1994 INTL. PULP BLEACHING CONF. - PAPERS. X. Yu J. L. Minor R. H. Atalla. M. M. Labbauf R. L. Farrell
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1 In: Proceedings of 1994 International pulp bleaching conference; 1994 June 13-16;Vancouver, B.C., Canada. Montreal, Quebec, Canada: Canada Pulp and Paper Association; 1994: EFFECT OF HEMICELLULASES ON UNBLEACHED SOFTWOOD KRAFT PULP X. Yu J. L. Minor R. H. Atalla USDA Forest Service Forest Products Laboratory Madison, Wisconsin M. M. Labbauf R. L. Farrell Sandoz Chemicals Biotech Research Corporation Lexington, Massachusetts ABSTRACT Hemicellulase enzymes removed small amounts of xylan or glucomannan and thereby increased the micropore size in the cell wall and allowed trapped lignin fragments to diffuse out. The hemicellulases also made lignin more accessible to bleaching chemicals. This was preferential to fibers with high lignin content, implying that the barrier removed by enzyme treatment was also a barrier to pulping. KEYWORDS: ENZYME PREBLEACHING, XYLANASE, MANNANASE, BLEACHING, LIGNIN DISTRIBUTION, KRAFT PULP, SIMONS STAIN, GRAFF C STAIN, ACCESSIBILITY INTRODUCTION Pretreating unbleached kraft pulp with polysaccharides minimizes or eliminates formation of undesirable chloro-organicbyproducts during chlorination [1,2]. Exactly how these enzymes break down lignin during bleaching, however, is not understood. If residual lignin is covalently bonded to hemicellulose, and chemical evidence indicates that it is [3], enzymatic cleavage of hemicellulose-glycosidicbonds could solubilize lignin with fewer lignin bonds cleaved by the bleaching agents. Physical association of hemicelluloses with cell wall lignin may pose a barrier to delignification by hindering the accessibility of bleaching reagents to lignin or restricting diffusion ofdegraded lignin from the cell wall. Enzymatic hydrolysis could remove this barrier. One hypothesis considers the reprecipitated xylan to be a physical barrier [4]. In softwood kraft pulps, the main hemicelluloses are xylan and mannan, thus, xylanase and mannanase are used to degrade xylan and mannan [5]. To determine the effect of xylanase and mannanase on kraft pulp, the study reported here used classical microscopy stain methods combined with chemical composition analyses and other physical techniques. The chemical and physical interpretation of the stain results was investigated. MATERIALS AND METHODS Enzyme Treatments Enzymes used for the treatment of unbleached kraft pulp samples included CARTAZYME HS 1 xylanase (Sandoz Chemicals Corporation, Lexington, MA) and a mannanase enzyme solution (Sandoz Chemicals Biotech Research Corporation, Lexington, MA). The starting unbleached kraft pulp was obtained from a mill in the northeastern United States and consisted primarily of spruce, fir, larch, and pine, with about 1% hardwood. The kappa number of the pulp was 24.3 ml/g. The pulp was treated with xylanase at three levels (1, 6, and 10 units/g), mannanase at two levels (5 and 10 units/g), and a mixture of the two enzymes (1 xylanase plus 5 mannanase units/g) (Table I). The pulp samples were incubated at 50 C, ph 4.8, for 2 to 18 h. The samples were then either washed with water or extracted with a 2.0% sodium hydroxide solution for 1 h at 70 C. Control pulp samples were prepared the same as enzyme-treated samples, except that the enzyme solution was replaced with water. The wet pulps were refrigerated until analyzed. Bleaching Experiments Control and enzyme-treated pulps were subjected to a standard C DED 1ED 2 bleaching sequence with 10% chlorine dioxide substitution in the chlorination stage. The C D stage was carried out at 3.5% consistency for 1 h at 25 C with active chlorine charge of 4.8% (% on pulp = 0.22 kappa number). Chlorine and chlorine dioxide were premixed before addition to the pulp samples. Extraction stages were carried out at 10% consistency and 70 C for 1 h with a caustic loading of 0.6 times the active chlorine used in the C D or D stages. During the first chlorine dioxide stage (D 1 ). pulp samples were bleached with 0.8% Cl0 2 and 0.35% NaOH at 10% consistency for 3 h at 70 C. The chlorine dioxide charge for the second stage (D 2 ) was 0.4% under the previously stated conditions. At three stages during the enzyme/chemical bleaching sequence, ISO brightness values were determined. Chemical Composition and Kappa Number Analyses Freeze-driedsamples were ground in a Wiley mill, hydrolyzed [6], and analyzed for the five major wood sugars [7]. The high performance liquid chromatography (HPLC) was performed with a Dionex model chromatograph using a CARBO-PAC PA-1 column. Kappa numbers were determined by the TAPPI microkappa number analysis method (UM246). The microkappa number was used because the available sample size was limited. 1The use of trade or firm names in this publication is for reader information and does not imply endorsement by the U.S. Department ofagriculture of any product or service. 63
