-H-tER-D-'E~EI{~S==-EM='PLAA~R~M~AG~•O~ND~E,: University Free State GEEN OMST Al'lOIGHEDE UIT DIE II~~~~~~~~~~~II~~ 34300000175681 BIBLIOTEEK VEHWYDER WORD NIE Universiteit Vrystaat lU1I1JL][ZA'{][ON OF WOOD-DECAY FlUNGI FOR BlOKRAFT PULPING OF SOFTWOOD by JACOBUS FRANCOIS WOLFAARDT Submitted in fulfilment of the requirements for the degree of PHILOSOPHIAE DOCTOR in the Department of Microbiology and Biochemistry, Faculty of Science, University of the Orange Free State, Bloemfontein, South Africa Promoter: Dr c.r, Rabie Co-promoter: Prof. M.J. Wingfield May 1999 " taking into consideration the whole of the southern part of Africa, there can be no doubt of its being a sterile country." Charles Darwin, Journal of Researches during the voyage ofB.M.S. Beagle (1837) This thesis is dedicated to my family CONTENTS ACKNOWLEDGEMENTS iv PREFACE v CHAPTERl APPLICATION OF FUNGI AND FUNGAL PRODUCTS IN BIOPULPING PROCESSES: A REVIEW 1 ABSTRACT 2 INTRODUCTION 3 BIOMECHAN1CAL PULPING 5 BIOCHEMICAL PULPING 8 Biosulphite pulping with white-rot fungi 8 Biokraft pulping with white-rot fungi 10 Organosolv pulping 11 BIOPULPING WITH CARTAPIP® 13 Biomechanical pulping 13 Depitching 15 Biochemical pulping 16 BIOPULPING OF NON-WOOD FIBRE 18 CONCLUSIONS 23 REFERENCES 25 CHAPTER2 A SURVEY OF SOUTH AFRICAN WOOD- INHABITING BASIDIOMYCETES AND CHARACTERIZATION OF CULTURED STRAINS 35 ABSTRACT 36 INTRODUCTION 37 MATERIALS AND METHODS 40 Collection of cultures 40 Enzymatic characteristics 42 RESUL TS AND DISCUSSION 43 Collection of cultures 43 Enzymatic characteristics 45 CONCLUSIONS 57 REFERENCES 58 ii CHAPTER3 ASSESSMENT OF WOOD-INHABITING BASIDIOMYCETES FOR BlOKRAFT PULPING OF SOF'fWOOD CHIPS 63 ABSTRACT 64 INTRODUCTION 65 MATERIALS AND METHODS 67 Fungi and inoculum 67 Wood and solid-substrate fermentation 67 Pulping conditions 68 Screening procedure 69 Pulp evaluation 70 RESULTS AND DISCUSSION 70 CONCLUSIONS 74 REFERENCES 77 CHAPTER4 EVALUATION OF THE MICRO CLIMATE, AND ENUMERA TION OF FUNGI IN A STORED SOFTWOOD CHIP PILE 81 ABSTRACT 82 INTRODUCTION '83 MATERIALS AND METHODS 85 Physical conditions 85 Microbial populations 87 RESUL TS AND DISCUSSION 88 Physical conditions 88 Microbial populations 94 CONCLUSIONS 96 REFERENCES 101 CHAPTERS COLONIZA TION OF FRESHLY CHIPPED SOFTWOOD BY WHITE-ROT FUNGI 104 ABSTRACT 105 INTRODUCTION 106 MATERIALS AND METHODS 108 Wood preparation 108 Enumeration of microbes 108 Biopulping 109 Extraction and analysis of monoterpenes 110 Influence of a-pinene on fungi III RESULTS AND DISCUSSION 113 Enumeration of microbes 113 Biopulping 114 Extraction and analysis ofmonoterpenes 116 Influence of a-pinene on fungi 119 CONCLUSIONS 120 REFERENCES 123 iii CHAPTER 6 KRAFT PULPING OF PINE WOOD, PRE-TREATED WITH A STRAIN OF STEREUM HIRSUTUM 125 ABSTRACT 126 INTRODUCTION 127 MATERIALS AND METHODS 129 Fungal pre-treatment 129 Pulping 130 Pulp evaluation 131 RESULTS AND DISCUSSION 132 Lignin content 132 Pulp yield and degree of polymerization 134 Alkali consumption 137 Pulping time 139 CONCLUSIONS 140 REFERENCES 141 SUMMARY 145 OPSOl\11VHNG 148 APPENDICES 151 APPENDIX A: ORIGIN OF FUNGAL STRAINS IN CULTURE COLLECTION 152 APPENDIX B: PHYSIOLOGICAL CHARACTERISTICS OF CULTURES 158 APPENDIX C: RESULTS OF THE FIRST SCREENING STEP TO SELECT STRAINS FOR KRAFT BIOPULPING 165 APPENDIX D: TEMPERATURES OBSERVED IN A COMMERCIAL CHIP PILE 172 APPENDIX E: LEVELS OF C02 OBSERVED IN A COMMERCIAL CHIP PILE 173 APPENDIX F: INFLUENCE OF CO2 ON THE BIOPULPING EFFICIENCY OF SELECTED STRAINS OF WlllTE-ROT FUNGI 174 APPENDIX G: INFLUENCE OF a-PINENE ON THE GROWTH OF FUNGAL STRAINS 177 iv ACKNOWLEDGEMENTS I wish to express my appreciation and gratitude to the following persons and institutions for their contributions to this project: Mr Andre Vlok for his vision to establish biotechnological research in Sappi. Leonie Bosman, Ilonka Haylett, Annalie Havenga, Annali Jacobs, JeffMale, Lynn Steyn and Dr. Sarel Venter (the team members at the CSIR) for the pleasant spirit of collaboration. The late Mr Steve Raubenheimer (Sappi, Research and Development) for his contribution in the management of this project. Annelie Lubben and Gert Marais (both of the CSIR) and Dr Derek Pegler (IMI) for the identification of some fungi. Dr Martie Smit, Carin Dunn and Leonie Engelbrecht (all from the UOFS) for assistance in the analysis of monoterpenes and evaluation of the influence of monoterpenes on fungi. Prof. Abrie van der Merwe and Mike Fair (both of the UOFS) for advice on statistical analysis. Dr Abraham Singels (previously of the UOFS) for assistance with modelling of biokraft pulping. Peter Merensky, Faan Jansen and Piet Steyn (Sappi Kraft, Ngodwana) for their cooperation during sampling and mill trials. Dr Deon van der Westhuizen for his assistance in the establishment of the culture collection and the sharing of his knowledge. Sappi Ltd. and the CSIR for the funding of this project. The Department of Water Affairs and Forestry for allowing us to collect fungi from areas under their control. My eo-promoter, Prof. Mike Wingfield, for his friendship and encouragement. My promoter, Dr Chris Rabie for his leadership and guidance throughout this project. v PREFACE The forest products industry is one of the most important earners of foreign exchange for South Africa (Kruger et al., 1995). Products such as mining timber, construction timber, veneer logs, particle and fibreboard and pulpwood chips make an important contribution to the GDP. However, the major focus of this industry is the production of pulp and paper with an annual pulp capacity of 2,4 million tons (Rockey, 1998). The pulping and paper-making process is a predominantly chemical process that utilizes biological raw materials (Ferris, 1997). During pulping, wood fibres are either separated by mechanical pulping, or lignin is dissolved by chemical cooking to free cellulose (Sjëstrëm, 1981). Roundwood can only be utilized directly, in one method of mechanical pulping (ground wood pulping), but chipped wood is used for other forms of pulping. Logs are, therefore, debarked and chipped before storage, because chips are more economical to handle than logs (Zabel & Morrell, 1992). Wood chips are then pulped by various methods, depending on the required pulp characteristics. Mechanical pulping yields pulp with a high yield and with low VI strength properties that can be used for newsprint (Sjostrom, 1981). Chemical pulp, on the other hand, has lower yields, but with better strength properties that are used for a wider range of paper products such as corrugated containers, fine paper and tissue. Many paper grades obligate the removal of residual lignin in pulp by bleaching with oxidizing chemicals. Bleached pulp and, sometimes, unbleached pulp are then formed into paper sheets with different strength and optical properties that are determined by the end use. Unfortunately, all of these processes generate potentially hazardous effluents. This results in considerable criticism against the pulp and paper industry (Bergbauer & Eggert, 1992). The paper and pulp industry relies on physical and chemical processes for most of the unit operations (Sjëstrom, 1981). However, biotechnology offers the industry the potential to produce higher quality products at reduced cost and with less environmental impact (Eriksson, 1991; Ferris, 1997; Kirk, 1989). These advantages are the result of more specific, but natural reactions that are catalyzed by microorganisms or their products (Eriksson, 1990). Only a limited number of biological systems are currently available to the industry (Ferris, 1997), but they have been researched for little more than 20 years (Shimada, 1996). The focus of this research has moved to different unit operations as determined by the needs of the industry. However, the rationale for all developments has been to save energy (Akhtar et al., 1997), improve product quality (Buchert et al., 1994; Popius-Levlin et al., 1997; Viikari et al., 1993), increase production (Lascaris et al., 1997) or reduce the environmental impact of conventional processes (Eriksson, vii 1991). Most of these processes utilize fungi or fungal products (Eriksson, 1991; Shimada, 1996). The justification for the use of fungi, lies in their ability to rapidly colonize lignocellulosic substrates (Kirk, 1989). Filamentous fungi have the ability to penetrate solid substrates (Messner & Srebotnik, 1994), and wood-inhabiting fungi are also able to produce important enzymes required for degradation of lignocellulose (Eriksson, 1990) and wood extractives (Blanchette et al., 1992b; Farrell et al., 1997; Fischer et al., 1996). The white-rot Basidiomycetes are, for example, the only group of organisms that can degrade lignin on a significant scale (Akhtar et al., 1997). The application of fungi or fungal products has been investigated for the following operations in the forest products industry: 4) Biological control of sap stain in felled lumber by application of Ophiostoma piliferum (Behrendt et al., 1995) and Phlebiopsis gigantea (Behrendt & Blanchette, 1997). o Improvement of debarking of logs through the application of P. gigantea (Behrendt & Blanchette, 1997). It Biological control of sap stain of wood chips by applying 0. piliferum (Farrell et al., 1994; Schmitt et al., 1998; Wall et al., 1995) «I Biological treatment of wood prior to pulping to improve mechanical and chemical pulping processes (Akhtar et a!., 1998; Iverson et al., 1997; Jacobs et a!., 1998; Jacobs-Young et al., 1998; Wall et al., 1994; Wall et al., 1995; Wall et al., 1996). viii o Biological treatment of wood for pitch control (Blanchette et al., 1992b; Farrell et al., 1997; Fischer et al., 1996; Haller & Kile, 1992; Qin & Chen, 1997; Wall et al., 1995). o Biological pre-treatment of non-wood fibre (Hatakka et al., 1996; Johnsrud et al., 1987; Sabharwal et al., 1996; Wolfaardt et al., 1998). e Modification of the papermaking properties of fibre by application of enzymes, to strengthen pulp webs and improve drainage on paper machines (Jeffries, 1992; Laleg & Pikulik, 1992; Lascaris et al., 1997; Sarkar, 1997). c§. ~ Northern Province S· o m Mpumalangag ~ Gauteng ::s-' ~ KwaZulu-Natal Southern Cape o~ Western Cape ~ ~#OCi>~ ...j:.>.... 