoxidized estolide as plasticizers.
1. Manufacture cheaper biobased detergents. Soaps and detergents industry is a multibillion-dollar business supplying products made using non-renewable petrochemicals and caustic and environmentally non-friendly methods. Microbial-produced biobased detergents can address these issues. However, they are comparatively more expensive. ARS researchers at Wyndmoor, Pennsylvania developed a flexible process economic model for approximating the production costs of the fermentative synthesis of sophorolipids – an emerging biobased detergent. The model showed that the starting-material cost is the overwhelming contributor to the total operating cost for sophorolipid production. The model also allows cost calculation for making sophorolipids using various different types of renewable feedstocks. A manuscript was in press, and is the subject of a special article in Inform magazine. The accomplishment is expected to provide an important guideline for the microbial-produced bioproducts industry to improve cost effectiveness.
2. Control-release of antimicrobial biosurfactant by varying the type of biopolymer films. Many biosurfactants have good antimicrobial activities allowing for their value-added application as antimicrobial agents in addition to traditional usage. To date many studies concentrate on assessing their antimicrobial activities in solutions. ARS researchers at Wyndmoor, Pennsylvania have investigated various types of biopolymers as carrier or scaffold for controlled release of the antimicrobial biosurfactants using sophorolipid as an example. The results showed that the relatively more water-liking (hydrophilic) biopolymers are better carriers or scaffold for the antimicrobial sophorolipid than the more water-hating (hydrophobic) ones. The finding can be used to make packaging films and tissue scaffold that can prevent bacterial and fungal growth. A great impact is expected in market expansion of the emerging biosurfactants into active food packaging and tissue regeneration areas.
3. Synthesis of highly modifiable functionalized fatty acids. While a number of technologically important materials are made from agriculture-sourced fatty acids, the chemical options for altering these starting materials are limited. New methods to introduce extra chemical functionality are therefore desirable to expand the possibilities for utilizing fats and oils. ARS researchers at Wyndmoor, Pennsylvania have attached a “springboard” group to one end of fatty acids. What this new unit does is permit modification with any of a wide number of chemical functionalities, such as naturally occurring amino acids. A whole new gallery of bio-based structures is therefore accessible. These new hybrid compounds should find use as building blocks for lubricants, surfactants, and polymers, as well as possibly in medicinal chemistry applications.
Xue, C., Solaiman, D., Ashby, R.D., Zerkowski, J.A., Lee, J., Lee, K. 2013. Study of structured lipid-based oil-in-water emulsion prepared with sophorolipid and its oxidative stability. Journal of the American Oil Chemists' Society. 90:123-132.