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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Bioenergy Research » Research » Research Project #436771

Research Project: Technologies for Improving Process Efficiencies in Biomass Refineries

Location: Bioenergy Research

2020 Annual Report

Objective 1: In collaboration with ARS plant production laboratories, identify agronomic practices that maximize the value to biorefiners of lignocellulosic feedstocks. Objective 2: Develop commercially-viable technologies to improve the commercial production of fermentable sugars from arabinoxylan in lignocellulosic biomass. Subobjective 2.A. Identification and subsequent characterization of glycosidic bonds that occur singly or in patterns that are unrecognized by arabinoxylan carbohydrases. Sub-objective 2.B. Characterize kinetic variation of soluble xylan products released from hydrothermal and acid catalyzed pretreatments. Sub-objective 2.C. Identify key and highly active enzymes for the hydrolysis of natural substrates that compose ß-xylan: a-L-arabinofuranosidases acting on arabinoxylan, aglucuronidases acting on uronoxylan, and ß-xylosidases acting on oligoxylans. Objective 3: Develop technologies to manage hydrolyzate inhibitors in commercialscale second generation biorefineries, including biorefineries utilizing significant water recycling. Sub-objective 3.A. Investigate multiple paths to engineering furan aldehyde tolerance in Saccharomyces cerevisiae for purpose of developing a platform biocatalyst. Sub-objective 3.B. Use biological inhibitor abatement to facilitate water recycling in conversion of biomass lignocellulose hydrolyzates.

Goal 1. Demonstrate that agronomic decisions directly affect biomass conversion yields to sugars and biofuels by changes in cell wall structure and composition. Goal 2.A. Show that plant cell wall xylan contains conserved glycosidic linkages across candidate biomass sources that are not hydrolyzed by commercially available enzymes. Goal 2.B. Establish that bimodal kinetic hydrolysis of xylan release, frequently observed for acidic and hydrothermal treatments is a result of compositional variation within the xylan structure. Goal 2.C. Discover key and highly active accessory enzymes for hydrolysis of heteroxylans by using activity on native substrates as a guide. Hypothesis 3.A. Application of multiple molecular methods, including increased expression of transcriptional regulators and engineering of a catabolic pathway, will yield yeast strains with increased tolerance to furan inhibitors. Goal 3.B. Determine the ability of an inhibitor-tolerant fungal strain that metabolizes fermentation inhibitors to facilitate reuse of process water streams.

