2011 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.
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 renewable technologies will ultimately strengthen our energy independence, improve sustainable agriculture, and provide economic support to rural communities. Specific examples of significant developments in 2011 include the following:
• The efficiency of a key enzyme involved in the production of lactic acid in Rhizopus is increased by site-directed modification of the protein. This modified enzyme is currently being tested to determine if it can result in increased synthesis of lactic acid.
• Efforts continued to further increase lactic acid production in the fungus Rhizopus by improving the ability of this organism to transport lactic acid across the cell membrane. Lactic acid normally requires special proteins to help facilitate the transport into and out of the cell, so we have isolated and characterized several genes from Rhizopus involved in this function.
• Several types of sugars and small sugar chains were modified using a novel water-based chemical process developed in our laboratory for the production of unique compounds known as C-glycosides. This work will help determine the potential of this "green" chemistry technology for the production of new renewable materials.
• Novel surfactants, used in manufacturing of detergents, have been prepared by combining our sugar C-glycosides together with fatty acids chains derived from plant oils. These long chain compounds, called keto-hydrazones, have considerable potential as renewable detergents.
• Methylated sugars, which are produced by several plants and microbes, have physical properties similar to light oils and greases. We have developed a recyclable method to chemically methylate sugars in a way that uses significantly less toxic chemicals and solvents than current methods.
• A rapid assay was developed for the detection of poly-malic acid (PMA). This method was tested in the examination of PMA production by genetically diverse strains of the fungus Aureobasidium pullulans.
• In collaboration with a visiting scientist from Rangsit University, Thailand, we examined genetically diverse strains of a fungus for the production of novel heavy oils that may have numerous industrial applications. Genetic groups were identified that produce a family of related oils.
• Enzymes that form a water-insoluble polymer were isolated from several different strains of the bacterium Leuconostoc mesenteroides and their products compared for chemical and physical properties. Based on these results, two strains were selected for further study, including the cloning of one of the genes responsible for the enzyme production.
• An enzyme from the bacterium Leuconostoc mesenteroides was isolated and found to make levan, a polymer of fructose. It will be used in further experiments for production of low-molecular weight sugars with unique properties.
Enzymes that produce insoluble gel-like polymers from sugar. Certain bacteria used in fermented foods, such as sauerkraut, 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, IL, have identified and characterized several novel enzymes from these food-grade bacteria that can form a related type of polymer that is insoluble in water. In addition, the gene for one of these enzymes has been cloned to allow further study of the enzyme. The unique gel-like structure of this polymer has potential for production of biodegradable fibers and films that can be used in a broad number of consumer applications. This work provides the foundation for developing new eco-friendly materials derived from renewable agricultural materials that expand economic opportunity and decrease dependence on foreign oil.
Improved enzyme efficiency for lactic acid production. The fungus Rhizopus is frequently used to convert, or ferment, sugars obtained from agricultural crops to lactic acid. This natural product has long been utilized by the food industry and more recently for the manufacture of environmentally friendly products, which include the biodegradable plastic poly-lactic acid and the solvent ethyl lactate. One of the key enzymes involved in the synthesis of lactic acid was found to be partially inhibited by a naturally occurring cellular compound, thereby preventing optimal effectiveness of the enzyme. USDA-ARS scientists with the National Center for Agricultural Utilization Research (NCAUR), Renewable Product Technology Research Unit in Peoria, IL, used genetic engineering to alter the protein structure of this enzyme to alleviate this inhibition. The improved efficiency of this modified enzyme provides a new approach to decrease production costs of lactic acid to the benefit of agricultural growers and ultimately the consumer.
Discovery of novel microbial oils. Certain microorganisms are known to produce oils that have potential as lubricants, bioactive compounds, and biodiesel replacements; however, the economic feasibility of generating these microbial oils using agriculturally-derived sugars is often limited by low production yields. In collaboration with a visiting scientist from Rangsit University, Thailand, USDA-ARS scientists with the National Center for Agricultural Utilization Research (NCAUR), Renewable Product Technology Research Unit in Peoria, IL, examined genetically diverse strains of the fungus Aureobasidium for the production of novel heavy oils that may have applications as biosurfactants, which can be used for production of detergents for cleaning, emulsification, foaming, wetting, and softening. Several genetic groups were identified that produce a family of related oils. This work allows researchers to more specifically direct further screening efforts on promising microbial oils and enhancing the likelihood of commercializing production of these oils from agricultural commodities and byproducts.
Microbial biosurfactant as renewable detergents. Many microorganisms synthesize a 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), Renewable Product Technology Research Unit in Peoria, IL, developed assay methods to identify and screen for biosurfactants produced by microbial isolates obtained from different ecological niches. This research provides the necessary tools to discover novel surfactants and more rapidly screen microbial strains with superior production capabilities.
