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United States Department of Agriculture

Agricultural Research Service

Research Project: Biorefining Processes

Location: Bioproducts Research

2013 Annual Report

1a. Objectives (from AD-416):
Objective 1: Develop enzyme-based technologies (based on cleaving specific covalent crosslinks which underlie plant cell wall recalcitrance) thereby enabling new commercially-viable* saccharification processes. Objective 2: Develop new enzyme-based technologies that enable the production of commercially-viable* coproducts such as specialty chemicals, polymer precursors, and nutritional additives/supplements from raw or pretreated lignocellulosic biomass. Objective 3: Develop pretreatment technologies that enable commercially-viable* biorefineries capable of utilizing diverse feedstocks such as rice straw, wheat straw, commingled wastes (including MSW), sorghum, switchgrass, algae, and food processing by-products. Objective 4: Develop new separation technologies that enable commercially-viable* and energy-efficient processes for the recovery of biofuels, biorefinery co-products, and/or bioproducts from dilute fermentation broths.

1b. Approach (from AD-416):
Novel enzymes for pretreatment of lignocellulosic feedstocks will be developed and improved by (1) creation of new genomic DNA libraries from diverse environments that are known to contain microbes that digest plant biomass, (2) development of novel rapid screening assays for identifying enzymes that have a specific activity, and (3) optimization of different enzyme cocktails for different biomass sources via multivariant, combinatorial optimization protocols. Greener routes toward production of styrene, terephthalic acid, vanillin and ferulic acid derivatives will be developed by a combination of biochemical and chemical synthetic pathways. Enzymes will be applied to created these bioproduct feedstocks. Engineering process models, economic analysis, and process parameters for developing integrated biorefineries using biomass from MSW and other under-utilized biomass sources as feedstock will be developed to create a source of cellulose that is consistent, easily converted to bioenergy and available during all seasons. Develop novel separation methods to reduce energy use and costs for recovering and purifying biofuels/bioproducts from low concentration fermentation broths, especially those resulting from lignocellulosic feedstocks where product concentrations are typically below (sometimes far below) 6 wt%. Replacing 5325-41000-046-00D (11/09).

3. Progress Report:
Scientists within the project discovered and characterized a bi-functional enzyme that hydrolyzes both mannans and mixed glucans, two distinct types of polysaccharides present in plant cell wall. Bi-functional enzymes enable two hydrolytic reactions to proceed more efficiently than corresponding individual reactions. In another aspect of the project, Western Regional Research Center(WRRC) and National Center for Agricultural Utilization Research (NCAUR) scientists collaborated to improve the thermal stability of ß-xylosidase. Both the WRRC and NCAUR high-activity ß-xylosidases have relatively low thermal and pH stability compared to the parent enzymes. Site-directed mutagenesis and directed evolution were used to improve the thermal stability of the WRRC ß-xylosidase. The enzyme was stable and highly active at temperatures 8°C higher than normal. This ß-xylosidase exhibited 1/3 lower end-product inhibition but had about 2/3 the activity of the very-highly active ß-xylosidase developed at NCAUR. Another aspect of the project focused on developing biobased caprolactone. Caprolactone is used in the manufacture of polyurethane and although it is biodegradable, it is petroleum-based. Biobased replacements for caprolactone were developed from derivatized glycerols and derivatized di-lactide. This work was performed in collaboration with an industrial partner and an invention disclosure was filed. Scientists within the unit received a 3-year, $479K NIFA grant to develop multi-functional enzymes with significantly better biomass-degrading properties. Multi-functional enzymes have multiple active sites. Work has shown that enzymes tethered to scaffolding proteins (self-assembling nanostructure chaperonin complex) can exhibit greater synergism than the same enzymes free in solution. The work is being initiated with cellulases and hemicellulases. This research is being performed in collaboration with multiple partners from both industry and a government agency (NASA).

4. Accomplishments
1. Adding value to almond byproducts. In California, the world’s leader in almond production with 85% of the international market, roughly ~3.3 billion lbs of almond waste (mostly hulls) and 1.3 billion lbs of almond shells are produced annually and generally sold as cattle feed. Researchers in Albany, California, working with the Almond Hullers & Processors Association and a commercial partner, created value-added uses for the sugars derived from these almond wastes. Extraction analysis showed that nonpareil almond hulls (which make up 75-80% of production) contain more than 30% simple sugars (glucose, fructose, sucrose) that can readily be obtained by hot-water extraction. This team, in collaboration with growers and processers throughout California, initiated a wide-spread sampling and analysis program to determine sugars and other components (including aflatoxin) in almond hulls based on their variety, locality, age, and harvesting/storage conditions. They then showed the industry that simple sugars derived from almond hulls are viable as natural food ingredients, such as a natural sugar in “nutrient bars” or could be converted to ethanol in commercially viable processes, potentially adding hundreds of millions of dollars to the value of these waste products.