Progress Report
This is a bridging project that was initiated on 7/25/2019, replacing 5010-41000-161-00D and reports current progress on continuing work on the same important objectives and incorporates the final report for that project plan. This is the final report for this project which terminates on 09/30/2020. See the report for the replacement project, 5010-41000-189-00D, “New Bioproducts for Advanced Biorefineries.” This project plan had the overarching goal to improve processing of lignocellulosic biomass for production of sugars and biofuels. The targeted feedstocks are agricultural residues (such as corn stover) and perennial grasses managed as bioenergy crops. Lignocellulosic biomass includes the leaves and stems of plants and is of interest as a feedstock for fuel and chemicals because it contains 60-70% carbohydrates. There is enough biomass to manufacture billions of gallons of biofuels or chemical intermediates without pulling land away from production crops and, thereby, promoting the rural economy. The carbohydrates are converted to fermentable sugars using cellulases and related enzymes. As the plant fibers serve structural roles, the biomass needs to be pretreated with a variety of chemical and/or thermal steps in order to prepare it for enzymatic conversion. This adds challenges for the fermentation step because side-products generated by pretreatment can stall the fermentation. The research targets were: (a) demonstration of ARS bioenergy crops for biofuel production; (b) strain development of robust yeast; (c) basic knowledge to improve enzymatic sugar yields; and (d) a novel technology to allow for recycling process water. This is the final report for this project plan and a complete summary. Bioenergy crops: perennials can supply 171 teragram/yr of biomass and when used to manufacture transportation fuel is 94% less carbon intensive than petroleum. ARS plant researchers have developed numerous bioenergy grasses. Yet, the following questions have not been addressed: how much ethanol can be produced per hectare, how does agronomics affect biofuel yields, and what is the relative importance of quality and production traits? Major results are summarized below. Napier grass (NG) grown in the Southeast: NG was field grown for several seasons under three management conditions. Biomass was processed using ARS inventions for ammonia-based pretreatment and a yeast created for fermentation of biomass sugars. It was demonstrated that a properly managed Napier grass field gives twice the ethanol yield (8.56 – 11.4 m3/ha) than corn at 200 bu/acre. Switchgrass (SG) grown in the Midwest: ARS plant breeders (Lincoln, Nebraska) have released a new bioenergy SG cultivar named Liberty. Liberty was compared to two widely planted cultivars of SG. SG from multiple seasons and production conditions were pretreated with low moisture ammonium hydroxide, enzymatically hydrolyzed, and the extracted sugars fermented to ethanol. Ethanol yields per hectare were highest for Liberty SG and up to 94% of the expected yield from a corn field harvested at 200 bushels of grain/acre. Pelleting grasses for better transport and storage: transport of baled or ground straw is volume limited, which drives up feedstock costs. Three ARS bioenergy crops were pelletized in a commercial feed mill and evaluated for bulk density and bioconversion to ethanol. It was determined that pelletizing allowed for efficient weight limited transport and did not negatively impact ethanol yields. This year’sNapier grass results were analyzed and published. The work was also broadened to include sweet sorghum and domestic sugarcane bagasse and to continue development on a novel pretreatment reactor/processed termed pressurized ammonia milling (PAM). A method for adapting these biomasses for dilute-acid pretreatment that involves biological removal of soluble sugars is also in development. Microbial strain development: Over the course of this project additional information has been gleaned on how varying inhibitor tolerant Saccharomyces strains (e.g. distillers’ yeast) growth characteristics and perhaps more importantly, genetic background, influence inhibitor tolerance. The importance of genetic background for tolerant strains was demonstrated through haploid strains (one copy each chromosome) derived from a diploid (two copies each chromosome) parental Saccharomyces strain with improved tolerance to lignocellulosic inhibitors possessing varying degrees of tolerance to lignocellulosic inhibitors in growth studies. The genome of this industrially relevant diploid parental strain has been sequenced and deposited into a public database. Haploid strains derived from the tolerant strain will enable the determination of traits involved in lignocellulosic inhibitor tolerance through genome sequencing in future work. These genetic resources are increasingly important to the efficient and cost-effective production of fuels and chemical using agricultural biomass as we have also determined that simple overexpression of enzymes and proteins called transcription factors, that act to increase the overall stress response in yeast, are not sufficient to overcome Saccharomyces yeast delayed onset of growth in the presence of lignocellulosic-derived inhibitors. This year, because overexpression of alcohol/aldehyde dehydrogenase enzymes were found to improve growth of Saccharomyces yeast strains, overexpression of a potential dehydrogenase enzyme isolated from a fungus able to use lignocellulosic inhibitors as an energy source was performed to characterize the enzyme for potential use in overcoming the growth lag in Saccharomyces. Bacterial oxidase enzymes that previously failed to improve growth characteristics of Saccharomyces in the presence of lignocellulosic inhibitors, were placed in Pseudomonas putida to determine if this bacterial species can provide a viable route to production of plastic precursors from a lignocellulosic inhibitor. The screening and characterization of the additional dehydrogenase enzyme and transformed Pseudomonas bacterial strains is ongoing. Process water reutilization: Water consumption is an important consideration for advanced biofuels production. The water footprint of a bioprocessing plant impacts site location, capital costs, and local environmental impact. Present-generation (corn-starch-to-ethanol) facilities directly reuse over 50% of their process water. For next-generation (biomass-to-ethanol) processes, however, the feasibility of recycling process water is unknown. A major obstacle to next-generation bioconversion is the presence of microbial inhibitors. The amount of process water that can be recycled will depend upon the inhibitor load, and re-use of process water will necessitate addressing inhibitors which may be concentrated by water recycle. As a test case, ARS fungus Coniochaeta ligniaria NRRL 30616 was used to biologically abate inhibitors in dilute-acid pretreated corn stover hydrolysate, allowing reuse of a portion of the liquor in subsequent fermentations. The hydrolysate was successfully reused five times in fermentations using ARS recombinant ethanologenic Escherichia coli, and bioabatement also enabled recovery of failed fermentations. Additionally, bioabatement allowed recycling of the spent liquor for use as process water for pretreatment of corn stover. Based upon the experience of the corn ethanol industry, technology for recycling process water will be a commercial necessity. This year, genes involved in inhibitor tolerance were identified and expressed for determination of their function. As part of an international collaboration, a fungus and two bacteria were mixed to break down wheat straw, and more than 50 new genes related to biomass consumption were identified. The fungus produced enzymes to degrade lignin and the bacteria could complement the breakdown of lignin with activities to break down other types of plant fibers. This work establishes a basis to exploit the inhibitor-tolerant phenotype of the fungus and use it as a source of genes and regulators to improve microbes used in biomass-to-chemicals processes. Platform method for formulating better cellulosic process enzymes: up to 40% of the carbohydrates contained in lignocellulose occurs as hemicellulose. Hemicellulose is chemically complex and incompletely characterized, which is a barrier to developing better enzyme mixtures to convert it to fermentable sugars at higher yields. To meet this analytical challenge, mass spectrometry techniques were developed that allowed for rapid structure analysis of short saccharide chains. Isolated xylan (a form of hemicellulose) from 10 different biomass sources were treated with commercial enzymes, and sugar chains that resisted digestion were analyzed for their structure. The dominant products contained an arabinose sugar (with 1-3 linkages), which could be eliminated by adding specific enzymes. A second side group consisting of a xylose-arabinose linkage was also identified, which represents an exciting opportunity because no known enzyme will break this bond. For switchgrass, such an enzyme could improve xylose yields 5-10%. This year, resistant sugar groups containing a different type of linkage (hexuronic acid) were structurally identified. This method represents a jump forward in enzyme development because it allows for specific targeting of enzyme candidate to promote enzyme digestion of hemicelluloses. In other work, ARS researchers addressed a classical phenomenon. When hemicellulose is treated hydrothermally, there is rapid release of xylose saccharide groups followed by a slow release. Fractions were collected during this bimodal time-course and analyzed for their structures. No structure distinct to each fraction was found, lending further credence to the hypothesis that the cause of fast and slow fractions is not determined at the molecular level.

