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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Bioenergy Research » Research » Research Project #436785

Research Project: Develop Technologies for Production of Platform Chemicals and Advanced Biofuels from Lignocellulosic Feedstocks

Location: Bioenergy Research

2020 Annual Report

Objective 1. Develop platform yeast technology to enable commercial conversion of lignocellulose-derived xylose to chemicals such as triacetic acid lactone (4- hydroxy-6-methyl-2-pyrone). Sub-objective 1.A. Develop an expanded xylose inducible expression system with various expression levels for tunable control of gene expression in Saccharomyces yeasts. Sub-objective 1.B. Generate a xylose-specific transporter that is not significantly inhibited by glucose. Sub-objective 1.C. Engineer industrial Saccharomyces cerevisiae strains to produce triacetic acid lactone from xylose. Objective 2. Develop technologies that enable the commercial production of itaconic acid (methylene succinic acid) from all the carbohydrates in lignocellulosic feedstocks. Sub-objective 2.A. Screen Aspergillus terreus strains for itaconic acid production from xylose and arabinose. Sub-objective 2.B. Adapt the best performing itaconic acid producing A. terreus strain to (i) dilute acid pretreated wheat straw hydrolyzate for inhibitor tolerance and (ii) high concentrations of itaconic acid for itaconic acid tolerance. Sub-objective 2.C. Optimize process parameters for batch production of itaconic acid from dilute acid pretreated wheat straw hydrolyzate by (i) separate hydrolysis and fermentation (SHF) and (ii) simultaneous saccharification and fermentation (SSF). Sub-objective 2.D. Demonstrate the batch itaconic acid production from wheat straw at a pilot scale (100 L). Objective 3. Develop technologies that enable the commercial production of xylitol from lignocellulosic hydrolyzates. Sub-objective 3.A. Optimize xylitol production by Coniochaeta ligniaria C8100, a fungal strain that produces xylitol from xylose but does not grow on xylose. Sub-objective 3.B. Clone and express heterologous xylose reductase in C. ligniaria C8100. Objective 4. Develop technologies that enable the commercial production of butanol from sweet sorghum bagasse. Sub-objective 4.A. Develop efficient pretreatment and enzymatic saccharification processes for generation of fermentable sugars from sweet sorghum bagasse. Sub-objective 4.B. Integrate enzymatic hydrolysis, fermentation and product recovery schemes for conversion of pretreated sweet sorghum bagasse to butanol. Sub-objective 4.C. Evaluate process economics of butanol production from sweet sorghum bagasse.

Hypothesis 1.A. Expressing xylose metabolism genes from tunable xylose-inducible expression modules will improve yield and productivity from both glucose and xylose. Hypothesis 1.B. Enhanced co-utilization of xylose and glucose will increase the xylose utilization rate. Goal 1.C. Integrate the genes required for triacetic acid lactone (TAL) production from xylose into an industrial S. erevisiae strain and produce TAL from lignocellulosic feedstocks. Goal 2.A. Through screening of Aspergillus terreus strains from varied sources, identify a strain that effectively produces itaconic acid from all sugars typically present in a lignocellulosic hydrolyzate. Goal 2.B. Determine if the mixed sugar utilizing and itaconic acid (IA) producing A. terreus strain will be able to tolerate the common fermentation inhibitors typically present in dilute acid hydrolyzates of lignocellulosic feedstock and high concentrations of IA through adaptive evolution. Goal 2.C. Develop efficient SHF or SSF process for itaconic acid production from pretreated lignocellulosic feedstocks. Goal 2.D. Scale up the itaconic acid product. Goal 3.A. Optimize xylitol production from hemicellulosic hydrolyzates by the inhibitor-tolerant fungus C. ligniaria C8100. Hypothesis 3.B. Increasing xylose reductase activity in C. ligniaria strain 8100 will enhance xylitol production from xylose by the recombinant fungal strain. Goal 4.A. Develop an optimized process of sweet sorghum bagasse pretreatment and enzymatic hydrolysis to release sugars that are efficiently fermented to butanol by Clostridium beijerinckii P260. Goal 4.B. Develop an integrated process for butanol production from pretreated sweet sorghum bagasse by combining enzymatic saccharification, fermentation, and product recovery. Goal 4.C. Perform economic analysis of conversion of sweet sorghum bagasse to butanol.

