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United States Department of Agriculture

Agricultural Research Service

2009 Annual Report

1a.Objectives (from AD-416)
The overall objective is to develop fermentation-based bioprocess systems that utilize the renewable agricultural fats, oils and coproducts (AFOC) as feedstocks to ultimately produce value-added bioproducts with enhanced properties and environmental benefits. Specific objectives are to expand the list of bioproducts producible via the fermentation of AFOC, to increase yields, expand variety, and improve properties of the target bioproducts via strain improvement, fermentation manipulation, and post-production modification; and to develop end-product applications for the bioproducts.

1b.Approach (from AD-416)
The capability of various AFOC to support cell growth of microorganisms that produce the target bioproducts -- rhamnolipids (RL), sophorolipids (SL), poly(hydroxyalkanoates) (PHA), gamma-polyglutamic acid and cyanophycin -- will be investigated. Genes needed for efficient production of bioproducts from AFOC will be identified, cloned, characterized and expressed. Chimeric genes and mutants having novel substrate specificity will be generated from PHA synthase genes with different PHA compositional profiles. Genes of enzymes or regulatory proteins involved in the biosynthesis of SL in Candida and RL in Pseudomonas will be cloned and characterized for subsequent protein and metabolic engineering to improve product variety and yield. Fed-batch and continuous culture techniques will be explored to increase the yields of bioproducts from fermentation of AFOC. Sophorolipids will be used to prepare new materials such as gemini surfactants, polymers of SL, value-added fatty acids, and bolaamphiphiles. Reactive functional groups, especially amino groups, will be incorporated to the sidechains of PHA for altered property and subsequent derivatization.

3.Progress Report
Achieved maximum poly-3-hydroxybutyrate (PHB) product yields (greater than 1 g/L) and maximum glycerol-substrate utilization (greater than 80%) to improve production economics through the development of a fermentation protocol where media salts were replenished in the fermentation process at the 10-L bench-top scale; studied the use of the glycerol-based PHB polymers as composite materials with oleic acid-based sophorolipids (SLs) to produce solution cast films and determined the effects of SL concentration on the material properties of the composites as well as testing these composites as delivery vehicles for SLs in antimicrobial (anti-acne) applications; investigated new PHA/SL films and nanoparticles for antimicrobial packaging and other uses; concluded the work on synthesis of methyl-branched medium-chain-length (mcl-) PHA polymers by exhaustive NMR and GC/MS analysis to conclusively show a new class of mcl-PHA polymers; developed new expression vectors capable of replicating in three host organisms for ease of experimental manipulation and for production of biopolymers and biosurfactants under different requirements; constructed and tested fusion genes of polyhydroxyalkanote biosynthesis to improve production efficiency; demonstrated the use of hydrolysates of rendered proteins for fermentative production of cyanophycin biopolymer; constructed and tested recombinant microbes harboring heterologous alpha-galactosidase genes for improved utilization of soy molasses in fermentation processes; and genetically engineered a rhamnolipid-producing nonpathogenic bacterium for improved production of the biosurfactant.

1. Produced New Nanoscale Glycerol-Based Polymers for Cellular Delivery of Bioactive Molecules: Currently glycerol, an abundant byproduct of biodiesel formation, can be fashioned into polyesters with dicarboxylic acids to form compounds having a highly branched structure and many crevices, thus allowing them to serve as delivery agents for the controlled release of therapeutic and other bioactive molecules. The problem is that the preparation of these compounds is done in a way that does not allow for the incorporation of other chemical groups that can fine-tune their properties and usefulness. To solve this problem, scientists at Eastern Regional Research Center, Wyndmoor Pa, have developed methods that permit the construction of highly branched glycerol polyesters in a stepwise way so that a variety of chemically reactive groups useful for carrying a wide-range of biologically active molecules can be selectively introduced or incorporated. The ultimate impact of the technology is an increased demand for agricultural products as a result of an improved economics for their utilization or processing brought about by the added value to glycerol and fatty acids derived from glycolipids that are produced by fermentation of vegetable oils.

6.Technology Transfer

Number of Active CRADAs1
Number of the New/Active MTAs (providing only)1
Number of Invention Disclosures Submitted1

Review Publications
Ashby, R.D., Wyatt, V.T., Foglia, T., Solaiman, D. 2008. Industrial Products from Biodiesel Glycerol. In: Hou, C.T., Shaw, J-F., editors. Biocatalysis and Bioenergy. Hoboken, NJ:John Wiley & Sons, Inc. p. 131-154.

Ashby, R.D., Solaiman, D.K.Y. 2008. Poly(hydroxyalkanoate) Biosynthesis from Crude Alaskan Pollock (Theragra chalcogramma) Oil. Polymers and the Environment. 16:221-229.

Stevens, E., Ashby, R.D., Solaiman, D. 2009. Gelatin Plasticized with a Biodiesel Coproduct Stream. Journal of Biobased Materials and Bioenergy. 3(1):57-61.

Ashby, R.D., Solaiman, D., Zerkowski, J.A. 2009. Production and Modification of Sophorolipids from Agricultural Feedstocks. In Hayes, D.G., Kitamoto, D., Solaiman, D.K.Y. and Ashby, R.D. (eds.) Bio-Based Surfactants and Detergents: Synthesis, Properties and Applications. Urbana, IL:AOCS Press. p. 29-49.

Singh, M., Milano, J., Stevens, E.S., Ashby, R.D., Solaiman, D. 2009. Gelatin films plasticized with a simulated biodiesel coproduct stream. eExpress Polymer Letters. 3(4):201-206.

Sujatha, K., Mahalakshmi, A., Solaiman, D., Shenbagarathai, R. 2009. Sequence analysis, structure prediction and functional validation of phaC1/phaC2 genes of Pseudomonas sp. LDC-25 and its importance in polyhydroxyalkanoate accumulation. Journal of Biomolecular Structure and Dynamics. 26(6):771-780.

Aneja, K.K., Ashby, R.D., Solaiman, D. 2009. Altered Composition of Ralstonia eutropha Poly(hydroxyalkanoate) through Expression of PHA Synthase from Allochromatium vinosum ATCC 35206. Biotechnology Letters. 31:1601-1612.

Last Modified: 4/18/2014
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