Location: Renewable Product Technology Research
2019 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
Progress was made on both objectives of the research project which focuses on developing new value-added products and reducing microbial contamination associated with biorefining and agriculture. This work contributes directly to the National Program 306 (Biorefining) Action Plan by addressing Component 3-Biorefining.
Specific examples of significant developments in FY2019 include the following:
Under Objective 1, progress was made on developing liamocins as novel antimicrobial products. Liamocins are microbial oils produced by certain strains of the fungus Aureobasidium and ARS scientists in Peoria, Illinois, have previously shown that they have antibacterial activity specific for species of Streptococcus, including important pathogens of cattle, swine and humans. We have further demonstrated that liamocins are able to inhibit biofilm production by Streptococcus mutans and Streptococcus sobrinus, organisms that are associated with dental plaque formation.
Under Objective 2, a novel antimicrobial polypeptide produced by a Lactobacillus strain was identified. This natural product was able to inhibit the growth of several Gram-positive bacteria and may have promising applications as an antibacterial agent for use with microbial fermentation technology. In addition, significant progress has been made on characterizing a new endolysin for control of bacterial contamination in biorefining facilities. Endolysins are a unique group of enzymes isolated from bacterial viruses that attack the bacterial cell wall and can reduce the presence of unwanted organisms without the use of antibiotics. This new endolysin was shown to have excellent stability and activity against common contaminants associated with fuel ethanol facilities. ARS scientists have also developed recombinant yeast strains that produce endolysins with specific antibacterial activity against Clostridium perfringens. Clostridium perfringens is the causative agent of necrotic enteritis, which is a significant problem to the poultry, pig and beef industry as well as foodborne and non-foodborne human disease. Work is currently being done with several collaborators to test the ability of these yeast strains to control presence of Clostridium perfringens in poultry. Finally, it was determined that inhibitory compounds produced by certain strains of Bacillus are not only able to prevent biofilm formation in fuel ethanol fermentations, but they are also able to inhibit Erwinia strains that are responsible for the cause of fire blight, a destructive disease of apple and pear trees.
Accomplishments
1. Improved alcohol tolerance by modified bacteria. While yeasts are traditionally considered for ethanol production, bacterial strains are often utilized for fermentations involving mixed sugars or synthesis of alternative biofuels such as butanol. Most bacteria are inhibited by the presence of alcohols, which limits their ability to accumulate product in high yields. ARS scientists in Peoria, Illinois, have developed technology that significantly improves the alcohol tolerance of bacteria. They first identified two bacterial strains found in wineries and fuel ethanol facilities that can survive high concentrations of ethanol. They then compared the genomes of these alcohol tolerant bacteria with other similar strains that were sensitive and found two genes, unique to the alcohol tolerant isolates, that are often associated with stress response. These genes were each expressed in alcohol sensitive bacteria and shown to confer higher tolerance to ethanol and butanol. The potential application of this work will lead to more efficient production strains that will benefit fuel ethanol producers.
Review Publications
Saunders, L.P., Bischoff, K., Bowman, M.J., Leathers, T.D. 2018. Inhibition of Lactobacillus biofilm growth in fuel ethanol fermentations by Bacillus. Bioresource Technology. 272:156-161. https://doi.org/10.1016/j.biortech.2018.10.016.
Leathers, T.D., Rich, J.O., Bischoff, K.M., Skory, C.D., Nunnally, M.S. 2019. Inhibition of Streptococcus mutans and S. sobrinus biofilms by liamocins from Aureobasidium pullulans. Biotechnology Reports. 21:e00300. https://doi.org/10.1016/j.btre.2018.e00300.
Qureshi, N., Harry-O'Kuru, R.E., Liu, S., Saha, B. 2018. Yellow top (Physaria fendleri) presscake: a novel substrate for butanol production and reduction in environmental pollution. Biotechnology Progress. 35(3):e2767. https://doi.org/10.1002/btpr.2767.
Qureshi, N., Saha, B.C., Klasson, K.T., Liu, S. 2018. Butanol production from sweet sorghum bagasse with high solids content: Part I – comparison of liquid hot water pretreatment with dilute sulfuric acid. Biotechnology Progress. 34(4):960-966. https://doi.org/10.1002/btpr.2639
Qureshi, N., Saha, B.C., Klasson, K.T., Liu, S. 2018. High solid fed-batch butanol fermentation with simultaneous product recovery: Part II - process integration. Biotechnology Progress. 34(4):967-972. https://doi.org/10.1002/btpr.2643
Hay, W.T., Fanta, G.F., Rich, J.O., Schisler, D.A., Selling, G.W. 2018. Antifungal activity of a fatty ammonium chloride amylose inclusion complex against Fusarium sambucinum; control of dry rot on multiple potato varieties. American Journal of Potato Research. 96(1):79-85. https://doi.org/10.1007/s12230-018-9683-8.
Lee, Y., Kim, T., Kim, Y., Lee, S., Kim, S., Kang, S., Yang, J., Baek, I., Sung, Y., Park, Y., Hwang, S., O, E., Kim, K., Liu, S., Kamada, N., Gao, N., Kweon, M. 2018. Microbiota-derived lactate accelerates intestinal stem cell-mediated epithelial development through the Gpr81. Cell Host and Microbe. 24(6):833-846. https://doi.org/10.1016/j.chom.2018.11.002.