2 TABLE I-SUGAR COMPOSITION AND LIGNINhe CONTENT OF ENZYME-TREATED PULPS Sugar composition (%) Sample Enzyme Dosage Time no. treatment (units/g) (h) W, E a Arabinan Galactan Glucan Xylan Mannan 1 Control 0 18 W Xylanase 1 18 W Xylanase 6 18 W Xylanase W Xyl. + man W Mannanase 5 18 W Mannanase W Control 0 18 E Xylanase 1 18 E Xylanase 6 18 E Xylanase E Xyl. + man E Mannanase 5 18 E Mannanase E Control 0 2 W Xylanase 1 2 W Xylanase 6 2 W Xylanase 10 2 W Xyl.+man W Mannanase 5 2 W Mannanase 10 2 W Control 0 2 E Xylanase 1 2 E Xylanase 6 2 E Xylanase 10 2 E Xyl.+man E Mannanase 5 2 E Mannanase 10 2 E aw, water wash after treatment; E, extraction with 2% NaOH after treatment. Microscopic Analysis Slides were prepared according to the TAPPI T401om-88 procedure. Graff C stain was purchased from Integrated Paper Service (Appleton, Wisconsin). Three drops of the stain were applied to the fiber field on a microscope slide. The wet fibers were covered with a cover glass and allowed to stand 1.5 min. The surplus stain solution was drained off, and the fibers were examinedimmediately. Direct blue 1 and direct orange 15 dyes for Simons stain were provided by Pylam Products Company, Inc. (GardenCity, New Jersey) under the commercial names Pontamine Sky Blue 6BX and Pontamine Fast Orange 6RN. Separate solutions, one consisting of 1% direct blue 1 dye and the other of 0.2% highmolecular-weight(>25,000) direct orange 15 dye, were prepared. The solutions were mixed in a ratio of 1:1. Eight drops of the mixed dye solution were applied to the fibers on a slide. The slide was dried at 75 C, washed with water, and examined. The quantitative microscopic analyses were performed following the TAPPI T401 om-88 procedure using an Olympus research microscope, model AH-2. Numerical Measurement of Color Photomicrographs of Graff C stained fibers were taken at preset locations on the slide for randomization. The L*a*b* system color values were measured from the photographs with a Minolta Chroma Meter (Ramsey, New Jersey) CR-200 at preselected points to provide further randomization. About data were obtained from each sample for analysis. (The b* values were not absolute, but relative. Because of the large head size on the Chroma Meter, we modified the procedure by measuring the colored fiber through a 3- by 6-mm hole in white paperboard.) 64
3 RESULTS AND DISCUSSION Chemical Changes After Enzyme Treatment The enzyme treatment did not result in a large hemicellulose loss. After xylanase treatment, the xylan content decreased from 6.8% of the total polysaccharides to 6.2%. After mannanase treatment, the mannan content decreased from 7.9% to a low of 6.8%. Xylanase and mannanase treatments each resulted in losses of their respective hemicelluloses on the order of 10%. The mannanase effected somewhat more of a loss of mannan than xylanase did of xylan. For both xylanase and mannanase, a high dosage of enzyme resulted in more xylan or mannan loss, and 18 h of treatment resulted in more xylan and mannan loss than 2 h. The alkali extraction removed somewhat more xylan after xylanase treatment, but the mannan content was unaffected by alkali extraction after mannanase treatment. Most of the lignin loss occurred with the alkali extraction, with or without enzyme treatment. The treatment with both xylanase and mannanase released slightly more lignin, especially at the higher enzyme dosages and 18 h treatment time. It appeared that the mixture of xylanase and mannanase resulted in more lignin loss than the additive effects of the individual treatments. The magnitude of the difference. however, was about that of the experimental error. TABLE II-BLEACHING RESULTS FOR SELECTED ENZYME TREATMENTS ISO brightness Enz Enz C DE Enzyme Xylanase Mannanase Enz C DE D 1E treatment (units/g) (units/g) C DE D 1E D 2 Control Xylanase Xylanase Xyl. + Man Xyl. + Man TABLE III-GRAFF C STAIN ANALYSIS OF ENZYME-TREATED PULP Fibers in each color category (%) Sample Enzyme Grayno. treatment Yellow Green Brown blue 8 Control Xylanase Mannase Bleaching Experiments The results of bleaching experiments with various enzyme treatments are shown in Table II. In terms of brightness gain, the improvement brought about by enzyme pretreatment was apparent after the C DE stage and carried through to the fully bleached pulp. This implied that the improved bleaching was related to an easier delignification in the first stage. Graff C Stain Graff C stain, an iodine stain consisting of potassium iodide, iodine, aluminum chloride, calcium chloride, and zinc chloride, is used extensively in microscopic fiber analysis [8,9]. This stain is