Location: Bioenergy Research
2016 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
Under Sub-objective 1.B, using a strain that was evolutionarily adapted for improved growth on xylose, the complete genome was sequenced and analyzed to determine the genetic changes that result in increased ability to use xylose. A mutation was identified in a gene that is known to play a role in xylose utilization. Experiments were conducted to determine if the newly identified mutation leads to even better xylose utilization than reported literature values.
Initial versions of the construct for identifying new xylose transporters 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 that show significant growth differences between repressed and induced conditions. This new version will allow identification of new xylose transporters.
Last fiscal year, a new xylose-fermenting yeast strain based on a Brazilian ethanol yeast was generated that performed well. During this fiscal year, haploid versions of the strain were created and screened for xylose metabolism. One strain with enhanced xylose metabolism was identified and its mating type was switched in order to create a diploid strain with the xylose fermentation genes integrated on both copies of chromosome 4. These strains were analyzed for xylose fermentation on pure sugars and were dilute acid pretreated and enzymatically saccharified switchgrass hydrolyzate.
To facilitate the identification of novel xylose transporters, a new test strain was developed based on the xylose-fermenting haploid Brazilian yeast strain we developed. This strain will serve as the parent for expressing and testing new transporters. Currently available strains do not metabolize xylose well and make it difficult to evaluate xylose transport. This new strain’s excellent xylose metabolism will allow improved identification of xylose transport.
Under Sub-objective 2.B., a mixed sugar (glucose, xylose, arabinose) utilizing itaconic acid producing Aspergillus NREL1972 strain could not grow and produce itaconic acid using dilute acid, liquid hot water, lime, and alkaline peroxide pretreated and enzymatically hydrolyzed wheat straw hydrolyzates at pH 3.1. Detoxification of the hydrolyzate with a fungal strain and by typical overliming did not improve the itaconic acid production. The Aspergillus strain was mutagenized by exposure to UV light and plated on dilute acid pretreated and enzymatically hydrolyzed wheat straw hydrolyzate gradient plates at pH 3.1. A few distinct colonies from the far right of the gradient plate were evaluated for itaconic acid production. The procedure was repeated several times but with no success so far. The strain was adapted in wheat straw hydrolyzate starting with 5% (v/v) at pH 3.1 and after growth transferred to the next concentration level in increments of 5% (v/v). The mutation and adaptation experiments will continue until a suitable mutant strain is obtained within this fiscal year. The effects of inhibitors and commercial enzyme preparations used for saccharification were evaluated at the level present in the hydrolyzate for inhibition of itaconic acid production from mixed sugars. Many of these were found to be strong inhibitors of itaconic acid production. The screening of UV mutants in gradient plates has been expanded to include tolerance to high concentrations of these inhibitors for production of itaconic acid from mixed sugars.
Under Sub-objective 4.A., production of butanol (a superior biofuel than ethanol with energy content close to gasoline on per gallon basis) from agricultural biomass is characterized by two important factors: i) generation of sugar degradation products during biomass pretreatment which inhibit butanol production; and ii) maximum acetone butanol ethanol (ABE) concentration in a batch reactor is limited to 20 g/L because of product (ABE) toxicity to the microbial culture. Increase in ABE production beyond 20 g/L can be achieved by simultaneous removal of ABE as it is produced (process integration). To reduce generation of inhibitory substances during dilute acid pretreatment so that a detoxification step is not required prior to fermentation while maximizing sugar yield, lignocellulosic biomass should be pretreated under optimized conditions of temperature, acid dose and duration of pretreatment. Sweet sorghum bagasse (86 g/L) was pretreated with dilute sulfuric acid (5.0 g/L) at 160 degrees Celsius for zero min holding time followed by enzymatic saccharification at pH 5.0 and 45 degrees Celsius for 72 h using a cocktail of 3 commercial enzyme (cellulase, ß-glucosidase, xylanase) preparations. The saccharified hydrolyzate contained 53.6 g total sugars (glucose, xylose, arabinose, mannose, and galactose) suggesting that almost complete saccharification occurred. This was followed by fermentation of the hydrolyzate using an ABE producing anaerobic bacterial strain. The culture (bacterial strain) produced 20.4 g total ABE per L with a productivity of 0.42 g/L.h indicating that the hydrolyzate as such was not inhibitory to the culture.