42 Enzymatic characteristics The oxidase reactions of strains were determined on duplicate plates of 1,5% malt extract agar containing 0,5 % gallie acid (GAM) or tannic acid (TAM). Plugs (5 mm diameter) were removed aseptically with a cork borer from one-week old cultures on malt extract agar (MEA) and inverted on GAM and TAM. Slow growing strains were only used when sufficient mycelium was produced to yield four inoculum plugs. Plates were incubated at room temperature (22°C) and reactions and growth determined after seven days. Instead of the six reaction types recorded by Davidson et al. (1938), we only distinguished between three reaction types, viz: No darkening of medium was regarded as a negative reaction (rated as 0), discolouration of the medium restricted to the area covered by mycelium was regarded as a positive reaction (rated as 1) and extension of discoloured zone beyond the mycelium was regarded as a strong positive reaction (rated as 2). Drop tests were performed in duplicate on one-week old cultures on 2 % MEA containing 10 % sucrose, neutralized with 2N KOR. Tests for cytochrome oxidase, laccase, peroxidase and tyrosinase were done by placing a drop of the following test solutions on the colony perimeter (Stalpers, 1978): Tetramethyl-p-phenylenediamine dihydrogen chloride (20 mg) in solution with 1° ml ascorbic acid (15 ppm in water) was used to test for the presence of cytochrome oxidase. The positive test for laecase was the development of a purplish colour on the colony when 0, IM a-naphtol in 96 % ethanol (aq) was added. Equal parts of freshly prepared 0,4 % hydrogen peroxide and 1 % pyrogallol in water turned yellowish brown in the presence of peroxidase. The 43 presence of tyrosinase was indicated by an orange-brown colour when 0, 1M p-cresol in 96 % ethanol (aq) was added to the mycelium. All reactions were read after 3 h, 24 hand 72 h. Tests on cytochrome oxidase were read after 20 to 30 minutes and compared with a blank control, because atmospheric oxidation also takes place and could interfere with the results after a longer period. Results of these tests were compared with results reported previously (Davidson et aI., 1938; Kaarik, 1965; Nobles, 1948, 1958; Stalpers, 1978; Van der Westhuizen, 1971). The different strains of fungi were grouped according to the production of oxidative enzymes, strength of oxidase reactions and growth rate on GAM and TAM. The growth rate for some groups was described as a range of possible measurements. RESULTS AND DISCUSSION Collection of cultures In total, 278 strains of wood-inhabiting Basidiomycetes were collected over a period of 27 months (Table 1; Appendix A). This collection represented 43 species from 13 families of Homobasidiomycetes and included two species that have, to our knowledge, not been recorded from South Africa, i.e. Gloeophyllum abietinum and Trametes nivosa. The fungi were collected from different areas and a variety of substrates (Figure 1; Table 1). 44 Table 1. Number of strains collected in different areas and from different hosts or substrates. Host / Substrate "e0e Collection area" ~ vo: Western Cape 10 13 41 11 5 43 2 Southern Cape 4 9 1 KwaZulu-Natal 9 2 1 5 22 Mpumalanga 15 5 13 3 6 2 Northern Province 5 2 14 25 3 Gauteng 6 1 TOTAL 39 26 57 12 31 105 8 a Refers to collection areas shown in Figure 1. b Exotic species occurring in commercial plantations. c Exotic species in commercial plantations and invaders of natural vegetation. The variety of habitats that was visited during collection and the differences in the weather experienced at the time were reflected by the species of fungi that were collected (Appendix A). Of the more abundant species that were collected, only Phellinus gilvus and Pycnoporus sanguineus were found in all the collection areas. Skeletocutis sp. was found only in the Western Cape, but species that were usually abundant such as Coriolus hirsutus, C. versicolor, Gloeophyllum spp. and Stereum hirsutum were not found in KwaZulu-Natal where it was hot and dry. Under these dry conditions, the predominant species were P. sanguineus and Trametes nivosa. The largest variety of fungal species was found on Quercus spp. and the fewest on Pinus spp. (Appendix A). The fungal diversity on Pinus spp. was restricted and 45 mostly yielded Gloeophyllum spp. that cause brown-rot. The large number of strains collected from softwood is a reflection on the effort that was made to collect as many cultures as possible from softwood for potential application in biopulping of pine wood (Table 1). However, the only white-rot fungal species that were collected on more than one occasion from softwood were P. sanguineus, S. hirsutum and T. nivosa. Enzymatic characteristics Characteristics that included the production of different enzymes, oxidase reactions as well as growth rates on GAM and TAMwere used to distinguish twelve groups of fungi (Table 2). The strength of the oxidase reactions was the most useful characteristic to differentiate between groups. The culture collection can, therefore, be divided into strong white-rot fungi (Group la, Ib, Ic, II and IV), weak white-rot fungi (Group V, VI and VII) and brown-rot fungi (Group VIII, IX, X and XI). Group III represents a group of brown and white-rot fungi that did not test positive for oxidase enzymes, but produced a positive Bavendamm reaction on GAM and TAM. Each of the fungal strains was assigned to a group on the basis of the enzymes that they produced and oxidase reactions (Table 3; Appendix B). Several species were represented by strains that were placed in more than one group, e.g., Pycnoporus sanguineus, Stereum hirsutum and Trametes nivosa. The variation within species should not be ascribed to a flaw in the assay, but is possibly due to genetic variation within species. Kaarik (1965) has demonstrated that large variation exists within 46 species by companng monosporic strains from the same sporocarp of Stereum sanguinolentum. TableZ. Criteria used for the grouping of wood-decay fungi and the number of strains occurring in each group. Enzymes" Gallic acid medium Tannic acid medium u .c"-" .el...:. ""-"o l. Q., ~ :l .c o a ol. :lZ la ± + + ± 2 0,0 - 6,5 2 0,0 - 11,0 173 lb ± + ± 2 0,0 - 6,5 2 0,0 - 6,5 3 Ic ± + ± 2 0,0 - 6,5 2 0,0 - 6,5 5 II ± ± + ± 2 0,0 - 18,5 1/2 11 - 33,0 13 III 2 0,0 - 4,5 0/1/2 0,0 - 7,5 5 IV ± + + ± 2 0,0 - 2,5 011 0,0 - 2,0 8 V ± ± ± ± 1 1,5 -3,0 ° 3,0 - 3,5 3VI ± + ± ° I 1 0,0 -6,0 0/1 0,0 - 3,0 4 VII + + + ± ° I 1 0,0 - 8,0 011 0,0 - 5,0 23 vm ± ± ° 8,0 - 24,5 ° 13 - 29,5 7IX ± ± ± 1,0 - 11 1,0-11,5 10 X + ± + + 11°0 18,5 - 30,0 ° 0,0 2 XI ± 1 10 0,0 - 8,5 11°0 0,0 22 a Presence of enzymes determined with drop tests (Stalpers, 1978): (+) enzyme present, (±) enzyme present for some strains and (-) enzyme absent. b Strength of oxidase reactions determined as follows: (0) no darkening of media, (1) darkened media not extending beyond mycelium, (2) darkened media extend beyond mycelium. c Number of strains that were identified to species level. 47 Our results were mainly in agreement with previous reports. Results of laecase tests were compared with previously published results, because it is the assay most frequently used by other researchers. The results of other enzyme tests were, therefore, not included in the comparison. Results of tyrosinase tests were not considered, as our results were ambiguous for some strains as experienced by Kaarik (1965) and Stalpers (1978). According to Davidson et al. (1938), significant differences occur between growth rates of strains grown on different sources of gallie and tannic acid. Strength of the oxidase reactions could, therefore, be compared but not the growth rates of fungi. Comparison of our results with those from previous studies was troublesome because the strength of oxidase reactions was reported according to different scales. Comparisons with previous studies are, therefore, based on our interpretation of published results (Davidson et al., 1938; Kaarik, 1965; Nobles, 1948, 1958; Stalpers, 1978; Van der Westhuizen, 1971). Table 3. Identity of fungal strains and grouping according to oxidase reactions and enzyme production. Number Orders, Families and Species" Croup" of strains AGARICALES Strophariaceae Hypholoma fasciculare (Huds. :Fr.) Kummer la 2 Tricholomataceae Cyptotrama asprata (Berk.) Redhead & Ginns VII 1 BOLETALES Coniophoraceae Coniophora olivacea (Fr. ) Karst. VI 1 48 Number Orders, Families and Species" Group" of strains GANODERMATALES Ganodermataceae Ganoderma applanatum (pers.: Wallr.) Pat. la 2 Ic 1 G. eurtisii (Berk.) MUIT. la G. lucidum (Leyss.: Fr.) Karst. IV VII 1 HERICIALES Gloeocystidiellaceae Laxitextum bieolor (pers.: Fr.) Lentz X HYMENOCHAETALES Hymenochaetaceae Phellinus gilvus (Schw.) Pat. la 7 PORIALES Coriolaceae Antrodia variiformis (Peck) Donk VII Bjerkandera adusta (Willd. : Fr.) Karst. la 2 VII 7 Coriolopsis polyzona (pers.) Ryvarden la 5 C. strumosa (Fr.) Ryvarden la 1 VII 1 Coriolus hirsutus (Wulf: Fr.) Quél. la 9 VII 1 C. pubeseens (Schum.: Fr.) Quél. la 1 C. versicolor (Wulf: Fr.) Quél. la 27 C. zonata (Nees: Fr.) Quél. la 1 Daedalea quereina L.: Fr. VIII Fomitopsis lilaeino-gilva (Berk.) Wright & Desehamps VIII IX 1 Gloeophyllum abietinum (Bull.: Fr.) Karst. XI 1 G. sepiarium (Wulf.: Fr.) Karst. III 1 IV 1 XI 14 G. trabeum (Pers.: Fr.) MUIT. III 1 XI 5 Hexagona rigida Berk. IV 1 Lenzites betulina (L.: Fr.) Fr. la 4 II 1 L. elegans (Spreng.: Fr.) Pat. Ic 1 IV 1 Nigroporus vinosus (Berk.) Murr. la 1 Phaeolus sehweinitzii (Fr.) Pat. Ic 49 Number Orders, Families and Species" Groupb of strains Pycnoporus coccineus (Fr.) Bond & Sing. la 3 P. sanguineus (L.: Fr.) MUIT. la 49 III IV 2 Trametes cingulata Berk. la 7 T glabrescens (Berk.) Fr. la 2 T nivosa (Berk.) MUIT. V 3 VI 1 VII 2 VIII 5 IX 6 Lentinaceae Lentinus stupeus Klotzsch la 5 L. villosus Klotzsch la 2 SCHYZOPHYLLALES Schizophyllaceae Schizophyllum commune Fr. IX 2 STEREALES Corticiaceae Pulcherricium caeruleum (Fr.) Parm. la Hyphodermataceae Schizopora paradoxa (Schrad. : Fr.) Donk III Meruliaceae Chondrostereum purpureum (Fr.) Pouz. la VII Stereaceae Stereumfulvum (Lév.) Sacc. VI VII S. hirsutum (Wild.: Fr.) S.F. Gray la 9 Jb II 10 S. illudens Berk. la 2 Ic S. ostrea (Blume & Nees: Fr.) Fr. la 3 Ib 1 VII S. rimosum Berk. la S. sanguinolentum (Alb. & Schw.: Fr.) Fr. Th a Classification of genera in orders and families follows the system of the Dictionary of the Fungi (Hawksworth et al., 1995). b Groups are based on the criteria defined in Table 2. 50 Unfortunately, only a small number of species in our collection have also been studied by other workers. Many similarities and a few discrepancies were noted between our results and those previously published, and these warrant further explanation. Some of the tested species were also included in one or more of the descriptions by Davidson et al. (1938), Kaarik (1965), Nobles (1948, 1958), Stalpers (1978) and Van der Westhuizen (1971). The following comparisons only note the characteristics that have been described previously: Bjerkandera adusta (Group la). The two strains that were examined produced diffusion zones on GAM that extended beyond the mycelium, interpreted as a strong reaction. Previous reports indicate negative or weak reactions (Davidson et al., 1938; Kaarik, 1965; Nobles, 1948; Van der Westhuizen, 1971). The positive laecase test suggests that strong reactions should be expected. (Group VII). Seven strains produced laecase and oxidase reactions similar to the descriptions of Davidson et al. (1938), Kaarik (1965), Nobles (1948, 1958) and Van der Westhuizen (1971). Chondrostereum purpureum (Group la). One strain produced laecase and oxidase reactions as described m literature (Davidson et al., 1938;Kaarik, 1965;Nobles, 1958; Stalpers, 1978). (Group VII). One of the tested strains showed a weak reaction on GAM and TAM. Previous results (Davidson et al., 1938; Kaarik, 1965) describe strong reactions, which is a result that is supported by the production of laccase. 