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United States Department of Agriculture

Agricultural Research Service


Location: Biobased and Other Animal Co-products Research

2013 Annual Report

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.

4. Accomplishments

Review Publications
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.

Ashby, R.D., Mcaloon, A.J., Solaiman, D., Yee, W.C., Reed, M.L. 2013. A process model for approximating the production costs of the fermentative synthesis of sophorolipids. Journal of Surfactants and Detergents. 16:683-691.

Last Modified: 10/20/2017
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