Location: Renewable Product Technology Research
2020 Annual Report
Objectives
Objective 1: Develop microbial and enzymic approaches that enable marketable value added products, including biofuels, from the conversion of biomass feedstocks. Subobjective 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 pureculture 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 is a bridging project that was initiated on 7/25/2019, replacing 5010-41000-164-00D and continuing important work on the same objectives. 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 (Quality and Utilization of Agricultural Products) Action Plan by addressing Problem Statement 3B Technologies that reduce risks and increase profitability in existing industrial biorefineries.
Specific examples of significant developments in FY2020 include the following:
Under Objective 1, progress was made on molecular characterization of a bacterial strain, called Clostridium tyrobutyricum, which we isolated from an ethanol production facility. This strain produces butyric acid from agricultural biomass residues. Butyric acid is a short-chain fatty acid that can be used as flavoring agents in feeds and foods. Butyric acid fermentation research provides a renewable bioproduction differing from the current chemical production route. We discovered that this strain also produces compounds with antibacterial activity against other bacteria. Complete genome sequencing is in progress and results will be used to guide further studies for improved butyric acid and novel antibacterial agents. We have also initiated studies with discovery of new antibacterial peptides through genome data mining of lactic acid bacterial genomes. We have selected a few potential peptides based on protein sequence similarities to current antibacterial peptides that are either being synthesized chemically or being produced by recombinant DNA technology. The resulting candidate peptides will be subjected to experimental assessments of their antibacterial properties against ethanol plant contaminants and poultry pathogens.
Under Objective 2, we have continued efforts with the development of endolysins as novel antimicrobials. 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. We have characterized numerous endolysins that have excellent antimicrobial activity against common contaminants associated with fuel ethanol facilities and have been working on methods for overexpression of these enzymes. In addition, we 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. We have recently developed methods to stabilize these endolysins so they can be tested for their ability to control Clostridium perfringens in poultry. Finally, we have made progress with the purification of new antimicrobial agent from certain strains of Bacillus that were shown to prevent biofilm formation in fuel ethanol fermentations and inhibit Erwinia strains that are responsible for the cause of fire blight, a destructive disease of apple and pear trees.
This annual report also serves as the final report of the project plan for 5010-41000-164-00D, which concludes a successful research undertaking that accomplished all of the project plan objectives and yielded important discoveries and new technologies. During the course of this project cycle, we developed numerous microbial and enzymic approaches for the conversion of biomass feedstocks to value-added products. Examples include improved methods for breaking down agricultural waste to fermentable sugars that can be used as building blocks for new products. This was accomplished primarily though the development of new enzymes including bacterial and fungal ferulate esterases and fungal laccases that attack the recalcitrant portions of the biomass. We also developed new fermentative methods to produce butyric acid, which is used in many industrial and food applications. Methods were developed to convert another agricultural waste product, called lignin, into valuable specialty chemicals. We also developed technology to produce novel polysaccharides that can be used in food and pharmaceutical applications. A new antimicrobial compound, called liamocins, was discovered and found to have activity against strains of bacteria implicated in numerous animal diseases and dental plaque formation.
There were several important discoveries that led to improved methods for industrial alcohol production. New alcohol (ethanol and butanol) tolerant bacterial strains/traits/genes were identified and used in the advancement of alcohol-producing strains for improved production efficiency. Novel antimicrobial technologies for biorefining and agricultural applications were developed. We extensively surveyed sources of microbial contamination in several fuel ethanol facilities and found that specific strains of bacteria accounted for most of the problems associated with production efficiency and failed fermentations. We then used these findings and designed fermentation infection models to develop innovative control strategies. One such technique involved utilizing specialized enzymes, called bacterial cell-wall hydrolyzing enzymes, that specifically target the outside protective layer of bacteria and kill them. Another method involved using a probiotic approach where beneficial bacteria were used to outcompete the detrimental strains without reducing the fermentation efficiency. All of these new technologies not only help farmers by creating new agricultural markets, but also provide economic benefits to producers and ultimately the consumers.
Accomplishments
1. Identification of microbial contaminants in fuel ethanol plants. Bacterial and fungal contamination is commonplace in biorefining facilities because it is not possible to maintain such large plants under microbiologically clean conditions. Chronic contamination reduces both the available sugars for conversion to ethanol and essential micronutrients required for optimal yeast growth, thus reducing profitability. It is important to understand what organisms are present throughout the production process in order to reduce the negative impact of contamination. Most studies to date have relied on isolating microbial contaminants at these facilities, but this does not always give an accurate portrayal of all the organisms that are present since many of the strains are difficult to propagate without the proper growing conditions. ARS scientists in Peoria, Illinois, utilized more sensitive culture-independent DNA sequencing methods to identify different types of microbial isolates found at likely points of contamination at numerous ethanol facilities. Several common areas in these facilities were determined to be potential sources of contamination. These results will be important to researchers developing improved methods to control microbial contamination in fuel ethanol production, ultimately resulting in superior conversion of corn to ethanol and economic security of rural communities.
Review Publications
Liu, S., Skory, C., Liang, X., Mills, D., Qureshi, N. 2019. Increased ethanol tolerance associated with the pntAB locus of Oenococcus oeni and Lactobacillus buchneri. Journal of Industrial Microbiology and Biotechnology. 46:1547-1556. https://doi.org/10.1007/s10295-019-02209-y.
Qureshi, N., Saha, B.C., Liu, S., Harry O Kuru, R.E. 2019. Production of acetone-butanol-ethanol (ABE) from concentrated yellow top presscake using Clostridium beijerinckii P260. Journal of Chemical Technology & Biotechnology. 95(3):614-620. https://doi.org/10.1002/jctb.6242.
Leathers, T.D., Saunders, L.P., Bowman, M.J., Price, N.P.J., Bischoff, K.M., Rich, J.O., Skory, C.D., Nunnally, M.S. 2020. Inhibition of Erwinia amylovora by Bacillus nakamurai. Current Microbiology. 77:875–881. https://doi.org/10.1007/s00284-019-01845-y.
Rich, J.O., Anderson, A.M., Leathers, T.D, Bischoff, K.M., Liu, S., and Skory, C.D. 2020. Microbial contamination of commercial corn-based fuel ethanol fermentations. Bioresource Technology. 11:100433. https://doi.org/10.1016/j.biteb.2020.100433.
