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United States Department of Agriculture

Agricultural Research Service

2007 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
Project scientists demonstrated the use of soy molasses (SM) as a nitrogen source in industrial fermentation; acquired and subcloned an alpha-galactosidase gene to promote SM utilization in poly(hydroxyalkanoate) (PHA)-producing organisms; cloned, sequenced and heterologously transferred rhamnolipid (RL) biosynthetic genes from a non-pathogenic bacterium (X); experimented with varying fermentor conditions to achieve a high RL production by bacterium X; introduced a new putative SL-biosynthetic gene into a yeast strain for production and testing of the gene product; screened a collaborator’s collection of Pseudomonas syringae promoters for use in gene expression; started developing a gene transfer system for Candida bombicola based on a collaborator’s vector; co-developed SL-embedded biofilms that inhibit acne bacterial growth; synthesized new derivatives of RLs with solubilizing units attached; selectively derivatized one of the unsaturated units of polyunsaturated hydroxy fatty acids produced by fermentation and readily separated the isomers: invented a way to make SLs more water-soluble without disrupting the SLs’ molecular architecture (lactone ring).

This paragraph serves to document research conducted under a reimbursable agreement #60-1935-3-0042 between ARS and CSREES-NRI (cf. 1935-41000-067-01R). Project researchers cloned genes coding for the PHA synthase (PHAS) of a photosynthetic bacterium; subcloned these genes and fragments for high expression of the proteins for crystallization study; transferred these genes into a soil bacterium to produce a biopolymer having short- and medium-chain-length monomers.

This paragraph serves to document research conducted under a reimbursable agreement #58-1935-4-0412 between ARS and University of Georgia (cf. 1935-41000-067-02S). Crystallographic work was hampered by problems obtaining highly pure proteins. Selenium-methionine-labeled PHAS isolated from E. coli failed to crystallize due to contaminants. Crystallization also failed with methylated PHASs or a PHAS with improved solubility. The ADODR visited the cooperator (fall of 2006) and conducted periodic conference calls to discuss research progress.

This paragraph serves to document research conducted under Cooperative Research and Development Agreement #58-3K95-5-1119 between ARS and an industrial partner (cf. 1935-41000-067-04T). SLs and RLs obtained from fermentation of fats, oils and their derivatives were prepared and sent to the cooperator who then evaluated their performance in the formulation of its commercial products.

This paragraph serves to document research conducted under Non-Funded Cooperative Agreement #58-1935-7-0731N between ARS and Binghamton University (cf. 1935-41000-067-05N). A surplus coproduct of biofuel production was successfully used as an inexpensive component for making biobased composite materials. A manuscript and an invention disclosure have been prepared.

Tailor-made functional fluids: Estolides are biobased short polyesters that show promise as functional fluids such as lubricants. They can be produced in good yield from fats and oils using acid catalysis methods. These methods, unfortunately, only produce a mixed product and are incompatible with many useful functional groups. Scientists at Eastern Regional Research Center, Wyndmoor, PA, have developed a new, milder method to form polyesters with building blocks obtained from a natural product (sophorolipid) made from fats and oils. "Designer" estolides with precisely known and desired structures can be tailor-made using the new method. This structural control will allow fine-tuning of the properties of these materials so that they can better be employed in areas such as coatings and thin films. (NP#306, Quality and Utilization of Agricultural Products; Component 2. New Processes, New Uses, and Value-Added Foods and Biobased Products; Problem Area 2b. New Uses for Agricultural By-products.)

From crude glycerol coproduct to biopolymer: Large amounts of crude glycerol are produced by the expanding biodiesel industry. The excess production of glycerol in turn retards the further expansion of the industry due to the need for costly purification and new glycerol outlets. Researchers at Eastern Regional Research Center, Wyndmoor, PA, showed that glycerol is useful to change the size (molecular weight or M.W.) of a water-soluble biopolymer called gamma-polyglutamic acid (gamma-PGA) produced by a bacterium. Glycerol media concentrations of approximately 8% resulted in gamma-PGA polymers with maximized molecular masses. This new use for glycerol, especially in its crude form, may provide a new outlet for this coproduct and ultimately benefit the biodiesel industry. Furthermore, the ability to tailor the molecular masses of gamma-PGA allows for specific targeting of its application, such as thickening agent in yogurt (requiring high-M.W. PGA) or yogurt-drink (low-M.W. PGA). (NP#306, Quality and Utilization of Agricultural Products; Component 2. New Processes, New Uses, and Value-Added Foods and Biobased Products; Problem Area 2b. New Uses for Agricultural By-products.)

Bioplastics from fermentation of Alaskan fish waste. The Alaskan fishing industry has been struggling with the accumulation of over 2.2 billion pounds of fish by-products per year. There is an urgent need to find new outlet for these coproducts. ARS scientists at the Eastern (Wyndmoor, PA) and the Western (Albany, CA) Regional Research Centers found that the crude fish oil derived from the Alaskan fishing industry could be used as a fermentation substrate in the production of biopolymers called polyhydroxyalkanoates (PHA). Depending on the specific organism used, PHA biopolymers were produced that exhibited either thermoplastic (meltable and formable) or elastomeric (rubberlike) characteristics. Because of their varying properties, these polymers have a potentially wide application base that could be attractive for use in industry. This research adds value to an Alaskan fish byproduct while lowering the production costs of PHA biopolymers. (NP#306, Quality and Utilization of Agricultural Products; Component 2. New Processes, New Uses, and Value-Added Foods and Biobased Products; Problem Area 2b. New Uses for Agricultural By-products.)

5.Significant Activities that Support Special Target Populations

6.Technology Transfer

Number of new CRADAs and MTAs4
Number of active CRADAs and MTAs4
Number of invention disclosures submitted1
Number of U.S. patents granted1
Number of non-peer reviewed presentations and proceedings8
Number of newspaper articles and other presentations for non-science audiences3

Review Publications
Panilaitis, B., Castro, G., Solaiman, D., Kaplan, D.L. 2007. Biosynthesis of emulsan biopolymers from agro-based feedstocks. Journal of Applied Microbiology. 102:531-537.

Solaiman, D., Ashby, R.D., Foglia, T.A., Marmer, W.N. 2006. Fermentative production of biopolymers and biosurfactants from glycerol-rich biodiesel coproduct stream and soy molasses. Hou, C.T., Shaw J-F., Editors. Biocatalysis and Biotechnology for Functional Foods and Industrial Products. Boca Raton, FL; CRC Press. p. 431-450.

Ashby, R.D., Cooke, P.H., Solaiman, D. 2007. Topographical Imaging as a Means of Monitoring Biodegradation of Poly(hydroxyalkanoate) Films. Polymers and the Environment. 15(3):179-187.

Solaiman, D., Ashby, R.D., Zerkowski, J.A., Foglia, T.A. 2007. Simplified soy molasses-based medium for reduced-cost production of sophorolipids by Candida bombicola. Biotechnology Letters. DOI 10.1007/S10529-007-9407-5.

Zerkowski, J.A., Solaiman, D. 2007. Polyhydroxy Fatty Acids Derived from Sophorolipids. Journal of the American Oil Chemists' Society. 84(5):463-471.

Last Modified: 4/21/2014
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