Location: Renewable Product Technology Research
2015 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." Portions of this work are a continuation of research performed under project 3620-41000-135-00D “Improving Biochemical Processes for the Production of Sustainable Fuels and Chemicals,” which was terminated in FY2014. 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 FY2015, ARS scientists made significant progress toward these objectives, as demonstrated by the following:
• Anticancer activity of a microbial oil (liamocins) was characterized.
• Methods were developed for production of liamocins from biomass.
• Methods were developed for enzymatic modification of schizophyllan.
• A novel high-oil and high-ammonia producing strain of Yarrowia lipolytica was identified for use as a protein expression platform.
• A novel artificial chromosome was produced and transformed into the yeast Saccharomyces cerevisiae.
• A gene encoding brazzein with yeast optimized codons was constructed in a yeast artificial chromosome for use in newly identified Yarrowia lipolytica strains.
• Enzymes used in deconstructing lignocellulosic biomass (e.g. xylanase and xylosidase) were isolated from thermophilic microorganisms, and tested for expression in recombinant hosts.
• Representative biofuel contaminant strains were identified.
• DNA from samples that were collected from various points in the fuel ethanol production process was isolated and will be used to identify all of the bacteria and fungi present in each sample.
• Novel inhibitors of biofilm formation by biofuel fermentation contaminants were identified.
• Genes fusing the catalytic domains of endolysin LysA and LysA2 were synthesized and expressed in E. coli.
Progress achieved during FY2015 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. Novel inhibitors of biofilm formation by biofuel contaminants. New methods are needed to control contaminants of fuel ethanol production, which reduce ethanol yields and can lead to stuck fermentations. ARS scientists in Peoria, Illinois identified novel potential inhibitors of biofilm formation by biofuel contaminants. Results enable the development of improved methods to control contamination of fuel ethanol production.
2. Enzyme-catalyzed conversion of lignin model compounds. The biorefining industry is still in need of biobased products produced from residual lignin in order to make more efficient use of the feedstock. ARS scientists in Peoria, Illinois examined a variety of laccase enzymes, both commercially prepared and crude extracts, for their ability to modify lignin model compounds. Both mediated and non-mediated laccase-catalyzed reactions were identified that converted into functional chemicals. Interestingly, the products produced by the concerted action of the laccase mediator system on the model substrates are the same as those produced by chemical catalytic approaches. The enzymatic approach affords the opportunity for a biological approach to convert lignin into valuable specialty chemicals that have use in a variety of industrial, consumer, and pharmaceutical applications.
3. Novel method for enzymatic modification of schizophyllan. New technologies are needed to convert agricultural materials into high-value bioproducts, to create new and expanded markets for agricultural commodities and reduce our dependence on imported petroleum. ARS scientists in Peoria, Illinois developed a novel method for the modification of the valuable biopolymer schizophyllan. Results will impact the development of new uses for biobased polymers produced from agricultural biomass.
4. Novel antibacterial protein from lactic acid bacteria. The emergence of antibiotic resistance has created a need for new bactericidal compounds to treat infections and control bacterial contamination. ARS scientists in Peoria, Illinois have discovered a novel enzyme that contains antibacterial activity against several pathogenic bacteria. The gene encoding this unique enzyme was isolated and a recombinant form produced that retained the antibacterial activity. The enzyme is an alternative to antibiotics in controlling contamination in the food and feed industries.
5. Characterization of new enzymes from thermophilic bacteria to release sugar from biomass. New enzymes that function under harsh industrial conditions of extreme temperature and pH are needed to help overcome some of the technical barriers to using agricultural residues as sources of fermentable sugars. ARS scientists in Peoria, Illinois collaborated with scientists at a university in South Dakota, to characterize the biomass-degrading enzymes from a novel thermophilic (high temperature) bacterium. Xylanases and xylosidases (enzymes which help break down the xylan component of biomass) were examined, and the enzymes were found to be active over a broad range of pH, and they were very stable at high temperatures. The enzymes from this organism enable the conversion of biomass to sugars for production of valuable fermentation products.
6. Application of yeast artificial chromosome for expression of heterologous proteins in Saccharomyces cerevisiae. Technologies that enable the synthesis of new coproducts during lignocellulosic biofuel production are needed to maintain the profitability and sustainable operation of biorefineries. ARS scientists in Peoria, Illinois used a genetic tool called a yeast artificial chromosome to express a heterologous protein in the yeast Saccharomyces cerevisiae. A gene encoding brazzein, a protein with potential as a low calorie artificial sweetener, was cloned into the artificial chromosome, and expressed in S. cerevisiae. These results enable the use of the vector system for expression of multiple genes in S. cerevisiae to produce high-value co-products during biofuel production.
7. Improving microbial production of oil and ammonia in the oil-producing yeast Yarrowia lipolytica. Technologies that enable the synthesis of new coproducts from lignocellulosic feedstocks are needed to maintain the profitability and sustainable operation of biorefineries. ARS scientists in Peoria, Illinois generated variants of the yeast Yarrowia lipolytica, an oil producing yeast capable of growth on a wide variety of substrates. Mutant strains of Y. lipolytica were selected for enhanced production of ammonia and oil, and these strains produced these coproducts by growing on a lignocellulosic feedstock made from coffee waste. These new strains have application in the biorefining industry for synthesis of value-added coproducts to biofuel production.
