Thomas W. Jeffries
USDA, FS, Forest Products Laboratory
One Gifford Pinchot Drive
Madison, Wisconsin 53705
Introduction
Paper manufacture is one of the largest industries in the United States. The US produced 71 million metric tons of paper and paperboard[1] with a wholesale value of over $47 billion in 1988,[2] and the pulp and paper industries in the US account for about $140 billion of the annual gross domestic product. The vast bulk consists of virgin fibers.
Only 27% of the total furnish for paper production in the U.S. consists of recycled or secondary fibers.[3,4] Virgin fibers are preferred for greater strength, fewer contaminants and more consistent properties. This situation, however, is changing due to environmental concerns. Increased demands on timber production have limited availability, and constraints on landfill siting have driven up the cost of waste paper disposal.
Paper production places a large demand on basic resources. It requires 3.3 tons of trees and 0.4 tons of petroleum to make one ton of paper from virgin materials.[5] These considerations have led to an increased emphasis on fiber recovery and recycle. Recycling fibers saves trees and energy, reduces waste effluents, and alleviates landfill problems.
Recycled fibers comprise up to a much larger fraction of the furnish in Japan, most of Europe and many other countries, and paper production in the United States is moving toward a 40% recycle rate by 1995. Even higher rates of fiber recycling might be practiced in the future, however, substantial capital investment and additional research and development will be required.
Paper pulp is, of course, a bio-based material comprised of cellulose, hemicellulose and lignin. As such, it is amenable to modification by microbial enzymes. Such treatments are potentially more environmentally benign than chemical processes, and the development of enzyme-based technology for the pulp and paper manufacture could become a major market segment for the enzyme industry. Because the technology for paper production is changing rapidly, this is a major prospective market for the enzyme industry.
Enzyme technologies
Several applications have been investigated for enzymatic treatments of pulp. These include modification of pulp properties such as improved fiber flexibility and fibrillation, decreased vessel picking from tropical hardwood pulps, improved drainage in recycled fibers, specific removal of xylan for dissolving pulp manufacture, facilitated bleaching of kraft pulp, enzymatic pulping of herbaceous fibers, enzymatic pitch removal, and facilitated contaminant removal from recycled fibers.
Fibrillation, inter-fiber bonding and strength enhancement
Paper strength, as measured by burst or tear indices is one of the most important properties. It is affected by the nature of the pulping process, the presence of additives, the fiber length, and the average chain lengths of the carbohydrate polymers. In general, chemical pulps such as kraft or sulfite make paper with better strength properties than mechanical pulps, and pulps made from softwoods are stronger than pulps made from hardwoods because the former have longer fibers. Recycled fibers are not as strong as virgin fibers due to a loss of hydratability, swelling and fiber flexibility that occurs in the paper forming and drying process.
Properly applied, microbial enzymes could enhance or restore fiber strength, reduce beating times, and increase inter-fiber bonding through fibrillation. It is important, however, to achieve these objectives while increasing drainage rates and avoiding cellulose depolymerization.
Beating or refining are mechanical processes to improve paper strength by enhancing fibrillation and inter-fiber bonding. Pulp fibrillation by cellulases was recognized as a means to enhance strength properties as early as 1959.[6]. Cellulases from Aspergillus niger were used to separate and rearrange paper fibrils This technology improved the strength of paper by increasing fiber contact and bonding. It was principally applied to cotton linters and other non-wood pulps. A process patented in 1968 used cellulases from a white-rot fungus, applied at a concentration of 0.1 to 1% (based on the dry wt of pulp), to reduce refining or beating time.[7]
The principal challenge in using enzymes to restore fiber bonding properties is to enhance fibrillation without reducing pulp viscosity. In one attempt to get around this problem, researchers used "cellulase-free" xylanase from mutants of Sporotricum pulverulentum and Sporotricum dimorphosphorum to defiberize pulp.[8,9,10,11] Relatively low xylanase treatments (0.001-0.1%, w/w) removed less than 2% of the total dry weight. The researchers employed mercuric chloride to inhibit cellulases. However, the mechanism or function of mercuric chloride is not entirely clear, nor is it likely that such a process would be environmentally acceptable. Enzymatic treatment reportedly improved defibrillation and fiber bonding while decreasing beating times. This treatment increased Schopper-Riegler (SR) index, and increased the water retention value abut 80%. At the same time it decreased viscosity about 30%, and decreased breaking length drastically. Some cellulases have been reported to accelerate digestion of pulps without damaging fibers. Nomura reported that cellulase (0.05-0.5%) plus cellobiase (0.005-0.015%) added to pulps in water, pH 4-7, 60deg.C facilitated fibrillation and pulp digestion without strength loss.[12] Clearly, this is an area that deserves more attention.