51 Coniophora olivacea (Group VI). Only one strain was tested and it showed a weak diffusion zone on TAM, compared to a reported (Kaarik, 1965) negative reaction. Coriolopsis polyzona (Group la). All five of the tested strains produced laecase as described by Stalpers (1978). Coriolus hirsutus (Group la). The nine strains in this group produced laecase and strong oxidase reactions. These results have previously been described by Káárik (1965), Nobles (1958) and Stalpers (1978) and by Davidson ef al. (1938), Kaarik (1965) and Nobles (1948) respectively. (Group VII). One strain showed a weak reaction on GAM. This is contrary to the characteristics of nine other strains that produced strong reactions, which is similar to all previous records (Davidson ef al., 1938; Kaarik, 1965; Nobles, 1948). Coriolus versicolor (Group la). Results of oxidase reactions and laecase production of 27 strains tested agree with those published (Davidson et al., 1938; Kaarik, 1965; Nobles, 1948, 1958; Stalpers, 1978; Van der Westhuizen, 1971). Coriolus pubeseens (Group la). The tested strain produced laccase, and strong oxidase reactions on GAM and TAM as described by Stalpers (1978) and Nobles (1948) respectively . .o.v~s.BIBLIOTEEK 52 Coriolus zonata (Group la). Laecase was produced, which is consistent with results published by Kaarik (1965) and Stalpers (1978). Positive oxidase reactions confirm those published previously (Davidson et al., 1938; Nobles, 1948). Daedalea quercina (Group VIII). The test for laecase was negative as described by Káarik (1965), Stalpers (1978) and Van der Westhuizen (1971). Negative oxidase reactions are consistent with previous studies (Davidson et al., 1938; Kaarik, 1965; Nobles, 1948). Fomitopsis lilacino-gilva (Group VIII and Group IX). The test for laecase showed negative results for the strains in both groups, as described by Stalpers (1978). Ganoderma applanatum (Group la and Group Ic). Strains in both groups tested positive for laecase and showed strong oxidase reactions. These results confirm those of Nobles (1948; 1958) and Stalpers (1978). Ganoderma curtisii (Group la). The tested strain produced oxidase reactions as described by Davidson et al. (1938). 53 Ganoderma lucidum (Group IV). The strong oxidase reaction on GAM is consistent with the results of Davidson et al. (1938). The single strain that was tested produced no reaction on TAM, although Davidson et al. (1938) described a weak positive reaction on TAM. All our tests for oxidative enzymes were positive, which contradicts results of the Bavendamrn reaction. (Group VII). The one strain that was tested, showed a weak reaction on GAM which is in contrast to the strong positive reaction reported by Davidson et al. (1938). However, all enzyme tests for this strain were positive, which supports the results of Davidson et al. (1938). Gloeophyllum sepiarium (Group III). One strain produced weak oxidase reactions despite the fact that Davidson et al. (1938) suggested that brown-rot fungi should not produce any reaction. (Group IV). One of the strains tested showed positive reactions to all enzyme tests as well as displaying diffusion zones on GAM and TAM. These reactions could be ascribed to non-enzymatic processes, such as oxidation by H202 (Ritschkoff & Viikari, 1991). (Group XI). Fourteen strains displayed negative oxidase reactions and tests for laccase, which is consistent with previous reports (Davidson et aI., 1938; Kaarik, 1965; Nobles, 1948, 1958; Stalpers, 1978; Van der Westhuizen, 1971). 54 Gloeophyllum trabeum (Group Ill). One strain showed a strong reaction on GAM, presumably also because ofH202 production. (Group XI). Five strains displayed negative oxidase reactions and as well as negative tests for laccase, which is consistent with previous reports (Davidson et al., 1938; Kaarik, 1965; Nobles, 1948; Stalpers, 1978; Van der Westhuizen, 1971). Hypholoma fasciculare (Group la). The two strains tested showed strong Bavendamm reactions and tested positive for laecase as described by Kaárik (1965). Lentinus villosus (Group la). Two strains tested positive for laecase as described by Stalpers (1978). Lenzites betu/ina (Group la and II). Strong oxidase reactions and positive tests for laecase were consistent with previous reports (Davidson et al., 1938; Kaarik, 1965; Nobles, 1948, 1958; Stalpers, 1978; Van der Westhuizen, 1971). Nigroporus vinosus (Group la). The one strain in this study tested positive for laecase as described by Stalpers (1978). 55 Phaeolus schweinitzii (Group Ic). The strain tested showed strong reactions on GAM and TAM. Only negative reactions or weak reactions have been described previously (Kaarik, 1965). All tests for oxidative enzymes, except for laccase, were positive. According to Nobles (1958), the Bavendamm reaction can indicate that either laecase or tyrosinase is present. Phellinus gilvus (Group la). Seven strains tested positive for laecase as reported by Stalpers (1978) and showed strong oxidase reactions similar to those reported previously (Davidson et al., 1938; Nobles, 1948). Pulcerricium caeruleum (Group la). One strain was collected and tested positive for laecase as described by Stalpers (1978). Pycnoporus coccineus (Group la). Three strains tested positive for laecase as described by Stalpers (1978). Pycnoporussanguineus (Group la, Group III and Group IV). All of the 52 tested strains were able to produce laecase as described by Stalpers (1978). Schizophyllum commune (Group IX). Neither of the two strains tested showed a reaction on TAM. Other reporters observed weak positive reactions (Davidson et al., 1938; Nobles, 1948). 56 Schizopara paradoxa (Group Ill). Contradictory results were obtained with the one strain that was tested. No enzyme tests were positive, but strong diffusion zones were produced. Stalpers (1978) reported the presence oflaccase, while Van der Westhuizen (1971) observed no reaction on TAMand a weak reaction on GAM. The species is described as a white-rot fungus by Van der Westhuizen (1971). Therefore, the Bavendamm test must be seen as most reliable for this strain. Stereum hirsutum (Group la, Group lb and Group II). The positive test for laecase and strong reáctions on GAM and TAMfor all 20 strains are consistent with the results of Kaarik and (1965) and Stalpers (1978). Stereum ostrea (Group la, Group lb and Group VII). The five strains from these groups all produced laecase as observed by Stalpers (1978). Stereum sanguinolentum (Group la). The single tested strain produced laecase and strong oxidase reactions, which are consistent with previously published results (Kaarik, 1965; Nobles, 1948; 1958; Stalpers, 1978). Trametes cingulata (Group la). Seven strains were tested and were laecase positive with strong oxidase reactions such as those described by Stalpers (1978) and Van der Westhuizen (1971 ). 57 CONCLUSIONS A culture collection was established by sampling from a variety of substrates, habitats and a diversity of climatic and topographic regions. As a result of this study, two species were recorded for the first time from South Africa. This survey also provides valuable additional information on the occurrence and distribution of wood- inhabiting Basidiomycetes. Current methods to identify species of wood-decay fungi in culture do not include the utilization of Bavendamm's test (Stalpers, 1978). The laecase test with guaiacol was introduced by Nobles (1958) to replace the cumbersome tests on GAM and TAM. More recently, tests with polymeric dyes were used to determine the type of decay caused by wood-inhabiting fungi (Cookson, 1995). The Bavendamm and drop tests are, however, still useful in characterizing cultures and as an instrument in the screening programme for biotechnological applications. It has become important to obtain as much information as possible in order to select fungi for biotechnological application. The growth of fungi on GAM and TAMcan, for instance, provide important information on the tolerance of fungi to phenolic compounds (Davidson et al., 1938), which would be valuable in the selection of fungi for degradation of phenolic pollutants (Collett, 1992). The variation in results of the present study and some disagreements with previously published studies (Davidson et aI., 1938; Kaarik, 1965; Nobles, 1948, 1958; 58 Stalpers, 1978; Van der Westhuizen, 1971) indicates that considerable variation exists between strains of the same species. The extent of genetic variation was also demonstrated by Kaarik (1965). It is, therefore, clear that as many strains aspossible should be included in screening trials. Progress in the field of biotechnology has brought new perspectives to the criteria previously used to characterize and group wood-decay fungi. These tests can indicate the potential of decay fungi to be used in industrial processes, while previous studies utilized these assays for identification purposes only. This study represents a contribution towards our understanding of the ability of South African wood-inhabiting fungi to produce lignin degrading enzymes and cause wood decay. REFERENCES AKHTAR,M., ATIRIDGE, M.e., MYERS, G.e., KIRK, T.K. & BLANCHETTE,R.A. 1992. Biomechanical pulping of loblolly pine with different strains of the white- rot fungus Ceriporiopsis subvermispora. Tappi J 75(2): 105-109. AKHTAR,M., BLANCHETTE,R.A. & KIRK, T.K. 1997. Fungal delignification and biomechanical pulping of wood. Adv. Biochem. Eng. Biotechnol. 57: 159-195. BAVENDAlvllvf, W. 1928. Uber das vorkommen und den nachweis von oxydasen bei holzzerstorenden pilzen. Z. Pflanzenkr. Pflanzenschutz 38: 257-276. 59 BERGBAUER,M., EGGERT,C. & KRAEPELIN,G. 1991. Degradation of chlorinated lignin compounds in a bleach plant effluent by the white-rot fungus Trametes versicolor. Appl. Microbiol. Biotechnol. 35: 105-109. BLANCHETTE,RA., BURNES, T.A., EERDMANS,M.M. & AKHTAR, M. 1992. Evaluating isolates of Phanerochaete chrysosporium and Ceriporiopsis subvermispora for use in biological pulping processes. Holzforschung 46: 109- 115. BLANCHETTE,RA., BURNES, T.A., LEATIffiAM, G.F. & EFFLAND,MJ. 1988. Selection of white-rot fungi for biopulping. Biomass 15: 93-101. COOKSON,L.l 1995. Reliability of Poly B-411, a polymeric anthraquinone-based dye, in determining the rot type caused by wood-inhabiting fungi. Appl. Environ. Microbiol. 61: 801-803. CROWDER,A.L., EDDY, W.W. & SETLIFF,E.C. 1978. 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FIELD,lA., DE JONG,E., FEIJOO-COSTAG, . & DEBONT,lA.M. 1993. Screening for ligninolytic fungi applicable to the biodegradation of xenobiotics. Tibtech 11: 44-49. GALENO,G.D. & AGOSIN, E.T. 1990. Screening of white-rot fungi for efficient decolourization of bleach pulp effluents. Biotechnol. Lett. 12: 869-872. HAWKSWORTH,D.L., KIRK,P.M., SUTION,B.C. & PEGLER,D.N. 1995. Ainsworth & Bisby's Dictionary of the Fungi, 8th ed. CAB International, Wallingford, UK. , JOB-CEl, c., KELLER,J. & JOB, D. 1991. Selective delignification of sulphite pulp paper: Assessment of 40 white-rot fungi. Mater. u. Organ. 26: 215-226. JOYCE, T.W. 1992. Applications of biotechnology in the manufacture of paper. Preprints: Papfor 92 Conference. St. Petersburg, Russia: 269-277. KAARI:K, A. 1965. The identification of mycelia of wood-decay fungi by their oxidation reactions with phenolic compounds. Stud For. Suec. 31: 1-80. KlRKPATRICKN, ., REID, LD., ZIOMEK,E. & PAICE,M.G. 1990. Biological bleaching of hardwood kraft pulp using Trametes (Coriolus) versicolor immobilized in polyurethane foam. Appl. Microbiol. Biotechnol. 33: 105-108. 61 MESSNER,K, SCIEFERMEIERM, ., SREBOTNIK,E. & TECHT, G. 1993. Bio-sulfite pulping: Current state of research. In: Lignin Biodegradation and Transformation: Biotechnological applications, Eds. 1.C. DUARTE, M.C. FERREIRA& P. ANDER,pp. 197-200. Forbitec Editions, Lisbon, Portugal. NOBLES,M.K 1948. Studies in forest pathology. VI. Identification of wood-rotting fungi. Can. J Res. 26: 281-431. NOBLES,M.K 1958. A rapid test for extracellular oxidase in cultures of wood- inhabiting hymenomycetes. Can. J. Bot. 36: 91-99. ONYSKO,KA. 1993. Biological bleaching of chemical pulps: A review. Biotechnol. Adv. Il: 179-198. REID, D.A. 1975. Type studies of the larger Basidiomycetes described from southern Africa. Contributions from the Bolus Herbarium 7. University of Cape Town, Cape Town, South Africa. REID, LD. 1991. Biological pulping in paper manufacture. Tibtech 9(8): 262-265. REID, LD. & PAICE,M.G. 1994. Biological bleaching ofkraft pulps by white-rot fungi and their enzymes. FEMS Microbial. Rev. 13: 369-376. RITscHKOFF,A-C. & VIlKARI, L. 1991. The production of extracellular hydrogen peroxide by brown-rot fungi. Mater. u. Organ. 