Source: FOREST PRODUCTS LABORATORY submitted to NRP
ENZYMATIC PROCESSING OF WOOD FIBER
Sponsoring Institution
Forest Service/USDA
Project Status
COMPLETE
Funding Source
USDA INHOUSE
Reporting Frequency
Annual
Accession No.
0197404
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jul 31, 2002
Project End Date
Jul 30, 2007
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
FOREST PRODUCTS LABORATORY
ONE GIFFORD PINCHOT DRIVE
MADISON,WI 53726
Performing Department
FOREST PRODUCTS LAB, MADISON LAB HQ - MADISON, WI
Non Technical Summary
Current fiber technology uses non-specific chemical and thermal processing that is inefficient and polluting. This research develops basic information and operating conditions needed for efficient and effective enzymatic and microbial processing of wood and wood fiber.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5110660100075%
5110660104025%
Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
0660 - Paper and pulp derived products;

Field Of Science
1040 - Molecular biology; 1000 - Biochemistry and biophysics;
Goals / Objectives
The objectives of this research are to identify, discover, or create enzymes for the treatment of wood and pulp fibers. For example, the pulp and paper industry uses xylanases to reduce the need for harsh oxidizing chemicals for pulp bleaching. Xylanase prebleaching decreases pollution and provides for more efficient use of chemicals. However existing xylanases are not ideally suited to the equipment and process in which they are used. Research to obtain xylanases that will work under more extreme conditions could broaden the applicability of this environmentally benign technology. In another application, cellulases have been developed for removing inks and contaminants from recycled fibers. Use of this technology enables more efficient use of our forest resources. The technology is not fully robust, so it is not as widely applied as it might be. Further research into enzyme mechanisms or processes could expand the use of recycled fibers and conserve timber while providing the public with material goods. Other possible applications include the use of laccases to modify fiber surfaces and properties and cellulases or cellulose binding domains to modify fiber properties.
Project Methods
This research will consist of basic and applied studies on the discovery and use of microorganisms and enzymes to treat virgin fibers, recycled fibers, and wood residues. Enzymes possessing the capacity to release lignin and chromophores from pulps will be sought, along with enzymes that might increase the strength of paper made from lower grade residuals and pulp. Enzymes will be purified and characterized, and cloned where it is deemed appropriate to better understand fundamental enzymatic mechanisms or to produce the enzyme in sufficient quantity for further characterization and study. Kinetic constants of the enzymes will be measured, and their modes of action will be assessed. The substrate range of some enzymes will be explored so that better predictions can be made of their effects on wood and wood fibers. The modification of wood and fiber surfaces provides great potential in developing new products. Specific removal or modification of components of the wood may be useful in producing new products some of which may be useful as chemicals or fuels, others as heavy metal or nutrient adsorbants and others having chemicals replaced by enzymatic action. Planned accomplishments include the following 1. Identify novel enzymes, cofactors and proteins that help to separate wood components and test if they will help release lignin and chromophores from pulps. 2. Improve fundamental understanding of how enzymes might be used to modify wood and fiber surfaces to attach various chemically defined functional groups. 3. Clone and express enzymes of potential interest to study them more easily and better understand their fundamental structural properties. 4. Create novel enzymes that combine a catalytic function with a sequence for immobilization on lignocellulosic residues.

Progress 07/31/02 to 07/30/07

Outputs
In FY2006 research focused on developing methods to refine wood into fermentable carbohydrates and paper products. Oxalic acid and diethyloxalate liberate hemicellulosic sugars from wood chips. Spruce, pine, red pine, oak, eucalyptus, maple and aspen have been tested. The carbohydrates obtained are approximately half free sugars and half sugar derivatives. Sugars released from the wood chips were concentrated by reverse osmosis to obtain solutions for ethanol fermentations. Acetic acid was partially separated in the concentration process. Sugars can be converted by microorganisms or chemical methods into a variety of important chemical products including ethanol for fuel. Both oxalic acid and diethyloxalate improve wood chips for thermomechanical pulping; making stronger paper products and requiring less energy to refine the chips to products. Up to 50% electrical refiner energy can be saved while making thermomechanical pulp that has superior strength. A patent on the new wood chip pretreatment using diethyloxalate (DEO) was filed. Application of the DEO pretreatment to chemical pulping and total saccharification of wood to sugars will be tested in the next year.

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, William; Bushle-Diller, Gisela; Ren, Xuehong. 2006. Enzymatic modification of fibers for the textile and forest products industries. In: J. V. Edwards, G. Buschle-Diller and S. C. Goheen, eds. Modified fibers with medical and specialty applications. Springer-Verlag. Pp. 189-206, Chapter 12.
  • Kenealy, W.R.; Akhtar, M.; Swaney, R.; Horn, E. 2006. Method for treating lignocellulose. PCT Application 2005 July 8.