1. Improved brewer’s yeast for advance ethanol production. Yeasts are used to convert sugars extracted from biomass into ethanol for production of advance biofuels. Among the major impediments for fermentation of these sugars are that they are unrefined and contain chemicals that block growth and fermentation by the yeast. However, not all yeasts are equal and some have a better ability to grow than others. The ARS Culture Collection in Peoria, Illinois, is among the greatest depository of Brewer’s yeast. This collection has been used to identify yeast strains with exceptionable ability to ferment unrefined biomass sugars. Furthermore, success has been achieved in laying the groundwork to understand which genes play a role in making the yeast hardy. The identified yeast strains should be directly helpful for ethanol producers interested in manufacturing advance biofuels and isolation of the specific gene(s) and their incorporation into commercial yeast strains could be used to improve their hardiness.

2. Rational method to improve sugar yield from biomass. Advanced biofuels are produced by fermenting sugars extracted from the stems and leaves of agricultural residues and bioenergy crops. Leaves and stems contain two major sources of carbohydrates: cellulose and hemicellulose. Cellulose is conveniently converted to glucose; the same sugar used to make sugarcane and corn ethanol. Hemicellulose is more problematic because it is comprised of a chain of a sugar named xylose. This chain also contains side groups of individual and short chain sugars and acetic acid groups. ARS researchers in Peoria, Illinois, have learned how to identify these side chains of sugars and are using them to formulate better enzyme mixtures. One such side chain discovered this year should enable sugar yields to be increased by up to 5%. This will eventually lead to lower production costs and facilitate processing of agricultural residues and bioenergy crops. It will directly benefit manufacturers of advance biofuels and corn farmers seeking a market for crop residues.

3. Greener way to produce sugars from biomass: agricultural residues and bioenergy crops can supply hundreds of millions of tons of sugars for making chemicals and biofuels. While biomass contains approximately 60% carbohydrates, these are hard to extract as sugars. The traditional method unlocks the sugars by heating biomass fibers in dilute sulfuric acid solution, followed by washing, and using enzymes to complete sugar recovery. This ends up being an expensive process because it generates excess waste and impairs reuse of process water. ARS researchers in Peoria, Illinois, are collaborating with scientists from the University of Illinois, Urbana, Illinois, to substitute water for acid. In the past when hot water was substituted for dilute acid, sugar yields were impaired. However, adding an additional milling step after treating with hot water allowed for these lost sugars to be recovered. Furthermore, the process was performed using industrial sized equipment, which reduces the risk for ethanol producers that wish to adopt the technology. This research promotes use of agriculture feedstocks for biofuels by making available a greener technology for use by current ethanol producers.