Drainage
It is also important to enhance the drainage rates of recycled fibers. Drainage rates determine the loss of water during paper formation. Recycled fibers tend to have much lower drainage rates than virgin fibers, and this slows down the paper making process. In the US, where manufactures use large quantities of virgin fibers, paper machines tend to be designed for such stocks. As larger quantities of recycled fibers are introduced into paper manufacture, drainage rates slow down and it is necessary to operate the machines at lower rates.
Enzyme treatments can improve the drainage rates of recycled fibers.[13] In one study, treating recycled fibers with cellulases and xylanases reduced the SR value (increased drainage) by 18-20%. Commercial enzymes such as Maxazyme 2000 (RAPIDASE), Cellulase 250 P (Genencor), and SP 249 (NOVO) were used to treat batches of recycled fibers on both lab and pilot scale. In general, pulp strength was reduced, but it could be improved by refining the pulp before enzyme treatment. Starch sizing likewise improved mechanical properties. These preparations all contained C1, Cx, and endoxylanase, so from these results, it is hard to determine which of the enzyme components were responsible for increasing freeness.
Modification of pulp properties
In some instances, enzymes can improve paper properties in specific ways. For example, tropical hardwoods contain large vessels that make a rough paper surface. During printing, these vessels prevent complete contact of the ink with the paper surface, and lead to an imperfect image. This can be particularly important in large image copy, and it is of increasing significance as Brazilian eucalyptus pulps are being used in larger amounts. Cellulase treatment can reduce vessel picking which occurs during printing. Pulps formed from tropical hardwoods can be smoothed by cellulase treatment. Treating with 0.01-5% cellulase SP 227, or BAN 204L (NOVO) reduces vessel picking by 85%.[14]
Xylanases can remove xylan from pulps without affecting other components. This is important in preparing dissolving pulps for rayon manufacture or for recovering hemicellulosic sugars while leaving the cellulose intact. For example, xylanase from Schizophylum commune reduced hemicellulose 22% in a delignified mechanical aspen pulp,[15] purified xylanase from Trichoderma harzianum reduced xylan content of unbleached kraft 25%,[16] and a cloned xylanase from Bacillus subtilis reduced xylan content of bleached hardwood pulp 20%.[17]
Enzymatic pulping
In general, enzymes cannot be used to pulp wood fibers because the lignin contents are too high. Microbial or enzymatic processes can, however, pulp herbaceous fibers. Microbial retting is an ancient process dating to the beginnings of civilization. Traditional retting uses mixed microbial populations with crude inocula. Fibers that are retted include flax, jute, coconut hulls[18] and bast. Contemporary practice uses selected microbial strains or purified enzymes.
