CHAPTER 3 BIOMECHANICAL PULPING OF ASPEN CHIPS: FUNGAL GROWTH PATTERN AND EFFECTS ON CELL WALL, FIBER, AND PULP

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1 27 In: Kirk, T. Kent; Chang, Hou-Min, eds. Biotechnology in pulp and paper manufacture-applications and fundamental investigations. Stoneham, MA: Butterworth-Heinemann; Chapter 3. CHAPTER INTRODUCTION BIOMECHANICAL PULPING OF ASPEN CHIPS: FUNGAL GROWTH PATTERN AND EFFECTS ON CELL WALL, FIBER, AND PULP MORPHOLOGY 1 I. B. Sachs G. F. Leatham G. C. Myers and T. H. Wegner Mechanical, semichemical, and chemical processes are those most frequently used in producing pulp. The mechanical action used in mechanical pulping tears individual fibers and portions of fibers or fiber bundles from the wood matrix. In semichemical pulping, wood chips are given a short exposure to a chemical pulping liquor and then passed through a mechanical refiner to separate the fibers. In chemical pulping, no mechanical action is needed to achieve fiber separation; therefore, kraft pulp is composed of smooth and largely undamaged fibers. Because of the disadvantages of current pulping methods (i.e., pollutant generation, high energy requirements), biologically aided processes are being investigated. Biomechanical pulping is an experimental process that uses a fungal treatment of chips as an alternative to chemicals prior to mechanical refining (4,7). The potential benefits of biomechanical pulping included saving energy during mechanical pulping 4, improving sheet strength properties 7, and reducing undesirable waste effluents 2,4. 1 This article was written and prepared by U.S. Government employees on official time, and it is therefore in the public domain and not subject to copyright.

2 28 Biotechnology in Pulp and Paper Manufacture The purpose of this investigation was to observe the effects of fungal treatment of aspen wood chips before and after mechanical pulping and compare the microscopic appearance of fungus-pretreated biomechanical pulp (BMP) with refiner mechanical pulp (RMP), stone groundwood (SGW) pulp, thermomechanical pulp (TMP), chemithermomechanical pulp (CTMP), neutral sulfite semichemical pulp (NSSC), and kraft pulp. In a recent study, we used P. chrysosporium to treat aspen wood chips in a specially constructed bioreactor 7. In this study, we used the scanning electron microscope (SEM) to obtain information on the ultrastructural changes in the wood structure caused by P. chrysosporium under conditions that model the bioreactor process. This report demonstrates the pattern of fungal growth on the surface and inside nutrientsupplemented aspen chips, lumina, and throughout the wood cell wall layers. It also demonstrates the characteristics of pulp prepared from fungal-treated chips (P. chrysosporium BKM-F-1767) as well as the effect of BMP and pulps made by other processes in paper production as seen in cross sections of 60-g/m 2 TAPPI handsheets. 3.2 MATERIALS AND METHODS Treatment of Wood Chips with Fungus, Phanerochaete Chrysosporium strain BKM-F-1767was grown on aspen (Populus tremuloides Michx.) wood chips supplemented with a chemically defined liquid medium 7. Approximately 4 kg (ovendry basis) of 19-mm wood chips at 60% moisture content were loaded into a reactor drum. A low-nitrogen, glucosecontaining liquid medium (including 2.5% glucose, 0.025% nitrogen, minerals, and vitamins) was added to the chips. The mixture of chemically defined medium plus chip was then steam-sterilized (121 C) for 90 min and then inoculated 7. Pulp Preparation. The BMP, RMP, NSSC, and kraft aspen pulps were prepared at the Forest Products Laboratory (FPL). The aspen TMP, CTMP, and SGW were obtained from commercial sources. Refiner mechanical pulp (RMP) was prepared by pulping wood chips in a 305-mm-diameter single rotating disk atmospheric refiner. The chips were reduced to coarse pulp during the first pass. Canadian Standard Freeness (CSF) was reduced to 110 ml by multiple passes of the pulp through the single disk refiner. Yield of RMP was 95%. The TMP and CTMP were produced at a commercial pilot plant facility. The TMP was produced at 138 kpa steam pressure in a 914 mm-diameter pressurized refiner followed by atmospheric refining in a second 914-mm-diameter refiner to 100 ml CSF. Yield of TMP was 95%.

