Progress 07/23/07 to 07/22/12
Outputs
OUTPUTS: Xylanases, the enzymes that degrade the major hemicellulosic polysaccharide of hardwoods and crop residues have been phylogenetically, biochemically and structurally characterized to understand classification, novel function and to identify biotechnological applications. Enzymes classified in glycosyl hydrolase (GH) family 30 (GH30) have been phylogenetically reclassified to reflect primary amino acid and structural similarities which were not previously identified. This lead to a new understanding of the breadth of enzymatic function assigned to this family and highlighted this enzyme family⿿s role in degradation of lignocellulosic biomass. The GH30 subfamilies that act on lignocellulose have been a focus of this problem area. In fiscal year 2012, the novel xylanase XynA1CD of Paenibacillus sp. JDR-2 was structurally characterized to initiate studies into its unique interactions with the complex polysaccharide substrate glucuronoxylan. This problem area will be continued in the new Research Work Unit Description. PARTICIPANTS: Dr. James F. Preston, Professor, Department of Microbiology and Cell Science, University of Florida, P.O. Box 110700 Gainesville, FL 32611 Dr. Edwin Pozharski, Assistant Professor, Department of Pharmaceutical Sciences, University of Maryland, 20 Penn St. Bldg HSF2, Rm 553, Baltimore, MD 21201 Dr. Jason C. Hurlbert, Assistant Professor, Department of Chemistry, Physics and Geology, Winthrop University, 301B Sims Science Building, Rock Hill, South Carolina 29733
Impacts
This research contributed to our understanding of enzymes that degrade hemicellulose so that they might be applied in bioconversion and biotechnology application.
Publications
- Chow, Virginia; Nong, Guang; St. John, Franz J.; Rice, John D.; Dickstein, Ellen; Chertkov, Olga; Bruce, David; Detter, Chris; Brettin, Thomas; Han, James; Woyke, Tanja; Pitluck, Sam; Nolan, Matt; Pati, Amrita; Martin, Joel; Copeland, Alex; Land, Miriam L.; Goodwin, Lynne; Jones, Jeffrey B.; Ingram, Lonnie O.; Shanmugam, Keelnathan T.; Preston, James F. 2012. Complete genome sequence of Paenibacillus sp. strain JDR-2. Standards in Genomic Sciences. 6: 1-10.
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Progress 07/23/07 to 07/22/12
Outputs
OUTPUTS: This project is complete. PARTICIPANTS: This project is complete. TARGET AUDIENCES: This project is complete. PROJECT MODIFICATIONS: This project is complete.
Impacts
This project is complete.
Publications
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Microbial inhibitors are chemicals that constitute one of the major roadblocks to bioprocessing woody biomass to bioenergy and bioproducts. Inhibitors prevent or slow microbial fermentation and/or bioconversion of cellulose and hemicellulose to value-added products such as biofuels, biopolymers and precursor chemicals for the growing industrial need for renewable platform chemicals. Although several microbial inhibitors, such as acetic acid and furfural, are known, microbial inhibitors in woody biomass extraction streams are largely unknown. Our lab has established a rapid bioassay system to locate and separate chemical inhibitors of two yeasts currently used for bioconversion of wood hydrolysates to ethanol and two bacteria that can synthesize biopolymers from suitable substrates. Experiments were conducted on a collection of polyhydroxyalkanoate (PHA) producing bacteria. Research focused on determination of PHA production from hexose and pentose carbon sources and spruce pre-pulping extracts. Conditions were established for PHA production from these organisms and methods were developed for their quantification. Samples were prepared for thermal-mechanical characterization. These organisms were also analyzed for sensitivity to a number of microbial inhibitors including furans, phenolics, phenolics acids and aliphatic organic acids. Experiments to determine the kinetics or carbohydrate utilization from pre-pulping extracts were performed. Events: Franz J. St. John, Jason C. Hurlbert, John D. Rice, James F. Preston and Edwin Pozharski, 2010, Ligand bound crystal structures identify the molecular interactions involved in the unique substrate specificity of glycosyl hydrolase family 30 glucuronoxylan xylanohydrolases. Lignobiotech One, March 28th - April 1st. - Oral presentation and a published abstract. Franz J. St John, 2010, Enzymatic deconstruction of glucuronoxylan for bioconversion to value-added products. AF&PA Agenda 2020/TAPPI May 11-12 - Oral presentation to an industrial liaison committee with a printed copy of the presentation. PARTICIPANTS: University of Florida, St John et al., Ligand bound crystal structures identify the molecular interactions involved in the unique substrate specificity of glycosyl hydrolase family 30 glucuronoxylan xylanohydrolases. University of Maryland, St John et al., Consolidation of glycosyl hydrolase family 30: a dual domain 4/7 hydrolase family consisting of two structurally distinct groups. TARGET AUDIENCES: Target audiences: Scientific community. Academic, governmental and commercial interests engaged in the development of renewable fuels based on cellulosic and hemicellulose sugars from lignocellulose. Efforts: Contributor to CAZypedia the online wiki for classification of carbohydrate-active enzymes.