Accomplishments
1. Improved industrial yeast strains for producing bio-ethanol from biomass-derived sugars. Industrial yeasts typically offer improved performance under harsh conditions found in an industrial setting. However, not all industrial yeasts perform well in the presence of additional inhibitors that are generated when producing sugars from biomass feedstocks. A yeast strain from a Brazilian fuel ethanol production facility was found to tolerate these inhibitors. ARS scientists in Peoria, Illinois, engineered the Brazilian yeast strain to express all of the proteins required for converting xylose to ethanol and then identified a strain with excellent performance. When hydrolyzed switchgrass was used as the source of sugars, this strain produced 30% more ethanol than the parent strain. Complete and efficient utilization of all biomass-derived sugars from any feedstock is important to achieve the highest ethanol production. The use of this new strain is expected to decrease the production cost for any process using biomass-derived sugars, thereby increasing profit.
None.
Review Publications
Mariano, A.P., Ezeji, T.C., Qureshi, N. 2015. Butanol production by fermentation: Efficient bioreactors. In: Snyder, S.W., editor. Commercializing Biobased Products: Opportunities, Challenges, Benefits, and Risks, 2015. RSC Publishing, Cambridge, United Kingdom. p. 48-70. doi: 10.1039/9781782622444-00048.
Lindquist, M.R., Lopez-Nunez, J.C., Jones, M.A., Cox, E.J., Pinkleman, R.J., Bang, S.S., Moser, B.R., Jackson, M.A., Iten, L.B., Kurtzman, C.P., Bischoff, K.M., Liu, S., Qureshi, N., Tasaki, K., Rich, J.O., Cotta, M.A., Saha, B.C., Hughes, S.R. 2015. Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates. Applied Microbiology and Biotechnology. 99(22):9723–9743.
Okonkwo, C.C., Azam, M.M., Ezeji, T.C., Qureshi, N. 2016. Enhancing ethanol production from cellulosic sugars using Scheffersomyces (Pichia) stipitis. Bioprocess and Biosystems Engineering. 39(7):1023-1032. doi: 10.1007/s00449-016-1580-2.
Saha, B.C., Qureshi, N., Kennedy, G.J., Cotta, M.A. 2016. Biological pretreatment of corn stover with white-rot fungus for improved enzymatic hydrolysis. International Biodeterioration and Biodegradation. 109:29-35. doi: 10.1016/j.ibiod.2015.12.020.
Liu, S., Skory, C., Qureshi, N., Hughes, S. 2016. The yajC gene from Lactobacillus buchneri and Escherichia coli and its role in ethanol tolerance. Journal of Industrial Microbiology and Biotechnology. 43(4):441-450. doi: 10.1007/s10295-015-1730-6.
Nichols, N.N., Saha, B.C. 2016. Production of xylitol by a Coniochaeta ligniaria strain tolerant of inhibitors and defective in growth on xylose. Biotechnology Progress. 32(3):606-612. doi: 10.1002/btpr.2259.
Galinda-Leva, L.A., Hughes, S.R., Lopez-Nunez, J.C., Jarodsky, J.M., Erickson, A., Lindquist, M.R., Cox, E.J., Bischoff, K.M., Hoecker, E.C., Liu, S., Qureshi, N., Jones, M.A. 2016. Growth, ethanol production, and inulinase activity on various inulin substrates by mutant Kluyveromyces marxianus strains NRRL Y-50798 and NRRL Y-50799. Journal of Industrial Microbiology and Biotechnology. 43(7):927-939. doi: 10.1007/s10295-016-1771-5.
