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United States Department of Agriculture

Agricultural Research Service

Research Project: INDUSTRIALLY ROBUST ENZYMES AND MICROORGANISMS FOR PRODUCTION OF SUGARS AND ETHANOL FROM AGRICULTURAL BIOMASS
2008 Annual Report


1a.Objectives (from AD-416)
Develop improved processes for converting herbaceous biomass to ethanol by incorporating new enzyme and biocatalyst technologies with modern pretreatment strategies. Evaluate potential for converting biomass derived sugars to hydrogen via fermentation.


1b.Approach (from AD-416)
Herbaceous biomass feedstocks will be converted to fermentable sugar mixtures using low-waste pretreatments and novel enzymatic preparations. The generated sugar mixtures will be fermented using recombinant microorganisms specifically engineered for producing ethanol from biomass sugars. Specific steps in this approach include: (1) working with plant breeders to develop cultivars especially suited for low-chemical usage, mild pretreatments, (2) generating new enzyme mixtures using genes recovered and over-expressed from highly active anaerobic fungi, (3) developing bioabatement methods for removing organic chemical that interfere with fermentation and, thereby, increasing the fermentability of the recovered biomass sugars, (4) engineering gram positive bacteria to selectively produce ethanol; members of this group have a long history of use in industrial fermentations, and (5) screen and evaluate hydrogen producing bacteria for capability to co-produce hydrogen from biomass feedstocks.


3.Progress Report
The overall goals of this project are to develop technologies for lowering the cost and increasing the efficiency with which biomass can be converted to ethanol and chemicals. The specific areas targeted for are improving the quality of energy crops for processing to biofuels, developing better enzymes for biomass processing, engineering strains for producing ethanol and lactic acid, and designing biological abatement strategies for selective removal of inhibitory chemicals.

Improving the quality of energy crops is a collaborative effort involving the Agricultural Research Service's (ARS) bioenergy crop research group. We have successfully developed a novel screening assay for measuring relative ethanol yield that is suitable for screening large collections of biomass samples. The assay has been applied to 107 switchgrass samples and has indicated a wide variation in ethanol yield. As part of this effort, an integrated process is being developed for converting energy crops to ethanol.

We have also discovered a biomass-related enzyme that has 10 times the activity of any reported previously. Why this matters: enzyme costs can be lowered for biomass processing by discovering and engineering more efficient enzymes. This enzyme catalyzes an important reaction, conversion of xylan-related polymers to xylose and arabinose, and it can also serve as a model for improving the activity of related enzymes. A detailed structural and kinetic model has been developed for the enzyme and its mechanism elicited with continuing work to understand what makes this particular enzyme so efficient. A patent has been filed, and the enzyme is in the process of being licensed. We have also identified 16 unique and previously unidentified polygalacturonase genes in Rhizopus oryzae. Polygalacturonase aids in the breakdown of pectin, a major component of plant cell walls. Fourteen of the sixteen unique enzymes are highly active and are expected to aide in biomass processing. Finally, we have discovered a new biomass related enzyme in Trichoderma reesei: glucuronic acid esterase enzyme.

Novel strains have been discovered and developed for removal of fermentation inhibitors. Biological removal of inhibitors relies on microorganisms to selectively remove chemicals from the hydrolysate. The microorganism being pursued is capable of removing a wide range of these chemicals from hydrolysates. Effectiveness has been demonstrated for treating corn stover hydrolysates based upon direct analytical measurements of inhibitors and in conjunction to ethanol fermentation. Most recently, we have developed a strain that does not metabolize xylose present in the hydrolysate, thereby increasing sugar yields. Bioabatement of inhibitors is the subject of an ARS patent. This research addresses NP 307, Component I.


