Enzymes Associated with Wood Decay and their Potential Uses in Industry

Size: px

Start display at page:

Download "Enzymes Associated with Wood Decay and their Potential Uses in Industry"

Herbert Johns
6 years ago
Views:

1 Enzymes Associated with Wood Decay and their Potential Uses in Industry Jessie A. Micales, Project Leader Center for Forest Mycology Research Forest Products Laboratory One Gifford Pinchot Dr. Madison, WI Wood decay fungi are the only organisms that can efficiently digest all three fundamental components ofwood: cellulose, hemicellulose and lignin. This ability makes them unique among living creatures and provides us with an opportunity to use their metabolism to our advantage. Our greatest need ofthese woody components is in paper production, an energy intensive process that has traditionally generated toxic by-products. By using the natural processes exhibited by wood decay fungi, we can make paper and other products more efficiently and with less impact on the environment. Wood decay fungi all belong to the group ofhigher fungi termed Basidiomycetes. This means that they produce their spores on microscopic club-like formations termed basidia. The spore-bearing surface can be in the form ofgills, as in the common commercial mushroom. Spores can also be born within pores, on teeth, or on flat surfaces ofvarious textures (smooth, warty, or leathery, for example). Mycologists use the microscopic morphology ofthe spores, the basidia, and the spore-bearing structures to identify wood decay fungi. This is often difficult and is quite an art! Techniques of molecular biology, including DNA sequencing, are being used increasingly for fungal identification and to study their biosystematics, thus revealing the true relationships among fungi. Wood decay fungi can also be separated by their metabolism. There are two major groups. Brown-rot fungi break down and metabolize only the hemicellulose and cellulose ofwood. They can modify the structure oflignin, but they can t really metabolize it as a carbon source. White rot fungi can digest and metabolize all three components. Some white rotters do this sequentially, breaking down the lignin initially before moving on to digest the hemicellulose and cellulose. These are the organisms ofmost interest to industry. The decomposition ofwood involves both enzymatic and nonenzymatic mechanisms, many ofwhich we arejust beginning to understand. Before we discuss the enzymes associated with, wood decay, we need to know a little bit about the substrates found within wood. The basic component ofwood is cellulose. Cellulose is composed oflong polymeric units ofglucose, a common six-carbon sugar, bonded together with b-1,4 glycosidic bonds (Figure 1). These long chains lie in parallel to each other and are cross-linked to make long microfibrils (Figure 2). Depending on the amount ofcross-linkages, the cellulose can be crystalline or amorphous. Wood decay

2 fungi attack primarily the amorphous areas. The highly cross-linked crystalline areas are very difficult to break down. Figure 1: Chemical structure ofcellulose showing polymer linkages and cross-linkages among glucose units. Figure 2: The cellulose chains are arranged in microfibrils. Crystalline areas exhibit extensive cross-linkages. The amorphous areas have fewer cross-links, allowing better access for the microbial enzymes to break the bonds between the glucose molecules. The enzymes that break down cellulose are termed cellulase. The activity of at least three different types of enzymes is needed for the degradation ofcellulose. Endoglucanases hydrolyze the internal b-1,4-glucosidic bonds, usually in the amorphous region ofthe cellulose, thus exposing new reactive ends ofthe cellulose chain. These end units are the site of action of cellobiohydrolases, which cleave offtwo-glucose units, termed cellobiose. The cellobiose is then further degraded into individual glucose

monomers by the activity of b-glucosidases. Wood decay fungi usually make multiple forms ofthese enzymes, termed isozymes, which are active under a variety of environmentalconditions.