Impacts
Knowledge: Fundamental differences in tolerance to microbial inhibitors and PHA production rates between select PHA producing bacteria were identified. Locating microbial inhibitors in wood hydrolysates for biomass conversion is a first step toward developing needed information for forest biorefineries that will impact (a) process removal of inhibitors from biomass extraction streams, (b) related costs and environmental impact of removing inhibitors, and (c) development of chemical markers for improvement of microbial strains. This research advances understanding of Actions: Bacteria were selected for preparation of germ-plasm and screening to genetically identify desired microbial inhibitor resistance characteristics. Similarly, bacteria were selected for use as target organisms for microbial strain improvement based on PHA production from commercial carbohydrates. Change in Knowledge: Two families of glycosyl hydrolase enzymes that are involved lignocellulose degradation were classified incorrectly and the correct classification was proposed and attributed to a specialized dual-domain structure protein-fold. Change in Action: The primary database which classifies glycosyl hydrolase enzymes has been revised to reflect the new information. This significantly increases the accuracy of microbial genome annotation efforts by allowing the correct protein family assignment. Change in Condition: These findings contribute to the field of gene/protein annotation.
Publications
- Illman, Barbara; Crooks, Casey. 2009. Microbial inhibitors: biorefinery roadblocks. In: 2009 TAPPI international bioenergy and bioproducts conference. 2009 October 14-16; Memphis, TN. Norcross, GA: TAPPI Press. ISBN: 978161567958
- St. John, Franz J.; Hurlbert, Jason C.; Rice, John D.; Preston, James F.; Pozharski, Edwin. 2010. Ligand bound crystal structures identify the molecular interactions involved in the unique substrate specificity of glycosyl hydrolase family 30 glucuronoxylan xylanohydrolases [abstract]. In: 1st Symposium on biotechnology applied to Lignocelluloses (Lignobiotech 1). 2010 March 28-April 1; Reims, France.
