Chapter 23
Possible Roles of Xylan-Derived Chromophores in Xylanase Prebleaching of Softwood Kraft Pulp 1
Enzymatic Degradation of Insoluble Carbohydrates Downloaded from pubs.acs.org by YORK UNIV on 12/02/18. For personal use only.
Ken Κ. Y. Wong, Patricia Clarke , and Sandra L. Nelson Chair of Forest Products Biotechnology, Department of Wood Science, Faculty of Forestry, University of British Columbia, 270-2357 Main Mall, Vancouver, British Columbia V6T 1Z4, Canada Xylanase treatment can directly brighten partially bleached kraft pulps but not brownstocks. It usually leads to a direct decrease i n the kappa number of the pulps. The degree of direct brightening seems to be correlated w i t h the amount of U V - a b s o r b i n g material solubilized from the pulps, rather than the amount of sugars. Previous results also showed that it d i d not correspond to the brightness gain after subsequent peroxide bleaching. There is therefore no evidence indicating that x y l a n - d e r i v e d chromophores are the m a i n target substrates during xylanase prebleaching of kraft pulp.
Xylanase prebleaching of kraft pulp has been implemented i n several kraft mills i n N o r t h America and Europe i n order to reduce the environmental impact of their bleach plant (1). This process enhances subsequent chemical bleaching, thereby reducing the chemical loading required to reach target p u l p brightness (2). The mechanism b y w h i c h xylanase enhances p u l p bleaching remains unclear. Kantelinen et al. (3) have proposed that the enzyme attacks xylan redeposited on the surface of pulp fibers at the end of the kraft cook. However, the removal of the surface xylan w h i c h is extractable w i t h dimethyl sulfoxide does not enhance bleaching (4). Other studies indicate that xylanase treatment of kraft pulp also leads to extensive depolymerization of xylan (5). To-date, four hypotheses have been made concerning the nature of the target xylan (6): • xylan i n lignin-carbohydrate complexes; • xylan that physically entraps residual lignin; • xylan that modifies fiber porosity; • xylan that is chromophoric. Current address: Pulp and Paper Research Institute of Canada, 3800 Wesbrook Mall, Vancouver, British Columbia V6S 2L9, Canada
0097-6156/95/0618-0352$12.00/0 © 1995 American Chemical Society
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A n objective of our w o r k was to eliminate one of these hypotheses, namely the role of xylan-derived chromophores i n xylanase prebleaching. It is hypothesized that chromophoric units derived solely from xylan are still carried on xylan polymers. These hypothetical polymers may be, at least i n part, h y d r o l y z e d by xylanase and they may contribute to the adsorptive and light-scattering properties of pulp, and thus its reflectance. Lignin-carbohydrate complexes differ i n that their chromophoric units are assumed to be derived from the lignin component. The hypothesis that xylan may contribute directly to the color of kraft p u l p is based on the observations of Harder and N o r r s t r ô m (7). These authors found that holocellulose develops color after kraft cooking, and thus estimated that about 10 % of the light absorption properties of kraft pulp could be attributed to carbohydrates. In addition, the reflectance of acellulose was reported to decrease after cooking w i t h xylan (8). W h e n cellulose was cooked w i t h sugars, the resultant decrease i n pulp brightness could be easily reversed by bleaching (9) and this decrease was more substantial if tannins were also present (8). Recent studies have also indicated that the kraft or alkaline cooking of monosaccharides produces aromatic compounds such as dihydroxybenzaldehyde, compounds based on benzendiols and acetyophenones (10), and h i g h molecular mass material that yields cresol, ethylphenols and phenol upon pyrolysis (11). Although xylanases are not k n o w n to directly modify lignin, which is considered to be the major contributor to p u l p color (7, 8,12,13), these enzymes might enhance p u l p bleaching by r e m o v i n g x y l a n - d e r i v e d chromophores. In the present work, this hypothesis on the mechanism of xylanase prebleaching is evaluated by examining the direct effects that xylanase has on different kraft pulps. Materials and Methods X y l a n a s e s . One