3 monomers by the activity of b-glucosidases. Wood decay fungi usually make multiple forms ofthese enzymes, termed isozymes, which are active under a variety of environmentalconditions. The second major component ofwood is hemicellulose, which is deposited on the surface ofthe cellulose fibrils. Hemicellulose is a more complicated polymer than cellulose and contains monomeric units offive- and six-carbon sugars, sugar acids, and acetyl esters. The most common components are glucose, galactose, mannose, glucuronic acid, xylose, and arabinose. The hemicellulose is composed of a backbone of sugars linked with b-1,4- bonds. There are also numerous sidechains bound covalently to backbone. The major hemicellulose in hardwoods is O-acetylglucuronoxylan, and the major hemicellulose in conifer wood is O-acetylgalactoglucomannan. A typical hemicellulose structure is shown in Figure 3. Figure 3 : Representative structure of hemicellose showing backbone and side chains. Hemicelluloses are degraded by hemicellulases, a family ofhydrolases that break down the multiple forms ofhemicellulose. These enzymes are quite specific for their substrates: xylanases break down hemicelluloses primarily formed from xylose, mannanases attack those that contain large numbers ofmannose molecules, etc. These enzymes break the bonds in the backbone ofthe polymeric chain, so they are termed endoxylanases and endomannanases. Another group of enzymes attack the side chains and are termed debranching enzymes. Many ofthese are esterases that attack ester bonds. The glycosidases break the oligomeric products ofthe endoenzymes into individual monomers. For the complete digestion ofthe hemicellulose O-acetylgalactoglucomannan, for example, a wood decay fungus needs five different. enzymes: 1) endomannanase, 2) a galactosidase, 3) acetylglucomannan esterase, 4) b-mannosidase, and 5) b-glucosidase. Each different type of hemicellulose requires the fungus to produce a unique set of enzymes for complete degradation. Multiple isozymic forms are usually present. The cellulose and hemicellulose are embedded in a matrix oflignin, a highly complex branched polymer ofvariously substituted phenylpropane units that are joined by C-C and C-O-C bonds. Due to the irregularity of its structure, lignin cannot be degraded by typical hydrolytic enzyme activity. Instead it is oxidized by highly reactive, nonspecific, low

4 molecular weight free radicals that are generated through the activity of lignin-degrading oxidases and peroxidases. The free radicals diffuse throughout the lignin, breaking bonds and oxidizing the lignin components. The enzymes involved with lignin degradation and free radical formation are the laccases, lignin peroxidases, and manganese peroxidases. One of the cofactors required for free radical generation by the enzymes is hydrogen peroxide. This is formed by peroxide-generating enzymes, such as glyoxal oxidase. The study of lignin degradation is quite complex and involves very complicated oxidative chemistry. White-rot fungi are the only organisms that can break down lignin in this manner. It is an aerobic process, requiring oxygen. So how can all of these various enzymes be used in industry? The biggest industrial use of wood components is the pulp and paper industry. In 1988, over 71 million metric tons of paper and paperboard were produced (Jeffries, 1996), and this quantity has steadily increased over the years. Paper is made in three basic steps. Initially, the cellulose fibers are separated from the wood during the pulping process. These fibers are then bleached and rearranged to form paper. The process is very energy intensive and has traditionally used many toxic chemicals, such as various forms of chlorine that formed dioxin as a byproduct. The use of enzyme technology in paper production has great potential for reducing energy requirements and eliminating the use of toxic chemicals. Some of the areas that are being researched for enzyme technology include pulp production, bleaching, enhanced drainage, fiber modification, resin and pitch control, and technologies associated with recycling, such as enzymatic deinking and contaminant removal. In these processes, the term bio is used when the entire fungus is involved in the process, such as biopulping and biobleaching. The term enzymatic is used when the enzymes are used in absence of the parent organism. Biopulping: The production of pulp is one of the most promising areas for the incorporation of a microbial process. Today, about 25% of pulp is produced by a mechanical process and 75% is formed by chemical pulping. During mechanical pulping, individual fibers are released through a grinding process. This is a very energy intensive and forms high yields but results in paper with fairly low strength properties. Chemical pulping, in which chemicals are combined with wood under high temperatures to improve efficiency, results in low yields of pulp but results in paper with greater strength. One danger of chemical pulping is the release of toxic chemicals and by-products. The development of biomechanical pulping has been extensively studied within the past decade. In this process, wood chips are predigested by selectively delignifying white rot fungi in order to remove the lignin from the wood. Once the lignin is removed, the fibers come apart more easily during mechanical pulping. This process is done by inoculating the wood chips with the decay fungus in a process referred to as solid state fermentation. During the research phase, hundreds of white-rot fungi were screened to find those with the best delignifying properties. Two of these organisms have been the focus of most subsequent research. Phanerochaete chrysosporium, initially isolated from