8. 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 pulp, mucilage, and lignocellulosic fractions from coffee waste. Optimal conditions for pretreatment of the feedstock and for fermentation were determined. Under the optimized conditions, the mutant strains enable the production of liquid fuels from a significant agricultural processing waste that creates environmental problems in coffee producing countries.
Review Publications
Hughes, S.R., Gibbons, W.R., Moser, B.R., Rich, J.O. 2013. Sustainable multipurpose biorefineries for third-generation biofuels and value-added co-products. In: Fang, Z., editor. Biofuels - Economy, Environment and Sustainability. Croatia: InTech. p. 245-267.
Hughes, S.R., Moser, B.R., Gibbons, W.R. 2014. Moving toward energy security and sustainability in 2050 by reconfiguring biofuel production. In: Songstad, D.D., Hatfield, J.L., Tomes, D.T., editors. Convergence of Food Security, Energy Security and Sustainable Agriculture. Heidelberg and New York, Springer. p. 15-30.
Liu, S. 2014. Proteomic analyses of ethanol tolerance in Lactobacillus buchneri NRRL B-30929. Proteomics. 14(21-22):2540-2544.
Hughes, S.R., Cox, E.J., Bang, S.S., Pinkelman, R.J., Lopez-Nunez, J.C., Saha, B.C., Qureshi, N., Gibbons, W.R., Fry, M.R., Moser, B.R., Bischoff, K.M., Liu, S., Sterner, D.E., Butt, T.R., Reidmuller, S.B., Jones, M.A., Riano-Herrera, N.M. 2015. Process for assembly and transformation into Saccharomyces cerevisiae of a synthetic yeast artificial chromosome containing a multigene cassette to express enzymes that enhance xylose utilization designed for an automated platform. Journal of Laboratory Automation. 20(6):621-635. doi: 10.1177/2211068215573188.
Manitchotpisit, P., Watanapoksin, R., Price, N.P., Bischoff, K.M., Tayeh, M., Teeraworawit, S., Kriwong, S., Leathers, T.D. 2014. Aureobasidium pullulans as a source of liamocins (heavy oils) with anticancer activity. World Journal of Microbiology and Biotechnology. 30(8):2199-2204.
Ezeji, T.C., Liu, S., Qureshi, N. 2014. Mixed sugar fermentation by Clostridia and metabolic engineering for butanol production. In: Qureshi, N., Hodge, D., Vertes, A., editors. Biorefineries: Integrated Biochemical Processes for Liquid Biofuels. Amsterdam, The Netherlands: Elsevier. p. 191-204.
Khatibi, P.A., Roach, D.R., Donovan, D.M., Hughes, S.R., Bischoff, K.M. 2014. Saccharomyces cerevisiae expressing bacteriophage endolysins reduce Lactobacillus contamination during fermentation. Biotechnology for Biofuels. 7:104. DOI: 10.1186/1754-6834-7-104.
Liu, S., Rich, J.O., Anderson, A.M. 2014. Antibacterial activity of a cell wall hydrolase from Lactobacillus paracasei NRRL B-50314 produced by recombinant Bacillus megaterium. Journal of Industrial Microbiology and Biotechnology. 42:229-235.
Tisserat, B., Reifschneider, L., Lopez Nunez, J.C., Hughes, S.R., Selling, G.W., Finkenstadt, V.L. 2014. Evaluation of the mechanical and thermal properties of coffee tree wood flour - polypropylene composites. BioResources. 9(3):4449-4467.
Hughes, S.R., Lopez-Nunez, J.C., Jones, M.A., Moser, B.R., Cox, E.J., Lindquist, M.R., Galindo-Leva, L., Rodriguez-Valencia, N., Tasaki, K., Brown, R.C., Darzins, A., Brunner, L., et al. 2014. Sustainable conversion of coffee and other crop wastes to biofuels and bioproducts using combined biochemical and thermochemical processes in a multi-stage biorefinery concept. Applied Microbiology and Biotechnology. 98(20):8413-8431.
Leathers, T.D., Bischoff, K.M., Rich, J.O., Price, N.P., Manitchotpisit, P., Nunnally, M.S., Anderson, A.M. 2014. Inhibitors of biofilm formation by biofuel fermentation contaminants. Bioresource Technology. 169:45-51.
Hughes, S.R., Qureshi, N. 2014. Biomass for biorefining: Resources, allocation, utilization, and policies. In: Qureshi, N., Hodge, D., Vertes, A., editors. Biorefineries: Integrated Biochemical Processes for Liquid Biofuels. Amsterdam, The Netherlands: Elsevier. p. 37-58.
Leathers, T.D., Sutivisedsak, N., Nunnally, M.S., Price, N.P., Stanley, A.M. 2015. Enzymatic modification of schizophyllan. Biotechnology Letters. 37(3):673-678.
Hughes, S.R., Reidmuller, S.B. 2015. Integrated automation for continuous high-throughput synthetic chromosome assembly and transformation to identify improved yeast strains for industrial production of biofuels and bio-based chemicals. In: Van den Berg, M.A., Maruthachalam, K., editors. Genetic Transformation Systems in Fungi. Cham, Heidelberg, New York, Dordrecht, London: Springer. p. 183-200.
Bhalla, A., Bischoff, K.M., Sani, R.K. 2014. Highly thermostable GH39 ß-xylosidase from a Geobacillus sp. strain WSUCF1. BMC Biotechnology. 14:963.