3 Biomechanical Pulping of Aspen Chips 29 The CTMP was produced by impregnating aspen wood chips with 4.6% Na 2 SO 3 and 2.0% NAOH, followed by pressurized refining at 138 kpa and atmospheric refining to 120 ml CSF. Yield of CTMP was 88%. The aspen SGW pulp was obtained from a commercial midwest source and refined to 115 ml CSF. Yield of SGW was 95%. Before cooking chips for the NSSC process, a pulping liquor of 13.5% Na 2 SO 3 and 4.5% Na 2 CO 3 was added to the chips in a liquor-to-wood ratio of 4 to 1. Digester temperature was raised from 80 C to 170 C in 90 min and held at 170 C for 170 min. The semichemically pulped wood chips were fiberized in a 203-mm-diameter single disk atmospheric refiner and refined to a nominal 230 ml CSF in a PFI mill. The NSSC pulp yield was 75.5%. A high-yield kraft pulp was prepared in a pulping digester. A pulping liquor consisting of 15% active alkali and 25% sulfidity was added to the wood chips in a liquor-to-wood ratio of 4 to 1. Digester temperature was raised from 80 C to 170 C in 60 min and held at 170 C for 60 min. The delignified chips were fiberized in a 203-mm-diameter single disk atmospheric refiner and refined to a nominal 315 ml CSF in a PFI mill. Yield of the kraft pulp was 55.8%. Handsheet Preparation and Testing. Handsheets of 60g/m 2 were prepared from each pulp according to TAPPI method T205. Burst, tear, and tensile indices were measured according to TAPPI methods T403, T414, and T404, respectively. Scattering coefficients and opacity were determined using an Elrephro 2 photometer. Density was measured according to TAPPI method T220. The SEM Observations. All preparations, except the dry handsheet samples, were dehydrated gradually through ascending ethanol solutions (5 min each) and critical point dried with CO The dried chips, pulps, and handsheets were cemented to aluminum stubs and sputter-coated with gold. Samples were photographed in a JEOL 840 SEM at an accelerating voltage of 20 kv. 2 The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the U.S. Department of Agriculture of any product or service to the exclusion of others which may be suitable.

4 30 Biotechnology in Pulp and Paper Manufacture Figure 3.1: Web-like network of hyphae on the chip surface 3 weeks after inoculating nutrient-supplemented aspen wood chips with Phanerochaete chrysossium strain BKM-F RESULTS Fungal Growth Viewed from the Surface of the Chip. Three weeks after inoculating nutrient-supplement aspen chips with P. chrysosporium, a web-like network of hyphae was evident over the chip surfaces (Figure 3.1). The aerial hyphae range in size from 0.7 µm to 3 µm diameter above the surface and from 3 µm to 4 µm diameter at the surface. Also evident on the chip surfaces were tightly organized bundles of 4 to 7 hyphae from which many hyphae were in contact with the hyphae network (Figure 3.2). Bundles of hyphae measured from approximately 8 to 21 µm in diameter. Within the lumen of the exposed vessels and fibers at the chip surface, filamentous hyphae were observed lying along the longitudinal axis of the wood cells. There was considerable erosion of the lumen surface where hyphae were present (Figure 3.3). Fungal bore holes apparently allowed the hyphae lateral movement through wood cell walls (Figure 3.4).

5 Biomechanical Pulping of Aspen Chips 31 Figure 3.2: Hyphal network (a) on wood chip surface in contact with hyphal bundles (b). Conjointly on the wood chip surface are fungal conidia (c), fibers (f), hyphae (h), and vessels (v). Figure 3.3: Erosion of cell wall (e) produced by enzymes secreted by fungal hyphae (h).

6 32 Biotechnology in Pulp and Paper Manufacture Figure 3.4: Bore hole (g) produced by fungal hypha (h) in the vessel wall (v). Throughout the vessel are collapsed fungal conidia (c). Figure 3.5: Normal rigid wood cell wall structure, cross section of an aspen wood chip.

7 Biomechanical Pulping of Aspen Chips 33 Fungal Growth Viewed from the Interior of the Chip. The establishment of the fungus within the wood chip resulted in bulk overall changes to the cell wall structure. Notable changes viewed in cross sections were swelling and softening (partial collapse) of the rigid wood cells (Figures 3.5 and 3.6). The fungus formed erosion troughs in some areas within the wood cells. Localized wood cell wall thinning and fragmentation and bore holes caused by the hyphae were visible. Wall material was removed near the hyphal tips and at points of close contact with the lateral surface of the hyphae. Hyphae within the interior of the chip were often enveloped in a slime sheath (Figure 3.7), similar to that reported in other fungi that may provide increased contact with the cell wall 8,9. Hyphae within the wood chip were generally larger in diameter than those on the surface of the chip. Many of the hyphae on the inside of the chip measured approximately 6 µm in diameter. The organized bundles of hyphae observed on the chip surface were not viewed within the wood chip. The hyphae in the lumens of the wood cells appeared to grow parallel to the long axis of the vessels and fibers, moving to adjacent cell constituents through walls through bore holes or natural openings, pits. Figure 3.6: Normal aspen wood cells modified by 3-week fungal treatment in a bench-scale biomechanical pulping process. Modifications seen in cross section are cell wall swelling (a), enzymatic softening resulting in the partial collapse of the tube-like cell structure (b), and localized areas of wall thinning (c), or fragmentation (d).

