SBIR-STTR Award

Natural flavor compounds find wide applications in the food, feed, cosmetic, chemical and pharmaceutical industry, and isoamyl acetate is an important flavor ester. In this research program, a low-cost process is proposed to convert glucose from renewable sources to isoamyl acetate using a high-yielding metabolically engineered E coli. Isoamyl acetate produced via chemical synthesis results in an undesirable racemic mixture. The fermentation route followed in this program will result in high yields of "natural" isoamyl acetate with additional usage in food industry along with other uses The proposed research will lay the foundation for isoamyl acetate biosynthesis at high yield from renewable sugars. Successful outcome of the project will provide a platform for the low-cost production of additional high-valued natural esters. OBJECTIVES: The first objective of this research will construct and characterize an improved metabolically engineered E.coli for high isoamyl acetate yields by overexpressing a more efficient alcohol transferase gene, ATF1 from yeast. The second objective is to develop high-cell-density fermentation technology that will utilize a low-cost source of glucose derived from corn and grain sorghum and other inexpensive nutrient sources. The third objective is to identify an effective separation process to recover the volatile isoamyl acetate APPROACH: The proposed research focuses on developing a process to produce high yields of isoamyl acetate using a proven and then an improved, genetically-stable metabolically engineered E coli. Renewable, inexpensive glucose derived from corn and grain sorghum will be used as fermentation feedstock. Operational parameters for efficient aerobic and anaerobic fermentation using both engineered E coli strains will be optimized. Fed-batch mode with cell mass recycle to obtain high-cell-density fermentation conditions will be developed. An efficient separation strategy to economically separate isoamyl acetate from the final fermentation broth will be determined and preliminary economic analysis will be performed. The proposed research methodology will result in a low-cost fermentation process for producing isoamyl acetate. PROGRESS: 2004/05 TO 2005/06 A new engineered E.coli production strain for improved isoamyl acetate by overexpressing a more efficient alcohol tranferase gene ATF1 in pBAD-TOPO plasmid has been developed. The improved metabolically engineered strain, E.coli YBS121, has been characterized and evaluated at the shake flask and at the 2 L fermenter level. Shake flask experiments were performed to evaluate the different media components; inexpensive fusel oil, sorghum glucose and CSL were found to effectively replace synthetic isoamyl alcohol, glucose and yeast extract, respectively. Batch fermentation experiments at 2 L fermenter level with different isoamyl alcohol addition patterns were performed and the conditions optimized. The target yield was successfully attained with fusel oil addition, and high-cell-density fermentation for increased isoamyl acetate yield was demonstrated. IMPACT: 2004/05 TO 2005/06 In this research program, a low-cost bio-based fermentation process to produce high yield of isoamyl acetate using engineered E coli and renewable sugars has been determined. Increased yield of 'natural'isoamyl acetate will lead to better production economics and wider application in food, feed, chemical and pharmaceutical industry. PUBLICATIONS: 2004/05 TO 2005/06 No publications reported this period PROGRESS: 2003/10/01 TO 2004/09/30 Good progress has been made in the Phase I project in the development of the improved engineered E.coli strain and in the experimental procedures to produce isoamyl acetate. A new engineered E.coli production strain for improved isoamyl acetate by overexpressing a more efficient alcohol tranferase gene ATF1 in pBAD-TOPO plasmid has been developed and characterized at the shake flask level. Issues relating to plasmid stability are being addressed. Experiments using glucose derived from corn and sorghum for isoamyl acetate production have been undertaken. Operational parameters for process optimization work of isoamyl acetate production under fed-batch and efficient dual-phase fermentation conditions will be determined in a 2 L fermenter using the improved E.coli YBS 121. An effective product recovery strategy will be identified and economic analysis of Phase I work will be executed. IMPACT: 2003/10/01 TO 2004/09/30 Production of a natural ester, such as isoamyl acetate, from renewable sugars and engineered E.coli will lead to a low-cost biobased process. The feasibility of producing high yields of isoamyl acetate will provide the framework for the bulk production of this high-valued specialty chemical

Agricultural mulch films are used for the production of vegetables and fruits. Thin plastic films (typically polyethylene) are spread along the rows of plants at the beginning of the growing season. Holes are made in the films to plant the seeds or seedlings. The use of mulch films speeds the ripening of crops, conserves moisture and fertilizer, and inhibits weed growth, fungus infection and insect infestation. At the end of the growing season (generally 3-5 months) the film, contaminated with dirt and vegetation, must be collected, transported and disposed. As the use of mulching films becomes more common, the disposal of the polyethylene film becomes a significant waste problem. The Phase I R&D project determined that a degradable agricultural mulch film from a poly(lactic acid)/starch blend could be produced and mechanical and exterior exposure field tests were conducted. The Phase II research program will focus on moving the polymer blending process to full-scale manufacturing equipment and procedures, optimizing the film manufacturing parameters and conduct large scale product field testing. Film product will be produced on a full-scale manufacturing line and the film tested for initial and aged mechanical properties, soil plot field application and exterior exposure performance. The development of a truly degradable mulching film will reduce labor and disposal costs for agricultural producers. The PLA/starch polymeric blend utilizes 99% biobased materials and its use will displace petroleum derived polyethylene mulch films. OBJECTIVES: A Poly(lactic acid)/starch polymeric blend has been developed which allows the replacement of PLA with starch while maintaining key mechanical properties. The Phase I project optimized the PLA/starch formulation and demonstrated that a thin plastic film could be processed on small scale film manufacturing equipment. The produced film was tested for mechanical and exterior exposure properties for use as an agricultural mulching film. The objective of the Phase II research project is to begin to scale up the polymeric blending process to production style and sized compounding equipment. PLA/starch formulation optimization work will continue to increase film strength, flexible and exterior performance properties. The next objective will be to produce a thin film product on a full-size film manufacturing processing line. The film produced from this objective will be applied to full-size soil test plots by standard mulch film application equipment and methods. The mulch films on a soil test plots will be monitored for mulching properties during typical growing conditions. APPROACH: The Poly(lactic acid)/starch blend has been developed which uses low levels of a crosslinking agent to maintain the polymeric blend's mechanical properties as the level of starch is increased. The major advantage of this approach is to reduce the blend's raw material cost well below that of using 100% PLA, which will allow the film product to be cost competitive with currently used polyethylene films. Plasticizers will be used to balance the film's strength and flexibility to provide the required mechanical properties for optimal film forming and usage as an agricultural mulch film. The polymeric blend will be compounded on a full-scale production compounding extruder, which is intended to alleviate the compounding difficulties encountered in the Phase I program. These blends will be processed on small-scale film production equipment to optimize the processing parameters and to determine the film's mechanical and physical properties. Full-scale film manufacturing will be conducted that will allow full-scale product and field tests. Initial film mechanical properties will be tested for each film sample and evaluated against standard polyethylene mulch films. Soil plot, film usage tests will be conducted to determine the film sample's performance and degradability characteristics