Location: Renewable Product Technology Research2012 Annual Report
1a. Objectives (from AD-416):
The broad goal of this project is to develop technologies for producing specialty/commodity chemicals and polymers from agriculturally derived carbohydrates. Objective 1: Develop commercially viable biocatalytic and chemical processes that enable the production of chemicals and monomers from agricultural feedstocks. Objective 2: Develop commercially viable biocatalytic processes that enable the production of novel biopolymers from agricultural feedstocks.
1b. Approach (from AD-416):
This research will specifically include the following strategies to achieve our objectives: (1) sequential metabolic engineering improvements of key metabolic steps of the fungus Rhizopus to enhance the production of carboxylic acids, including malic, fumaric, and lactic acid, which are all used as chemical feedstock in numerous manufacturing applications; (2) modification of carbohydrates through novel water based methods for the production of functional products, such as surfactants or detergents; (3) screening for superior isolates of the fungus Aureobasidium and fermentation optimization for improved bioproduction of the polyester, poly malic acid, which has the potential to be used as a biocompatible polymer; and (4) strain selection, fermentation optimization, and genetic modification of the bacterium Leuconostoc to enable enhanced production of a water insoluble polymer, alpha D glucan, which can be used in the production of polymer fibers and films. Accomplishing these objectives will allow for the development of new and improved methods for producing sustainable chemicals and polymers that can be employed in everyday consumer products.
3. Progress Report:
Annual progress was made on all four subobjectives of this project, which addresses research needs to discover and develop commercially viable biobased materials and conversion processes; and to improve biobased material performance and processing through enhanced knowledge of their structure/property relationships. These technologies will ultimately strengthen our energy independence, improve sustainable agriculture, and provide economic support to rural communities. Specific examples of significant developments in FY12 include the following: • The fungus Rhizopus was genetically modified using ARS technology to significantly enhance the production of fumaric acid. This work is expected to be a significant contribution towards the development of improved technologies for producing fumaric acid, thereby benefiting the agricultural grower and ultimately the consumer. • A library of new compounds was prepared using ARS patented technology to chemically link different sugars to numerous types of chemical groups. These unique compounds have potential as microbial inhibitors, detergents, and lubricants. • Production yields of microbial biosurfactants, which can be used for the manufacturing of specialty detergents, were significantly increased through improvements in culture conditions using novel yeast strains obtained by ARS scientists. The improved yields will allow more testing for application studies. • Novel microbial isolates obtained from soil were found to produce methylated polysaccharides by fermentation. These methylated sugars have potential as replacements for petroleum-based solvents, oils, and liquid fuels. • Modified chitin oligosaccharides were synthesized using ARS patented technology and are being tested as potential chitinase inhibitors for the study of plant defense mechanisms against fungal infections. • Several sugar-based glycosidase inhibitors produced by microbial fermentation were identified as potentially valuable anti-microbial and/or anti-insecticidal compounds. • Genetically diverse strains of a fungus Aureobasidium were screened for the production of a novel biopolymer that may have applications in second-generation bioplastics. Genetic groups were identified that produce high levels of this polyester. This work has potential impact for research to develop value-added products from agricultural commodities and byproducts. • Two key enzymes in the production of water-soluble and water-insoluble glucans from the bacterium Leuconostoc mesenteroides were cloned and characterized. It was found that the two enzymes interact in combination to synthesize graft copolymers that are unique from the products made from individual enzymes. Evidence suggests that this could be used to create a spectrum of novel structures with varying physical properties. • A novel bacterium was isolated from a fermented sugary beverage and found to be an exceptionally good producer of insoluble glucan. The genome has been sequenced, and multiple polysaccharides have been isolated and partially characterized.
1. Enzymes that produce insoluble gel-like polymers from sugar. Certain bacteria used in fermented foods are able to produce long polymers of glucose from cane or beet sugars. These polymers, called dextran, are typically water-soluble and are utilized in a large number of industrial, medical, and food applications. USDA, ARS scientists with the National Center for Agricultural Utilization Research (NCAUR), Renewable Product Technology Research Unit in Peoria, Illinois have identified and characterized several novel enzymes from these food-grade bacteria that can form similar glucose-based polymers that are insoluble in water. Genes for a number of these enzymes were cloned and modified to produce polymers with differing chemical and physical properties. Evidence indicates that using combinations of these enzymes can produce a spectrum of related polymers, ranging from very insoluble, gel-like materials, to partially soluble materials with nanoparticle-like properties. These polymers have potential for production of biodegradable fibers and films that can be used in a broad number of consumer applications. These polymers also provide the foundation for developing new eco-friendly materials derived from renewable agricultural materials that expand economic opportunity and decrease dependence on foreign oil.