- West, Abby L.; St. John, Franz; Lopes, Pedro E.M.; MacKerell, Jr., Alexander D.; Pozharski, Edwin; Michell, Sarah L.J. 2010. Holo-Ni(II)HpNikR is an asymmetric tetramer containing two different nickel-binding sites. Journal of American Chemical Society. 132: 14447-14456.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Value prior to pulping (VPP) is a strategic approach to producing fuel ethanol from the hemicelluloses generated by the industrial pulp and paper process, creating higher value products without negatively affecting paper quality. The VPP project goal is to demonstrate the technical and commercial feasibility of introducing a new value stream into existing pulp and paper mills, essentially transferring extracted hemicellulose to a liquid transportation fuel. The hemicellulose extract will be hydrolyzed and conditioned to remove inhibitors. The hemicellulose fraction will consist of pentose and hexose sugars in proportions dependent on the extraction chemistry and the wood species. Experimental microorganisms are evaluated for efficient and rapid fermentation of five-carbon sugars to ethanol. Microbial inhibitors in the hemicellulose stream constitute one of the major roadblocks to bioprocessing woody biomass to ethanol in the VPP strategy and to precursor chemicals to meet the growing industrial need for renewable platform chemicals. Inhibitors prevent or slow microbial fermentation or bioconversion and require expensive removal. Although several microbial inhibitors, such as acetic acid and furfural, are known, microbial inhibitors in woody biomass extraction streams are largely unknown. Our lab has established a rapid, simple bioassay system to locate and separate chemical inhibitors of two yeasts currently used for bioconversion of wood hydrolysates to ethanol in the VPP strategy and two bacteria that can synthesize biopolymers from suitable substrates. Pyranose oxidase is a peroxide-producing enzyme that growing evidence indicates has a role in wood decay by certain fungi. It is also the major glucose-oxidizing enzyme in ligninolytic cultures of Phanerochaete chrysosporium. Furthermore, the enzyme is of interest for numerous biotechnological applications, analytical methods, and the synthesis of novel chemicals. We constructed a vector for the heterologous expression of the protein from P. chrysosporium. Fully active recombinant oxidase was produced at approximately 270 mg/l medium. An N-terminal T7-tag along with a C-terminal His6-tag afforded simple one-step purification. Kinetic analysis of the pure recombinant pyranose oxidase shows that alternate electron acceptors are better substrates than oxygen. Observing the differences between brown and white rot colonization of wood, we chose to isolate and characterize pyranose 2-oxidase from the brown rot fungi Gloeophyllum trabeum. We used the expression system developed by Kersten and colleagues to produce recombinant enzyme for comparative analysis. We observe that while many characteristics are conserved with pyranose oxidases from white rot fungi, some aspects are distinct. This is the first molecular genetic analysis of a pyranose oxidase from a brown rot fungus.
Impacts
The bioassay for microbial inhibitors is a needed tool for VPP forest biorefineries that will impact fundamental research and industrial processes in several areas, including (a) process removal of inhibitors from biomass extraction streams, (b) related costs and environmental impact of removing inhibitors, and (c) development of chemical markers for improvement of microbial strains. Production of recombinant protein at high levels allows both basic and applied studies that are not otherwise possible with the native fungal hosts. We have developed an efficient production system for active recombinant pyranose oxidase from P. chrysosporium. Our studies indicate that peroxide production (from oxygen) may not be the only role for the enzyme. Certain quinones are better substrates than oxygen, suggesting that the reduction of quinones to hydroquinones may be an important role for pyranose oxidase. The quinones that are most efficiently reduced are consistent with the metabolism of quinones that are derived from lignin during ligninolysis.
Publications
- Dietrich, Diane; Crooks, Casey. 2009. Gene cloning and heterologous expression of pyranose 2-oxidase from the brown-rot fungus, Gloeophyllum trabeum. Biotechnology Letters. 31: 1223-1228.
- Illman, B.L. 2009. Fermentation of biomass to ethanol: adding value prior to pulping. In: Proceedings Biofuels and bioenergy: a changing climate. 2009 July 30 ⿿ August 2. International Energy Agency Bioenergy Symposium. University of British Columbia-Forestry: Vancouver, Canada. 55.
- Pisanelli, Ines; Kujawa, Magdalena; Spadiut, Oliver; Kittl, Roman; Halada, Petr; Volc, Jindrich; Mozuch, Michael D.; Kersten, Philip; Haltrich, Dietmar; Peterbauer, Clemens. 2009. Pyranose 2-oxidase from Phanerochaete chrysosporium ⿿ expression in E. coli and biochemical characterization. Journal of Biotechnology. 142: 97-106.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: We are leading the fermentation portion of a large multidisciplinary Value Prior to Pulping (VPP) Consortium focused on the conversion of forest biomass to fuel ethanol in a Forest Biorefinery. Novel yeast and bacterial strains are evaluated for fermentation of hemicellulosic hydrolysates from four wood species to produce high yield ethanol, while leaving cellulose to produce a commercially acceptable pulp. The best microorganism(s) will be identified for fermenting solutions of mixed pentoses and hexoses that are predominately glucurono-xylan in hardwoods and galactoglucomannan and arabino-xylan in softwoods. Additionally, we are developing nanocatalysts for converting cellulose to useful monomers, including biofuels such as ethanol. The desired catalyst properties include, acid-stability to withstand carbohydrate hydrolysis conditions, efficient kinetics for hydrogenation of monomers, a colloidal state for interaction with wood at the nanoscale, and magnetic susceptibility (conferred by carriers) for improved catalyst recovery. To meet these challenges, our focus is on catalysts made of iron-carbon core-shell nanoparticles decorated with ruthenium crystals.