26: 157-167. SPIKER, 1.K, CRAWFORD,D.L. & CRAWFORD, R.L. 1992. Influence of 2,4,6- Trinitrotoluene (TNT) concentration on the degradation of TNT in explosive contaminated soils by the white-rot fungus Phanerochaete chrysosporium. Appl. Environ. Microbial. 58: 3199-3202. 62 STALPERS,lA. 1978. Identification of wood-inhabiting fungi in pure culture. Studies in Mycology 16. CBS, Baarn, Netherland. TALBOT,P.H.B. 1951. Studies of some South African resupinate Hyrnenomycetes II. Bothalia 6: 1-116. TALBOT,P.R.B. 1958. Studies of some South African resupinate Hymenornycetes II. Bothalia 7: 131-187. VAN DERBYL, P.A. 1924. Descriptions of additional South African Polyporeae. S. Afr. J Sci. 21: 308-313. VANDERBYL, P.A. 1928. Aantekeninge oor enige Suid-Afrikaanse swamme. S. Afr. J Sci. 25: 181-184. VAN DER WESTHUIZEN,G.e.A. 1971. Cultural characters and carpophore construction of some poroid Hyrnenomycetes. Bothalia 10: 137-658. VANDERWESTHUIZENG, .C.A. & EICKER,A. 1994. Mushrooms of Southern Africa, Struik Publishers, Cape Town, South Africa. WALL,M.B., CAMERON,D.e. & LIGHTFOOTE, .N. 1993. Biopulping process design and kineties. Biotech. Adv. 11: 645-662. WORRALL,ll, ANAGNOSTS, .E. & ZABEL,R.A. 1997. Comparison of wood decay among diverse lignicolous fungi. Mycologia 89(2): 199-219. WOLFAARDTl,F., BOSMAN,lL., JACOBS,A., MALE, lR. & RABIE, CJ. 1996. Bio- kraft pulping of softwood. In: Biotechnology in the Pulp and Paper Industry: Recent advances in applied and fundamental research, Eds. E. Srebotnik & K. Messner, pp. 211-216. Facultas Unversitatsverlag, Vienna, Austria. 63 CHAPTER3 ASSESSMENT OF WOOD-INHABITING BASIDIOMYCETES FOR BlOKRAFT PULPING OF SOFTWOOD CHIPS Stereum ostrea 64 ABSTRACT Wood-inhabiting Basidiomycetes have been screened for vanous industrial applications in the pulp and paper industry and it is evident that different fungi need to be used to suit the specific requirements of each application. Special screening techniques have, therefore, been developed to select fungi for biopulping that selectively degrade lignin and are able to grow under specific environmental conditions. This study assesses the suitability of a collection of 278 strains of South African wood-decay fungi for the pre-treatment of softwood chips prior to kraft pulping. The influence of these fungi on kappa number, yield and strength properties of pulp was evaluated. A number of these strains were more efficient in reducing kappa number than the frequently used strains of Phanerochaete chrysosporium and Ceriporiopsis subvermispora. Seven of the tested strains, including six strains of Stereum hirsutum and a strain of an unidentified specie were able to reduce the kappa number significantly without a significant influence on the pulp yield. Treatment of wood with two strains of S. hirsutum, one strain of Peniophora sp. and a strain of an unidentified sp. resulted in paper with improved strength properties. 65 INTRODUCTION White-rot fungi are the most efficient degraders of lignin (Kirk et al., 1980) and they are, therefore, probably also the most suitable organisms to be utilized in an industrial process that requires delignification (Messner & Srebotnik, 1994). White-rot fungi are not only capable of producing lignin degrading enzymes, but are also able to penetrate the substrate to transport enzymes into material such as wood chips (Messner & Srebotnik, 1994). A variety of possible applications for these fungi exist, including bio-chemical pulping processes. The potential benefits of bio-chemical pulping include decreased lignin content of pulp, reduction of pulping time, reduced consumption of bleaching chemicals (Messner & Srebotnik, 1994; Wall et al., 1996) and an increase in the strength properties of pulp (Oriaran et aI., 1990, 1991). The biological treatment of wood, prior to chemical pulping, has not been investigated to the same extent as biomechanical pulping (Messner & Srebotnik, 1994) and biokraft pulping is almost unexplored (Chen & Schmidt, 1995). Only a small number of publications deal with kraft pulping of degraded wood (Oriaran et al., 1990, 1991; Wall et al., 1996), although kraft pulping accounts for more then 80 % of the world's annual pulp production (Sjëstrom, 1981). To our knowledge, no results on the evaluation of fungi for biokraft pulping of softwood have been published. It has been suggested that different biotechnological applications have specific requirements (Job-Cei et al., 1991) and that it is difficult to predict the effect of biological treatment on pulping (Messner & Srebotnik, 1994). Our approach was, therefore, to evaluate the potential of a South African collection of wood-inhabiting 66 Basidiomycetes as biopulping organisms. By testing the ability of strains to produce lignin degrading enzymes (Stalpers, 1978) and by means of tests for oxidase reactions (Davidson et al., 1938), fungi with the ability to degrade lignin were selected for further screening (Wolfaardt et al., 1993). Many different screening procedures have been developed to select organisms with appropriate characteristics. These methods include differential staining and microscopy (Otjen et al., 1987), scanning and transmission electron microscopy (Job- Cei et al., 1991), determination of weight loss (Job-Cei et al., 1991, Blanchette et al., 1992) and analysis of degradation products (Otjen et al., 1987, Bechtold et aI., 1993). Unfortunately, none of these methods can be used to accurately predict the effect of fungal treatment on chemical pulping; possibly because an improvement in pulping could simply be ascribed to an improvement in cooking liquor penetration (Oriaran et al., 1991). The purpose of this study was to evaluate a number of South African strains of lignin-degrading fungi for potential application as a pre-treatment of softwood chips prior to kraft pulping. We chose to do mini pulping trials and to rank strains according to their improvement of the resultant pulp. A number of criteria routinely used in the pulping industry were applied to determine the effect of fungal treatment. These parameters included kappa number, pulp yield and paper strength. 67 MATERIALS AND METHODS Fungi and inoculum Two hundred and seventy eight strains of wood-inhabiting Basidiomycetes representing 44 genera were collected in South Africa (Chapter 2). The different strains were deposited in the culture collection of the Division of Food Science and Technology at the Council for Scientific and Industrial Research (CSIR). All of these strains as well as strains of Phanerochaete chrysosporium and Ceriporiopsis subvermispara were used in screening trials. Inoculum was produced by growing fungi on 1,5 % malt extract agar (MEA) plates. Plugs overgrown with mycelium were used to start pre-inoculum in 200 ml liquid medium containing 3 % (v/v) corn steep liquor (CSL) and 1 % (w/v) sucrose. The pre-inoculum was incubated in stationary culture for seven days at 28°C before being homogenized with an Ultra Turrax (Janke Kunkel). The homogenized culture (20 ml) was inoculated into 200 ml CSL and sucrose medium in 500 ml baffled flasks. Cultures were incubated at 28°C on a rotary shaker at 100 rpm for seven days, after which they were again homogenized to produce inoculum for wood chips. Wood and solid-substrate fermentation Softwood chips (40 % Pinus patuIa, 40 % P. elliottii and 20 % P. taeda) from Sappi's Ngodwana mill were dried for five days at 50°C to a moisture content of 3 % and stored at room temperature. The dried wood was placed in 2 L bioreactors (100 gI reactor) and supplemented with 160 ml of the CSL and sucrose medium to provide additional carbon and nitrogen sources (Kirk et al., 1976). The wood was 68 autoclaved for 15 min (121 "C, 100 kPa) and inoculated with 20 ml homogenized inoculum, which brought the moisture content to 60 %. The control treatment consisted of 20ml CSL and sucrose medium that was added to the wood in stead of inoculum. The treated wood was incubated for three or eight weeks at 28°C and a relative humidity of 85 % to 95 %. This experimental procedure allowed control of solid-substrate fermentation conditions such as temperature and moisture, wood sterilization, nutrient supplementation and a treatment period of three weeks. These factors would, however, have important economic implications to be considered during scale-up. Pulping conditions Batches of up to 20 chip samples (50 g dry weight/ sample) were cooked in stainless steel mesh bags in a mini (20 L) digester. In the present study, the effect of biopulping was observed only when the active alkali charges and liquid to wood ratios that are used in mills were modified. This could possibly be ascribed to the heterogeneous cooking batch, or to a greater consumption of cooking chemicals by decayed wood (Hunt, 1978; Hunt & Hatton, 1979). The active alkali charge and liquid to wood ratio were, therefore, increased to 32 % and 10,0: 1 respectively. The digester was charged with kraft cooking liquor (32 % active alkali, 25 % sulphidity) at a liquid to wood ratio of 10: 1. The pulping conditions were: ambient to 170°C in 90 min. and 90 min. at 170 °C. 69 Screening procedure All the fungal strains In the collection (280 including strains of P. chrysosporium and C. subvermispora) were subjected to initial screening on wood chips. Practical considerations limited the second screening step to 20 % of the total number of strains. Initial treatments were done in duplicate, and the best 20 % of strains selected on their ability to reduce the kappa number of pulp. These strains were all able to reduce kappa number by 13 % after three weeks or by 23 % after eight weeks in one of the duplicate treatments (Appendix C). These levels of reduction were selected as In a second screening step, 36 selected strains were evaluated in three different experiments, because of restricted space in the digester (Table 1). A control sample was included in each experiment as reference and each treatment replicated three times. A randomized block design was used for each experiment. Replications of treatments were included in separate cooking batches that were analyzed as different blocks. Table 1. Trial design for the pulping of treated wood to. evaluate different fungal strains. Replications (Blocks) Samples 1 2 3 A 14 fungal treatments Cook 1 Cook2 Cook 3 + Control A Experiments B 7 fungal treatments Cook4 Cook 5 Cook 6 + Control B C 15 fungal treatments Cook 7 Cook 8 Cook 9 + Control C 70 Pulp evaluation To determine yield, pulp was dried at 55 oe until the weight remained constant. Pulp yields were calculated to reflect losses caused by fungal treatment as well as losses incurred during pulping. Kappa numbers were determined according to Tappi Test Method, T236. The reduction in kappa numbers and pulp yields were calculated as a percentage of the controls that were included in each experiment. Pulp from all three replications was bulked to obtain enough material for strength tests. Handsheets were made from the pulp and bursting strength (Tappi Test Method, T403) and tearing strength (Tappi Test Method, T220) determined. Strength ratings were calculated by expressing the average of bursting