purified xylanase and two commercial xylanases were used i n the work. Enzyme A was Irgazyme 40, a crude Trichoderma xylanase commercially produced by Genencor (San Francisco, U S A ) . Enzyme Β was Pulpzyme H B , a bacterial xylanase commercially produced by N o v o N o r d i s k (Bagsvaerd, Denmark). Enzyme E , a 22 k D a xylanase belonging to Family 11 of glycanases, was purified from Trichoderma harzianum E58 as reported by Maringer et al. (14). Kraft P u l p s . Samples of brownstock and oxygen delignified kraft p u l p derived from western hemlock (Tsuga heterophylla) were k i n d l y provided by H o w e Sound P u l p and Paper (Port M e l l o n , Canada). The kraft pulps derived from Douglas-fir (Pseudotsuga menzeisii) and western red cedar (Thuja plicata) were provided by Fletcher Challenge (Crofton, Canada). P u l p B l e a c h i n g . Xylanase treatment was carried out at 10 % p u l p consistency and 50 °C for 1 h. Its start p H , adjusted using H S 0 , was 7 for Enzymes A and B , and 4.8 for Enzyme E. The enzymes were each loaded at 2
4
354
ENZYMATIC DEGRADATION OF INSOLUBLE CARBOHYDRATES
200 nkat xylanase/g pulp, w i t h the activity assayed at the start p H . The dinitrosalicylic acid-based assay of Bailey et al. (25) was used w i t h citrate buffer at p H 4.8 and phosphate buffer at p H 7. The enzyme was replaced w i t h water, or boiled enzyme when specified, i n the control treatment. Most of the data presented for Enzymes A and Β were collected as parts of a survey on the effects that different xylanases have on the bleaching of different kraft pulps (26). Chemical bleaching was carried out using peroxide (2 % H 2 Q 2 ; 2 % N a O H ; 0.05 % M g S 0 - 7 H 0 ) at 10 % pulp consistency and 80 °C for 3 h. A 30 m i n . chelation stage was always carried out before peroxide bleaching using 1 % N a - E D T A - 2 H 0 at a start p H of 5.5, 3 % consistency and 50 °C, and it was followed by p u l p washing. Oxygen delignification of the brownstock derived from Douglas-fir was carried out i n a M a r k IV reactor (Quantum Technol. Inc., Twinsburg, U S A ) using 1.5 % N a O H and 0.3 % M g S 0 - 7 H 0 at 70 psi oxygen, 10 % consistency and 90 °C for 1 h. A l l chemical loadings were based on the dry weight of the pulp. A n example of a bleaching sequence is O X Q P , where Ο = oxygen delignification, X = xylanase treatment, Q = chelation, and Ρ = peroxide bleaching. 4
2
4
2
2
2
A n a l y t i c a l T e c h n i q u e s . The brightness of p u l p handsheets ( C P P A Standard C.5) was measured using a Technibrite T B - l c instrument (Technidyne, N e w Albany, U S A ) , and the lignin content was quantified by determining the microkappa number ( T A P P I Useful M e t h o d U M 250, 1991). Filtrates from the enzyme treatment were analyzed for the solubilization of U V - a b s o r b i n g material and sugars. A b s o r p t i o n at 280 n m was measured after appropriate dilution of the filtrate to give an absorbance ranging from 0.3-0.7, and the relative absorption was then calculated by m u l t i p l y i n g the absorbance w i t h the d i l u t i o n factor and subtracting the enzyme blank. Total sugars were quantified using the phenol-sulfuric acid method (27), w i t h the filtrate diluted eight folds and results verified using samples spiked w i t h a k n o w n quantity of xylose. Results and Discussion O u r study examined the direct effects that three xylanases have on brownstock, oxygen delignified and peroxide bleached kraft pulps derived from three softwood species. P u l p handsheets were made immediately after the enzyme stage and after subsequent peroxide bleaching. They were compared w i t h pulp that had undergone a control treatment, carried out under the same conditions. To simplify the discussion, differences i n pulp brightness and kappa number were considered to be substantial when they were greater than 0.5 % ISO and 0.5 p t , respectively. For brownstocks derived from Douglas-fir and hemlock, treatment w i t h xylanase d i d not lead to a direct increase i n p u l p brightness as compared to the control treatment (Figure 1). In most of these trials, a subsequent peroxide stage indicated that bleaching had been enhanced by the enzyme treatment. The exceptions were the pretreatment of the brownstock derived from Douglas-fir w i t h Enzymes A and B, although