sensitive to the chemical composition of fiber. For unbleached kraft pulp, the stained fiber color ranges from yellow through green to blue depending on the degree of cooking. Raw or slightly cooked fibers with high lignin content stain yellow, medium cooked fibers with medium lignin content stain green to brown, and well-cooked fibers with little or no lignin content stain gray to blue [10]. This stain was used to analyze the enzyme-treated pulp by counting the number of fibers in each color category (Table III). After enzyme treatment, the percentage of yellow fibers decreased from 20.3% to 17.2% for xylanase and to 13.2% for mannanase, while the percentage of green fibers increased from 15.4% to 17.6% for xylanase and to 22.2% for mannanase. The relative number of brown fibers and gray-blue fibers remained unchanged after either xylanase or mannanase treatment. For both xylanase and mannanase treatment, changes occurred between the yellow and green fibers. These changes indicated that lignin was lost primarily from high lignin content fibers. Fig. 1. The L*a*b* color chart. Numerical Measurement of Color Instead of sorting the fibers by color subjectively, color can be expressed numerically according to a system adopted by the International Commission on Illumination. In their L*a*b* system, color is expressed in three dimensions as L*, a*, and b* values. The numerical scales for a* and b* are shown in Figure 1 [11]. 65
4 The b* value decreases from positive values to negative as the color changes from yellow to blue. This is similar to the fiber color stained with Graff C stain, which is yellow when the lignin content is high and blue when the lignin content is low. Therefore, the b* value of a Graff C stained fiber indicates lignin content-the higher positive b* values represent high lignin content, and the negative b* values represent low lignin content. The exact color of fibers stained with Graff C stain depends on the structure and conformation ofthe polysaccharides as well as the lignin content (Yu, in preparation). Nevertheless, for a given pulp, the influence of lignin on the color appeared to dominate, and we used the b* value to describe the lignin content change in kraftpulp after enzyme treatment (Table IV, Fig. 2). TABLE IV-AVERAGEb* VALUE OF ENZYME-TREATED PULPS Sample Enzyme Average Average no. treatment b* value lignin content a 8 Control Xylanase Mannanase a Kappa no./6. Afterenzyme treatment, the average b* values decreased from 3.3 to 2.9 for xylanase and to 2.4 for mannanase. The b* distribution showed that for both xylanase and mannanase, b* distribution narrowed at the expense of fibers with higher b* values. These results corroborated those ofthe more subjective visual examination oftable III. The b* value analysis also suggested that upon enzyme treatment, the lignin content distribution narrowed at the expense of fibers with the highest lignin content. Accessibility Changes and Simons Stain Deuterlum Oxide Exchange Deuterium oxide exchange measures fiber accessibility. It is generally believed that hydroxyl hydrogens in the less crystalline regions and can the surface of the crystalline regions of cellulose are accessible to deuterium oxide, while the hydroxyl hydrogens in the highly crystalline regions are not [12]. After treatment with deuterium oxide, the accessible hydroxyl hydrogens exchanged with deuterium, and the hydroxyl absorption peak in the infrared moved from 3300 cm -1 to 2500 cm -1. The inaccessible, unexchanged hydroxyl absorption remained the same. Therefore, the ratio of absorbance at 2500 cm -1 (A2500) to absorbance at 3300 cm -1 (A3300) indicated the fiber s accessibility (Table V). Enzyme treatment did not increase the A2500/A3300 ratio, but rather decreased it slightly. Thus. neither xylanase nor mannanase opened the crystal lattice in the fiber. The slight decrease of A2500/A3300 after enzyme treatment was probably due to the loss of some xylan and mannan that was accessible to deuterium oxide in the control pulp. Simons Stain Simons stain [9,13,14] consists of two direct dyes: direct blue 1, which has a well-defined structure with a molecular weight of , and direct orange 15, a polymeric mixture containing a high molecular weight fraction. This stain is relatively independent of the chemical composition of the fiber but is sensitive to its physical structure. The Simons stain is used to measure the size of the fiber cell wall micropores the same way that the solute exclusion technique does. The accessibility measured is not the same as that measured by deuterium oxide exchange. Fibers with pores accessible to the high molecular weight fraction of the orange dye stain orange or yellow. Fibers with 66 Fig. 2. Distribution of b* color values of kraft pulps stained with Graff C stain.