Progress Report
This is a bridging project that was initiated on 7/25/2019, replacing 5010-41000-163-00D and reports current progress on continuing work on the same important objectives and incorporates the final report for that project plan. The overall goal of this bridged project was to develop bioprocess technologies for production of platform chemicals (triacetic acid lactose, xylitol and itaconic acid) and advanced biofuel (butanol) from lignocellulosic feedstocks. Significant progress was made on all objectives, all of which fall under National Program 306, Component 3: Biorefining. Objective 1: Expression systems available for this microorganism rely mainly on constitutive promoter systems, which burden the cell with unnecessary and wasteful use of limited resources to produce high levels of proteins, even when they are not required. To address this problem, a synthetic promoter was created that is activated by xylose. It is a critical enabling technology by allowing for cells to regulate gene expression in response to xylose availability and to more efficiently ferment both glucose and xylose. The USDA was awarded a U.S. patent with foreign rights protection for this technology. To further expand the utility of this expression system, an expanded set of tools was developed that also controls the amount of protein made. The ability to control both the amount of protein produced, and when it is produced, allows fine-tuning of cellular pathways, resulting in more efficient sugar metabolism. These new tunable promoters are being used by other ARS researchers and collaborators. The expression systems described above were used to engineer robust, industrial Brewer’s yeasts that produce triacetic acid lactone (TAL). TAL is a potential platform chemical that can serve as an intermediate for a wide range of synthesized products. The robust industrial yeast strains developed in this project will serve as a starting point for engineering TAL production from renewable agricultural materials. A yeast strain was constructed for identifying and analyzing new xylose transporters to further improve the rate of xylose fermentation. Industrial ethanol yeast strains were engineered to express two differing xylose metabolic pathways. The best fermenting strain was investigated to identify haploid cells from the parent diploid. The newly generated, stable, haploid strains are robust, inhibitor tolerant strains that are easy to modify genetically and grow well on xylose. The best performing strain was engineered to remove seven sugar transporter genes, which resulted in lack of growth on glucose, fructose, and xylose. This fiscal year the yeast strain used for testing sugar transporters was improved further. The gene for galactose uptake (GAL2) was deleted and two of the other transporter deletions were remade to improve the growth of the strain. New transporter genes from xylose-utilizing yeasts were expressed in the test strain and several strains resumed the ability to grow on xylose as the sole carbon source. These novel transporter genes were characterized for their ability to transport additional sugars in addition to xylose. Objective 2: Itaconic acid (IA) is a building block platform chemical that is produced using glucose by fermentation with Aspergillus terreus. However, lignocellulosic biomass has potential to serve as low cost source of sugars. The goal is to identify an A. terreus strain that ferments all sugars typically present in a lignocellulosic hydrolysate, especially pentose sugars. One hundred A. terreus strains obtained from the ARS Culture Collection (NRRL), Peoria, Illinois, were evaluated for IA production from glucose, xylose and arabinose. Twenty strains showed good production of IA from the sugars. Six of these strains were evaluated for production of IA from wheat straw hydrolysate (WSH) and one strain was selected to use for further study. Effective utilization of all softwood derived sugars such as glucose, mannose and galactose by the fungus for production of IA will lower the cost of its production. Twenty fungal strains were evaluated for IA production from mannose and galactose. One strain produced high concentrations of IA from mannose (highest titer reported so far). This strain can be used to produce IA from the sugars derived from woody biomass especially softwood. The fungal strain could not grow and produce IA from WSH even at 100-fold dilution. The effects of acetic acid, furfural, hydroxymethyl furfural, metal ions and three commercial enzyme preparations (cellulase, beta-glucosidase, xylanase) were evaluated for inhibition of IA production from mixed sugars. Acetic acid, furfural, manganese and enzyme preparations were found to be strong inhibitors of IA production. Aeration, pH, other culture conditions were subsequently optimized. There results were used in further studies to lessen inhibition and be used to rate various