23. WONG ET AL.
Xylanase Prebleaching of Softwood Kraft Pulp
355
the former enzyme was found to enhance p u l p brightness when a second peroxide stage was used (26). There is therefore no evidence that xylanase directly brightens brownstocks when used under conditions i n w h i c h it enhances subsequent peroxide bleaching. However, a direct decrease i n kappa number was observed i n most cases (Figure 1), the exceptions being the pretreatment of hemlock brownstock w i t h Enzymes Β and E . Other trials w i t h Enzyme Ε indicated that it can directly decrease the kappa number, but not increase the brightness, of the brownstock derived from hemlock as w e l l as that from red cedar (Table I, trials l a , 2a & 3). Boiled enzyme controls were also performed for the experiments carried out with Enzyme E , and the results were essentially indistinguishable from those obtained with the water controls (data not shown).
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Table I. The direct effects that Enzyme Ε has on various unbleached and partially bleached kraft pulps
23.
WONG ET AL·
Xylanase Prebleaching of Softwood Kraft Pulp
357
In general, our results agree with most of the other work reported on the direct effects that xylanase has on kraft pulps. Direct decrease i n kappa number has been reported by several workers (18-23), but the case for direct brightening remains debatable. Direct brightening by xylanase was not found i n a hardwood kraft pulp by Paice et al. (28) nor i n an oxygen delignified softwood pulp by Pedersen et al. (24). However, it was reported in an oxygen delignified hardwood pulp by Pedersen et al. (24), and i n both hardwood and softwood pulps by Yang and Eriksson (29). In our work with softwood brownstocks, it is not clear w h y the direct decrease i n kappa number was not associated w i t h direct p u l p brightening. More w o r k is therefore required to characterize the materials solubilized from the pulp. Our results on partially bleached kraft pulps suggest that they can be directly brightened by xylanase (Figure 2). Both Enzymes A and Β directly brightened two peroxide bleached p u l p s , whereas o n l y E n z y m e A brightened the oxygen delignified pulp. For Enzyme A , a direct decrease i n the kappa number of the p u l p was also observed. The scatter plot summarizing the results clearly shows that partially bleached pulps were directly brightened but not brownstocks. A l t h o u g h the degree of direct brightening d i d not correspond to the brightness gain after subsequent peroxide bleaching (26), the direct effects that xylanase has on more fully bleached p u l p warrant further investigation i n order to determine whether the enzyme can further brighten the pulp.
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358
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Trials have been carried out on QPP-bleached p u l p derived from hemlock using the purified xylanase, Enzyme Ε (Table I, trials l b & 2b). The results indicate that Enzyme Ε cannot directly brighten bleached kraft p u l p derived from hemlock, even though it can enhance the bleaching of the brownstock (Table I, trials l a & 2a). Indeed, this enzyme was found to be unable to directly brighten peroxide bleached pulps from three w o o d species and oxygen delignified p u l p derived from hemlock (Table I, trials 4-7). The absence of direct brightening i n these cases could be due to several factors, such as the difference i n the enzyme, pulp or start p H used. O n the other hand, it is also possible that the removal of metal ions during bleaching has reduced the hydrolytic efficiency of this enzyme, as has been reported for a similar xylanase purified from T. reesei (25). A n unexpected correlation was found between the amounts of U V absorbing material s o l u b i l i z e d from p u l p and the degree of direct brightening (Figure 3), but these two variables were not correlated w i t h the direct decrease i n kappa number (Figures 1 & 3). It w o u l d appears that U V absorbance might be more indicative of lignin than kappa