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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Bioenergy Research » Research » Research Project #438816

Research Project: New Bioproducts for Advanced Biorefineries

Location: Bioenergy Research

2021 Annual Report

Objective 1: Generate enzymes required to hydrolyze recalcitrant xylan structures to increase sugar availability for biorefinery processes. Objective 2: Develop an improved biorefinery process for production of itaconic acid from lignocellulose. Objective 3: Develop a biorefinery process for production of butyric acid from lignocellulose. Objective 4: Develop technologies that enable non-Saccharomyces yeast-based processes for bioconversion of lignocellulose to advanced biofuels and value-added bioproducts. Objective 5: Enable production of biocontrol co-products to add value to biorefinery process streams.

The last few decades have seen a dramatic growth in biofuels and bioproducts. Bioethanol accounts for over one-third of United States corn consumption and bio-based products (e.g., apart from ethanol) add $369 billion and 1.5 million jobs to the national economy (USDA, 2015). Yet the transition to lignocellulose feedstocks, with an estimated availability of 1 billion tons per year, has been slow and halting. One reason for their slow adoption has been a lack of bioproducts. The objectives of this plan share the common goal of developing microbial bioproducts for advanced bioenergy plants. Sugar conversion efficiency will be increased by using sophisticated analytical techniques to identify recalcitrant xylan structures and to use this knowledge for enzyme discovery. Structural analysis of carbohydrates is technically challenging and will rely on novel methods developed by ARS researchers. It is proposed to convert the generated sugars using either bacterial, yeast, or fungal cultures to butyrate, itaconate, and lipids. Butyrate is a widely used commodity chemical, itaconate can be used to manufacture bio-plastics, and lipids can be used either as a bioproduct or as a feedstock to manufacture biodiesel or green diesel. Finally, a unique set of Pseudomonas of proven efficacy as biocontrol agents for effectively combating fungal potato dry rot (and other plant diseases) will be evaluated for valorizing agriculture and lignocellulose associated process streams. The biocontrol agent is a substitute for azole-based chemicals and, so, this goal is medically beneficial in combating overuse of anti-fungal azole-based chemicals, which are blamed for raising fungal resistance in clinical settings. Taken together, the success of this project will advance the use of lignocellulose to the benefit of the U.S., especially the rural economy, and advance national environmental goals.

