Location: Renewable Product Technology Research
2016 Annual Report
Objectives
Objective 1: Develop microbial and enzymic approaches that enable marketable value-added products, including biofuels, from the conversion of biomass feedstocks. Sub-objective 1.1: Production and utilization of microbial oils. Sub-objective 1.2: Develop microbial catalysts to produce value-added proteins as co-products of biofuel production. Sub-objective 1.3: Biochemical conversion of agricultural feedstocks to butyric acid.
Objective 2: Improve fermentation processes by controlling microbial contamination in commercial biorefineries. Sub-objective 2.1: Develop molecular tools to characterize the microbial communities (planktonic and biofilm) of commercial biorefineries. Sub-objective 2.2. Develop novel antibacterial agents effective against common bacterial contaminants.
Approach
Reducing the economic risks of biorefining by diversifying the portfolio of marketable biobased products and by improving the efficiencies of processes for producing them from agricultural materials will enable the growth and sustainability of biorefining. Research will develop biological approaches to creating new products from agricultural feedstocks, and on reducing the incidence of operating disruptions at commercial biorefineries.
Growth of the ethanol-based biorefining industry is hindered by gasoline blending rates and limited uses for distillers grains. Novel products from renewable biomass-based feedstocks could enable additional revenue streams in commercial biorefineries, but technical challenges still exist for biorefineries that want to manufacture new products and co-products for a variety of consumer, food, and industrial applications. Research will focus on the development of three classes of value-added biobased products: oils, proteins, and chemicals.
Fermentations at commercial biofuel biorefineries are not performed under pure-culture conditions, and a variety of Gram-positive and Gram-negative bacteria as well as yeast have been isolated from fuel ethanol fermentations. Lactic acid bacteria are generally considered to be the primary contaminants of corn-based fuel ethanol facilities, and it is anticipated that they will also infect the fermentation unit operation of future biorefineries employing a wide variety of biomass-based feedstocks. Our previous research was selective for bacterial strains that are readily cultured under laboratory conditions, and was successful in identifying hundreds of bacterial strains and their impact on Saccharomyces cerevisiae production of ethanol from corn mash. Research will characterize the microorganisms that contaminate commercial fermentation facilities, and on the development of new intervention strategies to control infections by planktonic and sessile (i.e. biofilm) bacteria.
Progress Report
This report documents accomplishments for the research project 5010-41000-164-00D, entitled "New biobased products and improved biochemical processes for the biorefining industry." Research focuses on two Objectives: 1. Develop microbial and enzymic approaches that enable marketable value-added products, including biofuels, from the conversion of biomass feedstocks; and 2. Improve fermentation processes by controlling microbial contamination in commercial biorefineries. In FY 2016, ARS scientists made significant progress toward these objectives, as demonstrated by the following activities:
• Aureobasidium pullulans strains and oleaginous yeasts were screened for growth and microbial oil production from agricultural biomass substrates.
• Methods were developed for genetic transformation of representative biofuel contaminant strains.
• Novel sources of a biomass degrading enzyme called feruloyl esterase were identified.
• Butyric acid production by fermentation of various biomass hydrolysates was examined.
• Proteins whose expression was altered in response to increasing concentrations of ethanol were identified in Lactobacillus buchneri.
• Genes responsible for bacterial ethanol tolerance in Lactobacillus buchneri were identified.
• Genes whose expression was altered in response to increasing concentrations of butanol were identified in Lactobacillus mucosae.
• A polyurethane chemical precursor was produced by fermentation of switchgrass acid hydrolysates using Kluyveromyces marxianus and Yarrowia lipolytica strains.
• A gene coding for a peptide sweetener was expressed in a yeast artificial chromosome as a potential biorefinery product.
• A two-stage fermentation system was used to convert sugars and protein in cocoa processing waste streams to oil and ammonia.
• Inulin from coffee processing waste was converted to ethanol by fermentation using Kluyveromyces marxianus strains.
• Samples were collected from multiple ethanol production facilities and DNA was isolated and used to identify the microbial diversity in the production process.
• Next generation sequencing was employed for the culture-independent identification of the microbiome of an ethanol production facility.
• Novel inhibitors of biofilm formation by bacterial contaminants of ethanol fermentations were characterized.
• A gene encoding a new lytic enzyme (phage endolysin) was cloned and expressed in Escherichia coli, and the recombinant enzyme shown to possess antibacterial activity.
• Genes fusing a yeast secretion signal to the catalytic domain of a phage endolysin were constructed for expression of the lytic enzyme in Saccharomyces cerevisiae.
Progress achieved during FY 2016 has potential scientific impact for researchers in industry, government, and academia and will facilitate development and improvement of efficient processes that lower the production costs of fuels and chemicals from renewable agricultural materials.
Accomplishments
1. Identification of bacterial contaminants of fuel ethanol production. Bacterial contamination of fuel ethanol fermentation reduces ethanol yields and can lead to stuck fermentations. Fundamental information on the types of bacteria that inhabit commercial fuel ethanol facilities is needed to develop intervention methods to control contamination. ARS scientists in Peoria, Illinois, conducted a two-year study of bacterial contaminants from a Midwestern dry grind corn fuel ethanol facility. A variety of species of bacteria were identified, but the study found that ethanol inhibition was related to acetic acid production by certain species of lactic acid bacteria. Results will impact the development of improved methods to control contamination of fuel ethanol production.
2. Novel sources of a biomass degrading enzyme. Novel enzymes are needed for improved degradation and modification of agricultural biomass. ARS scientists in Peoria, Illinois, genetically classified strains of the fungus Aureobasidium and examined them for production of an enzyme called feruloyl esterase, which is involved in deconstruction of lignocellulosic biomass. Certain genetic groups of the fungus were identified as superior sources of this enzyme. This work provides new information that facilitates the identification of new sources of industrial enzymes for application in the biorefining industry.