and tearing strengths of the treated sample as a percentage of the average of bursting and tearing strengths of the control. The data from each experiment were statistically analyzed in a two-way analysis of variance. The multiple comparisons of treatment means were done with Tukey's method (Winer, 1971) at the 5 % level. RESULTS AND DISCUSSION The initial screening of 280 strains resulted in the selection of 36 strains that were evaluated in a second screening step (Table 2). The reference strains, P. chrysosporium and C. subvermispara were not selected, because they failed to reduce kappa number by the required 13%. A number of the strains that were able to cause a notable reduction in kappa number in the first screening step, were not as effective in the second cycle of experiments. This could possibly be ascribed to some strains that 71 were selected as result of incubation for eight weeks during the initial screening step (Appendix C). The long incubation could have allowed better wood colonization in some samples. Considerable variation between cooking batches were also observed in the initial screening, possibly because these batches were composed of a heterogeneous combination of treatments. The variability in results obtained in the initial screening was eliminated to a large extent in the second screening step. The randomized block experimental design compensated for differences between cooking batches, and the composition of batches was less heterogeneous. In some cases no significant differences were found between the experimental blocks that represented different cooks. Twenty-two of the strains tested in the second screening, resulted in a significant modification of lignin, as reflected by reduction in kappa number of the pulp (Table 2). Stereum hirsutum was very efficient in reducing kappa numbers, with all nine tested strains causing significant (p :::;0,05) reductions. Pycnoporus sanguineus was less efficient, with only two out of eight tested strains causing significant reductions. Two out of five strains of Corio/us hirsutus tested, caused significant reductions. The most efficient strains were P. sanguineus (WR 398), Pycnoporus sp. (WR 270) and S. hirsutum (WR 310). These strains were able to reduce the kappa number by 18,1 %, 18,5 % and 18,5 % respectively, compared to the control sample, but also caused significant losses in pulp yield (Table 2). The most promising results were obtained with S. hirsutum (WR 3), which reduced the kappa number by 15,2 % without causing a significant reduction of pulp yield. 72 Table 2. Influence of treatment of wood chips with selected fungal strains on the lignin content and yield of laaft pulp. Kappa lJ. Kappa Pulp yield s Yield Treatment Strain" number" (%r (g/lOO 2: wood)" (%t Control A 31,3 42,2 Coriolus hirsutus WR83 28,6 -8,6 39,6* -6,2 Pycnoporus coccineus WR 132 29,0 -7,5 42,1 -0,2 Pycnoporus sanguineus WR93 28,8 -8,1 39,9* -5,5 WR114 27,5* -12,2 37,8* -10,3 -.<... WR130 29,2 -6,7 38,8* -8,1=c:oJ WR131 28,7 -8,3 38,3* -9,2 oet: WR 170 29,0 -7,4 39,7* -5,9 c:oJ Co Stereum hirsutum WR3 26,5* -15,2 40,8 -3,3~ 1'<1 WR9 27,0* -13,7 40,3 -4,5 WR25 27,6* -11,7 40,9 -3,0 WR91 28,0* -10,7 40,4 -4,3 WR95 26,5* -15,3 40,2* -4,7 WR 156 27,7* -11,5 40,7 -3,6 Trametes glabrescens WR 120 29,8 -4,8 40,2* -4,7 Control B 31,3 41,9 ~ Coriolus hirsutus WR61 29,7 -5,0 38,8* -7,4.e... WR 141 29,2 -6,8 40,8 -2,6 ec:oJ Lentinus stupeus WR24 29,5 -5,6 38,7* -7,6ot: Pycnoporus sanguineus WR 124 28,8 -7,9 39,0* -6,9 ec:oJ~, WR 146 29,4 -6,0 38,2* -8,8 1'<1 Stereum hirsutum WR22 27,8* -11,3 41,0 -2,8 Stereum ostrea WR19 29,6 -5,3 39,0* -6,9 Control C 32,1 41,8 Coriolopsis polyzona WR308 29,6 -7,8 40,6 -2,9 Coriolus hirsutus WR407 27,7* -13,9 36,1* -13,6 WR255 27,7* -13,8 36,7* -12,2 Ganoderma curtisii WR349 28,3* -11,9 36,6* -12,4 U Gymnopilus sp. WR351 27,8* -10,6 35,5* -15,1.=... Lentinus villosus WR339 28,1* -12,5 37,2* -11,0 ec:oJ Lenzites betulina WR402 27,7* -13,9 37,2* -11,0ot: Peniophora sp. WR286 26,7* -17,0 36,7* -12,2 ce:oJ~, Pycnoporus sanguineus WR398 26,3* -18,1 35,8* -14,4 1'<1 Pycnoporus sp. WR270 26,2* -18,5 34,9* -16,5 Stereum hirsutum WR297 26,8* -16,7 36,6* -12,4 WR310 26,2* -18,5 37,6* -10,1 Trametes cingulata WR340 27,1* -15,6 33,9* -18,9 WR345 28,5* -11,2 35,9* -14,1 Unidentified sp. WR251 28,0* -12,8 40,0 -4,3 a Culture numbers of strains maintained in the collection of wood-inhabiting fungi at the CSIR. b Average of three replications. *c Change calculated as the percentage reduction compared to the control treatment of the experiment.Significantly different from the control treatment at the 5 % level by Tukey's test. 73 Of the 22 strains that were able to cause a significant reduction in kappa number, only seven did not cause a significant loss in the yield (Table 2). These strains were six strains of S. hirsutum (WR 3, WR 9, WR22, WR 25, WR 91 and WR 156) and a strain of an unidentified specie (WR 251). The identity of strain WR 251 is in doubt. It was originally identified as Laetiporus sulphureus (Fr.) MUIT. miniatus (Jungh.) Imaz. on the basis of the dried fruit body, but these results indicate that it could be a white-rot fungus with some selective lignin-degrading potential. We, therefore, regard this strain as an unidentified specie. The pulp yields (Table 2) appear to be low, compared with pulp yields obtained on a commercial scale. These yields should, however, be compared with those of the control samples that were also low and were possibly related to specific pulping conditions. Handsheets were made from biopulp produced by the seven fungal strains that did not reduce the yield significantly, as well as four other promising strains and of the control treatments. The strength of biopulp from the unidentified specie (WR 251), Peniophora sp. (WR 286) and two strains of S. hirsutum (WR 156 and WR 310) was better than that of pulp from untreated wood (Table 3). The strongest pulp was produced from treatment with S. hirsutum (WR 310) that was 5 % stronger than the control. This improvement in strength can be ascribed to the large improvement in the bursting index and to a small increase in the tearing index. In general, the bursting strength of hand sheets from biopulp was higher and the tearing strength lower than those of the controls. 74 Table 3. Influence of treatment of wood chips with eleven selected fungal strains on bursting and tearing indices and the strength rating of k.raft pulp. Bursting index Tearing index Strength rating" Treatment Strain" (MNIkg) (Nm1Ikg) (%) Control A 6,4 15,4 100 -< Pycnoporus sanguineus WR1l4 6,2 14,3 94.c... Stereum hirsutum WR3 6,2 13,8 92~ E WR9 6,5 14,6 96·~C WR25 6,7 14,4 97 C~o WR91 6,5 14,4 96 ~ WR95 6,3 15,5 100 WR 156 6,7 15,6 102 = Control B 6,4 15,4 100ë. Stereum hirsutum WR22 6,5 14,5 96 ~~ Control C 6,4 12,5 100 U ë. Peniophora sp. WR286 6,9 13,1 104~ ~ Stereum hirsutum WR310 6,9 12,7 105 Unidentified sp. WR251 6,8 12,4 101 a Culture numbers of strams maintained In the collection of wood-inhabiting fungi at the CSIR. b Average of bursting and tearing strengths expressed as a percentage of the burst and tearing strengths of the controls. CONCLUSIONS The collection of wood-inhabiting Basidiomycetes provided us with new strains of previously tested species as well as some species that have not previously been tested for use in biopulping. The strategy, to collect new strains with the aim of selecting superior ligninolytic strains, was followed by De Jong et al. (1992). This strategy proved to be successful in the present study because a number of strains gave better results than the reference strains. One strain each of the widely used biopulping species, P. chrysosporium (ATCC 32629) and C. subvermispora (CZ-3) (Akhtar et al., 1996), were included in the initial screening trials. However, these strains were 75 not able to reduce the kappa number by the required 13 % within three weeks (Appendix C). A number of strains from certain species (e.g., Pycnoporus sanguineus and Stereum hirsutum) were collected and included in the screening trials. These strains were included, because of the abundance of the species in nature, but also because of the diversity that exists within species (Otjen et aI., 1987; Chapter 2). Two strains, S. hirsutum (WR 156) and unidentified specie (WR 251), were identified as potential biopulping fungi that will now be tested in scale-up and optimization experiments. The mini pulping of the large number of samples was time consuming and labour intensive; however, it allowed us to screen different strains for a specific application (kraft pulping) and on a specific substrate (pine wood). The treatment of the specific substrate is important, since different rates of delignification have been described for different substrates (Otjen et al., 1987; Homolka et al., 1994). Pulping conditions similar to those used at pulp mills have been used for the pulping of fungal treated hardwood (Oriaran et al. 1991; Wall et al. 1996). However, the present study required special pulping conditions to accommodate the large number of samples generated by the screening trials. A change from these experimental pulping conditions to mill conditions will, therefore, be necessary and it will be necessary to optimize conditions for the large scale pulping of fungal treated wood and to do an economic evaluation of the process. 76 The results of this study showed that most of the 38 strains tested, caused a reduction in pulp yield of treated wood, possibly due to degradation of cellulose as non-selective delignification took place. Fungi capable of "selective delignification" are, therefore, regarded as the most suitable organisms for biopulping (Otjen et al., 1987). Several fungi are able to degrade lignin with some degree of selectivity (Blanchette et al., 1988), however absolute selectivity as reported for Ganoderma australe (Bechtold et al., 1993) appears to be unattainable (Erikson & Kirk, 1986). Selectivity of delignification has, for example, been shown to change, depending on the substrate and environmental conditions (Otjen et al., 1987). Pulp yields are often based on the oven dry weight of treated chips that are placed into the digester (Oriaran et al., 1990) and it is then possible to observe an increase in yield due to an increase in holocellulose to lignin ratio in the digester (Oriaran et aI., 1991). However, mass balance calculations based on the weight of wood before fungal treatment showed a yield loss (Oriaran et al., 1990). The increased pulp strength observed in this study, is consistent with the results ofkraft pulping of fungal treated hardwoods (Oriaran et al., 1990, 1991). The change in strength properties, demonstrates that non-selective delignification was caused by fungal treatment. The increase in bursting strength and decrease in tearing strength indicate an improvement in fibre flexibility and a reduction in fibre length respectively (Chen & Schmidt, 1995). 