8 34 Biotechnology in Pulp and Paper Manufacture Figure 3.7: Hyphal tip (h) enveloped by a slime sheath(s). Figure 3.8: Hyphal (h) enzyme action dissolve the warts (t) of the warty layer 2.

9 Biomechanical Pulping of Aspen Chips 35 Ultrastructural Change in Cell Components and Wall Layers. The enzymes of P. chrysosporium dissolved and reduced the size of the warty layer 6 (Figure 3.8). The fungus frequently moved along the same axis as the vessel or fiber, and the secreted enzymes etched the S 3 and S 2 layers 11 (Figure 3.9). The decay appeared to be more of the corrosive type, there was no honeycombing. Bore holes frequently occurred in the walls of the wood cells. The organism appeared to readily penetrate the S 1, primary wall 10 and middle lamella (Figure 3.10). Figure 3.9: Hyphal enzyme action on the S 2 cell. Hypha (h). and S 3 layers of the aspen wood Figure 3.10: Hyphal (h) penetration of the S 1 primary wall and middle lamella (p) of the aspen wood cell.

10 36 Biotechnology in Pulp and Paper Manufacture Figure 3.11: BMP. Uniform length of fibers (a) with abundant fibrillation (b). Figure 3.12: BMP. Fibrillation (f) woolly-like, looser, and bulkier.

11 Biomechanical Pulping of Aspen Chips 37 Figure 3.13: BMP. Higher magnification of fibrillation. Figure 3.14: RMP. Fibers (a) narrow, of different lengths, stiff with moderate fibrillation (b). Debris (c).

12 38 Biotechnology in Pulp and Paper Manufacture Figure 3.15: SGW. Fibers (a) are of various lengths and widths, stiff with reduced fibrillation (b). Debris (c). Figure 3.16: TMP. Fibers (a) are of various lengths, stiff with moderate fibrillation (b).

13 Biomechanical Pulping of Aspen Chips 39 Figure 3.17: CTMP. Fibers (a) are of various lengths, usually longer than RMP, stiff with moderate fibrillation (b). Characteristics of BMP Pulp Compared with Pulps Produced by Other Pulping Processes. Aspen pulps and 60-g/m 2 TAPPI handsheets produced from BMP were compared with those produced by other pulping processes. Those included other mechanical pulping processes (SGW, RMP, TMP, CTMP), semichemical pulping (i.e., NSSC), and kraft pulp ing. The pulps showed differences in fiber length, stiffness, and compressibility. Mechanical processes - When fiberized, BMP pulp emerged woollylike, looser, bulkier, with fibers less variable in length and with abundant fibrillation (Figures 3.11, 3.12, 3.13). In contrast to BMP, RMP fibers were not as wide, appeared stiffer, were of different lengths, and had moderate fibrillation (Figure 3.14). The SGW pulp fibers (Figure 3.15) showed reduced fibrillation, debris, and stiff fibers of various lengths. The TMP and CTMP pulp fibers were of various lengths and longer than RMP with moderate fibrillation and appeared stiff (Figures 3.16, 3.17). These fibers were more twisted than the BMP fibers. Semichemical processes - The NSSC pulp (Figure 3.18) for the most part exhibited fewer stiff fibers of various lengths, with more compressibility and conformability when compared to the mechanically processes pulps. Compared to BMP, NSSC pulp fibers were not as compressed and more variable in length. Chemical (kraft) processes - Kraft pulp ing produced more uniform separated collapsed fibers with abundant fibrillation. BMP pulp appeared very similar to the kraft pulp (Figure 3.19).