no., where a reaction w i t h permanganate is the basis for measuring l i g n i n . It is therefore possible that some of the UV-absorbing material present i n p u l p do not react w i t h permanganate or that some of the material reacting w i t h permanaganate do not absorb i n the U V range. For example, compounds other than lignin might be oxidized by permanganate. These oxidizable compounds, along w i t h certain l i g n i n structures, might have little absorbance i n the visible range and thus further complicate the relationship between pulp brightness and kappa no. These considerations could help explain how Enzyme A can decrease the kappa no. of different pulps to the same extent while solubilizing different amounts of U V absorbing material, and how Enzymes Β and Ε can decrease the kappa no. i n three cases without s o l u b i l i z i n g large amounts of U V - a b s o r b i n g material. O u r results indicate that xylanases m a y vary i n their ability to decrease the kappa number of kraft pulps, as has been reported previously (22). Xylanases may also vary i n their ability to solubilize sugars from pulp (23,26,27). In our work, Enzyme A was found to be consistently more effective than Enzyme Β or Ε (Figure 4). The relationship between the solubilization of sugars or U V - a b s o r b i n g material and the change i n the brightness or l i g n i n content of p u l p may be further clarified if these variables are examined i n a specific pulp by changing enzyme loadings or reaction times. However, it may be difficult to resolve the relatively small changes i n pulp brightness and lignin content. A l t h o u g h our w o r k does not eliminate the possibility that x y l a n derived chromophores occur i n kraft pulp, it does suggest that they are not the sole explanation for the mechanism of xylanase prebleaching of kraft pulps. One reason is that when direct pulp brightening was observed i n partially bleached pulps, the increase i n pulp brightness was not the same as that achieved after additional peroxide bleaching (26). Furthermore, since direct brightening is generally associated w i t h some decrease i n kappa number, it w o u l d seem that the solubilization of x y l a n - d e r i v e d
23. WONG ET AL.
Xylanase Prebleaching of Softwood Kraft Pulp
359
chromophores always leads to some solubilization of residual l i g n i n or lignin-carbohydrate complexes. A n alternative explanation is that o x i d a t i o n b y permanganate does not d i s t i n g u i s h x y l a n - d e r i v e d chromophores from lignin. This possibility reflects the limitations of the approach used i n the present study as well as the techniques available for differentiating l i g n i n - c o n t a i n i n g components from x y l a n - d e r i v e d chromophores. There is also a lack of methods to examine the effects that 'non-chromophoric* xylan has on the light-scattering properties of p u l p . It appears that further investigation of the other three hypotheses concerning the mechanism of xylanase prebleaching may be more fruitful. Recent reports have indicated that larger lignin molecules can be extracted from kraft pulp w i t h alkali after a treatment w i t h xylanase (28,29) and that xylanase can reduce the molecular mass of the U V - a b s o r b i n g material i n xylan-containing extracts from kraft pulps (30).
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Conclusions • • • • •
Xylanases varied i n their ability to directly decrease the kappa number or increase the brightness of kraft pulp Direct decrease i n kappa number was observed i n most cases Direct brightening was only observed i n partially bleached kraft pulp Direct brightening seemed to be correlated w i t h the solubilization of UV-absorbing materials rather than the solubilization of sugars Evidence d i d not indicate that xylan-derived chromophores constitute the sole target substrate during xylanase prebleaching
Acknowledgments We thank the P u l p and Paper Research Institute (Vancouver, Canada) for the use of their equipment for handsheet making, optical testing and
