Intellectual Merits: This Small Business Innovation Research (SBIR) Phase II project will address further development of findings from the Phase I Project addressing development of advanced biopesticides by simple and scalable modification of sophorolipids. The yeast Candida bombicola produces sophorolipids (SLs) in volumetric yields of ~ 300 g/L. The Phase I program demonstrated that by simple chemical modification of unrefined natural SLs, a series of five highly active lead compounds were identified which possess broad spectrum activity against all three major fungal groups that cause serious diseases in commercially important plants. By amidation of the SL fatty acid carboxyl group (e.g. - NH2CH2CH2N(CH3)2), or by reduction of the SL-fatty acid double bond, derivative activity against pathogens greatly increased. Minimum inhibitory concentration (MIC) values of amide SL-derivatives were generally on a par with tested commercial fungicides. Broader Impacts/Commercial Potential: The broader impacts of this research address the market pull for green agricultural products by developing a bio-pesticide produced via an efficient microbial fermentation followed by simple chemical modification to improve the performance of nature?s molecules. The goal is to create a superior bio-pesticide product that does not harm the environment, is safe for farmers that regularly handle these materials, and to provide safe food for consumers. SyntheZyme bio-pesticides will contribute to the on-going green food-production revolution. Their introduction into the market is expected to replace an increasing fraction of synthetic chemical pesticides during season-long disease control programs. Furthermore, new safe bio-pesticides are needed to replace chemical pesticides now banned due to tightened regulations and increased concerns about their pollution and health hazards.
Due to the pollution and health hazards of many chemical pesticides, regulators have increased safety targets. For example, in the European Union, 600 of 1,000 actives have recently been delisted. Persistence of many chemical pesticides due to their chemical stability is a major problem that is overcome by biopesticides that are readily degradable in soil and water environments. Furthermore, the world population is growing and is expected to reach 9+ billion by 2050. This translates into a need for 100% more food than is currently produced. During the Phase II program, SyntheZyme developed a suite of green biopesticide agricultural products that are produced via an efficient microbial fermentation followed by simple and scalable chemical modification. This strategy allowed SyntheZyme to successfully improve the performance of nature’s molecules. Superior biopesticide products with commercially viable cost-performance were developed that have thus far proved to be not harmful to the environment and safe for farmers that regularly handle these materials. SyntheZyme biopesticides will contribute to the on-going food-production revolution. Their introduction into the market is expected to replace an increasing fraction of synthetic chemical pesticides during season-long disease control programs. Students participating in this SBIR Phase II program included undergraduates, masters and Ph.D. students. These students gained important training in green chemistry and the development of safe agricultural practices. The major accomplishments of the Phase II program are summarized below. A process was developed whereby readily renewable glucose and a vegetable oil were converted by yeast fermentation to sophorolipids. An optimized fermentation process was developed that gave commercially viable volumetric yields (260 g/L) and productivity (1.3 g/L/h). The methods used can be further up-scaled to commercial fermenters producing products at 1000 ton quantities. Furthermore, an all aqueous process was developed for the efficient isolation/purification of sophorolipids from the fermentation medium. Simple chemical methods were developed to manufacture modified sophorolipids. By combining modified sophorolipids with other safe ingredients, formulated products were developed that enhance both the concentration of modified sophorolipids that can be dissolved in product solutions as well as the antimicrobial activity of compounds. A set of modified derivatives were shown by in-vitro (using isolated organisms) assays to have commercially viable activity against commercially important plant pathogenic fungal strains such as A. tomatophilia, A. solani and Botrytis cinerea, respectively. Plant pathogenic activities up to 50 times better than competitive products were demonstrated. In addition to plant fungal pathogens, in vitro studies also showed that SyntheZyme’s modified sophorolipid products have valuable inhibitory properties against bacterial plant pathogens such as Pseudomonas syringae, Xanthomonas campestris and Acidovorax carotovorum. Results from in vitro evaluations were further tested using plant systems (in planta) that were purposely infected with a plant pathogen. Results thus far obtained are promising but work remains to be performed on a wider range of plant systems and fungal/bacterial plant pathogens. Further work on field trials will be continued in partnerships developed with potential product manufacturers. Evaluation of the biodegradation and apparent toxicity of a series of modified sophorolipids was determined by a standard laboratory test method that simulates an environment that might be found in runoff water ecosystems. Products developed were found to meet the criteria of being "ultimately biodegradable." Furthermore, tests thus far give no indication of product toxicity. A cost model developed by the project team showed that at commercial scale production levels, products developed in this program have a total projected cost of $1.29-1.43 per pound. This cost is well in line with that required for commercialization.