Progress Report
Objective 1: A major technical barrier to production of chemicals and fuels from cellulosic biomass sugars is the difficulty and cost involved in extracting the sugars. The two sources of sugars in biomass are cellulose and hemicellulose. Broad efforts by industry and government labs continue to be devoted to developing better cellulases. We are investigating better enzymes for hemicellulose breakdown (e.g., hemicellulases). Effective hemicellulases are essential for good yields of total sugars because 30% of the plant carbohydrates are made from xylose and because incomplete digestion of the hemicellulose interferes with that of cellulose. The fundamental challenge to making effective hemicellulases is that the backbone for this polymer is decorated with side groups (e.g., organic acids and sugars) that block digestion. In prior work, we have used advanced chemical techniques to identify the most troublesome of these blockers and also identified multiple crude enzyme formulations with the sought-after activity, albeit in very small amounts. This year, enzyme mixtures were fractionated, and the fractions tested to ensure the critical activity was reproducibly present within certain fractions. The next step will be to purify these enzymes and determine fine kinetic properties and protein sequence. We are also working to develop novel saccharides as a functional food ingredient for promoting healthy gut microbes. Objective 2: Itaconic acid is a potential chemical feedstock with utility for production of polymers and solvents. We have assembled a group of world-class fungi that selectively produce itaconic acid at high titers and yields. The major technical barrier to its production from cellulosic biomass is that certain metals and other non-organic material present in biomass suppresses itaconic acid production. The inorganic material includes metals such as sodium, potassium, magnesium, calcium, iron, manganese and aluminum and non-metals such as phosphorus and sulfur. Generally, the inorganic matter is physically or chemically bound to biomass structure. Demineralization of wheat straw was performed by soaking wheat straw with dilute acid at ambient temperature. The leachate was separated from the solid and analyzed for metal content using inductively coupled plasma-optical emission spectrometry. The phosphate and sulfate contents were determined by ion chromatography. The demineralization process was found to be effective in removing inorganic elements from wheat straw. In an unexpected finding, removing the minerals also enhanced the enzymatic hydrolysis efficiency of liquid hot water pretreated wheat straw. Objective 3: Butyric acid is a low-weight organic acid. While 80 thousand metric tons of it are used each year by everything from the perfume to textile industries, it also can be chemically upgraded to fuel ingredient for jet fuel, which is a 23 billion gallon per year market. However, production by fermentation needs further refinement to make it commercially attractive to reach its potential market. An ARS researcher recently isolated a microorganism that has superior butyrate yields compared to other reported strains. Our overall goal is to integrate it into an advanced fermentation system that will allow for very high productivity and titers. In the first year of this project, fermentations were optimized for pH and temperature. Under optimized fermentation conditions of pH and temperature, the microbial strain produced butyric acid at a faster rate. In this process, butyric acid productivity has been improved by over 300% (a factor of 3). Additional work is being undertaken to improve the process. Objective 4: We are working on yeast fermentation processes that convert sugars into single cell oils instead of ethanol; these oils can potentially be used to produce biodiesel and renewable jet fuel. Also, if the sugars used for fermentation are sourced from plant stalks and leaves, the biofuels could be labelled as advanced, which opens up markets and carbon credits. A technical barrier is finding the right yeast that grow and produce lipids when fed unrefined biomass sugars at a high enough concentration to achieve high oil titers. We are developing a systematic method for the rapid screening of oil-producing yeast from the ARS Culture Collection (NRRL, Peoria, Illinois). We have developed a 48 well format to test for growth on concentrated biomass sugars with continuous monitoring of growth. A single-sample analytical method has been developed that allows for determination of total oil production, fatty acid content, total yeast biomass, and sugar utilization. The single-sample method has been broadened to measure content in yeast strains that produce colorants as well. The next step will be applying the method to screen 100 yeast strains from the culture collection. We were also able to successfully evaluate one oil-producing yeast at a commercial development facility using cellulosic sugars prepared at high concentrations in a large continuous reactor. In other research, working with collaborators we discovered that the major enzyme for producing cellulosic sugars can be made to last longer simply be flushing the top of the mixing tank used for biomass digestion with nitrogen gas, which is expected to reduce the costs of sugar production. Objective 5: Fusarium dry rot, incited by Fusarium sambucinum, causes greater potato losses than any other postharvest disease. Over 80% of pathogenic strains are resistant to thiabendazole. There is a need to develop non-azole alternatives since use in agriculture may threaten efficacy of medically useful azoles. Three Pseudomonas sp. antagonistic to F. sambucinum have been discovered and evolved by us to be desiccation tolerant (U.S. patent issued April 2021), a trait allowing a shelf stable product for farmers to conveniently apply. The triculture biological control agent (BCA) protects potatoes against sprouting and several fungal diseases--dry rot, late blight, pink rot, and Pythium leak. Evolution yielded nine desiccation tolerant (DT) biological control agent strain variants (three for each of three parents). Based on single strain growth data, the three-strain combinations most likely to populate together in successful cocultures were predicted. Best combinations were confirmed by testing 12 of 27 possible combinations on synthetic and hydrolysate media and measuring cell yield and equality of co-cultured biocontrol agent (BCA) strain populations. In studies supported by the ARS State Potato Research Program, cocultures produced on biomass hydrolysate media with optimized pH and sugar content performed as well as when produced on synthetic media, achieving 67-81% disease reduction in first year lab and small pilot studies; second year studies are in progress. Additionally, surfactants compatible with the biocontrol agent (some organic) were identified and improved spray coverage when tested in lab and small pilot assays of bioefficacy against dry rot. Rehydration solution components were optimized and found to extend bioefficacy of the dry stored BCA to 4-6 mo. We are evaluating a new osmoprotectant which may further extend BCA dry storage viability to 6-12 mo. Production of a broad-spectrum antifungal agent on low cost renewable substrates and improved dry storage formulations of the biocontrol product are expected to lower costs and expedite application by growers. This new technology benefits agriculture by providing an antifungal microbial alternative to azole chemicals no longer effective due to pathogen resistance and by serving as a potential co-product of a renewable lignocellulose biorefinery with the effect of boosting economic feasibility.

1. Scale up for oil production from yeast. U.S. airlines have committed to reduce carbon dioxide emissions by 50% in 2050. This has created pent up demand for renewable jet fuel products in the 23 billion gallon per year traditional jet fuel market. ARS researchers at Peoria, Illinois, have assembled a collection of yeast that covert agriculture waste to oil, which is expected to be used to manufacture biodiesel or renewable jet fuel. One of these yeasts was recently used for a pilot demonstration for conversion of sugarcane bagasse to yeast oil at a commercial development center. The yeast produced 7.8 g/l of oil. The results demonstrated that the yeast is robust for growth and oil production in a large-scale operation. The project supports agriculture processors interested in converting underutilized agricultural residues to a value-added and green biofuel.

2. Biocontrol agent formula for rapid launch of disease protection on storage potatoes. Each year we consume 110 pounds of potatoes per person. Chemicals have historically been used to protect stored potatoes from decay. However, many of these chemicals have lost efficacy against postharvest diseases due to resistance among the causative pathogens. In addition, the chemicals face increased regulation because they are similar to formulations used to treat clinical fungal infections in humans. As alternatives to chemical disease control, ARS researchers at Peoria, Illinois, have developed microbes (e.g. biological control agents) that, when applied to potatoes, prevent rotting and even delay sprouting. A major commercial barrier of the microbe is shelf-life because biocontrol requires microbes that can be dried and revived when needed. ARS researchers have developed strains able to survive the drying process and have additionally formulated a special solution which boosts reactivation of the dried microbes, especially after they have been stored over six months. Long shelf-life simplifies supply logistics and is necessary for commercialization. This research was partially funded by the ARS-State Partnership Potato Research Program to address a critical gap in the disease protection of the U.S. potato crop.