14 40 Biotechnology in Pulp and Paper Manufacture Comparison of Handsheets. Aspen BMP produced a stronger handsheet than did aspen TMP and SGW pulp (Table 3.1). NSSC pulps appeared to be superior to BMP in handsheet properties. Of all the pulps, kraft pulp had the highest sheet-strength properties. To gain insight and visually assess how the fiber morphology in these pulps may have contributed to sheet-strength properties, we examined cross sections of handsheets. Handsheets made from mechanically processed pulps showed uncollapsed fibers leading to poor conformability and reduced bonding (Figures 3.20, 3.21, 3.22, 3.23). The NSSC and kraft processed pulp gave handsheets that exhibited fibers of enhanced compressibility and conformability (Figures 3.24, 3.25). Handsheets prepared from BMP visually resembled the kraft handsheets, exhibiting good compressibility and conformability of the fibers (Figures 3.26). Figure 3.18: NSSC. Fibers (a) are of various lengths and stiff with moderate fibrillation (b).

15 Biomechanical Pulping of Aspen Chips 41 Figure 3.19: KRAFT. Fibers (a) are uniform in length, separated, not stiff with abundant fibrillation (b). Table 3.1 Comparison of Yield and Handsheet Data for Different Pulping Processes

16 42 Biotechnology in Pulp and Paper Manufacture Figure 3.20: SGW handsheet. Uncollapsed stiff fibers (a) contribute to poor bonding (b). (cross section) Figure 3.21: RMP handsheet. Uncollapsed stiff fibers (a) contributing to poor bonding (b). (cross section) Figure 3.22: TMP handsheet. Uncollapsed stiff fibers (a) contributing to poor bonding (b). (cross section)

17 Biomechanical Pulping of Aspen Chips 43 Figure 3.23: CTMP handsheet. Uncollapsed stiff fibers (a) contributing to poor bonding (b). (cross section) Figure 3.24: NSSC handsheet. Uncollapsed stiff fibers (a). However, many fibers are collapsed (d) contribute to good bonding. (cross section) Figure 3.25: KRAFT handsheet. Good bonding (c) due to good fiber collapse and compressability (d). (cross section)

18 44 Biotechnology in Pulp and Paper Manufacture Figure 3.26: BMP handsheet. Good bonding (c) due to good fiber collapse and compressability (d). (cross section) 3.4 DISCUSSION The ultrastructural results obtained with the scanning electron microscope have helped improve our understanding of the progression of P. chrysosporium hyphae and its modification of aspen wood chips during a 3-week pretreatment before mechanical pulping. Chip surface colonization, surface penetration, interior colonization, partial degradation of internal cell walls, which was initiated at the lumen surface, were observed. The tightly organized bundles of hyphae observed on the chip surfaces have been reported 13 in other fungi, but not in P. chrysosporium. The function of these remain to be established for this fungus 12. As expected, hyphae entered and advanced into the chips through vessels or fiber lumens as well as directly through the cell walls, using both natural wood pits 1 and fungal bore holes. The number of bore holes in a cell wall appeared to be proportional to the length of time that hyphae had been in the vicinity of the cell. More bore holes were observed at the chip surface than in the interior, and interior cells containing more hyphae also contained many bore holes. It is not known if the thread-like hyphae observed were penetration forms as suggested by Eriksson et al. 3 Attack of the chip by P. chrysosporium was preferentially from the lumen, presumably using secreted enzymes capable of lateral diffusion. Hyphae were much more frequently observed lying in erosion troughs along the lumen walls than within the cell wall layers. The distinct erosion zones near hyphal tips suggest that the tips were a significant initial source of enzymes. The enzyme action by P. chrysosporium appeared most extensive when hyphae directly contacted the wood cell wall. It is likely that the diffusion of the secreted enzymes by P. chrysosporium is directed or aided in some way by the slime sheath, as suggested by Jutte et al. 5 and Palmer et al. 2 for other fungi.

19 Biomechanical Pulping of Aspen Chips 45 Our observations suggest that the fungal treatment is likely to involve enzymatic softening and swelling of wood cell wall fibrils as well as the thinning and fragmentation of the wood cell walls. This leads to a BMP that produces a handsheet visually resembling a handsheet produced from kraft pulp and approximately equal in strength to CTMP. ACKNOWLEDGMENTS We thank Marguerite S. Sykes and Louis C. Lunte, Jr., of the Forest Products Laboratory (FPL) for technical assistance, T. Kent Kirk, (FPL), John W. Koning, Jr., University of Wisconsin Biotechnology Center (UWBC), and Richard R. Burgess, UWBC, for valuable discussions. The work was funded in part by a Biopulping Consortium involving 17 pulp and paper companies and related companies, the UWBC, and the FPL. REFERENCES

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