2. Enzymatic breakdown of covalent linkages in plant cell wall biopolymers. Utilization of biomass requires a cascade of enzymes, and a key process involves cleavage of the covalent bonds crosslinking individual polymers in plant cell wall. Agricultural Research Service scientists at Albany, California, applied cloning techniques to produce and test mportant new enzymes to break down crosslinks in various natural biopolymers. The research has helped industrial partners to improve processing of forage crops into biofuels and feed ingredients. These processes add value to underutilized agricultural wastes and forage crops.

3. Biomass degradation by enzyme nanoassemblies. The breakdown of lignocellulosic biomass into simple sugars requires many different enzymes which add to the overall cost of conversion. ARS scientists at Albany, California, assembled different enzyme combinations onto nanoscale ring scaffolds. They demonstrated that enzymes mounted onto the scaffolds had higher specific activities relative to enzymes free in solution. This research will lead to decreased enzyme usage and, hence, improved process economics. The ultimate goals is to create environmentally chemicals using enzymes and not harsh chemicals.

4. Selection and characterization of novel Uronate Dehydrogenase enzymes. Conversion of sugars and sugar acids to value-added sugar dicarboxylic acids, a Department of Energy top-ten target for biobased building blocks, can provide a bio-based chemical platform for polymers. The use of enzymes for this conversion is environmentally relatively benign and potentially can be more cost effective than harsh chemical procedures such as nitric acid oxidation. Researchers at Albany, Californa, identified five uronate dehydrogenase genes from three different bacterial genera that converted sugar uronic acids to sugar dicarboxylic acid building blocks. The kinetic and biophysical properties of these enzymes were extensively characterized, showing that this process may be commercially viable because it results in chemicals that can be used to make environmentally friendly plastics.

5. Engineering a GH43 xylosidase enzyme for improved thermal stability. GH43 enzymes are used to convert woody biomass including rice and wheat straw into simple sugars and value added products. Researchers at the Western Regional Research Center in Albany, California, in collaboration with researchers at the Peoria labs, used the enzyme engineering technique of directed evolution to improve the thermal stability of a highly active xylosidase by up to 8 degrees celcius. Improved thermostability facilitates commercial viability by (1) lowering enzyme cost, and (2) allowing the process to take place at elevated temperatures where reaction rates are higher and the risk of bacterial contamination lessened. Several commercial partners have indicated interest in these enzymes for improving their biorefinery strategies.

Review Publications
Wong, D., Tenkanen, M., Vrsanaka, M., Sika-Aho, M., Puchart, V., Penttila, M., Salohelmo, M., Biely, P. 2012. Xylanase XYN IV from Trichoderma reesei showing exo- and endo-xylanase activity. FEBS Journal. 280: 295-301.

Orts, W.J. 2008. Self-assembled films of cellulose nanofibers and poly (o-ethoxyaniline). Colloid and Polymer Science. 286(11):1265-1272.

Lee, C.C., Kibblewhite, R.E., Wagschal, K.C., Li, R., Orts, W.J. 2012. Isolation of alpha-glucuronidase enzyme from a rumen metagenomic library. Protein Journal. 31(3):206-211.

Holtman, K.M., Kodama, A.B., Klamczynski, A., Flynn, A., Bozzi, D.V., Torres, L., Franquivillanueva, D.M., Mao, J., Glenn, G.M., Orts, W.J. 2012. Thermal properties of Poly(ethylene terephthalate) recovered from municipal solid waste by steam autoclaving. Journal of Applied Polymer Science. 126(5):1698-1708. doi: 10.1002/app.36752.

Bilbao-Sainz, C., Chiou, B., Glenn, G.M., Gregorski, K.S., Williams, T.G., Wood, D.F., Klamczynski, A., Orts, W.J. 2013. Solid lipid particles in lipid films to control diffusive release of 2-heptanone. Pest Management Science. 69(8):975-982. DOI:10.1002/ps.34162.

Wong, D., Chan, V.J., Liao, H., Zidwick, M. 2013. Cloning of a novel feruloyl esterase gene from rumen microbial metagenome and enzyme characterization in synergism with endoxylanases. Journal of Industrial Microbiology. 40:287-295.

Lee, C.C., Wagschal, K.C., Ruiping, L., Robertson, G.H., Kibblewhite, R.E., Orts, W.J. 2012. Isolation and characterization of a novel GH67 a-glucuronidase from a mixed culture. Journal of Industrial Microbiology and Biotechnology. 39:1245-1251. DOI:10.1007/s10295-012-1128-7.

Jordan, D.B., Lee, C.C., Wagschal, K., Braker, J.D. 2013. Activation of a GH43 ß-xylosidase by divalent metal cations: Slow binding of divalent metal and high substrate specificity. Archives of Biochemistry and Biophysics. 533:79-87.

Last Modified: 09/23/2017
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