77 The results presented in this paper demonstrate the importance of screening to select superior fungal strains for use in biopulping. Under the specific pulping conditions of the screening trials, 38 strains of white-rot fungi were tested that were more suitable than the reference strains of P. chrysosporium and C. subvermispora. The importance of mini pulping experiments to select the strains with the greatest benefit in a biokraft pulping process was also evident. REFERENCES AKHTAR,M., KIRK, T.K. & BLANCHETIER, A. 1996. Biopulping: An overview of consortia research. In: Biotechnology in the pulp and paper industry: Recent advances in applied and fundamental research. Proceedings of the Sixth International Conference on Biotechnology in the Pulp and Paper Industry. Eds E. Srebotnik & K. Messner, pp. 187-192. Facultas-Universitatsverlag, Vienna, Austria. BECHTOLD,R, GONZALEs,A.E., ALMENDROS,G., MARTINEZ,M.l & MARTINEZ, A.T. 1993. Lignin alteration by Ganoderma australe and other white-rot fungi after solid-state fermentation of beech wood. Holzforschung 47(2): 91-96. BLANCHETTE,RA., BURNES, T.A., EERDMANS,M.M. & AKmAR, M. 1992. Evaluating isolates of Phanerochaete chrysosporium and Ceriporiopsis subvermispora for use in biological pulping processes. Holzforschung 46(2): 109-115. 78 BLANCHETTER, A., BURNES,T.A., LEATHAMG, .F. & EFFLAND,M.l 1988. Selection of white-rot fungi for biopulping. Biomass 15: 93-101. CHEN, Y. & SCHMIDT,E.L. 1995. Improving aspen laaft pulp by a novel, low- technology fungal pretreatment. Wood Fiber Sci. 27(2): 198-204. DAVIDSON,RW., CAMPBELL,W.A. & BLAISDELL,DJ. 1938. Differentiation of wood-decaying fungi by their reactions on gallie or tannic acid medium. J. Agric. Res. 57: 683-695. DE JONG, E., DE VRIES, F.P., FIELD,lA., VANDERZWAN,RP. & DE BONT, lA.M. 1992. Isolation and screening of basidiomycetes with high peroxidase activity. Mycol. Res. 96(12): 1098-1104. ERIKSSON,K.-E. & KIRK, T.K. 1986. Biopulping, biobleaching and treatment of laaft bleaching effluents with white-rot fungi. In: Comprehensive biotechnology in industry, agriculture and medicine. Ed. M. Moo-Young, pp. 271-294. Pergamon Press, Oxford, UK. HUNT,K. 1978. How rot-decayed wood affects chipping and laaft pulping. Pulp Pap. Can. 79(8): 65-68. HUNT, K. & RATTON, lV. 1979. Predicting the laaft pulping behavior of decayed pulpwood. Pulp Pap. Can. 80(3): 55-57. HOMOLKA,L., NERUD,F., KOFRONOVA0, ., NOVOTNA,E. & MACHURovA, V. 1994. Degradation of wood by the basidiomycete Coriolopsis occidentalis. Folia Microbiol. 39(1): 37-43. JOB-CEl, C., KELLER, l & JOB, D. 1991. Selective delignification of sulphite pulp paper: Assessment of 40 white-rot fungi. Mater. u. Organ. 26(3): 215-226. 79 KIRK, T.K., CONNORS,W.l & ZEIKUS, lG. 1976. Requirement for a growth substrate during lignin decomposition by two wood rotting fungi. Appl. Environ. Microb. 32(1): 192-194. KIRK, T.K., HIGUCHI,T. & CHANG,H. 1980. Lignin biodegradation: Summary and perspectives. In: Lignin biodegradation: Microbiology, chemistry, and potential applications Vol 2. Eds. T.K. Kirk, T. Higuchi & H. Chang, pp. 235- 243. CRC Press Inc, Boca Raton, USA. MESSNER,K. & SREBOTNIKE, . 1994. Biopulping: An overview of developments in an environmentally safe paper-making technology. FEMS Microb. Rev. 13: 351-364. ORlARAN,T.P., LABOSKY,P. & BLANKENHORNP, .R. 1990. Kraft pulp and paper making properties of Phanerochaete chrysosporium-degraded aspen. Tappi J 73(7): 147-152. ORlARAN,T.P., LABOSKY,P. & BLANKENHORNP, .R. 1991. Kraft pulp and paper making properties of Phanerochaete chrysosporium-degraded red oak. Wood Fiber Sci. 23(3): 316-327. OIJEN, L., BLANCHETTER, ., EFFLAND,M. & LEATHAM,G. 1987. Assessment of 30 white-rot basidiomycetes for selective lignin degradation. Holzforschung 41 (6): 343-349. SJOSTROM,E. 1981. Wood chemistry: Fundamentals and Applications, 2nd ed. Academic Press, San Diego, USA. SIALPERS,lA. 1978. Identification of wood-inhabiting fungi in pure culture. Studies in Mycology 16. CBS, Baarn, Netherland. 80 WALL, M.B., STAFFORD,G., NOEL, Y., FRITZ, A, IVERSON,S. & FARRELL,R.L. 1996. Treatment with Ophiostoma piliferum improves chemical pulping efficiency. In: Biotechnology in the pulp and paper industry: Recent advances in applied and fundamental research. Proceedings of the Sixth International Conference on Biotechnology in the Pulp and Paper Industry. Eds E. Srebotnik & K. Messner, pp. 205-210. Facultas-Universitatsverlag, Vienna, Austria. WINER,B.l 1971. Statistical principles and experimental design, 2nd ed. Me Graw- Hill Book Co., USA WOLFAARDT, r.r., BOSHOFF, I.E., BOSMAN, J.L., RABIE, CJ. & VAN DER WESTHUIZEN,G.C.A 1993. Lignin degrading potential of South African wood-decay fungi. In: FEMS Symposium: Lignin Biodegradation and Transformation. Book of Proceedings. Eds. J.C. Duarte, M.C. Ferreira & P. Ander, pp. 67-68. Forbitec Editions, Lisbon, Portugal. 81 CHAPTER4 EVALUATION OF THE MICROCLIMATE, AND ENUMERA TION OF FUNGI IN A STORED SOFTWOOD CHIP PILE Outside chip storage at Sappi Ngodwana 82 ABSTRACT Storage of wood chips is preferred to storage of round wood. The deterioration of chips, however, causes considerable problems in chip piles. Factors contributing to deterioration have, therefore, been studied to manage chip storage. The requirements of biopulping processes have renewed interest in the microelimate and microbial populations in these piles. The aim of this study was to investigate conditions such as temperature, moisture, C02 and microbial populations that develop over a three-week period in a softwood chip pile of 3000 tons, and to assess the suitability of the chip pile for colonization of a biopulping fungus. The results show that zones formed within the pile where temperature, CO2 concentration, moisture and microbial contamination varied. The high temperatures that developed in some areas in the chip pile would make a large volume (29 %) of the chip pile unsuitable for colonization by mesophilic white-rot fungi. The moisture content in 24 % of the chip pile reached 55%, but is not expected to have a large impact on biopulping. The areas of high temperature and high moisture were also overlapping. Special management practices would, however, be required to produce a suitable environment in the chip pile for colonization by biopulping fungi. High levels of C02 (12,7 %) accumulated for a short period in some areas, but results have shown that biopulping could still be effective at these levels. 83 INTRODUCTION Storage of wood chips is preferred to storage of logs, because they are more economical to handle, degradation is reduced and smaller storage areas are required (Zabel & Mo rrell , 1992). Pulping properties such as colour and fibre yield of chips can, however, deteriorate rapidly when the chip piles are not managed carefully (Bergman & Nilsson, 1979; Hulme, 1979; Vanderhoff, 1992). Chip deterioration is mostly caused by sap stain and decay fungi. In order to assist with the management of outside chip storage (OCS), several studies have evaluated factors that contribute to chip deterioration (Bergman & Nilsson, 1979; Fuller, 1985; Hatton & Hunt, 1972; Tarocinski, 1976). These investigations emphasized the importance of microelimate and microbial populations in chip pile management (Tarocinski, 1976) as well as the interactions between these factors. The changes in chip piles can be described in terms of respiration of wood cells, chemical oxidation and microbe activity (Hulrne, 1979). Initially, respiration of living parenchyma cells in green wood causes an increase in temperature and carbon dioxide concentration (Springer & Hajny, 1970). Air is moved by convection in the centre of the pile as it is heated, cooler air is drawn in through the sides and humidified as it passes over moist chips. The movement of moisture then redistributes heat by absorbing it during evaporation and emitting it during condensation (Hulme, 1979). An increase in temperature to 40°C leads to auto oxidation of carbohydrates that becomes increasingly important as temperatures continue to rise (Springer et al., 1971). The heat lost to the surroundings eventually 84 equals the heat generated so that stability is reached (Springer et al., 1971). At this point heat generated by microbes would maintain the temperature. Although microorganisms are already present at the time of chipping, their contribution to heat generation only becomes important when sufficient microbial biomass has been produced (Springer & Hajny, 1970; Springer et al., 1971). Biopulping has been defined as the treatment of wood chips with fungi to improve pulping processes (Akhtar et al., 1998). This fungal treatment can be described as a solid-state fermentation process during which asepsis or control of contaminating microbial populations, aeration and temperature is of utmost importance. Factors such as carbon dioxide concentration, moisture content and temperature influence the degradation of wood (Fuller, 1985; Nishimoto et al., 1975) and it can, therefore, be expected that these factors will have a considerable impact on the activity ofbiopulping fungi in chip piles. Recent advances in biopulping research have renewed interest in the chemical and physical changes occurring in chip piles (Akhtar et al., 1998; Eriksson, 1998; Wall et al., 1993). Suppression of biopulping fungi by populations of naturally occurring microorganisms can also play an important role in proposed biopulping processes (Akhtar et al., 1998; Messner & Srebotnik, 1994). It is, therefore, important to study all these conditions on the specific chip pile where such an operation is envisaged. During a project to evaluate the potential of a biopulping process for a kraft mill, a comprehensive study was done on physical and microbiological parameters in 85 the OCS. This paper presents the results of this study on an un-inoculated softwood chip pile at Sappi's Ngodwana mill in Mpumalanga, South Africa. MATERIALS AND METHODS Physical conditions The study of chip pile conditions commenced on May 3rd 1994 (early winter) in a section of a chip pile that was stacked by a traversing stacker. The traversing direction was east to west, and the temperature was monitored in the centre and the southern (shaded) half of the pile. The chip pile consisted of mixed softwood [Pinus patula (40 %), P. elliottii (40 %) and P. taeda (20 %)] that was chipped (18 mm) within three weeks after felling. Trees were less than 15 years old. The chip pile contained c.a. 3000 tons of wood (oven dry) and was 12 m high and 28 m wide at the base. Nine sampling points were used in the chip pile and ambient conditions were monitored from one point outside the chip pile (Figure 1). Temperature was measured with constantan-copper thermocouples. Carbon dioxide was monitored with a Lira 202 analyzer (MSA Instruments, Pittsburgh, USA), by pumping air from the chip pile through 4 mm copper tubing. Temperature and CO2 were monitored at two-hour intervals for 24 hours after stacking of the pile commenced. Monitoring for periods of 24 hours was repeated one, two and three weeks after stacking. A factorial experiment with a completely randomized design was used to determine the influence of storage time on temperature and CO2 at nine different positions in the chip pile. 86 Three factors were considered: age of chip pile (one, two and three weeks), time of day (day or night) and position in the chip pile. The observations made from 08:00 to 18:00 were used as six replications for day observations and observations made from 20:00 to 06:00 as replications for night observations. One-way analysis of variance was done and means were compared using Tukey's test (Winer, 1971). North face South face Side elevation Figure 1. Chip pile dimensions (in metres) and monitoring positions within the pile. Five chip samples (100 g oven dry) were collected at two-hourly intervals during the stacking of the chips to determine the initial moisture content of wood. Two replicate samples were also collected during reclamation of the chip pile from the area close to each of