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

Location: Bioenergy Research

2019 Annual Report

Objectives
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.

Approach
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. cerevisiae 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 production process from one L to 100 L. 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
The overall goal of this project is to develop commercially targeted, integrated bioprocess technologies for production of platform chemicals (triacetic acid lactose, xylitol and itaconic acid) and advanced biofuel (butanol) from lignocellulosic feedstocks. The project emphasizes microbiologically based approaches to overcome technical constraints that impede industrial applications. It addresses and facilitates the elimination of microbial and fermentation related challenges associated with the production of platform chemicals and advanced biofuels from lignocellulose based feedstocks. Significant progress was made on all objectives, all of which fall under National Program 306, Component 3: Biorefining. Specific examples of progress in FY 2019 include: Objective 1: The genome of an inhibitor-tolerant fungus was previously sequenced and analyzed. The fungus, discovered by an ARS scientist in Peoria, Illinois, is able to consume the major inhibitors in lignocellulosic hydrolysates. Using the genome and gene expression data, ARS scientists identified new sugar transporters for cell uptake of xylose, a biomass-derived sugar. New transporters were tested for their ability to increase uptake of xylose in Saccharomyces yeasts. Two of the newly discovered transporters showed xylose transport ability. From three new classes of promoters that function at different phases of cell metabolism, one promoter was previously shown to increase production of a platform chemical (triacetic acid lactone). Gene expression from the promoters was further characterized, showing that increased production was due to more efficient timing of enzyme expression to achieve maximum activity of the enzyme. A tunable set of xylose inducible gene expression tools was further developed this year. The toolset was characterized to analyze xylose-induced gene expression throughout a batch culture. Stable gene expression was observed throughout the cell growth phase of the culture, after which, gene expression started to decrease. It was also shown that extremely low levels of xylose were needed to fully induced gene expression, indicating that xylose could be used as an inexpensive inducer at industrial scale. Objective 2: Itaconic acid is one of the 12 identified building block platform chemicals that can be produced by fermentation. We made substantial progress in removing the specific components of wheat straw hydrolyzate that prevent its production using an itaconic acid producing fungus. The levels of the inhibitory components removed were also determined, providing information needed to develop a commercial process using the fungus. In addition to typical fermentation inhibitory compounds, a metal (manganese) present in wheat straw hydrolyzate was found to be a strong inhibitor of itaconic acid production. A novel medium was then developed for production of itaconic acid which alleviated the strong inhibitory effect of manganese. The fungus was able to tolerate manganese very well in the newly developed medium for efficient production of itaconic acid. Egg shell powder, a food industrial waste, was used successfully as a detoxifying and buffering agent for efficient production of polymalic acid by a fungal strain from barley straw hydrolyzate. Polymalic acid and its component L-malic acid have a wide range of applications in the food, pharmaceutical, agriculture and chemical industries. Objective 3: Coniochaeta ligniaria strain C8100 does not grow on xylose, instead converting xylose to xylitol which is secreted. Culture conditions were investigated to identify important factors for xylitol production. Appropriate temperature and pH ranges were identified and xylitol production was measured both in culture medium and in sugars obtained by hydrolysis of corn stover. The addition of an aromatic chemical increased xylitol production. The xylose reductase gene from another fungus was cloned into the strain and increased yield by 20% in rich medium and 11% in corn stover hydrolysate. Objective 4: The production of butanol requires 3 operations, namely feedstock (sweet sorghum bagasse) hydrolysis to sugars, fermentation of sugars to butanol, and butanol (product) recovery from the fermentation broth. During the prior years we developed two processes: Process 1, where 2 operations were combined; and Process 2, where three operations were combined. Process 1 involves separate hydrolysis, combined with fermentation and product recovery. Process 2 involves simultaneous saccharification, fermentation, and product recovery. For these two processes, the cost of butanol production from sweet sorghum bagasse was estimated. For both processes, economic analysis was performed for a plant capacity of 100 thousand tons of butanol production per year. The cost of production of butanol was projected to be $3.48 per gallon for Process 1. Sweet sorghum bagasse cost was considered to be $50 per ton. The production cost is affected by feedstock price, enzymes (to hydrolyze feedstock), fermentation and recovery process used. For this process, the total cost of feedstock, enzymes, and other chemicals was $46.6 million per year. The utilities and other operating costs were at $50.1, and $46.3 million per year, respectively. For Process 2 in which all three operations were combined, the production cost was estimated to be $3.28 per gallon. In this process, the total cost of feedstock, enzymes and other chemicals was $46.6 million per year. Utilities were at $50.1 million per year, and the other operating costs were $43.4 million per year. The operating cost was reduced by $3 million per year. Effect of sweet sorghum bagasse price on butanol production cost was also projected. If sweet sorghum bagasse price is reduced to $30 per ton, butanol production cost can be reduced to $2.89 per gallon using Process 2.

