Location: Renewable Product Technology Research
2017 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 2017, ARS scientists in Peoria, Illinois, made significant progress toward these objectives, as demonstrated by the following activities:
• Production of novel antibacterial oils from agricultural biomass was optimized.
• A novel pigment-producing bacterial species, Bacillus nakamurai, was described.
• Production of the biopolymer schizophyllan from corn fiber was optimized.
• Butyric acid production by fermentation of various biomass hydrolysates was examined.
• Production of 2,5-furandimethanol, a commodity platform chemical, from switchgrass acid hydrolysates was demonstrated in engineered yeast strains.
• Inulin from coffee processing waste was converted to ethanol by fermentation using Kluyveromyces marxianus strains.
• Samples were collected from multiple ethanol production facilities and deoxyribonucleic acid (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.
• A new lytic enzyme (phage endolysin) was cloned, expressed, and characterized.
• Evaluated biological activity of novel antimicrobial compounds against a variety of bacteria, yeast, and fungi, including plant and animal pathogens.
Progress achieved during FY 2017 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. Optimization of novel antibacterial oil production from biomass. Liamocins are novel antibacterial oils produced by the fungus Aureobasidium pullulans, with specificity for Streptococcus spp., including pathogens that cause important infections of swine and dairy cows. Recently, it was shown that A. pullulans produces liamocins when grown on a variety of sugars and polyols; however, production from agricultural biomass substrates had not previously been reported. Among five biomass substrates tested, pretreated wheat straw appeared to be promising as an abundant, low-value agricultural byproduct. Twenty-seven strains from phylogenetic clades 8, 9, and 11 were surveyed for production of liamocins from wheat straw and sucrose. Liamocin yields were highest from strains in clade 11, and higher from cultures grown on sucrose than wheat straw. However, when supplementary enzymes were added to the fermentation, liamocin production on wheat straw was equivalent to that on sucrose. Furthermore, it was determined that liamocins produced from wheat straw were under-acetylated, resulting in higher levels of liamocin species with the highest activity against Streptococcus. Production from such low-cost substrates might be particularly appropriate for bulk agricultural applications, such as in dairy cattle dips for prevention of mastitis caused by Streptococcal infections.
2. Resolving bacterial contamination of industrial fermentations with beneficial bacteria. Bacterial contamination of fuel ethanol fermentation reduces ethanol yields and can lead to stuck fermentations. Novel alternatives to antibiotics are needed to control bacterial contamination in industrial fermentations. ARS scientists in Peoria, Illinois, examined several hundred microorganisms as potential treatments to mitigate the deleterious effects of harmful bacterial contaminants. Dozens of beneficial bacteria were identified that fully mitigated a harmful contamination. This research could eliminate the use of antibiotics used in industrial fermentations and improve the quality of the animal feed co-product from biofuel production; thereby, could mitigate the emergence and spread of antibiotic-resistance.
3. Application of lytic enzyme 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, used a protein (known as a lytic enzyme) that possesses antibacterial activity and tested it for its ability to mitigate the effects of contamination in cellulosic ethanol production. Treatment with the enzyme reduced the concentration of lactic acid bacteria and increased ethanol yield in experimentally infected fermentations of corn fiber. Lytic enzymes have application in the fuel ethanol industry as an alternative to antibiotics for prevention and control of bacterial contamination.
4. Novel biomass and food processing enzyme. The growth and sustainability of the biorefining industry requires the development of more value-added products from abundant agricultural feedstocks, including lignocellulosics. ARS scientists in Peoria, Illinois, discovered a highly active bacterial ferulate esterase that is produced by a generally recognized as safe microorganism; thereby, it has added potential applications in food processing. This enzyme is currently used to convert low valued lignicellulosic biomass, including the lignin in corn fiber and corn stover, into ferulic acid, a high-value specialty chemical. Such enzymes have application in the biorefining industry in the breakdown of biomass, as well as the synthesis of valuable coproducts.
Review Publications
Leathers, T.D., Nunnally, M.S., Stanley, A.M., Rich, J.O. 2016. Utilization of corn fiber for production of schizophyllan. Biomass and Bioenergy. 95:132-136.
Hughes, S.R., Qureshi, N., Lopez-Nunez, J., Jones, M.A., Jarodsky, J.M., Galindo-Leva, L.A., Lindquist, M.R. 2017. Utilization of inulin-containing waste in industrial fermentations to produce biofuels and bio-based chemicals. World Journal of Microbiology and Biotechnology. 33(4):78. doi:10.1007/s11274-017-2241-6.
Dunlap, C.A., Saunders, L.P., Schisler, D.A., Leathers, T.D., Naeem, N., Cohan, F.M., Rooney, A.P. 2016. Bacillus nakamurai sp. nov., a black pigment producing strain. International Journal of Systematic and Evolutionary Microbiology. 66(8):2987-2991. doi: 10.1099/ijsem.0.001135.
Price, N.P.J., Bischoff, K.M., Leathers, T.D., Cosse, A.A., Manitchotpisit, P. 2016. Polyols, not sugars, determine the structural diversity of anti-streptococcal liamocins produced by Aureobasidium pullulans strain NRRL 50380. Journal of Antibiotics. 70(2):136-141. doi: 10.1038/ja.2016.92.
Galinda-Leva, L.A., Hughes, S.R., Lopez-Nunez, J.C., Jarodsky, J.M., Erickson, A., Lindquist, M.R., Cox, E.J., Bischoff, K.M., Hoecker, E.C., Liu, S., Qureshi, N., Jones, M.A. 2016. Growth, ethanol production, and inulinase activity on various inulin substrates by mutant Kluyveromyces marxianus strains NRRL Y-50798 and NRRL Y-50799. Journal of Industrial Microbiology and Biotechnology. 43(7):927-939. doi: 10.1007/s10295-016-1771-5.
Liu, S., Qureshi, N., Hughes, S.R. 2017. Progress and perspectives on improving butanol tolerance. World Journal of Microbiology and Biotechnology 33(3):51. doi: 10.1007/s11274-017-2220-y.