the nine temperature and CO2 probes for determination of moisture content. Samples were also collected from the surface of the chip pile close to the top, where free water was visible after three weeks. Chips were dried at 50°C 87 for 72 h and weighed to determine moisture content on a dry weight basis. One-way analysis of variance was done and means compared using Tukey's test. Microbial populations Five chip samples (c.a. 50 g) were collected in duplicate at two-hourly intervals during the stacking of the chips, to enumerate the fungal populations on fresh wood. Pieces of wood (approximately 9 x 2 x 1 mm) were removed from 100 chips in each sample and were placed onto 1,5 % potato dextrose agar (Difco) (PDA) and incubated at 25 oe. Samples were collected during reclamation of the chip pile from the area close to the temperature and CO2 probes. Three of these chip samples (from position 5, 6 and 7) were discarded due to exceptionally large mite infestations in those areas. Duplicate chip samples were also collected from the north face, south face and top of the chip pile to enumerate the fungi occurring on the surface of the DeS. Two hundred pieces of wood per sample were picked at random and plated onto PDA (five pieces per plate). Half of each sample (100 pieces) was incubated at 25 oe to allow development of mesophilic and thermotolerant fungi, and the other half incubated at 54 oe to allow growth of thermophilic fungi. The total number of each fungal species occurring on the wood was counted and in some cases a piece of wood yielded more than one species. Identification was based on macroscopic, microscopic and culture morphology. Non-sporulating cultures were purified and exposed to near-ultra violet radiation at 25 oe to allow sporulation. 88 RESULTS AND DISCUSSION Physical conditions Analysis of vanance showed that the two-factor interactions between temperature and the age of the chip pile, time of day and monitoring position were significant (p ~ 0,01). Mean day-time and night-time temperatures within the oes changed significantly over three weeks, although the changes were small (Table 1, Appendix D). Three distinct temperature zones, which could be observed by using nine sampling points, already developed after one week within the chip pile (Figure 2). Significant differences were found between temperatures of these zones, although significant differences in temperature were also observed within zones (Table 2). Significant, but small differences (~ 1,3 Oe) were observed between the day and night temperatures at the outer positions in the pile, but except for position 2, day and night temperatures in the core positions did not differ significantly (Table 3). Table 1. Influence of chip age on temperatures in outside chip storage (OeS). Day temperatures (OC) Night temperatures (OC) Age OCSa Ambient" OCSa Ambient" 1 Week 38.9 a 15.4 38.5 a 8.4 2 Weeks 39.6 b 2l.3 39.4 b 9.6 3 Weeks 36.8 c .1. 3.4 34.7 c l.3 a Means of SIX measurements at rune posiuons. Means ill the same column followed by the same letter do not differ significantly (p S; 0,01; Tukey's test). b Means of six measurements at one position outside chip pile. 89 Temperature zones • 44,7 - 55,8°C • 27,6 - 40,8°C .47-214°C Figure 2. Extent of temperature zones that had developed in the chip pile after storage for three weeks. Numbers 1 to 10 indicate sampling positions. Table 2. Mean temperature eC) at different positions in a chip pile at weekly intervals. Temperature Monitoring A2e of chip pile Zone position 1 Week 2 Weeks 3 Weeks 1 5l,6a 55,8a 5l,2bc 2 52,la 53,9a 50,3c Hot 3 5l,6a 55,6a 53,8a 4 44,7b 54,9a 52,5ab 5 40,()c 36,6b 30,9d Warm 6 40,8c 36,Ob 30,9d 7 32,7d 30,7c 27,6e 8 20,ge 21,4d 15,2f Cool 9 13,6f 11, lf 4,7h Ambient ll,9f l6,4e 723g a, b, c, d, e, f. g, h. Means in the same column followed by the same letter do not differ significantly (p s0,01; Tukey's test) 90 Table 3. Mean temperature at different positions during day-time and night -time observations. Monitoring Time of measurement position Day temperatures Night temperatures (OC) (OC) 1 52,9a 53,Oa 2 52,5a 51,7b Inner positions 3 53,6a 53,3a 4 50,5a 50,9a 5 36,5a 35,2b Outer positions 6 36,5a 35,2b 7 30,4a 30,2a 8 19,4a 18,9b 9 10,3a 9,3b Ambient 16,4a 6,4b a, b. Means in the same row followed by the same letter do not differ significantly (p s 0,01; Tukey's test) The highest CO2 measurement of 12,7 % was made after one week at position 4 (Appendix E). It is possible that the CO2 levels were higher during the first week after stacking, but conditions in the chip pile were not observed on a daily basis. Analysis of variance showed that the three-factor interaction between the age of the chip pile, time of day and monitoring position had a significant (p s 0,01) influence on C02 concentration. The weekly CO2 levels decreased significantly (p s 0,01) in the centre, bottom (positions 3 and 4) of the pile (Table 4). In adjacent positions, this reduction only became significant after three weeks, possibly because the initial levels of CO2 were lower. The largest reductions in CO2 concentrations were observed in the core of the chip pile. Significant differences between day and night-time CO2 concentrations were observed only in the centre, bottom of the chip pile (positions 3 and 4 after one week and position 4 after three weeks) (Table 5). 91 Table 4. Weekly CO2 concentrations (%) at different positions, monitored in the day and night over three weeks of chip pile storage. Monitoring Day Ni~ht position 1 Week 2 Weeks 3Weeks 1 Week 2 Weeks 3Weeks 1 1,6a 1,4ab 0,9b 1,3a 1,2ab 0,7b 2 2,3a 1,9a 1,2b 1,8a 1,7a l,lb 3 5,9a 2,6b 1,4c 3,5a 2,4b 1,6c 4 9,6a 3,lb 2,3c 4, 8a 3,Ob 1,6c 5 0,8a 0,8a 0, 5a 0,8a O,7ab 0,2b 6 1, la 0,8a 0,5a 0,9a 0,6ab 0,2b 7 0,9a 0,5a 0,3a 0,7a 0,4ab O,lb 8 0,3a 0,2a 0,2a 0,4a 0,2a O,Oa 9 0,2a O,la 0, la 0,2a O,la O,Oa Ambient O,la O,la O,la 0,2a O,la O,Oa a, b, c. Means of SiXreplications, Means for day-urne observations ill the same row, and means for night -time observations in the same row that is followed by the same letter do not differ significantly (p s 0,01; Tukey's test) Table 5. CO2 concentrations (%) at different posrttons in the day and night, monitored weekly over three weeks of chip pile storage. Monitoring 1 Week 2 Weeks 3 Weeks position Day Night Day Night Day Night 1 1,6a 1,3a 1,4a 1,2a 0,9a 0, 7a 2 2,3a 1,6a 1,9a 1,7a 1,2a 1, la 3 5,8a 3,5b 2,6a 2,4a 1,5a 1,6a 4 9,6a 4,8b 3, la 3,Oa 2,3a 1,6b 5 0,8a 0,8a 0,8a 0,7a 0,5a 0,2a 6 1, la 0,9a 0,8a 0,6a 0,5a 0,2a 7 0,9a 0,7a 0,5a 0,4a 0,3a O,la 8 0,3a 0,4a 0,2a 0,2a 0,2a O,Oa 9 0,2a 0,2a O,la O,la O,la O,Oa Ambient O,la 0,2a O,la O,la O,la O,Oa a, b. Means of SIXreplications, Observations for the same ChIPpile age and ill the same row that is followed by the same letter do not differ significantly (p s; 0,01; Tukey's test) The significant reduction of CO2 concentrations during the night in the lower centre of the pile could possibly be ascribed to increased aeration of the chip pile. The possibility that the increased influx of air was temperature driven, was 92 investigated by plotting ambient temperature and CO2 concentrations at these positions against time (Figure 3). The reduction of CO2 concentrations with reduction in ambient temperature points to a system where aeration of the chip pile is driven by a temperature gradient between the ambient and the inside of the chip pile. Temperatures in the central positions of the chip pile did not differ significantly, therefore, the gradient changed because of fluctuations in ambient temperature. The relationship between CO2 concentrations, at those positions where significant differences were observed between ambient temperature during the day and night was, therefore, tested. Analyses showed that correlation coefficients were 81 %, 77 % and 76 % for positions 4 and 3 after one week and position 4 after three weeks respectively. In the three-week old chip pile, the moisture content at only two positions differed significantly (p s 0,01) from the moisture content of chips that were collected during stacking (Figure 4). The moisture content of the sample collected from the top, where free water was visible on the chip surface, was 9 % higher than the initial moisture content of 56 %. The driest chips were collected from the centre, bottom (position 4) of the oes, where the moisture content was reduced by 28 %. The drying of chips in this area can be ascribed to moisture migration within the pile. The most important area to which moisture was transferred, was the top of the chip pile creating the wet lens as described by Hulme (1979). 93 1"'[C02] ....Temperature 20 c 6 ~ 15 .e~s 4... 10 0~ Q, -'-:le.....e ......0 ~ U2 .......5 20 1Week, Position 4 12 .-.. U 15 10 ~ .. ~.. 8 '0--:le.s ...... .~~.. 10 0....6 U Q .......e,~ 4 ~ 5 2 - 20 33 Weeks, Position 4U 0--~ 15... 2.C=.IS. 0~ 10 --':le........Qe, ...0~ ~ . U...... 5 1 0 0 0 2 4 6 8 10 12 14 16 18 20 22 Time (hours) Figure 3. Change in ambient temperature and CO2 concentration at positions where significant differences between day and night time temperatures were observed. Positions where these observations were made are shown in Figure 1. 94 70 a 65 --. 'e;R '.-...' 60 C .Qc.. J. 55 be b 0~ e be QJ 1.0 .=... 50.Il 60 ~ 40 ~ 20 o Ascomycetes, Ascomycetes, Basidiomycetes, Basidiomycetes, Basidiomycetes, Hardwied Softwood Softwood Hardwood Dead wood Taxonomic atIiliation and habitat offungi Figure 4. Ability of different groups of wood-inhabiting fungi to grow in an environment saturated with a-pinene. (a, b, c: Columns with the same letter do not differ significantly, p ~ 0,05, apriori contrast test) CONCLUSIONS In the past, poor colonization of fresh wood by biopulping fungi was only ascribed to competition with contaminating microbes (Akhtar et al., 1998; Messner & Srebotnik, 1994; Wall et al., 1993). Volatile oleoresin fractions were, on the other hand, seen as compounds that play a doubtful role in the resistance mechanism of live trees against fungal attack (Cobb et al., 1968; De Groot, 1972; Pearce, 1996; Rudman, 1965). The present study has demonstrated that a-pinene play an important role in 121 the inhibition of biopulping fungi and that no single factor is responsible for the inadequate colonization of wood chips. Colonization of softwood chips and degradation of lignin by biopulping fungi improved when the chips were treated with steam before inoculation. The best results were obtained when chips were autoclaved. Unfortunately, this method can not be applied on an industrial scale, because of the high energy costs that will be required for steam generation (Wall et al., 1993). Steam treatments under atmospheric pressure for as little as ten minutes also improved the biopulping effect significantly. It has been demonstrated under laboratory conditions (Wall et al., 1993) and with mill trials (Akhtar et al., 1998) that steaming under atmospheric pressure could be a commercially viable alternative to autoclaving. The cost effectiveness of steam treatments should be evaluated during the scale-up ofbiopulping processes. Steaming is not the only possible solution to problems that are experienced when freshly cut chips are inoculated. Results of this study showed that saprophytic Basidiomycetes, which are frequently used for biopulping (Messner & Srebotnik, 1994; Wall et al., 1993; Chapter 3), are the group of fungi that are the most susceptible to inhibition by a.-pinene. Ascomycetes were more tolerant to inhibition by a.