Progress 10/01/04 to 09/30/05

Outputs
In FY2005 research focused on methods to refine wood to obtain fermentable carbohydrates and paper products. Oxalic acid pulping is a method developed to produce stronger paper products and save electrical refiner energy when producing thermomechanical pulps. We have demonstrated that oxalic acid pulping also liberates hemicellulosic sugars from wood chips. The carbohydrates obtained are approximately half free sugars and half oligosaccharides. The release of sugars from the wood chips provides for an additional product from the same starting material. Sugars can be converted by microorganisms or chemical methods into a variety of important chemical products including ethanol for fuel. A new wood chip pretreatment was developed using diethyloxalate (DEO). The DEO is injected as a liquid but permeates the wood chip as a vapor. DEO was added to preheated wood chips where it reacts with the water in the wood chips to deposit oxalic acid. Upon further heating the oxalic acid releases hemicellulosic sugars which can be recovered prior to making paper products. The DEO pulping pretreatment works on both hardwoods and softwoods. The glucose recovered from the wood chips represents the glucose from the hemicellulose and the cellulose appears to be unharmed by the process. The strength of most paper products depends on the integrity of the cellulose. Pretreated softwoods release a mixture of mannose, galactose, glucose, xylose and arabinose. Pretreated hardwoods produce mostly xylose and smaller amounts of the other sugars. Both softwoods and hardwoods release acetic acid as an additional product. The release and recovery of hemicellulosic sugars is considered a method of forest biorefining where sugars and acetic acid can be obtained in addition to paper products.

Impacts
A forest biorefining concept that produces paper products with greater energy efficiency and the additional products acetic acid and fermentable sugars is being developed. The forest materials will be used for traditional paper products, but will also help reduce the need for petroleum by providing feedstocks that can be converted into chemical building blocks and fuel ethanol.

Publications

  • Kenealy, William R.; Hunt, Chris; Horn, Eric; Houtman, Carl. 2004. A new mechanism of biopulping: attachment of acid groups on fiber. In: Ninth International conference on biotechnology in the pulp and paper industry; 2004 October 10-14; Durban, South Africa. pp. 97-99.
  • Kenealy, W.R.; Laplaza, J.; Jeffries, T. 2005. A new biorefining method of generating chemicals and paper from wood chips (abstract). In: Industrial microbiology & biotechnology conference SIM meeting program and abstracts; 2005 August 21-25; Chicago IL. Fairfax, VA: Society for Industrial Microbiology: S101.
  • Jeffries, Thomas W.; Kenealy, William R.; Laplaza, Jose M.; Horn, Eric G. 2005. Biorefining for recovery of fuels, fibers and chemicals from wood (abstract). 2005. In: 1st International workshop on biorefining, 2005 July 20-21; Washington, DC. Washington, DC: U.S. Department of Energy: 2F.
  • Weimer, P.J.; Koegel, R.G.; Lorenz, L.F.; Frihart, C.R.; Kenealy, W.R. 2005. Wood adhesives prepared from lucerne fiber fermentation residues of Ruminococcus albus and Clostridium thermocellum. Applied Microbiology and Biotechnology. 66: 635-640.
  • Kenealy, William R.; Destree, Joseph C. 2005. Time- and Cost-Saving Apparatus for Analytical Sample Filtration. Research Note FPL-RN-0298. Madison, WI: U.S. Department of Agriculture, Forest Service, Forest Products Laboratory: 2 p.


Progress 10/01/03 to 09/30/04

Outputs
In FY2004 research focused on using enzymes, organisms and the reactions that organisms use to alter the properties of fiber to be used in the pulp and paper industry. The biopulping organisms Ceriporiopsis subvermispora and Phanerochaete chrysosporium were found to alter wood by attaching or creating oxalate esters on the fiber. The placement of oxalate esters on fiber causes a localized increase in osmotic pressure leading to the influx of water. This is a mechanism of softening (and hydrating) the wood for further enzymatic degradation. Other wood degrading fungi are being tested for evidence of similar wood degrading mechanisms. The correlation between oxalate production, enzymes and genes for oxalate production, and the biopulping effect was demonstrated for P. chrysosporium. Methods of chemically or enzymatically attaching oxalate to fiber are being explored to alter fiber by attachment of carboxylic acid groups. Additional effort was focused on establishing scale up equipment for microbial growth and protein production.

Impacts
The alteration of the surfaces of fiber can have great impact on the strength of products and direct in which products the fiber can be used. Attachment of carboxylic acid groups to fiber will increase the water holding capacity of the fibers and increase the strength of the bonding of the fiber in products. The results of these studies may provide new pulping processes and enhanced recovery of sugars prior to pulping via biorefining. The impact of these studies will be in developing new fibers, and new processes that will efficiently use available fiber.