3. Novel ferulate esterase for complete hydrolysis of plant cell wall polymers. To fully release all the sugars available in lignocellulosic biomass, new enzymes are needed to break the bonds between lignin and the hemicellulose component. Ferulate esterases are hemicellulase accessory enzymes that complement the activity of xylanases and pectinases to degrade the hemicellulose portion of the plant cell wall. ARS scientists in Peoria, Illinois, have discovered a highly active ferulate esterase from a bacterium called Lactobacillus fermentum. The gene for this enzyme was used to make a recombinant form of the ferulate esterase that showed high activity against several artificial substrates. This enzyme has application in biofuel production as well as in the pharmaceutical, polymer, and detergent industries.
4. Application of lytic enzymes to control contamination of cellulosic ethanol fermentations. Lactic acid bacteria frequently contaminate commercial fuel ethanol fermentations, reducing yields and decreasing profitability of biofuel production. ARS scientists in Peoria, Illinois, and Beltsville, Maryland, purified four proteins (known as lytic enzymes) that possess antibacterial activity and tested their ability to mitigate the effects of contamination in cellulosic ethanol production. Treatment with each enzyme reduced the concentration of lactic acid bacteria and increased ethanol yield in experimentally infected fermentations. Lytic enzymes have application in the fuel ethanol industry as an alternative to antibiotics for prevention and control of bacterial contamination.
5. Production of ethanol from byproducts of coffee processing. Economically producing fuels and chemicals from agricultural residues requires a microorganism to fully utilize all available sugars derived from biomass. Kluyveromyces marxianus is a yeast that can use a variety of sugars to make ethanol. ARS scientists in Peoria, Illinois, evaluated the ability of mutant strains of K. marxianus to make ethanol from inulin, a major polysaccharide derived from coffee processing waste. One strain was capable of converting coffee inulin to ethanol. Application of this strain will aid in the disposal of waste products from coffee processing, which will yield significant economic and environmental benefits.
None.
Review Publications
Rich, J.O., Leathers, T.D., Bischoff, K.M., Anderson, A.M., Nunnally, M.S. 2015. Biofilm formation and ethanol inhibition by bacterial contaminants of biofuel fermentation. Bioresource Technology. 196:347-354.
Rich, J.O., Anderson, A.M., Berhow, M.A. 2016. Laccase-mediator catalyzed conversion of model lignin compounds. Biocatalysis and Agricultural Biotechnology. 5:111-115.
Liu, S., Bischoff, K.M., Anderson, A.M., Rich, J.O. 2016. Novel feruloyl esterase from Lactobacillus fermentum NRRL B-1932 and analysis of the recombinant enzyme produced in Escherichia coli. Applied and Environmental Microbiology. 82(17):5068-5076. doi: 10.1128/AEM.01029-16.
Bischoff, K.M., Leathers, T.D., Price, N.P.J., Manitchotpisit, P. 2015. Liamocin oil from Aureobasidium pullulans has antibacterial activity with specificity for species of Streptococcus. Journal of Antibiotics. 68:642-645. doi: 10.1038/ja.2015.39.
Sutivisedsak, N., Leathers, T.D., Biresaw, G., Nunnally, M.S., Bischoff, K.M. 2016. Simplified process for preparation of schizophyllan solutions for biomaterial applications. Preparative Biochemistry and Biotechnology. 46(3):313-319.
Bischoff, K.M., Zhang, Y., Rich, J.O. 2016. Fate of virginiamycin through the fuel ethanol production process. World Journal of Microbiology and Biotechnology. 32(5):76. doi: 10.1007/s11274-016-2026-3.
Doll, K.M., Walter, E.L., Bantchev, G.B., Jackson, M.A., Murray, R.E., Rich, J.O. 2016. Improvement of lubricant materials using ruthenium isomerization. Chemical Engineering Communications. 203(7):901-907.
Qureshi, N., Dien, B.S., Saha, B.C., Iten, L., Liu, S., Hughes, S.R. 2015. Genetically engineered Escherichia coli FBR5 to use cellulosic sugars: Production of ethanol from corn fiber hydrolyzate employing commercial nutrient medium. European Chemical Bulletin. 4(3):130-134. https://doi.org/10.17628/ecb.2015.4.130-134
Lindquist, M.R., Lopez-Nunez, J.C., Jones, M.A., Cox, E.J., Pinkleman, R.J., Bang, S.S., Moser, B.R., Jackson, M.A., Iten, L.B., Kurtzman, C.P., Bischoff, K.M., Liu, S., Qureshi, N., Tasaki, K., Rich, J.O., Cotta, M.A., Saha, B.C., Hughes, S.R. 2015. Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates. Applied Microbiology and Biotechnology. 99(22):9723–9743.
Liu, M., Bischoff, K.M., Gill, J.J., Mire-Criscione, M.D., Berry, J.D., Young, R., Summer, E.J. 2015. Bacteriophage application restores ethanol fermentation characteristics disrupted by Lactobacillus fermentum. Biotechnology for Biofuels. 8:132.
Leathers, T.D., Price, N.P.J., Bischoff, K.M., Manitchotpisit, P., Skory, C.D. 2015. Production of novel types of antibacterial liamocins by diverse strains of Aureobasidium pullulans grown on different culture media. Biotechnology Letters. 37(10):2075-2081. doi: 10.1007/s10529-015-1892-3.
Liu, S., Skory, C., Qureshi, N., Hughes, S. 2016. The yajC gene from Lactobacillus buchneri and Escherichia coli and its role in ethanol tolerance. Journal of Industrial Microbiology and Biotechnology. 43(4):441-450. doi: 10.1007/s10295-015-1730-6.