Pectinolytic enzymes secreted by the soft-rot bacterium Erwinia carotovora or Erwinia chrysanthemi[19] cause maceration of bast fibers. Endo-pectate lyase and endo-pectin lyase are most important with the optimum ratio of the two enzymes being about 30:1. During the pulping of Edgeworthia papyrifera, endo-pectate lyase elutes a lignin carbohydrate complex (LCC) rich in L-arabinose and D-galactose while endo-pectin lyase elutes mainly D-xylose.[20] Alkaline presoaking greatly enhances enzymatic activity.[21] It decreases the Canadian Standard Freeness and the shives content, and it improves sheet formation.[22] Enzymatic pulps prepared with endo-pectate lyase enzymes produce bulkier paper with higher opacity and better printability than pulps prepared from the same stock by a soda ash process. The strength of paper made from enzymatic pulp is comparable to that from the chemical process.[23]
Commercial enzymes such as cellulases, hemicellulases, pectinases and other polysaccharidases have been applied to flax at various levels and compared to traditional retting methods.[24] The chief disadvantage of the traditional method is the bad odor that develops in the retting tanks, the handling of the retted flax and the discharge of the effluent. The biochemical method avoids many of these problems. If the green flax is dried artificially at high temperatures, it is difficult for the enzymes to penetrate the outer cuticle, but low temperature drying does not have this effect. Enzymatic retting is faster and produces fewer odors, but may not be economically competitive with more traditional methods. Further development is, therefore, required. Chemical and enzyme retting have both been carried out on a semi-industrial scale, and the characteristics of the fibers produced by these two methods are not significantly different.[25]
In more recent years, a few fundamental studies have been initiated on the biochemical retting process. These employ purified enzymes on defined substrates, and characterization of the resulting products. A purified endo-polygalacturonase from Aspergillus preparation will release three size classes of polysaccharides from flax.[26] In order to ensure maximum strength of the thread manufactured from retted flax, only a small fraction of the pectins belonging to the fiber bundles need to be hydrolyzed. Some advantage might be gained, therefore, in using enzyme preparations with better specificity.
In developing nations, and particularly in countries where forest stands are endangered from over exploitation, better use might be made of herbaceous fibers for paper production. Such feedstocks should be amenable to biochemical pulping, and the resulting processes should give higher yields with fewer environmental problems. Clearly, however, much more work needs to be done in this area before biochemical pulping of herbaceous fibers will see wide application.
Resin hydrolysis and pitch control
Certain types of cellulosic pulps - such as sulfite pulps and various mechanical pulps - have high resin contents. These resins can interfere in the pulp manufacture process and can also impart negative properties to the final product.
Mechanical pulping, either alone or combined with chemical processes - such as alkaline sulfite soaking to make chemithermomechanical pulp (CTMP) - is widely used in pulp manufacture because pulp yields are high (85 to 95%), and the resulting fibers are useful for manufacturing fluff pulps for tissues, towels, sanitary articles or disposable diapers.
CTMP manufacture is carried out at pH 4 to 9 and the components in the wood undergo few changes. The pulp therefore has a high resin content which, for the purpose of fluff pulp manufacture, can interfere with its absorptive properties. When resins are present, the rate at which a pulp will absorb liquid is a function of pH. At low pH, the absorption time increases dramatically.
Figure 1. Absorption time of reference pulp and lipase-treated pulp as a function of the liquid pH. (From reference 27).
Problem lipids are triglyceride esters of fatty acids. They can be removed by solvent extraction or by strong alkali, but the former process is not practicable, and the latter results in yield losses or discoloration.