23. WONG ET AL.
Xylanase Prebleaching of Softwood Kraft Pulp
361
oxygen delignification. W e also thank Drs. R . P . Beatson (Canfor R & D Centre, Vancouver) and H . Worster (Fletcher Challenge, Vancouver) for arranging the supply of pulp samples, and Drs. E. de Jong and J.N. Saddler for their critique of this manuscript. This work was partly financed by a grant to J.N. Saddler and K . K . Y . W o n g from the Science Council of British C o l u m b i a and a grant to J . N . Saddler a n d C . Breuil from the N a t u r a l Sciences and Engineering Research Council of Canada. Literature Cited 1. Jurasek, L.; Paice, M.G. Proc: Int. Symp. Pollution Prevention Manuf. Pulp Pap. 1992, pp 105-107. 2. Viikari, L.; Kantelinen, Α.; Sundquist, J.; Linko, M . , FEMS Microbiol Rev. 1994, 13, 335-350. 3. Kantelinen, Α.; Hortling, B.; Sundquist, J.; Linko, M . ; Viikari, L. Holzforschung 1993, 47, 318-324. 4. Holm, H.C.; Skjold-Jørgensen, S.; Munk, N.; Pedersen, L.S. Proc. PanPac. Pulp Pap. Technol. Conf. 1992, A, 53-57. 5. Paice, M.G.; Gurnagul, N.; Page, D.H.; Jurasek, L . Enzyme Microb. Technol. 1992, 14, 272-276. 6. Wong, K.K.Y.; Saddler, J.N. Crit. Rev. Biotechnol. 1992, 12, 413-435. 7. Harder, N.; Norrström, H . Tappi 1969, 52(9), 1712-1715. 8. Bard, J.W. Pap. Trade J. 1941, 113(12), 29-34. 9. Pigman, W.W.; Csellak, W.R. Techn. Assoc. Pap. 1948, 31, 393-399. 10. Forsskåhl, I.; Popoff, T.; Theander, O. Carbohydr. Res. 1976, 48, 13-21. 11. Ziobro, G.C. J. Wood Chem. Technol. 1990, 20, 133-149. 12. Schwartz, H.; McCarthy, J.L.; Hibbert, H . Pap. Trade J. 1940, 111(18), 3034. 13. Kimble, G.C. Pap. Trade J. 1942, 115(3), 37-46. 14. Maringer, U.; Wong, K.K.Y.; Saddler, J.N.; Kubicek, C.P. Biotechnol. Appl. Biochem. 1995, 21, 49-65. 15. Bailey, M.J.; Biely, P.; Poutanen, K. J. Biotechnol. 1992, 23, 257-270. 16. Nelson, S.L.; Wong, K.K.Y.; Saddler, J.N.; Beatson, R.P. Pulp Pap. Can. 1995, 96, in press. 17. Dubois, M.; Gilles, K.A.; Hamilton, J.K.; Rebers, P.A.; Smith, F. Anal. Chem. 1956, 28, 350-356. 18. Paice, M.G.; Bernier, R., Jr.; Jurasek, L. Biotechnol. Bioeng. 1988, 32, 235-239. 19. Yang, Y.,L.; Eriksson, K.-E.L. Holzforschung 1992, 46, 481-488. 20. Allison, R.W.; Clark, T.A.; Wrathall, S.H. Appita 1993, 46, 269273,281. 21. Patel, R.N.; Grabski, A.C.; Jeffries, T.W. Appl. Microbiol. Biotechnol. 1993, 39, 405-412. 22. Pekarovicová, Α.; Rybáriková, D.; Kosík, M . ; Fiserová, M . Tappi J. 1993, 76(11), 127-130. 23. Saake, B.; Clark, T.; Puls, J. Holzforschung 1995, 49, 60-68. 24. Pedersen, L.S.; Kihlgren, P.; Nissen, A.M.; Munk, N . ; Holm, H.C.; Choma, P.P. TAPPI Proc. - Pulp. Conf. 1992, 1, 31-37.
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25. Buchert, J.; Viikari, L. Int. Pulp Bleaching Conf. - Preprints 1994, pp 59-62. 26. Buchert, J.; Ranua, M . ; Kantelinen, Α.; Viikari, L . Appl. Microbiol. Biotechnol. 1992, 37, 825-829. 27. Gilbert, M . ; Yaguchi, M . ; Watson, D.C.; Wong, K.K.Y.; Breuil, C.; Saddler, J.N. Appl. Microbiol. Biotechnol. 1993, 40, 508-514. 28. Hortling, B.; Korhonen, M . ; Buchert, J.; Sundquist, J.; Viikari, L. Holzforschung 1994, 48, 441-446. 29. Suurnäkki, Α.; Kantelinen, Α.; Buchert, J.; Viikari, L. Tappi J. 1994, 77(11), 111-116. 30. Yokota, S.; Wong, K.K.Y.; Saddler, J.N.; Reid, I.D. Pulp Pap. Can. 1995, 96(4), 39-41. RECEIVED July 13, 1995