Review Publications
Hollingshead, A.K., Olsen, N.L., Thornton, M., Miller, J., Schisler, D.A., Slininger, P.J. 2020. Evaluation of biological control agents and conventional products for post-harvest application on potato (Solanum tuberosum L.) to manage leak. American Journal of Potato Research. 97:477-488.
Ispirli, H., Bowman, M.J., Skory, C.D., Dertli, E. 2021. Synthesis and characterization of Bifidogenic raffinose-derived oligosaccharides via acceptor reactions of glucansucrase E81. LWT - Food Science and Technology. 147. Article 111525.
Kenar, J.A., Felker, F.C., Singh, M., Byars, J.A., Berhow, M.A., Bowman, M.J., Moser, J.K. 2020. Comparison of composition and physical properties of soluble and insoluble navy bean flour components after jet-cooking, soaking, and cooking. LWT - Food Science and Technology. 130. Article 109765.
Hay, W.T., McCormick, S.P., Hojilla-Evangelista, M.P., Bowman, M.J., Dunn, R.O., Teresi, J.M., Berhow, M.A., Vaughan, M.M. 2020. Changes in wheat nutritional content at elevated [CO2] alter Fusarium graminearum growth and mycotoxin production on grain. Journal of Agricultural and Food Chemistry. 68(23):6297-6307.
Saha, B.C., Kennedy, G.J. 2020. Optimization of xylitol production from xylose by a novel arabitol limited co-producing Barnettozyma populi NRRL Y-12728. Preparative Biochemistry and Biotechnology.
Liu, S., Skory, C.D., Qureshi, N. 2020. Ethanol tolerance assessment in recombinant E. coli of ethanol responsive genes from Lactobacillus buchneri NRRL B-30929. World Journal of Microbiology and Biotechnology. 36. Article 179.
Qureshi, N., Lin, X., Liu, S., Saha, B.C., Mariano, A.P., Polaina, J., Ezeji, T.C., Friedl, A., Maddox, I.S., Klasson, K.T., Dien, B.S., Singh, V. 2020. Global view of biofuel butanol and economics of its production by fermentation from sweet sorghum bagasse, food waste, and yellow top presscake: Application of novel technologies. Fermentation. 6(2). Article 58.
Cheng, M., Kadhum, H., Murthy, G., Dien, B.S., Singh, V. 2020. High solids loading biorefinery for the production of cellulosic sugars from bioenergy sorghum. Bioresource Technology. 318. Article 124051.
Cheng, M., Sun, L., Jin, Y., Dien, B.S., Singh, V. 2020. Production of xylose enriched hydrolysate from bioenergy sorghum and its conversion to ß-carotene using an engineered Saccharomyces cerevisiae. Bioresource Technology. 308. Article 123275.
Dos Santos, A.C.F., Ximenes, E., Thompson, D.N., Ray, A.E., Szeto, R., Erk, K., Dien, B.S., Ladisch, M.R. 2020. Effect of using a nitrogen atmosphere on enzyme hydrolysis at high corn stover loadings in an agitated reactor. Biotechnology Progress. 36(6). Article e3059.
Slininger, P.J., Cote, G.L., Shea-Andersh, M.A., Dien, B.S., Skory, C.D. 2020. Application of isomelezitose as an osmoprotectant for biological control agent preservation during drying and storage. Biocontrol Science and Technology. 31(2):132-152.
Jia, Y., Kumar, D., Moser, J.K., Dien, B.S., Singh, V. 2020. Recoveries of oil and hydrolyzed sugars from corn germ meal by hydrothermal pretreatment: a model feedstock for lipid-producing energy crops. Energies. 13(22). Article 6022.
Liu, S., Qureshi, N., Bischoff, K., Darie, C.C. 2021. Proteomic analysis identifies dysregulated proteins in butanol-tolerant gram-positive Lactobacillus mucosae BR0713-33. ACS Omega. 6(5):4034-4043.
Saha, B.C., Kennedy, G.J. 2020. Production of xylitol from mixed sugars of xylose and arabinose without co-producing arabitol. Biocatalysis and Agricultural Biotechnology. 29. Article 101786.
Singh, M., Bowman, M.J., Berhow, M.A., Price, N.P., Liu, S.X. 2021. Application of near infrared spectroscopy for determination of relationship between crop year, maturity group, location, and carbohydrate composition in soybeans. Crop Science. 61(4): 2409-2422.