This has led to the development of processes based on microbial lipases. Incubating CTMP in the presence of lipase decreases absorption time of the resulting paper - particularly at low pH (Fig. 1). Moreover, lipase treatment increases the network strength and the specific volume.[27]
Resinous materials from pine groundwood pulps can deposit on stock chests and press rolls during paper manufacture, resulting in pitch spots and holes in the paper web.[28] It is well known that seasoning the wood chips or adding talc can reduce pitch troubles, but seasoning can require long storage times and often leads to discoloring. Colorless strains of the resin-digesting fungus, Ophistoma, have been selected and developed to remove pitch during chip storage without degrading pulp properties.[29] While this is a satisfactory biological process, it does not work well in all climates or at all times during the year. For this reason, an enzymatic process has been developed to remove pitch. A lipase obtained from Candida cylindrica when added to the groundwood stock chest at a rate of 3 ppm (based on dry groundwood pulp) reduces pitch problems and talk consumption considerably.[30]
Pitch in sulfite pulps can also present problems during paper manufacture. Pitch deposits on exposed parts of the paper machine such as air foils or machine wire can degrade the product and impair production. Chlorinated triglycerides are particularly troublesome. Formation of pitch deposits originates during bleaching when the double bonds of pitch triglycerides are chlorinated. The chlorinated pitch then is released from fibers and accumulates in the water system where it accumulates deposits through poorly understood processes. Fisher and Messner have found that by treating unbleached sulfite pulps at 2.9% consistency with 0.02% (vol/vol) Resinase A (Novo) for 2 h at 37 deg.C followed by alkaline extraction, they can remove 86% of the triglycerides from sulfite pulp.[31] The enzyme absorbs rapidly to pulp fibers and it is not really possible to recycle them for reuse. On the other hand, their absorptive properties enable application at low pulp consistencies, and the enzymes remain active and attached during various treatment and washing stages.[32] This process has been scaled-up in a 12 ton per day pulp trial, and has been shown to remove 90% of the triglycerides in a very short period of time.[33]
Enzymatic pretreatments for bleaching
The most common application of enzymes in pulping is to enhance bleaching. At least 15 patents or patent disclosures dealing with enzymatic treatments to enhance bleaching of kraft pulps were submitted between 1988 and 1993. In addition, there have been numerous presentations in conference proceedings. The reason for this interest lies in the extreme economic importance of kraft pulping and the environmental effects of chlorine bleaching. Most proceedings reports and many of the patent disclosures have been reviewed in earlier publications,[34,35,36,37] so the present report will concentrate on more recent literature.
The kraft process accounts for 85% of the total pulp production in the United States, and it is the largest component of paper manufacture world-wide. Bleached kraft pulp is a major, relatively high-value component of that total. Kraft pulping removes lignin, dissolves and degrades hemicellulose without damaging cellulose. The kraft process results in excellent pulp from a wide variety of wood species. Kraft pulp fibers are flexible, readily hydrated, and the pulp matrix is accessible to enzymes.
Unfortunately, degradation products generated during pulping become trapped in the matrix and impart a brown color to kraft pulp. Cooking consumes pulping chemicals, and residual xylan (along with covalently-linked degradation products) precipitates on the surfaces of the cellulosic fibers.[38,39 ] The chromophores are believed to be composed of residual lignin and carbohydrate degradation products.[40] They are hard to extract because they are covalently bound to the carbohydrate moieties in the pulp matrix.[41,42,43]
Manufacturers use elemental chlorine (Cl2) and chlorine dioxide (ClO2) to bleach the chromophores, and then they extract the pulp to make white paper. The cost of Cl2 is about $12 to $15 per metric ton of pulp, but because this results in the production of chlorinated aromatic compounds, alternative bleaching agents such as O2, ClO2 or H2O2 are employed. These can be several times more expensive than Cl2. Bleaching substantially increases the value of the product, so the additional expense can be justified. As an example, unbleached kraft pulp for shipping bags costs $550 per ton, but bleached kraft pulp for the same product costs $800 per ton.
Chlorine bleaching can create environmental problems. Cl2 makes toxic and recalcitrant chlorinated aromatic hydrocarbons (including small amounts of dioxin), and pulp manufacturers must use special processes to remove them from effluent streams. Consumers are becoming increasingly wary of chlorine bleached papers for products such as tissues, coffee filters, diapers, paper cups and paper plates. Moreover, environmental regulations have largely eliminated chlorine bleaching as an acceptable process in several European countries.