biomass sources for IA production. Significant progress was made identifying the specific components and inhibitory concentrations of components in WSH that prevent IA production by the fungal strain. One strong inhibitor was found to be manganese. It was found that phosphate limitation in the medium mitigates manganese suppression of IA production. Building on this, a balanced medium was developed using response surface methodology in which the fungal strain was able produce IA with high yield in the presence of manganese. In this fiscal year, the performance of the new medium was verified by comparing the IA production in five different media with or without manganese. The IA production was successfully demonstrated at 100 L scale in stainless steel fermenter with 100 mg manganese per liter. The effect of high glucose concentration up to 160 g per liter on IA production by the fungal strain was studied. Objective 3: Biomass is a source of sugars for production of fuels and chemicals, but the sugars obtained from biomass are contaminated with inhibitory chemicals. Those inhibitors interfere with the microbes that convert biomass sugars to fuel and chemical products. Our group identified a soil fungus that has innate ability to not only tolerate but also metabolize numerous inhibitors. The tolerant strain was engineered to produce xylitol, a sweetener used in candies and personal care products, from a biomass sugar, xylose. Xylitol is a naturally occurring sweetener that has been shown to improve dental health and prevent ear infections while having 40% fewer calories than sucrose (table sugar). Appropriate culture conditions were identified for xylitol production, and a gene (xylose reductase) from another fungus was added into the tolerant microbe and increased xylitol yield by 11% from biomass sugars and by 20% from a rich growth medium. The chemical method of producing xylitol has high energy and cost demands. Xylitol is also difficult to extract from natural sources, and so a biobased method to produce xylitol is desirable. In FY 2020, work to produce xylitol using the inhibitor-resistant fungus was extended to begin to identify genes that are important when the fungus is exposed to fermentation inhibitors. The sugars obtained from biomass are contaminated with inhibitory chemicals; therefore, microbial resistance to the inhibitors is important for a biomass-based process. Objective 4: Sweet sorghum is a commercial sugar crop adapted for growth in northern climates, able to grow in poor soils, and that requires low inputs compared to corn. Bagasse (SSB) is the stem material after soluble sugars have been pressed out. Production of butanol from SSB requires milling, pretreatment, enzymatic hydrolysis, fermentation, and recovery. Pretreatment was performed using only hot water at 190 degrees C. Following pretreatment, SSB was hydrolyzed using commercial enzymes and a sugar concentration over 100 g per liter were achieved with yield of 96%. The hydrolysate was successfully fermented to butanol. Traditionally, the unit operations mentioned above are separated which adds to capital costs. The possibility of reducing costs by combining operations was evaluated. For this reason, two schemes were employed: i) separate hydrolysis followed by combined fermentation and recovery (SHFR); and ii) simultaneous saccharification, fermentation, and recovery (SSFR). In the SHFR process, fermentation and recovery are combined (merging two units), while in the SSFR, enzymatic saccharification, fermentation, and product recovery are combined into one-unit operation (consolidating three units into one). In the first scheme, enzymatic hydrolysis was performed at its optimal temperature and pH (45 degrees C and pH 5.0). In the second scheme, the saccharification was performed at the fermentation temperature (35-37 degrees C) and pH (5.0-5.2). These results were used to estimate the economics for a 170,000 tons of solvent per year. The SHFR process resulted in the butanol production price of $3.54 per gal. Employing SSFR process resulted in the butanol production price of $3.26 per gal. It showed that combining all three units (simultaneous saccharification, fermentation, and recovery) into one unit, reduces the price of butanol by $0.28 per gal. The butanol price was shown to be very sensitive to the cost of SSB. This fiscal year, deep eutectic solvent (DES) was used for pretreatment of corn stover. It can be recycled, reused and is environmentally friendly. DES did not inhibit hydrolytic enzymes as well as butanol producing bacterium. Butanol production from waste yellow top (Physaria fendleri) presscake was studied. It was obtained from yellow top seeds after oil extraction. The presscake was pretreated and enzymatically saccharified to sugars and fermented to butanol with good yields in batch reactors. A cost estimation was performed for butanol production from yellow top.