4.Accomplishments
1. NEW STRAIN FOR IMPROVING FERMENTABILITY OF BIOMASS SUGARS. Inhibitors arising during conversion of biomass to sugars are impediments to fermentative production of ethanol. A microorganism has been discovered that removes these inhibitory chemicals, rendering the hydrolysates readily fermentable to ethanol. However, this microorganism also metabolizes xylose, which is present in appreciable amounts in the treated hydrolysate. To overcome this, a strain was constructed that does not metabolize xylose. This strain has been demonstrated to improve the fermentability of hydrolysates while retaining the xylose content for improved ethanol yields. This research addresses NP 307, Component I, Problem Area "Ethanol cannot be produced from any agricultural feedstock today at a selling cost competitive with petroleum sources."

2. NEW PRETREATMENT METHOD DEVELOPED FOR SWITCHGRASS. Switchgrass has been viewed as one of the most promising candidates for an energy crop, but more efficient cultivars are needed for conversion to ethanol. Pretreating the biomass to deconstruct the cell wall structure is necessary prior to enzymatic hydrolysis and ethanol fermentation to achieve appreciable product yields. A new alkaline pretreatment method was developed to accomplish this. Glucose and xylose yields are on the order of 80% based upon beginning carbohydrates, and the hydrolysates are readily fermented by Saccharomyces cerevisiae without need for additional processing other than alkaline recovery. This process is envisioned to lower the cost for processing switchgrass to ethanol. This research addresses NP 307, Component I, Problem Area "Ethanol cannot be produced from any agricultural feedstock today at a selling cost competitive with petroleum sources."

3. ISOLATION OF HYDROLYTIC ENZYMES FROM RHIZOPUS ORYZAE. New sources of enzymes are needed that are more efficient at converting biomass into fermentable sugars. The filamentous fungus R. oryzae contains a large and diverse complement of biomass degrading enzymes that could improve the efficacy and efficiency of biomass degradation. Sixteen unique, previously unidentified polygalacturonase genes (polygalacturonase aids in the breakdown of pectin, a major component of plant cell walls) were identified, isolated, expressed, and initial biochemical characterization performed. Fourteen of the sixteen unique enzymes were highly active and provide a unique view to the evolutionary history and development of fungal polygalacturonase enzymes. These enzymes are expected to have value as processing aids in biomass degradation. This research addresses NP 307, Component I, Problem Area "Ethanol cannot be produced from any agricultural feedstock today at a selling cost competitive with petroleum sources."

4. THE BIFUNCTIONAL BETA-D-XYLOSIDASE/ALPHA-L-ARABINOFURANOSIDASE FROM SELENOMONAS RUMINANTIUM IS THE BEST CATALYST KNOWN (KCAT, KCAT/KM) FOR PROMOTING HYDROLYSIS OF 1,4-BETA-D-XYLOOLIGOSACCHARIDES. Highly active biomass conversion enzymes are required for economical and efficient saccharification of agricultural biomass. The title enzyme is attractive for this purpose, but further data is needed to determine the source of its excellent kinetic properties. Functional attributes of the enzyme were determined. Active-site amino acid residues that are involved in substrate distortion and increasing enzyme activity have been identified, active-site amino acid residues that are involved in preferring xylose glycosides over arabinose glycosides have been identified, and enzyme-inhibitor complexes that could be formed in saccharification processes have been demonstrated. Methods employing aminoalcohol inhibitors for isolating the binding activities of the two sugar binding sites of the enzyme’s active site have been developed, and the X-ray structure of the enzyme in complex with an aminoalcohol inhibitor was completed and reported. Because of its high catalytic activity and ease of production, the title enzyme is estimated to cost less than one cent per gallon of ethanol produced in processes that employ enzymatic saccharification of biomass. This research addresses NP 307, Component I, Problem Area "Ethanol cannot be produced from any agricultural feedstock today at a selling cost competitive with petroleum sources."