Numerous reports have shown that xylanases can reduce chemical demand in subsequent bleaching reactions.[44,45] Treatments are effective on both hardwoods and softwoods, but they have a greater effect on hardwood kraft pulps. The hydrolysis of hemicellulose appears to partially loosen the pulp structure, thereby enhancing lignin extraction, reducing chemical consumption, and attaining higher brightness.[46] Endoxylanases are much more effective than mannanases. Moreover, mannanase treatments have very little effect on handsheet properties, whereas xylanase treatments cause the pulp to become more beatable and increase paper strength. The amount of hemicellulose solubilized by xylanase increases with the kappa number or pulp yield, but the amount of mannan solubilized by mannanase does not increase. Interestingly enough, the reduced chlorine demand following enzyme treatment cannot be explained simply on the basis of the hemicellulose solubilized.[47] Although not all mannanases are equally effective, the mannanase from Trichoderma reesei has been shown to act synergistically with xylanases to enhance pulp bleaching.[48]
Purity of the xylanase is very important; cellulase should not be present. If cellulases are present, pulp viscosities decrease. In the absence of cellulase, enzyme treatment increases viscosity because the lower molecular weight xylans are removed. The mechanical strength of fibers is not affected when pure xylanases are employed. Treating pulps with cloned xylanase can avoid viscosity losses.[49] This, however does not mean that paper strength properties are unaffected. Roberts et al.[50] showed that when a cellulase-free xylanase from Saccharomonospora viridis was used to selectively remove 20% of the residual xylan in a kraft birch pulp, burst strength and long span tensile strength decreased. The results indicated that xylanase activity can weaken inter-fiber bonding although it might not weaken the fibers themselves.
Effective xylanases should possess several properties. First, they should be stable on kraft pulps. Some xylanase preparations non-specifically absorb to pulp fibers and are inactivated by degradation products from kraft pulping.[51] Second, they should have a neutral to alkaline pH optimum and good thermal stability.[52] The pulp is hot (75deg.C) when it first comes out of the stock washers, and residual alkali leaks out of the pulp during enzyme treatment. The pH of even well-washed pulp stocks can shift dramatically. Third, factors affecting the interaction of the enzymes with the pulps are important. These include the effective molecular weight, net ionic properties, and specific action pattern.[53]
The mechanism of enzymatic prebleaching is not completely understood. Kantelinen et al. suggested that precipitated xylan blocks the extractability of lignin from the kraft fibers.[54] Xylan is dissolved in the kraft cooking liquors, but as the cook proceeds, the alkali concentration decreases, leading to a reprecipitation of xylan on the fiber surface. Chromophores generated during the kraft cook cross-react with the xylan and are either physically occluded by or chemically bound to the reprecipitated xylan.
Paice et al. have shown that the prebleaching effect on black spruce pulp is associated with a drop in the degree of polymerization, even though the xylan content decreases only slightly.[55] A similar but smaller effect is seen with mannanase treatment. Prebleaching appears to result from depolymerization, but not necessarily solubilization of the xylan-derived hemicellulose components. This suggests that cleaving xylan to allow release of covalently bound chromophoric material is more important than removing a physical barrier to extraction.
Xylanases show different effects that appear to vary with their properties. For example liberation of reducing sugars by a xylanase from T. reesei having a pI of 5.5 correlated well with its bleaching effect, but a pI 9.0 xylanase from this same organism achieved high brightness with less than 2% of xylan hydrolysis when the treatment was carried out at pH 7.0.[56] Pulp fibers are generally negatively charged at neutral pH due to the presence of carboxylic acid groups. Therefore, a protein with a pI of 9.0 would possess a net positive charge at pH 7 and would absorb to the fibers. Even though its activity might be low at this pH, it could still be effective; whereas, a protein with a pI of 5.5 would have a net negative charge and might not absorb at all.
Skjold-Jørgensen et al. have examined some of the basic mechanisms involved in xylanase bleaching.[57] They found that xylanase treatment decreased the chlorine demand (aCl2) for a batch kraft pulp by 15%, but decreased aCl2 continuous cooked kraft pulp by only 6 to 7%. In a continuous digester, the solubilized xylan is carried away in the cooking liquor and does not precipitate to a great an extent. Therefore the greater bleaching effect on a batch-cooked pulp is consistent with a role for reprecipitated xylan in preventing chromophore removal. The xylan present in kraft pulp consists of two fractions: one which is soluble in dimethyl sulfoxide (DMSO), and the other which is degraded by xylanase. While DMSO treatment does not lead to an increase in bleachability, xylanase treatment does. This indicates that the DMSO-insoluble xylan fraction is more important for the xylanase bleaching effect. One would expect that the DMSO-soluble xylan is either of a lower molecular weight or is not chemically cross-linked into a DMSO-insoluble polymer such as residual lignin. These findings suggest that xylanases facilitate the extraction of lignin by breaking covalent bonds between insoluble xylan and lignin or chromophoric materials.
