Location: Renewable Product Technology Research
2018 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 research project 5010-41000-164-00D, and both objectives contribute to National Program 306, Component 3, Biorefining. Progress on this project focuses on Problem 3B, technologies that reduce risks and increase profitability in existing biorefineries. Specific examples of progress include the following:
• Under Sub-objective 1.1, production of liamocins was further characterized. Liamocins are novel antibacterial agents with specificity for species of Streptococcus, including pathogens of cattle and swine. Liamocin yields and chemical structures were characterized over time in an optimized production medium. Results will facilitate the commercial development of liamocins.
• Under Sub-objective 1.3, genes related to ethanol tolerance from Lactobacillus buchneri were identified and characterized in recombinant host. Results will enable engineering approaches to improve ethanol tolerance of microbial biocatalysts.
• Under Sub-objective 2.1, strains of fuel ethanol contaminants were identified that can be distinguished one from another in mixed biofilm cultures. Results will enable further studies to determine the role of microbial biofilms in the persistent contamination of fuel ethanol facilities, which causes economic losses in this industry.
• Under Sub-objective 2.1, methods were developed to inactivate the antibiotic virginiamycin. Virginiamycin is used to control contamination in fuel ethanol production, and residues may occur in coproducts.
• Under Sub-objective 2.1, the microbial community present in process samples from both dry grind and wet mill factories were identified by a state-of-the-art DNA sequencing approach. Results will enable the development of novel intervention strategies for controlling microbial contamination in industrial processes.
Accomplishments
1. Alternative to antibiotics for control of fuel ethanol contaminants. Most fuel ethanol facilities use baker’s yeast to ferment sugars from agricultural products to alcohol. Contamination in large-scale production plants is unavoidable, so efforts usually focus on controlling levels of the bacteria. These naturally occurring bacteria compete for the same sugars that are used for production of the ethanol and they often synthesize by-products that inhibit the ability of the yeast to grow. Chronic and acute contamination problems significantly reduce the economic viability of the U.S. fuel ethanol industry. While antibiotics can be used to control the contamination, the overuse of antibiotics to combat these infections has led to antimicrobial resistance and the presence of antibiotic residues in fuel ethanol coproducts. ARS scientists in Peoria, Illinois, developed an alternative method to control contamination using beneficial bacteria similar to probiotics. These bacteria are able coexist with yeast and inhibit the detrimental effects of contaminating strains. These findings will allow ethanol producers to improve the efficiency of their fermentation and reduce the use of antibiotics in their plants.
2. Sustainable production of butyric acid. Butyric acid is a short-chain fatty acid that can be used as flavoring agents in feeds and foods. It can also be incorporated into perfumes, pharmaceuticals, plastics, and textile auxiliaries. Butyric acid has historically been produced via a petrochemical route, but the biological production of butyric acid addresses sustainability concerns and satisfies consumer preferences when used as food additives or cosmetic products. ARS scientists in Peoria, Illinois, used agricultural residues including wheat straw, corn fiber as well as paper mill sludge (PMS) to produce butyric acid via a microbial fermentation route. The pulp and paper making process produces about 300–350 million tons of PMS every year and the majority is disposed of by landfill. The research could lead to applications in recycle and reuse of paper mill waste.
Review Publications
Liu, S., Duncan, S., Qureshi, N., Rich, J.O. 2018. Fermentative production of butyric acid from paper mill sludge hydrolysates using Clostridium tyrobutyricum NRRL B-67062/RPT 4213. Biocatalysis and Agricultural Biotechnology. 14:48-51. https://doi.org/10.1016/j.bcab.2018.02.002.
Leathers, T.D., Rich, J.O., Nunnally, M.S., Anderson, A.M. 2017. Inactivation of virginiamycin by Aureobasidium pullulans. Biotechnology Letters. 40(1):157-163. doi: 10.1007/s10529.
Leathers, T.D., Price, N.P.J., Vaughn, S.F., Nunnally, M.S. 2017. Reduced-molecular-weight derivatives of frost grape polysaccharide. International Journal of Biological Macromolecules. 105:1166-1170. doi: 10.1016/j.ijbiomac.2017.07.143.
Rich, J.O., Bischoff, K.M., Leathers, T.D., Anderson, A.M., Liu, S., Skory, C.D. 2017. Resolving bacterial contamination of fuel ethanol fermentations with beneficial bacteria – an alternative to antibiotic treatment. Bioresource Technology. 247:357-362. https://doi.org/10.1016/j.biortech.2017.09.067.
Leathers, T.D., Skory, C.D., Price, N.P.J., Nunnally, M.S. 2017. Medium optimization for production of anti-streptococcal liamocins by Aureobasidium pullulans. Biocatalysis and Agricultural Biotechnology. 13:53-57. doi: 10.1016/j.bcab.2017.11.008.
Alpdagtas, S., Yücel, S., Kapkaç, H.A., Liu, S., Binay, B. 2018. Discovery of an acidic, thermostable and highly NADP+ dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929. Biotechnology Letters. 40(7): 1135-1147. doi: 10.1007/s10529-018-2568-6.
Montipó, S., Ballesteros, I., Fontana, R.C., Liu, S., Martins, A.F., Ballesteros, M., Camassola, M. 2017. Integrated production of second generation ethanol and lactic acid from steam-exploded elephant grass. Bioresource Technology. 249:1017-1024. doi: 10.1016/j.biortech.2017.11.001.
Bischoff, K.M., Brockmeier, S.L., Skory, C.D., Leathers, T.D., Price, N.P.J., Manitchotpisit, P., Rich, J.O. 2018. Susceptibility of Streptococcus suis to liamocins from Aureobasidium pullulans. Biocatalysis and Agricultural Biotechnology. 15:291-294. doi: 10.1016/j.bcab.2018.06.025.