4. New co-product to increase value of sorghum: an economic obstacle to producing cellulosic biofuels is the low market price of bioethanol relative to its operating and very high capital costs. ARS researchers in Peoria, Illinois, working with University of Illinois scientists in Urbana, Illinois, solved this problem by developing a new process that allows for extracting glucose and xylose separately. Glucose is conveniently fermented to ethanol. The xylose syrup was concentrated to achieve 66 grams of xylose per liter. The concentrated syrup was next converted to a nutritional product using an engineered Brewer’s yeast. The product was beta-carotene, which is used by our bodies to produce vitamin A. The maximum product titer was 114.5 mg/L. This research will be of interest to those work on commercializing cellulosic biofuels and more generally agriculturally based refiners and farmers looking to find new markets for their crops.

Review Publications
Nichols, N.N., Mertens, J.A., Dien, B.S., Hector, R.E., Frazer, S.E. 2019. Recycle of fermentation process water through mitigation of inhibitors in dilute-acid corn stover hydrolysate. Bioresource Technology. 9:100349.
Hector, R.E., Mertens, J.A., Nichols, N.N. 2019. Development and characterization of vectors for tunable expression of both xylose-regulated and constitutive gene expression in Saccharomyces yeasts. New Biotechnology. 53:16-23.
Jimenez, D., Wang, Y., de Mares, M., Cortes-Tolalpa, L., Mertens, J.A., Hector, R.E., Lin, J., Johnson, J., Lipzen, A., Barry, K., Mondo, S.J., Grigoriev, I.V., Nichols, N.N., Van Elsas, J.D. 2019. Defining the eco-enzymological role of the fungal strain Coniochaeta sp. 2T2.1 in a tripartite lignocellulolytic microbial consortium. FEMS Microbiology Ecology. 96(1). Article fiz186.
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.
Dien, B.S., Anderson, W.F., Cheng, M., Knoll, J.E., Lamb, M., O Bryan, P.J., Singh, V., Sorensen, R.B., Strickland, T.C., Slininger, P.J. 2020. Field productivities of Napier grass for production of sugars and ethanol. ACS Sustainable Chemistry & Engineering. 8(4):2052-2060.
You, J., Johnston, D., Dien, B.S., Singh, V., Engeseth, N.J., Tumbleson, M., Rausch, K.D. 2020. Effects of nitrogenous substances on heat transfer fouling using model thin stillage fluids. Food and Bioproducts Processing. 119:125-132.
Cheng, M., Wang, Z., Dien, B.S., Slininger, P.J., Singh, V. 2019. Economic analysis of cellulosic ethanol production from sugarcane bagasse using a sequential deacetylation, hot water and disk-refining pretreatment. Processes. 7(10): 1-15.
Chen, M.H., Dien, B.S., Lee, D.K., Singh, V. 2019. Sugar production from bioenergy sorghum by using pilot scale continuous hydrothermal pretreatment combined with disk refining. Bioresource Technology. 289:121663.
Jordan, J.H., Easson, M.W., Dien, B., Thompson, S., Condon, B.D. 2019. Extraction and characterization of nanocellulose crystals from cotton gin motes and cotton gin waste. Cellulose. 26(10):5959-5979.
Berhow, M.A., Singh, M., Bowman, M.J., Price, N.P.J., Vaughn, S.F., Liu, S.X. 2020. Quantitative NIR determination of isoflavone and saponin content of ground soybeans. Food Chemistry. 317:126373.
Johnson, E.T., Bowman, M.J., Dunlap, C.A. 2020. Brevibacillus fortis NRS-1210 produces edeines that inhibit the in vitro growth of conidia and chlamydospores of the onion pathogen Fusarium oxysporum f. sp. cepae. Antonie van Leeuwenhoek. 113:973-987.
Saha, B.C., Kennedy, G.J., Bowman, M.J., Qureshi, N., Dunn, R.O. 2018. Factors affecting production of itaconic acid from mixed sugars by Aspergillus terreus. Applied Biochemistry and Biotechnology. 187(2):449-460.
Dunlap, C.A., Bowman, M.J., Rooney, A.P. 2019. Iturinic lipopeptide diversity in the Bacillus subtilis species group – important antifungals for plant disease biocontrol applications. Frontiers in Microbiology. 10:1794.
Dunlap, C.A., Bowman, M.J., Zeigler, D.R. 2020. Promotion of Bacillus subtilis subsp. inaquosorum, Bacillus subtilis subsp. spizizenii and Bacillus subtilis subsp. stercoris to species status. Antonie Van Leeuwenhoek. 113:1-12.
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.