Location: Bioenergy Research
2017 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 lactone, 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 four sub-objectives, all of which fall under National Program 213, Component I: Biochemical Conversion.
Under Sub-objective 1.A, initial versions of the construct to be used for identifying new xylose transporters (per Sub-objective 1.B) leaked expression of the marker gene, making it impossible to determine if a transporter improved xylose uptake into the cell. New versions of the marker were developed last fiscal year that show significant growth differences between repressed and induced conditions. Further modifications were made this year to the marker and strain, and growth conditions for testing new xylose transporters using this system have been identified.
To facilitate the identification of novel xylose transporters, an alternative test strain is being developed based on the xylose-fermenting haploid yeast strain we developed. During the last fiscal year, genes associated with hexose transport were deleted and this fiscal year genes responsible for uptake were deleted. The final strain is being used as the parent for expressing and testing new transporters. Putative transporters from recently identified xylose-fermenting yeast strains were identified and engineered for expression in the test strain. Two transporters from Spathaspora xylofermentans, when expressed in the parent test strain, resulted in growth on xylose medium. The parent strain was unable to grow using xylose. These new transporters are being evaluated for use to enhance xylose utilization for the production of bioethanol.
An ethanol yeast strain which displays good performance under inhibitory conditions was evaluated further to identify a genetic component for ethanol tolerance, acetic acid tolerance, furfural tolerance, and carbon source utilization in this strain. Forty haploid strains derived from ten starting cells are being evaluated for tolerance to the compounds mentioned above. Identification of the best performing strain selects for strains carrying genes with beneficial mutations that enhance the utilization of biomass-derived xylose.
Under Sub-objective 2.C, significant progress was made toward itaconic acid (a building block platform chemical with wide applications for the manufacture of various synthetic resins, coatings, polymers and clear plastics) production from lignocellulosic hydrolyzates by a fungal strain. The fungal strain could not grow and produce itaconic acid from dilute acid pretreated and enzymatically saccharified wheat straw hydrolyzate. The hydrolyzate contained significant quantities of a compound at a concentration that was totally inhibitory to itaconic acid production by the fungus. The effects of other compounds present in the hydrolyzate on growth and itaconic acid production by the strain were investigated. Several factors such as pH, temperature and aeration affecting the production of itaconic acid by the fungal strain were investigated. A convenient and reproducible microtiter plate based method for fermentative production of itaconic acid was developed which will greatly aid in screening, developing mutants and optimizing various parameters for itaconic acid production by the fungal strain. One suitable fungal strain was obtained by evaluating twenty fungal strains for production of itaconic acid from mannose, a major sugar component of woody biomass. This strain can be used to produce itaconic acid from the sugars derived from woody biomass especially softwood.
Under Sub-objective 3.B, the effects of several factors including temperature and pH on the production of xylitol (a naturally occurring sweetener that has 40% fewer calories than table sugar and has been shown to improve dental health and prevent ear infections) from biomass derived sugar xylose by a fungal mutant strain were investigated. Engineering this strain to carry an extra gene increased the xylitol yield by 11-22%. The fungus withstands inhibitory compounds that are commonly found in sugars obtained from fibrous biomass and thus has useful properties for biomass utilization. This inhibitor-tolerant strain produced from 0.34 to 0.71 g xylitol per g of xylose.
Under Sub-objective 4.B, butanol (a superior biofuel than ethanol per gallon basis and has excellent gasoline blending properties) was produced from sweet sorghum bagasse at high solid loading. Sweet sorghum bagasse at 25% solid loading was first pretreated with liquid hot water at a high temperature followed by two stage enzymatic hydrolysis using commercial enzyme preparations. The first stage hydrolyzate (hydrolyzate I) contained about 90 g sugars per liter. The left over residue was then subjected to second stage enzymatic hydrolysis which yielded about half of the sugars obtained in first stage hydrolysis (hydrolyzate II). The fermentation of hydrolyzate II by an acetone butanol ethanol (ABE) producing anaerobic bacterium was started with simultaneous removal of ABE by energy efficient vacuum technique. Then the hydrolyzate I was added in a fed-batch mode to the fermentation vessel, and the fermentation and simultaneous recovery of ABE were continued until all sugars were utilized by the bacterium. Simultaneous removal of ABE by vacuum greatly helped to alleviate the strong inhibitory effects of ABE to the culture. In this process an ABE yield of 0.39 g per g sugar with high productivity was achieved.