Publications

  • Kenealy, William R.; Jeffries, Thomas W. 2003. Rapid 2,2-bicinchoninic-based xylanase assay compatible with high throughput screening. Biotechnology Letters. 25: 1619-1623.
  • Kenealy, William R.; Dietrich, Diane M. 2004. Growth and fermentation responses of Phanerochaete chrysosporium to O2 limitation. Enzyme and Microbial Technology. 34: 490-498.
  • Hunt, Chris; Kenealy, William; Horn, Eric; Houtman, Carl. 2004. A biopulping mechanism: creation of acid groups on fiber. Holzforschung. 58: 434-489.
  • Clausen, Carol A.; Kenealy, William R. 2004. Scaled-up remediation of CCA-treated wood. In: Proceedings of the environmental impacts of preservative-treated wood; 2004 February 8-11; Orlando, FL. Gainesville, FL: Florida Center for Environmental Solutions: 71-80.
  • Kenealy, William; Klungness, John; Tshabalala, Mandla; et al. 2004. Laccase modification of the physical properties of bark and pulp of loblolly pine and spruce pulp. In: Saha, B.C.; Hayashi, K., eds. ACS Symposium Series 889; Lignocellulose biodegradation. Washington, DC: American Chemical Society: Chapter 7: 126-138
  • Kenealy, W.; Hunt, C.; Dietrich, D.; Houtman, C.; Cullen, D.; Horn, E. 2004. Oxalate attachment and wood swelling by biopulping fungi [abstract]. In: SIM annual meeting program and abstracts; 2004 July 25-29; Anaheim, CA. Fairfax, VA: Society for Industrial Microbiology: P40: 94.
  • Kenealy, William; Klungness, John; Tshabalala, Mandla; et. al. 2003. Modification of lignocellulosic materials by laccase. In: Proceedings of the 2003 TAPPI fall technical conference; 2003 October 26-30; Chicago, IL. Atlanta, GA: TAPPI Press: Available: CD Rom-ISBN: 1-930657-30-7. Available online: http://www.tappi.org. Session 17-1.


Progress 10/01/02 to 09/30/03

Outputs
In FY2003 research focused on using enzymes and organisms to alter the properties of fiber to be used in the pulp and paper industry. Laccase is a commercially available enzyme with potential to reduce the lignin content of pulps, bleach pulps or attach (graft) compounds to the pulp fiber. Various substrates of the enzyme laccase imparted different properties to the enzyme plus substrate treated fibers. The ability of the surface to act as a Lewis acid or base could be changed depending upon the substrate used. The hydrophobicity and hydrophilicity of the fiber could also be altered. Optimizing the attachment of substrates and determining the changes imparted will determine if there is an economical benefit. The biopulping organisms Ceriporiopsis subvermispora and Phanerochaete chrysosporium were found to alter wood by attaching oxalate esters onto the fiber. Methods of chemically or enzymatically attaching oxalate to fiber are being explored to alter fiber by attachment of carboxylic acid groups.

Impacts
The alteration of the surfaces of fiber can have great impact on the strength of products and direct in which products the fiber can be used. Laccase has been used to attach various substrates to thermomechanical pulp and some of these have indicated strength increases. Attachment of carboxylic acid groups to pulp should increase the water holding capacity of the fibers and increase the strength of the fiber. Other additions to the fiber could be made if sufficient densities of carboxylic acids can be attached. The impact of these studies will be in developing new fibers that will efficiently use available fiber.

Publications

  • Kenealy, William R.; Jeffries, Thomas W. 2003. Enzyme processes for pulp and paper: a review of recent developments. In: Goodell, Barry; Nicholas, Darrel D.; Schultz, Tor P., eds. ACS Symposium Series 845; Wood deterioration and preservation: Advances in our changing world. Washington, DC: American Chemical Society: Chapter 12: 210-239.
  • Kenealy, William R.; Klungness, John; Tshabalala, Mandla A.; Akhtar, Masood; Horn, Eric; Gleisner, Roland; Buschle-Diller, Gisela. 2003. Modification of the physical properties of lignocellulosic materials by laccase [abstract]. In: Abstracts of papers, 225th ACS national meeting; 2003 March 23-27; New Orleans, LA. Washington, DC: American Chemical Society: CELL 17.
  • Kenealy, William R.; Buschle-Diller, Gisela; Elder, Thomas J. 2003. Modification pulp fibers by laccase treatment [abstract]. In: Abstracts of papers, 225th ACS national meeting; 2003 March 23-27; New Orleans, LA. Washington, DC: American Chemical Society: CELL 122.
  • Buschle-Diller, Gisela; Kenealy, William R.; Wu, Ye; Zulaica-Villagomez, Hilda; Elder, Thomas J. 2003. Analysis of laccase-modified pulp fibers [abstract]. In: Abstracts of papers, 225th ACS national meeting; 2003 March 23-27; New Orleans, LA. Washington, DC: American Chemical Society: CELL 123.
  • Hunt, Chris; Kenealy, William; Houtman, Carl. 2003. Understanding wood chemistry changes during biopulping. In: Proceedings of the 23th ISWPC International symposium on wood and pulping chemistry; 2003 June 9-12; Madison, WI. Madison, WI: University of Wisconsin-Madison, Department of Forest Ecology and Management. II: 169-172.