1. Scale up of itaconic acid production using metal tolerant medium. Itaconic acid (an important building block platform chemical with a variety of applications) is currently produced industrially from glucose by a fungal fermentation. In order to expand the use of itaconic acid, its production cost must be lowered. Waste agricultural residues have the potential to serve as a low-cost source of sugars for itaconic acid production. ARS researchers in Peoria, Illinois, discovered that a specific metal typically present in agricultural residues suppresses itaconic acid production by the fungus completely. A metal tolerant medium that had been developed for production of itaconic acid was successfully demonstrated to be highly effective at 100-liter scale using a typical commercial stainless-steel fermenter. This medium will allow the fungal strain to produce itaconic acid from lignocellulosic biomass without the strong inhibitory effect of the metal. The importance of this work is it allows ethanol and other grain processors to add a higher value chemical to their product portfolio and when agricultural residues are used as the source of sugars it will directly benefit corn, sorghum, and wheat farmers. National Corn Growers Association, National Corn Refiners Association, National Sorghum Producers and Association of Wheat Growers will potentially benefit from this research.

2. Waste yellow top presscake as a low value feedstock for biofuel butanol production. Yellow top (Physaria fendleri) is a plant that belongs to the mustard family. The seed of the plant is rich in oil that can replace imported castor oil, which is extracted from a seed that is also the source of the poison ricin. Economics require finding a use for the presscake left after extraction of the oil. ARS researchers in Peoria, Illinois, were able to utilize waste pressed cake to successfully produce sugars upon pretreatment and enzymatic hydrolysis. The sugars were successfully fermented to acetone-butanol-ethanol (ABE). Butanol is used as a fuel and chemical. Conversion of waste yellow top presscake to butanol represents a new sustainable source of this chemical/biofuel and improves the economics of a safe replacement for castor oil. Production of butanol from waste yellow top presscake would benefit the transportation industry and the farmers who grow yellow top.

3. A tool for improving yeast for fuel ethanol production. Xylose is an abundant sugar naturally present in most agricultural materials. For the past 30 years, scientists have been working to develop Brewer’s yeast that can ferment this sugar in order to make it economically feasible to produce ethanol from unutilized crop residues (such as corn stalks and leaves). While successful, the rate of fermentation is still low by industrial standards. It is thought that the key to speeding up fermentation is to get xylose into the yeast cell faster. Transport of sugars is accomplished by proteins referred to as transporters. ARS researchers in Peoria, Illinois, have developed a yeast that can be used to discover new xylose transporter proteins. The yeast has been made unable to grow on xylose by eliminating eight genes. Now when transporter genes are put into the yeast, xylose transporters can be conveniently identified as those that allow for growth on xylose. This yeast has already been used to discover two novel xylose transporters. As an example of the potential impact, improving the rate of fermentation rate by 10% will allow a 40 million gallon per year ethanol plant to produce four million extra gallons. Increasing fuel ethanol production directly benefits the farmers by creating demand for their unused agricultural residues.

4. Identification of genes for degrading plant biomass. Renewable conversion of plants to products begins with breaking down the fibers in plant material. In nature, fungi do the heavy lifting for biomass breakdown. ARS scientists in Peoria, Illinois, collaborated with the Joint Genome Institute and scientists from Colombia and The Netherlands to decode the genome of a unique fungus for that natural process. This is the first in-depth genome/transcriptome analysis in an understudied group of fungi with powerful machinery for breaking down plant fibers. More than 50 new genes related to biomass breakdown were identified. This fungus has an arsenal of genes and enzymes needed to break down biomass fibers. The genes and enzymes can be “borrowed” to break down plant polymers and make value-added fuels and chemicals. This research and these results will benefit producers of renewable products, who seek effective enzymes to deconstruct the fibers in plant biomass.

Review Publications
Saha, B.C., Kennedy, G.J. 2019. Efficient itaconic acid production by Aspergillus terreus –Overcoming the strong inhibitory effect of Manganese. Biotechnology Progress. 36(2):e2939.
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.
Mondo, S.J., Jimenez, D.J., Hector, R.E., Lipzen, A., Yan, M., Labutti, K., Barry, K., Dirk Van Elsas, J., Grigoriev, I.V., Nichols, N.N. 2019. Genome expansion by allopolyploidization in the fungal strain Coniochaeta 2T2.1 and its exceptional lignocellulolytic machinery. Biotechnology for Biofuels. 12:229.
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.
Yegin, S., Saha, B.C., Kennedy, G.J., Berhow, M.A., Vermillion, K. 2020. Efficient bioconversion of waste bread into 2-keto-D-gluconic acid by Pseudomonas reptilivora NRRL B-6. Biomass Conversion and Biorefinery. 10(2):545-553.
Mariano, A.P., Braz, D.S., Venturelli, H.C., Qureshi, N. 2020. In Vertes, A. A, Green energy to Sustainability: Strategies for global industries. John Wiley & Sons Ltd., Hoboken, NJ, USA. Clostridia and process engineering for energy generation. p.239-267.
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.