2. Microbial biosurfactants as renewable detergents. Annual U.S. production of surfactants, one of the primary components of detergents and personal care products, is approximately 7.7 billion pounds with almost 50% being produced from petrochemicals. Certain microorganisms are able to synthesize a similar class of compounds known as biosurfactants, which can be used for production of detergents for cleaning, emulsification, foaming, wetting, and softening. Biosurfactants have several advantages over surfactants chemically synthesized from petro-chemicals, such as lower toxicity and higher biodegradability, but have traditionally been limited by the high cost of production due to low yields by existing microbial strains. USDA, ARS scientists with the National Center for Agricultural Utilization Research (NCAUR) in Peoria, Illinois identified a new yeast strain that produces significant yields of a novel sophorolipid, which is a sugar-based biosurfactants, that provides improved detergent capabilities for certain applications. This research allows production of surfactants from renewable agricultural material, thereby having the potential to decrease production of petroleum-based detergents that rely on limited supplies of oil.
3. Improved efficiency for fumaric acid production. The fungus Rhizopus is frequently used to convert, or ferment, sugars obtained from agricultural crops to fumaric acid. This natural product is used extensively by the food industry and for the manufacturing of synthetic resins. The world demand for fumaric acid is in excess of 90,000 tons/yr and is expected to increase with the development of new conversion technologies that allow fumaric acid to be used for numerous applications. However, improved technologies are necessary to minimize production costs. USDA, ARS scientists with the National Center for Agricultural Utilization Research (NCAUR), Renewable Product Technology Research Unit in Peoria, Illinois, in collaboration with scientists at the Ohio State University, Columbus, Ohio, significantly increased fumaric acid production by modifying Rhizopus to express a novel enzyme that enhances the metabolic conversion pathway in this fungus. This discovery improves our current technology for producing fumaric acid, thereby benefiting the agricultural grower and ultimately the consumer.
4. Improved microbial strains for poly-malic acid production. Poly(malic acid) or PMA is a water-soluble biopolymer that has numerous potential applications in the biomedical and pharmaceutical industries. PMA can be produced from sugars obtained from agricultural crops by a yeast-like fungus called Aureobasium, but industrial production is typically limited by low yields. USDA, ARS scientists with the National Center for Agricultural Utilization Research (NCAUR), Renewable Product Technology Research Unit in Peoria, Illinois, in collaboration with a visiting scientist from Rangsit University, Thailand, examined PMA production by genetically diverse isolates of Aureobasidium and discovered novel isolates that produce high levels of this polyester. This work provides new information that allows further development towards the commercialization of microbial production of PMA from agricultural commodities and byproducts.
Adeuya, A., Price, N.P. 2012. Enumeration of hydroxyl groups of sugars and sugar alcohols by aqueous-based acetylation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry. 26:1372-1376. DOI: 10.1002/rcm.6234.
Dunlap, C.A., Schisler, D.A., Price, N.P., Vaughn, S.F. 2011. Cyclic lipopeptide profile of three Bacillus subtilus strains; antagonists of Fusarium head blight. Journal of Microbiology. 49:603-609. DOI: 10.1007/s12275-011-1044-y.
Manitchotpisit, P., Skory, C.D., Leathers, T.D., Lotrakul, P., Eveleigh, D.E., Prasongsuk, S., Punnapayak, H. 2011. a-Amylase activity during pullulan production and a-Amylase gene analyses of Aureobasidium pullulans. Journal of Industrial Microbiology and Biotechnology. 38:1211-1218. DOI: 10.1007/s10295-010-0899-y.
Burch, A.Y., Browne, P.J., Dunlap, C.A., Price, N.P., Lindow, S.E. 2011. Comparison of biosurfactant detection methods reveals hydrophobic surfactants and contact-regulated production. Environmental Microbiology. 13:2681-2691. DOI: 10.1111/j.1462-2920.2011.02534.x.
Manitchotpisit, P., Skory, C.D., Peterson, S.W., Price, N.P., Vermillion, K., Leathers, T.D. 2012. Poly (beta-L-malic acid) production by diverse phylogenetic clades of Aureobasidium pullulans. Journal of Industrial Microbiology and Biotechnology. 39(1):125-132. DOI: 10.1007/s10295-011-1007-7.
Hernandez-Hernandez, O., Cote, G.L., Kolida, S., Rastall, R.A., Sanz, M. 2011. In vitro fermentation of alternansucrase raffinose acceptor products by human gut bacteria. Journal of Agricultural and Food Chemistry. 59(20):10901-10906.
Price, N.P., Ray, K.J., Vermillion, K., Dunlap, C.A., Kurtzman, C.P. 2011. Structural characterization of novel sophorolipid biosurfactants from a newly-identified species of Candida yeast. Carbohydrate Research. 348:33-41. DOI: 10.1016/j.carres.2011.07.016.
Cote, G.L., Skory, C.D. 2011. Cloning, expression, and characterization of an insoluble glucan-producing glucansucrase from Leuconostoc mesenteroides NRRL B-1118. Applied Microbiology and Biotechnology. 93:2387-2394. DOI: 10.1007/s00253-011-3562-2.
Arca, M., Sharma, B.K., Price, N.P.J., Perez, J.M., Doll, K.M. 2012. Evidence contrary to the accepted Diels-Alder mechanism in the thermal modification of vegetable oil. Journal of the American Oil Chemists' Society. 89:987-994.
Selling, G.W., Hojillaevangelist, M.P., Evangelista, R.L., Isbell, T., Price, N.P., Doll, K.M. 2013. Extraction of proteins from pennycress seeds and press cake. Industrial Crops and Products. 41(1):113-119.