Accomplishments
1. Efficient production of itaconic acid by relieving the strong inhibitory effect of a metal. Itaconic acid (a building block platform chemical with a variety of industrial 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, found that a specific metal typically present in agricultural residues inhibits the itaconic acid production by the fungus completely. A novel growth medium was then developed for production of itaconic acid which relieved the strong inhibitory effect of the metal. The fungus was able to tolerate the metal very well in the newly developed medium. This new medium is expected to perform well for production of itaconic acid in the presence of the metal.

2. Production of xylitol by a novel inhibitor tolerant microbe. Production of xylitol by a novel inhibitor-tolerant microbe. Xylitol is a naturally-occurring sweetener that has 40% fewer calories than table sugar and has been shown to improve dental health and prevent ear infections. These desirable traits support use of xylitol in pharmaceutical and personal-care products, and as an alternative sweetener in gums and mints. Xylitol is difficult to extract from natural sources and, because the current chemical method of production has high energy and cost demands, a biological route to xylitol is desirable. ARS scientists in Peoria, Illinois, measured the effect of several factors on xylitol production using a microbe that makes xylitol from a sugar, xylose, found in biomass. The microbe has intrinsic resistance to inhibitors of the types encountered in converting biomass to fuels and chemicals. Production of xylitol from fibrous biomass would be a new use for agricultural residues that are typically viewed as low-value.

Review Publications
Jin, Q., Qureshi, N., Wang, H., Huang, H. 2019. Acetone-butanol-ethanol (ABE) fermentation of soluble and hydrolyzed sugars in apple pomace by Clostridium beijerinckii P260. Fuel. 244:536-544. https://doi.org/10.1016/j.fuel.2019.01.177.
Yegin, S., Saha, B.C., Kennedy, G.J., Leathers, T. 2019. Valorization of egg shell as a detoxifying and buffering agent for efficient polymalic acid production by Aureobasidium pullulans NRRL Y–2311–1 from barley straw hydrolysate. Bioresource Technology. 278: 130-137. https://doi.org/10.1016/j.biortech.2018.12.119.
Cheng, C., Tang, R., Xiong, L., Hector, R.E., Bai, F., Zhao, X. 2018. Association of improved oxidative stress tolerance and alleviation of glucose repression with superior xylose-utilization capability by a natural isolate of Saccharomyces cerevisiae. Biotechnology for Biofuels. 11:28. https://doi.org/10.1186/s13068-018-1018-y.
Qureshi, N., Saha, B.C., Klasson, K.T., Liu, S. 2018. Butanol production from sweet sorghum bagasse with high solids content: Part I – comparison of liquid hot water pretreatment with dilute sulfuric acid. Biotechnology Progress. 34(4):960-966. https://doi.org/10.1002/btpr.2639
Qureshi, N., Saha, B.C., Klasson, K.T., Liu, S. 2018. High solid fed-batch butanol fermentation with simultaneous product recovery: Part II - process integration. Biotechnology Progress. 34(4):967-972. https://doi.org/10.1002/btpr.2643
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. https://doi.org/10.1007/s12010-018-2831-2
Mertens, J.A., Kelly, A., Hector, R.E. 2018. Screening for inhibitor tolerant Saccharomyces cerevisiae strains from diverse environments for use as platform strains for production of fuels and chemicals from biomass. Bioresource Technology. 3:154-161. https://doi.org/10.1016/j.biteb.2018.07.006.
Quarterman, J.C., Slininger, P.J., Hector, R.E., Dien, B.S. 2018. Engineering Candida phangngensis – an oleaginous yeast from the Yarrowia clade – for enhanced detoxification of lignocellulose-derived inhibitors and lipid overproduction. Federation Of European Microbiological Societies Yeast Research. 18(8):foy102. https://doi.org/10.1093/femsyr/foy102.
Nichols, N.N., Hector, R.E., Frazer, S.E. 2019. Genetic transformation of Coniochaeta sp. 2T2.1, key fungal member of a lignocellulose-degrading microbial consortium. Biology Methods and Protocols. 4:1-5. https://doi.org/10.1093/biomethods/bpz001.
Nichols, N.N., Hector, R.E., Frazer, S.E. 2019. Factors affecting production of xylitol by the furfural-metabolizing fungus Coniochaeta ligniaria. Current Trends in Microbiology. 12: 109-119.
Qureshi, N., Harry-O'Kuru, R.E., Liu, S., Saha, B. 2018. Yellow top (Physaria fendleri) presscake: a novel substrate for butanol production and reduction in environmental pollution. Biotechnology Progress. 35(3):e2767. https://doi.org/10.1002/btpr.2767.
Saha, B.C., Kennedy, G.J. 2019. Phosphate limitation alleviates the inhibitory effect of manganese on itaconic acid production by Aspergillus terreus. Biocatalysis and Agricultural Biotechnology. 18:101016. https://doi.org/10.1016/j.bcab.2019.01.054.
Glover, K.D., Kleinjan, J., Jin, Y., Osborne, L., Ingemansen, J., Turnipseed, E., Dykes, L. 2019. Registration of ‘Focus’ hard red spring wheat. Journal of Plant Registrations. 13(1):63-67. https://doi.org/10.3198/jpr2018.05.0029crc.