5 TABLE V-DEUTERIUMOXIDE EXCHANGE WITHIN ENZYME-TREATED PULPS Enzyme Standard Sample no. treatment A2500/A3300 deviation Water washed 1 Control Xylanose Mannanase Alkali extracted 8 Control Xylanase Mannanase aratio of D-O/H-O stretching frequency intensities. Sample no. TABLE VI-SIMONS STAIN OF ENZYME-TREATED PULPS Fibers in each color category (%) Enzyme treatment Green Yellow Water washed 1 Control Xylanase Mannananase Alkali extracted 8 Control Xylanase Mannananase pores accessible to the blue dye, but not accessible to the high molecular weight fraction of the orange dye, stain blue [14]. In a given fiber, there may be some areas that are accessible to the high molecular weight orange fraction while some areas are inaccessible. Such fibers will stain a mixture of orange and blue, and will appear green (Table VI). These data showed an increase in fiber accessibility to the high molecular weight fraction of the orange dye after both xylanase and mannanase treatment, and reflected an increase in the fiber pore size. This increased accessibility suggested removal of some physical hindrance in the fiber. CONCLUSIONS Treating unbleached kraft pulp with xylanase or mannanase under conditions that facilitate chemical bleaching releases small amounts of the respective sugars and lignin. Microscopic examination of the pulps using Graff s C stain as an indicator of lignin shows that the lignin released by xylanase and mannanase is preferentially released from fibers with a high lignin content. This implies that the enzymes help release lignin that is relatively resistant to pulping. The high lignin content fibers may come from compression wood. The inhibited pulping may result from chemical bonding between lignin and carbohydrates. from xylan and glucomannan forming a physical barrier to diffusion, or from a combination of the two. The Simons stain test shows that fiber pore size increases after xylanase or mannanase treatment. This supports the theory that a physical barrier is at least in part responsible for inhibited lignin removal. Enzyme treatment does not increase the accessibility of the cellulose crystallites to deuterium oxide. REFERENCES 1. TURNER, J.C., SKERKER, P.S., BURNS, B.J., HOWARD J.C., ALONSO, M.A., and ANDRES, J.L., Bleaching With Enzymes Instead of Chlorine, Tappi J. 75(12):83-89 (1992). 2. SKERKER, P.S., BEERMAN, N., LABBAUF, M.M., MCCARTHY, P., and FARRELL, R.L., Practical Bleaching Using Xylanses: Laboratory and Mill Experience With CARTAZYME HS TM in Reduced and Chlorine-Free Bleach Sequences, Proceedings, TAPPI Pulping Conference, Boston, MA. (1992). 3. MINOR, J.L., Chemical Linkage of Polysaccharides to Residual Lignin in Loblolly Pine Kraft Pulps, J. Wood Chem. Technol. 6(2): (1986). 4. KANTELINEN, A., HORTLING, B., SUNDQUIST, J., LINKO, M., and VIIKARI, L., Proposed Mechanism of the Enzymatic Bleaching of Kraft Pulp With Xylanses, Holzforschung 47(4): (1993). 5. CLARK. T.A., STEWARD, D., BRUCE, M., MCDONALD, A., SINGH, A., and SENIOR, D., Improved Bleachability of Radiata Pine Kraft Pulps Following Treatment With Hemicellulolytic Enzymes, Appita 44(6): (1991). 6. SAEMAN. J.F., MOORE, W.E., MITCHELL, R.L., and MILLETT, M.A., Techniques For the Determination of Pulp Constituents by Quantitative Paper Chromatography, Tappi 37(8): (1954). 7. PETERSEN, R.C. and SCHWANDT, V.H., Wood Sugar Analysis by Anion Chromatography, J. Wood Chem. Technol. 11(4): (1991). 8. GRAFF, J.H., New Stains and Their Use For Fiber Identification, Paper Trade J. 100(16):45-50(1935). 9. ISENBERG, I.H., Pulp and Paper Microscopy, 3rd ed, The Institute of Paper Chemistry, Appleton, WI, p (1967) 10. GRAFF, J.H., Color Atlas for Fiber Identification, The Institute of Paper Chemistry, Appleton, WI. (1940). 11. Precise Color Communication, Minolta Camera Co., Ltd. p. 10 (1989). 12. NISSAN, A.H., and HUNGER, G.K., Accessibility of Cellulose, Ency. of Polylmer Sci. and Technol. 3: SIMONS, F.L., A Stain For Use in the Microscopy of Beaten Fibers, Tappi 33(7): (1950). 14. YU, X., MINOR. J.L., and ATALLA, R.H., Mechanism of the Action of Simons Stain (To be published). 67
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