5 wood chip piles, is uniquely adapted for growth in this type ofenvironment. It is thermophilic, so it can tolerate the large amounts of heat that are generated in large chip piles. It is primarily effective at delignifying hardwoods. Ceriporiopsis subvermispora is another selective delignifier. It is mesophilic and requires lower temperatures, but it can degrade both hardwood and softwood lignin. During the basic research phase of biopulping, it was learned that the wood chips need to be surface disinfested to prevent the growth ofmold fungi that outcompete the biopulping organisms. Disinfestation was initially done by soaking the chips in sodium bisulphate, but it was later determined that a 15 second exposure to steam was just as effective. The levels of inoculum required for the chip pile were reduced to only 5 grams of fungal preparation per ton of wood by the addition ofunsterilized corn steep liquor to the inoculum. This high sugar compound gives the fungi an initial boost, resulting in rapid growth and colonization ofthe chip pile. Fungal growth also required oxygen, proper levels of moisture (about 60% by weight) within the wood chips, and temperature regulation ofthe chip pile to prevent the thermal death of the fungi. During scale-up, a process was developed where the wood chips moved from a storage hopper onto a conveyer belt on which they were quickly steamed. They were then allowed to cool and then moved onto a second conveyer, on which they were inoculated. From there they were dumped into the chip pile that was ventilated with humidified air from below. This aeration maintained the chip pile at the proper temperature, removing heat as it was formed. The fungi were also provided with the proper amount of oxygen and moisture. The experimental chip pile formed at the Forest Products Laboratory contained 50 tons of chips and measured 4 X 9 X 21 m! It took workers 24 hours, working night and day, to process this many chips! After two weeks of fungal growth, the wood chips were pulped and the resultant test sheets of paper analyzed. The sheets made through biopulping showed an energy savings of about 30% and increased tensile strength. Some research has also been done on biochemical pulping. This process has not been as extensively studied as biomechanical pulping, but also seems to hold promise. The fungal pretreatment seems to increase the porosity ofthe wood, allowing more efficient penetration of the pulping chemicals throughout the wood structure. Cooking time was reduced from 90 to 30 minutes, thus resulting in large energy saving. The process is still in the research phase. Enzymatic Bleaching: The most common application of enzyme technology, rather than the use of the whole organism, in the pulp and paper industry is the process ofbleaching. The majority ofpulp produced in the U.S. is made by the Kraft pulping process. In this type of chemical pulping, lignin is removed from the wood, and hemicellulose is dissolved and degraded without damaging the cellulose. The pulp has excellent physical properties and results in the production ofhigh quality, high strength paper. Unfortunately, many ofthe degradation products become trapped within the pulp, giving it a brownish color. This is removed through a bleaching process. Traditionally various forms of chlorine were used for bleaching, but these have been replaced by alternatives, such as oxygen, hydrogen

6 peroxide, and chlorine dioxide, due to environmental considerations. Xylanases have been shown to reduce the amount of chemical required for bleaching. The exact mechanism of this is not known, but it is thought that the partial hydrolysis ofthe hemicellulose partially loosens the pulp structure, enhancing the removal oflignin and other colored compounds (chromophores), reducing chemical consumption, and resulting in pulp with higher brightness and better strength. Endomannanases are also useful but not as efficient as the xylanases. The process must be limited because the best results occur when the hemicellulose is depolymerized (i.e. the bonds are broken) but not actually removed from the cellulose fibers. The xylanases most useful for enzymatic bleaching have low molecular weights so they can move easily among the fibers and can work in the appropriate range ofph. The enzymes must also be thermally stable so that they do not denature at high temperatures, and they should have an alkaline isoelectric point so that they can bind to the fibers that are negatively charged during the conditions used in the pulping process. Enhanced Drainage: In order to make paper, the fibers in the pulp are suspended in water. This slurry is then deposited on a screen to make paper. The water runs through the screen and the fibers are pressed and dried to form paper. The rate of drainage of the water through the screen can greatly influence the speed, and thus the cost, of paper production. This is especially important when recycled fiber is used since contaminants in the recycled fiber can significantly slow drainage rates. Recycled fibers treated with a mixture ofxylanase and cellulase increased drainage by as much as 20%. Commercial enzymes are available for this process. Many of them are derived from bacteria rather than wood decay fungi because ofthe large quantities ofenzymes that can be generated inexpensively and rapidly by bacterial fermentation. Fiber Modification: Specific modifications can be made to pulp fibers by enzymatic treatments. Xylanases have been used to remove xylan from pulp without affecting other components. This is important during the process of rayon manufacture. Cellulases have been used to reduce the size ofvessels found in tropical hardwoods, such as eucalyptus, that can make the surface of the paper rough and uneven. By smoothing the surface of the paper, the ink can be deposited in a more uniform layer leading to better print resolution. Enzymes have also been used for biochemical pulping ofnonwoody fibers, such as those found in kenaf, flax, jute, and coconut hulls. This process is termed retting and can be done with whole organisms or isolated enzymes. Enzymatic retting is faster and produces fewer odors than the traditional microbial retting methods. Resin Hydrolysis and Pitch Control: Softwoods, the principle source of pulp in the U.S., contain resins and other extractives that can interfere with paper manufacture. The presence of resins, or pitch, can interfere