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of the New MTAs (providing only)5
Number of New Patent Applications Filed1
Number of Non-Peer Reviewed Presentations and Proceedings7
Number of Newspaper Articles and Other Presentations for Non-Science Audiences1
Number of Other Technology Transfer3

Review Publications
Dien, B.S., Ximenes, E.A., O'Bryan, P.J., Moniruzzaman, M., Li, X., Balan, V., Dale, B., Cotta, M.A. 2008. Enzyme characterization for hydrolysis of AFEX and liquid hot-water pretreated distillers' grains and their conversion to ethanol. Bioresource Technology. 99(12):5216-5225.

Anderson, W.F., Dien, B.S., Brandon, S.K., Peterson, J. 2008. Assessment of bermudagrass and bunch grasses as feedstock for conversion to ethanol. Applied Biochemistry and Biotechnology. 145(1-3):13-21.

Srinivasan, R., Dien, B.S., Rausch, K.D., Tumbleson, M.E., Singh, V. 2007. Fiber Separated from Distillers Dried Grains with Solubles as a Feedstock for Ethanol Production. Cereal Chemistry. 84(6):563-566.

Kim, Y., Mosier, N.S., Hendrickson, R., Ezeji, T., Blaschek, H., Dien, B.S., Cotta, M.A., Dale, B., Ladisch, M.R. 2008. Composition of corn dry-grind ethanol by-products: DDGS, wet cake, and thin stillage. Bioresource Technology. 99(12):5165-5176.

Nichols, N.N., Sharma, L.N., Mowery, R.A., Chambliss, C.K., Van Walsum, G.P., Dien, B.S., Iten, L.B. 2008. Fungal metabolism of fermentation inhibitors present in corn stover dilute acid hydrolysate. Enzyme and Microbial Technology. 42(7):624-630.

Liu, S., Dien, B.S., Nichols, N.N., Bischoff, K.M., Hughes, S.R., Cotta, M.A. 2007. Coexpression of pyruvate decarboxylase and alcohol dehydrogenase genes in Lactobacillus brevis. FEMS Microbiolology Letters. 274(2):291-297.

Jordan, D.B., Braker, J.D. 2007. Inhibition of the two-subsite beta-d-xylosidase from Selenomonas ruminantium by sugars: competitive, noncompetitive, double binding, and slow binding modes. Archives of Biochemistry and Biophysics. 465(1):231-246.

Jordan, D.B., Li, X. 2007. Variation in relative substrate specificity of bifunctional beta-D-xylosidase/alpha-L-arabinofuranosidase by single-site mutations: roles of substrate distortion and recognition. Biochimica et Biophysica Acta. 1774(9):1192-1198.

Jordan, D.B. 2008. Beta-D-xylosidase from Selenomonas ruminantium: catalyzed reactions with natural and artificial substrates. Applied Biochemistry and Biotechnology. 146:137-149.

Brunzelle, J.S., Jordan, D.B., McCaslin, D.R., Olczak, A., Wawrzak, Z. 2008. Structure of the two-subsite beta-D-xylosidase from Selenomonas ruminantium in complex with 1,3-bis[tris(hydroxymethyl)methylamino] propane. Archives Of Biochemistry and Biophysics. 474(1):157-166.

Hughes, S.R., Dowd, P.F., Hector, R.E., Panavas, T., Sterner, D.E., Qureshi, N., Bischoff, K.M., Bang, S.B., Mertens, J.A., Johnson, E.T., Li, X., Jackson Jr, J.S., Caughey, R.J., Riedmuller, S.B., Bartolett, S., Liu, S., Rich, J.O., Farrelly, P.J., Butt, T.R., Labaer, J., Cotta, M.A. 2008. Lycotoxin-1 insecticidal peptide optimized by amino acid scanning mutagenesis and expressed as a co-product in an ethanologenix Saccharomyces cerevisiae strain. Journal of Peptide Science. 14(9):1039-1050. Available: http://www3.interscience.wiley.com/cgi-bin/fulltext/119030240/PDFSTART.

Last Modified: 4/16/2014
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