It may be possible to identify enzymes that have highly specific effects on chromophores without substantially affecting xylan at all. Patel et al. have recently described four xylanases from Streptomyces roseiscleroticus that release chromophores and reduce the kappa number of hardwood and softwood kraft pulps. All four enzymes possess endo-xylanase activity, but some result in greater kappa reduction and others release more chromophores. Characterization of the chromophoric materials by reverse-phase HPLC indicated that compounds absorbing strongly in the visible region were relatively hydrophobic.[58]
Enzymatic enhancement of contaminant removal
Printing and writing grades used in offices are among the most valuable papers manufactured and sold. They amount to about 25% or 20 million tons per year, and are a rapidly-growing segment of the paper industry. About 88% of un-segregated office waste paper (OWP) is composed of chemical fibers.[59] Less than 10% of office waste papers are recycled back into printing and writing grades. The recycling rate could increase several fold to about 40% or about 8 million tons if an effective means were found to release toners from office waste. High-quality office waste papers consisting largely of uncoated, bleached kraft paper can be segregated.
Mixed waste papers present technical and economic challenges to the paper recycler, and of the wide variety of fibers and contaminants present in the paper stock, toners and other non-contact polymeric inks from laser-printing processes are the most difficult to deal with.[60] Toners and laser printing inks are synthetic polymers with embedded carbon black that do not disperse readily during conventional pulping processes. Moreover, they are not readily removed during flotation or washing. Because of these problems, recycled papers contaminated with non-contact inks have a lower value. For example, laser-free waste computer printout paper wholesales for about $266/ton whereas computer paper containing laser-printed toners has a wholesale value of about $170/ton.[61] With effective toner removal, the total value of office waste paper could increase about $575 million annually, and avoided landfill disposal costs would amount to about $240 million annually
Conventional deinking uses surfactants to float toners away from fibers, high temperatures to make toner surfaces form aggregates, and vigorous, high intensity dispersion for size reduction. Most of the deinking chemicals and high-energy dispersion steps are expensive. Deinking chemicals are used a 1 to 3% by weight of paper, and they cost between $1 and $3 a pound. The high-energy dispersion step is both capital and energy intensive and can also reduce fiber length. The total cost of conventional deinking amounts to $10 to $100/ton of processed pulp.
Enzymes can also enhance deinking of recycled fibers. Treating used paper with cellulases, hemicellulases or pectinases prior to deinking results in a brighter pulp of higher quality,[62] and cellulases have been used to deink newsprint.[63] Alkaline lipases will facilitate the removal of lipid-based offset printing inks.[64,65]
Microbial enzymes have also been shown to enhance the release of toners from office waste.[66] When cellulases and xylanases were applied to xerographic-printed papers in a medium consistency mixer, they released toner particles and facilitated subsequent flotation and washing steps. In comparison to water deinked pulps, enzymes released 95% of the residual toner particles from recycled fibers. The amounts of enzymes required were highly cost effective with conventional deinking chemicals.
Conclusions
Enzymes can be used in many stages of pulp and paper processing. These include enhancing digestion, improving drainage, increasing fiber flexibility, selectively removing xylan, removing pitch, facilitating bleaching, and removing contaminants. Enzymes can be used to fibrillate chemical pulps and herbaceous fibers. By combining enzymatic and chemical treatments the fiber properties can be improved while maintaining high pulp yields. The value added by enzyme treatments can readily justify the enzyme cost in many instances, so enzyme technology should see wide use in the future.
For comments or further information write to Tom
Jeffries: twjeffri@facstaff.wisc.edu