Biofilm formation by bacterial contaminants of food processing facilities. Bacterial contaminants cause significant problems in sugar refineries and food processing facilities where these microorganisms grow as biofilms that tightly adhere to surfaces and result in "slime" production, which result in significant economic losses for these industries. It has been suggested that the ability of contaminants to form biofilms is dependent on production of certain polysaccharides that serve as cementing agents to strengthen the biofilm. Understanding how these biofilms are formed is integral to developing new and improved strategies to reduce bacterial contamination. USDA-ARS scientists with the National Center for Agricultural Utilization Research (NCAUR), Renewable Product Technology Research Unit in Peoria, IL, compared biofilm formation among different types of bacteria that contaminate sugar processing facilities and found that the ability to form biofilms was not dependent solely on the types of polysaccharides produced by the bacteria. This work provides new information that allows further development of research methods and approaches for control of bacterial contamination of industrial processes.
Improved technologies for production of detergents from agricultural sugars. 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. Methods to improve current manufacturing technologies and develop superior specialty surfactants using renewable agricultural materials as feedstocks are necessary to meet the growing demand for this market. USDA-ARS scientists with the National Center for Agricultural Utilization Research (NCAUR), Renewable Product Technology Research Unit in Peoria, IL, have developed environmentally-friendly techniques to link different chemical groups to sugars and small sugar chains for the production of several novel specialty surfactants. These sugar-based surfactants typically contain a fatty acid chain that is obtained from natural plant oils (e.g., corn oil, sesame oil, and olive oil), have considerable promise as biobased detergents, and are economically competitive with existing production methods. This discovery allows the "green" production of surfactants almost entirely from renewable agricultural materials, thereby having the potential to decrease production of petroleum-based detergents that rely on limited supplies of oil.
Faber, T.A., Bauer, L.L., Price, N.P., Hopkins, A.C., Fahey, Jr., G.C. 2011. In vitro digestion and fermentation characteristics of Temulose molasses, a co-product of fiberboard production, and select Temulose fractions using canine fecal inoculum. Journal of Agricultural and Food Chemistry. 59:1847-1853.
Price, N.P., Naumann, T.A. 2011. A high-throughput MALDI-TOF mass spectrometry-based assay of chitinase activity. Analytical Biochemistry. 411(1):94-99.
Ruiz-Matute, A.I., Brokl, M., Sanz, M.L., Soria, A.C., Cote, G.L., Collins, M.E., Rastall, R.A. 2011. Effect of dextransucrase cellobiose acceptor products on the growth of human gut bacteria. Journal of Agricultural and Food Chemistry. 59:3693-3700.
Finkenstadt, V.L., Cote, G.L., Willett, J.L. 2011. Corrosion protection of low-carbon steel using exopolysaccharide coatings from Leuconostoc mesenteroides. Biotechnology Letters. 33:1093-1100.
Faber, T.A., Hopkins, A.C., Middlebos, I.S., Price, N.P., Fahey, Jr., G.C. 2010. Galactoglucomannan oligosaccharide supplementation affects nutrient digestibility, fermentation end-product production, and large bowel microbiota of the dog. Journal of Animal Science. 89:103-112.
Kurtzman, C.P., Price, N.P., Ray, K.J., Kuo, T. 2010. Production of sophorolipids biosurfactants by multiple species of the Starmerella (Candida) bombicola yeast clade. FEMS Microbiology Letters. 311(2):140-146.
Isenberg, S.L., Brewer, A.K., Cote, G.L., Striegel, A.M. 2010. Hydrodynamic versus size-exclusion chromatography characterization of alternan and comparison to off-line MALS. Biomacromolecules. 11:2505-2511.
Price, N.P., Hartman, T.M., Faber, T.A., Vermillion, K., Fahey, Jr., G. 2011. Galactoglucomannan oligosaccharides (GGMO) from a molasses byproduct of pine (Pinus taeda) fiberboard production. Journal of Agricultural and Food Chemistry. 59:1854-1861.
Manitchotpisit, P., Price, N.P., Leathers, T.D., Punnapayak, H. 2011. Heavy oils produced by Aureobasidium pullulans. Biotechnology Letters. 33(6):1151-1157.
Carpenter, C.A., Kenar, J.A., Price, N.P. 2010. Preparation of saturated and unsaturated fatty acid hydrazides and long chain C-glycoside Ketohydrazones. Green Chemistry. 12(11):2012-2018. DOI: 10.1039/c0gc00372g.
Chen, W., Qu, D., Zhai, L., Tao, M., Wang, Y., Lin, S., Price, N.P., Deng, Z. 2010. Characterization of the Tunicamycin gene cluster unveiling unique steps involved in its biosynthesis. Protein & Cell. 1(12):1093-1105. DOI: 10.1007/s13238-010-0127-6.
Leathers, T.D., Bischoff, K.M. 2011. Biofilm formation by strains of Leuconostoc citreum and L. mesenteroides. Biotechnology Letters. 33(3):517-523.