7 with a paper s ability to absorb water - important for certain types of paper, such as paper towels, tissues, toilet paper and disposable diapers. Resinous materials can also deposit on the press rolls of the paper machine. This can result in the formation of holes or spots on the paper that subsequently passes over this part ofthe machine. Colorless strains of a nondecay fungus, Ophiostoma piliferum, are commercially available under the trade name Cartapip. This organism has been used as a pretreatment to reduce pitch. The fungus is inoculated into chip piles during storage and grows rapidly throughout, removing pitch from the wood chips. Since the fungus is colorless, its rapid colonization ofthe chips prevents growth of contaminating stain fungi that impart a bluish or grayish color to the chips. The Cartapip treatment thus gives the final pulp a brighter color, reducing the need for bleaching by preventing the growth of sapstain fungi. Enzymatic treatments have also been used for pitch removal. These are usually bacterial- or yeast-derived lipases and can be very effective at removing pitch from different types ofpulp. Recycling Technologies: The use ofrecycled fiber greatly complicates the production ofpulp and. the manufacture ofpaper. Recycled material contains numerous contaminants, including ink, adhesives, sizing materials, waxes, toners, and surfactants. All ofthese must be removed and can present specific problems to recycling technology. Microbial cellulases seem particularly effective at removing ink and toner from recycled fiber. The mechanism for this is not entirely understood, although treatment with cellulase seems to reduce the surface area of the fiber and decrease the number ofpoints in which it is bound to the contaminant. Cellulase activity also increases the drainage rate of recycled fibers and thus improves separation technologies based on differential flotation. This is done by placing the fibers in a very dilute suspension and adding surfactants to increase wetting. After treatment with cellulases, the suspension is aerated and the hydrophobic bubbles trap the toner and ink particles and move them away from the fibers to the top of the tank where they are removed by skimming. Toner fibers with fewer fibers attached to them are more likely to be carried to the surface during flotation. Enzymatic treatment has been shown to increase the brightness of recycled pulp and to reduce the biological oxygen demand in the wastewater. Enzymatic techniques are more efficient than chemical deinking technologies. Bioremediation : The final process that will be discussed is bioremediation. This is not involved with pulp and paper production but is becoming increasingly used to clean up contaminated industrial sites. The one-electron oxidation chemistry that is involved in lignin degradation is a very nonspecific reaction. The free radicals generated by the lignin-degrading enzymes can break down a large variety ofpolycyclic aromatic hydrocarbons including PCPs, dioxins, and azo dyes. Bioremediation is defined as the use ofthe degradative abilities of microorganisms to decontaminate soil, wood, sludge, or water that contains hazardous compounds. Many highly toxic compounds can be broken down to carbon dioxide and water through the activity ofwhite-rot fungi. Many different species and strains ofwhite rot fungi have been screened to find the most efficient organisms for the degradation of

8 specific compounds. Bioremediation can be done by bioaugmentation, in which the contaminated material is inoculated with a specific strain of fungus. Another technique is biostimulation, in which the natural microflora ofa site is given stimulatory nutrients to increase their activity and subsequent breakdown ofcontaminating materials. Bioremediation is becoming a frequently used technique and has been used to clean up EPA Superfund sites. Certain fungi also concentrate heavy metals and can be used to decontaminate soils or other materials contaminated by mercury, lead and other toxic metals. The enzymes used in all ofthe processes have certain characteristics in common that make them useful in industry, either in an in vivo solid state fermentation process or in the form ofisolated enzymes. The enzymes must be produced inexpensively in large amounts and must be able to function in a fairly crude form without extensive purification. They must be stable under temperatures in which they will operate, which might be quite extreme, but they also need to be stable for storage at room temperature. The application of enzyme technology must fit in well with the preexisting industrial process; industry cannot afford to retool or reengineer an entire technology. The overwhelming consideration is price. The price ofthe final product must be competitive with materials generated by conventional processes. In many cases, industrial-enzymes are currently produced most efficiently bacteria although some specialized enzymes, such as the ligninases, can be generated only from decay fungi. Molecular techniques, such as genetic engineering, are being researched to make enzyme production from wood decay fungi more efficient. The future of enzymatic technology in industry is unlimited. As the world becomes more heavily populated, more polluted, and more dependent on a limited amount ofresources, we must use every opportunity provided to us from the microbial world to maximize efficiency and decrease harm to the environment. References and Further Reading:

10 University of Wisconsin-La Crosse

Biobleaching in dissolving pulp production

Proceedings of the 6th International Conference on Biotechnology in the Pulp and Paper Industry: Advances in Applied and Fundamental Research Biobleaching in dissolving pulp production L.P. Christov 1