Impacts
General principles learned from iron-carbon-ruthenium nanocatalysts may be applicable to diverse catalytic processes needed in the forest biorefinery. The VPP fermentation work has enabled increased collaboration with universities, industry, DOE, and AF&PA for biorefinery research. Using estimates developed from the pulp and paper industry, when this technology is completely deployed it could enable production of 1.6 to 2.4 billion gallons of ethanol, 260 to 400 million gallons of acetic acid and provide $740 to $1,110 million in new revenue to the pulp and paper industry.
Publications
- Illman, Barbara. 2008. Update on microorganism technologies, projects at US Forest Products Lab (abstract). In: Proceedings 2008 International Bioenergy and Bioproducts Conference abstract book. 2008 August 27-29; Portland, OR. Atlanta, GA: TAPPI Press.
- Mozuch, Michael D.; Braden, Drew; Dumesic, James A.; Kersten, Philip J. 2008. Nanocatalysts for wood conversion [abstract]. In: Abstracts for the 2008 international conference on nanotechnology for the forest products industry; 2008 June 25-27; St. Louis, MO: Atlanta, GA: TAPPI Press.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Biorefining wood into paper and chemicals is not as easy as making a single traditional paper product. Paper is made from the cellulose- containing fractions of wood and processing may remove lignin and hemicellulose components. The yield and composition of the product depend upon the type of paper being produced. The paper process often alters the noncellulose fractions, making them less suitable for conversion to other valuable products. This chapter will review methods of obtaining hemicellulosic sugars and investigate the potential for obtaining sugars by a wood pretreatment before it is made into pulp. As an example of hemicellulose removal the results of a new pretreatment using diethyl oxalate are also described. Hemicellulose removal by this system provides other benefits to the pulping process. Biorefining extracts hemicellulosic sugars from wood chips prior to being refined into paper products. A process was developed where pretreatment chemicals are dispersed throughout the wood chips as a vapor and not by solution. Up to 14% of the weight of the wood chips was removed by the method. Liberated sugars support growth of yeasts. A variety of enzymes are available for the surface modification of cellulosic fibers, both in the area of textile applications and for pulp and paper applications. Enzymatic treatment conditions are milder, less damaging for the fiber, and are environmentally friendly while producing effects comparable to chemical treatments. Surface modifications can be achieved by oxidative and/or hydrolytic enzymes. Some of the enzymatic processes have recently attained commercial importance and more systems are being developed. Current research on the application of oxidoreductases and hydrolases that are valuable for textile and forest products industries was presented in a synthesis chapter of the important book Modified Fibers with Medical and Specialty Applications. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A forest biorefining concept is being developed. Wood chips are treated to recover hemicellulosic sugars and also produce paper or other products which are altered in their properties. Thermomechanical pulps are made stronger with greater energy efficiency and the additional products, acetic acid and fermentable sugars are recovered. Chemical pulps can also be made with the recovery of even greater amounts of fermentable carbohydrate as a second product. The conversion of all the wood to fermentable sugars and the fractions not made into paper will provide a renewable source of sugars which can be converted to ethanol, lessening our need for petroleum.
Publications
- Kenealy, W.R.; Horn, E.; Houtman, C.J.; Laplaza, J.; Jeffries, T.W. 2007. Obtaining value prior to pulping with diethyl oxalate and oxalic acid. In: Proceedings of the 10th International Congress on Biotechnology in the Pulp and Paper Industry. 2007 June 10-15: Madison, WI. Madison, WI: Omni Press. BioP/R 1.2: 25
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