Accomplishments
1. Improved production of butanol to benefits farmers. Butanol is an advanced biofuel that packs 30% more energy than ethanol on per gallon basis and has excellent blending properties with gasoline. Agricultural Research Service scientists in Peoria, Illinois, developed an integrated fermentation and simultaneous product recovery process that enables 18% more production of butanol from sweet sorghum bagasse that will benefit US farmers, biofuel, and transportation industries.
2. Microtiter plate as microbioreactors for production of itaconic acid. Itaconic acid is a building block platform chemical which is currently produced industrially from corn-derived glucose by fermentation with a fungus. Lignocellulosic biomass has the potential to serve as a low cost source of sugars for production of itaconic acid; however, the fungus could not grow and produce itaconic acid from lignocellulosic biomass hydrolyzates. Agricultural Research Service scientists in Peoria, Illinois, have developed a micro-scale fermentation method for production of itaconic acid in microtiter plate microbioreactors. The new technique is very useful as a convenient, reliable and much cheaper way to investigate the reasons for inhibition of growth and itaconic acid production by the fungus and greatly aid in developing mutant strains, screening and optimization of itaconic acid production.
3. Itaconic acid production from mannose, a sugar component of woody biomass. Itaconic acid has gained importance as a fully sustainable building block platform chemical for wide applications for the manufacture of various synthetic resins, coatings, polymers and clear plastics. It is currently produced industrially from corn-derived glucose by fermentation with a fungus. However, the production cost of itaconic acid must be lowered in order to expand its market. Agricultural Research Service scientists in Peoria, Illinois, found a fungal strain that can be used for production of itaconic acid from mannose in good yield. This fungal strain can be used in woody biomass conversion to itaconic acid. Efficient utilization of mannose together with glucose will lower the production cost of itaconic acid from woody biomass.
Review Publications
Saha, B.C., Kennedy, G.J., Qureshi, N., Cotta, M.A. 2017. Biological pretreatment of corn stover with Phlebia brevispora NRRL-13108 for enhanced enzymatic hydrolysis and efficient ethanol production. Biotechnology Progress. 33(2):365-374.
Saha, B.C. 2017. Emerging biotechnologies for production of itaconic acid and its applications as a platform chemical. Journal of Industrial Microbiology and Biotechnology. 44(2): 303-315. doi: 10.1007/s10295-016-1878-8.
Hector, R.E., Mertens, J.A. 2017. A synthetic hybrid promoter for xylose-regulated control of gene expression in Saccharomyces yeasts. Molecular Biotechnology. 59(1):24-33. doi: 10.1007/s12033-016-9991-5.
Solana, M., Qureshi, N., Bertucco, A., Eller, F. 2016. Recovery of butanol by counter-current carbon dioxide fractionation with its potential application to butanol fermentation. Materials. 9(7):530-540.
Jimenez, D.J., Hector, R.E., Riley, R., Lipzen, A., Kuo, R.C., Amirebrahimi, M., Barry, K.W., Grigoriev, I.V., Dirk van Elsas, J., Nichols, N.N. 2017. Draft genome sequence of Coniochaeta ligniaria NRRL 30616, a lignocellulolytic fungus for bioabatement of inhibitors in plant biomass hydrolysates. Genome Announcements. 5(4):e01476-16. doi: 10.1128/genomeA.01476-16.
Qureshi, N., Liu, S., Hughes, S., Palmquist, D., Dien, B., Saha, B. 2016. Cellulosic butanol (ABE) biofuel production from sweet sorghum bagasse (SSB): Impact of hot water pretreatment and solid loadings on fermentation employing Clostridium beijerinckii P260. BioEnergy Research. 9(4):1167-1179. doi: 10.1007/s12155-016-9761-z.
Liu, S., Qureshi, N., Hughes, S.R. 2017. Progress and perspectives on improving butanol tolerance. World Journal of Microbiology and Biotechnology 33(3):51. doi: 10.1007/s11274-017-2220-y.
Hughes, S.R., Qureshi, N., Lopez-Nunez, J., Jones, M.A., Jarodsky, J.M., Galindo-Leva, L.A., Lindquist, M.R. 2017. Utilization of inulin-containing waste in industrial fermentations to produce biofuels and bio-based chemicals. World Journal of Microbiology and Biotechnology. 33(4):78. doi:10.1007/s11274-017-2241-6.
Saha, B.C., Kennedy, G.J., Qureshi, N., Bowman, M.J. 2017. Production of itaconic acid from pentose sugars by Aspergillus terreus. Biotechnology Progress. 33(4): 1059-1067. doi: 10.1002/btpr.2485.