-pinene than saprophytic Basidiomycetes. Cobb et al. (1968) also demonstrated that four Ophiostoma and Ceratocystis species were more tolerant to inhibition by terpenes than the Basidiomycete Heterobasidion annosum. This might indicate that ascomycetous fungi are more suitable for application as biopulping agents on 122 softwood than saprophytic Basidiomycetes. They are, however, not known as efficient degraders of lignin (Crawford & Crawford, 1980). Inoculation of untreated chips with P. sanguineus was more successful than inoculation with S. hirsutum, demonstrating that some fungi within the groups described in this study, are better able to compete with contaminants or tolerate wood extraetives better than others. De Groot (1972) demonstrated that the biopulping fungus Phlebiopsis gigantea was more tolerant to monoterpenes than fungi from other taxonomic groups. Phlebiopsis gigantea was described as a pioneer fungus and should, therefore, be more tolerant to inhibitory compounds. It was also noted that species such as Phanerochaete chrysosporium were better able to colonize fresh chips than Ceriporiopsis subvermispora (Wall et al., 1993). Costly treatment processes for chips could, therefore, be avoided by the correct choice of a biopulping organism. Fungi that are tolerant to wood extractives could be selected on the basis of in vitro experiments (Cobb et al., 1968). The competitive ability of biopulping fungi could be enhanced by developing the correct formulation of inoculum. Inoculum consisting of fragmented mycelium instead of spores can for instance improve the competitive ability of biopulping fungi by reducing the lag phase (Wall et al., 1993). 123 REFERENCES AKHTAR,M., KIRK, T.K. & BLANCHETTER, .A. 1996. Biopulping: An overview of consortia research. In: Biotechnology in the pulp and paper industry: Recent advances in applied and fundamental research. Proceedings of the Sixth International Conference on Biotechnology in the Pulp and Paper Industry. Eds. E. Srebotnik & K. Messner, pp. 187 - 192. Facultas-Universitatsverlag, Vienna, Austria. AKHTAR,M., LENTZ, MJ., SWANEY,RE., SCOTT,G.M., HORN, E. & KIRK, T.K. 1998. Commercialization of biopulping for mechanical pulping. Proceedings of the 7th International Conference on Biotechnology in the Pulp and Paper Industry, Vancouver, Canada. Vol. A: 55-58. BARZ, W. & WELTRING,K.-M. 1985. Biodegradation of aromatic extractives of wood. In: Biosynthesis and biodegradation of wood components, Ed. T. Higuchi, pp. 607-664. Academic Press, Orlando, USA. COBB,F.W., KRSTIC,M., ZAVARIN,E., & BARBER,HW. 1968. Inhibitory effects of volatile oleoresin components on Fomes annosus and four Ceratocystis species. Phytopathol. 58: 1327-1335. CRAWFORDD, .L. & CRAWFORDR, L. 1980. Microbial degradation of lignin. Enzyme Microb. Techno!. 2: 11-22. DAVIDSON,RW., CAMPBELL,W.A. & BLAlSDELL,DJ. 1938. Differentiation of wood-decaying fungi by their reactions on gallie or tannic acid medium. J Agric. Res. 57: 681-695. 124 DE GROaT, R.C. 1972. Growth of wood-inhabiting fungi in saturated atmospheres of monoterpenoids. Mycologia 64: 863-870. K.MRIK A. 1965. The identification of mycelia of wood-decay fungi by their oxidation reactions with phenolic compounds. Stud For. Suec. 31: 1-80. MESSNER,K. & SREB01NIK,E. 1994. Biopulping: An overview of developments in an environmentally safe paper-making technology. FEMS Microb. Rev. 13: 351 - 364. MlRov, N.T. 1961. Composition of gum turpentines of pines. Us. Dep. Agr. Tech. Bull. 1239: 158p. PEARCE,R.B. 1996. Tansley Review No. 87. Antimicrobial defences in the wood of living trees. New Phytol. 132: 203-233. RUDMAN,P. 1965. The causes of natural durability in timber. XVIII. Further notes on the fungitoxicity of wood extractives. Holzforschung. 19: 57-58. SJOSTROM,E. 1981. Wood chemistry: Fundamentals and Applications, 2nd ed. Academic Press, San Diego, USA. SPRINGER,E.L. & HAlNY, G.J. 1970. Spontaneous heating in piled wood chips. I. Initial mechanism. Tappi. J 53(1): 85-86. WALL,M.B., CAMERON,D.C. & LIGHTFOOTE, .N. 1993. Biopulping process design and kinetics. Biotech. Adv. Il: 645-662. WINER,B.J. 1971. Statistical principles and experimental design, 2nd ed. Me Graw- Hill Book Co., USA. ZAR, J.H. 1984. Biostatistical analysis, 2nd ed. Prentice-Hall Inc., Englewood Cliffs, USA. 125 CHAPTER6 KRAFT PULPING OF PINE WOOD, PRE-TREATED WITH A STRAIN OF STEREUM HIRSUTUM Continuous kraft digester at Usutu 126 ABSTRACT The successful application of biopulping in most pulping processes has not been exploited to the same extent as for kraft pulping. The influence of biopulping of pine wood on kraft pulping parameters was, therefore, investigated. Pinus patuIa wood chips were pre-treated with a selected strain of Stereum hirsutum. Wood was pulped on a small scale and the pulping conditions were varied. Lignin content, yield and viscosity of the pulp were evaluated and the alkali consumption determined. The relationships between these parameters were used to model a biokraft pulping process. Fungal pre-treatment reduced kappa number and yield, but not the degree of polymerization of cellulose. Alkali consumption increased when fungal pre-treated wood was pulped. This study showed that biopulping can reduce the kappa number of pulp or reduce the pulping time, but pulp yield is also reduced and chemical consumption increased. The implementation of biopulping on industrial scale would consequently be determined by the specific requirements of a mill that enables it to exploit specific economic benefits. 127 INTRODUCTION Biopulping has been defined in the narrow sense as the pre-treatment of wood with lignin degrading fungi before the mechanical or chemical production of pulp (Messner et al., 1992). Wood chips are usually pre-treated with fungi in a solid- substrate fermentation process (Reid, 1989). Extensive screening has resulted in the identification of a number of white-rot fungi that are, to some extent, selective degraders of lignin (Blanchette et al., 1992b; Job-Cei et al,. 1991; Messner & Srebotnik, 1994; Otjen et aI., 1987). The result of degradation by these fungi is wood in which the lignin has been utilized or modified. Consequently the efficiency of chemical pulping (Scott et aI., 1995; Wall et aI., 1996) or fibre separation during mechanical pulping is improved (Blanchette et al., 1992a). The successful application of biological treatment prior to mechanical pulping is the result of eight years of research by two international consortia based at the Forest Products Laboratory, Madison, Wisconsin (Akhtar et al., 1996). This research led to the issuing of a patent (Blanchette et al., 1991) and to mill scale trials (Akhtar et aI., 1996). These investigations showed that pre-treatment of loblolly pine chips with Ceriporiopsis subvermispora resulted in energy savings of 38 %. The pulp produced during this process had improved strength properties, with an increase in burst and tear indices (Akhtar et al., 1996). The pre-treatment of wood with white-rot fungi prior to chemical pulping has not been researched to the same extent as biomechanical pulping (Reid, 1991). 128 However, Messner et al. (1992) and Scott et al. (1995) demonstrated the potential of biopulping in a sulphite processes. Fungal pre-treatment of birch chips produced pulp with a reduced kappa number and improved brightness, but with decreased strength properties (Messner et al., 1992). Pre-treatment of loblolly pine chips with Ceriporiopsis subvermispara resulted in pulp with a reduced kappa number or pulping time (Scott et al., 1995). Kraft pulping accounts for 80 % of the chemical pulp production in the world (Sjëstrëm, 1981). Biokraft pulping has, however, been restricted to studies using blue stain fungi (Wall et a/., 1994; 1996), or those utilizing white-rot fungi on hardwood (Oriaran et al., 1990; 1991). Valuable information does, however, exist on the kraft pulping properties of softwood decayed by white-rot fungi (Hunt, 1978b; 1978c). These studies have focussed on the utilization of wood from decadent stands for kraft pulping. The degradation of this wood occurred under uncontrolled conditions and results can, therefore, only be applied to biopulping to a limited extent. From the studies on the kraft pulping of decayed wood, it is clear that several pulping parameters and pulp properties need thorough investigation to allow economic evaluation of a biokraft pulping process. The most obvious benefit of fungal pre- treatment, is the reduction of lignin content (Oriaran et a/., 1990; 1991) or, alternatively, the pulping time (Oriaran et al., 1991; Scott et al., 1995). These improvements also appear to be associated with negative changes in pulp yield (Oriaran et al., 1990; Hunt, 1978b) and chemical consumption (Hunt, 1978a; 1978c). 129 The aim of this study was, therefore, to determine the influence of fungal pre-treatment in a controlled environment on biokraft pulping parameters and pulp properties. In this study, Pinus patuIa chips were pre-treated with Stereum hirsutum (WR 95). This white-rot fungus was selected in earlier studies, where it showed potential for application in biokraft pulping (Chapter 3). MA TERIALS AND METHODS Fungal pre-treatment Pinus patuIa chips were collected at Sappi's Ngodwana kraft mill, dried to 3 % moisture content and stored until used. Thirty kilograms (dry weight) of chips were prepared in 25 L polypropylene bioreactors (3 kg/reactor) by adjusting moisture content to 60 % with a 3 % (v/v) corn steep liquor and 1 % sucrose solution. Corn steep liquor, containing 50 % solids, was obtained from African Products (p.O. Box 554, Gerrniston 1400, South Africa). The wood was autoclaved on two consecutive days for one hour. Inoculum of Stereum hirsutum (WR 95) was produced as described for screening trials (Chapter 3). Fragmented mycelium was used to inoculate chips at a dosage of 3g dry weight per kilogram wood. Cultures were incubated for nine weeks and aerated with humidified air (31°C, 72 % relative humidity, 500 ml/min) that was pre-sterilized using an air incinerator (New Brunswick, U.s.A.). An equivalent mass of uninoculated chips with sterile growth medium was used as a control. Chips were 130 harvested and fungal activity was halted by drying at 55 oe to a moisture content of 3 %. Weight loss of the wood that occurred during fungal pre-treatment was determined to use as a correction factor in the calculation of final pulp yield. Pulping Pre-treated wood was bulked and divided into 800 g samples and pulped in a 15 L rotating digester at 170 oe. In order to obtain the same pulping conditions, a control sample (100 g) in a canister was included in the digester with the fungal pre- treated sample. The different active alkali charges, liquor to wood ratios and pulping times that were used for different treatments are shown in Table 1. The series of pulping experiments was started by pulping wood under conditions similar to those used at the Sappi Ngodwana mill. These conditions were: 22 % active alkali, 5,4 : 1 liquor to wood ratio and 85 min at the pulping temperature (Table 1). At first, only the liquor to wood ratio was changed to 4,5 : 1 and 7,5 : 1. When it was found that alkali consumption increased for the fungal pre-treated wood, the alkali charge was also increased and tested with different liquor to wood ratios. A reduced pulping time of 65 min and 75 min at 170 oe was used for the last pulping treatments (Table 1). 131 Table 1. Different pulping treatments to determine the optimal conditions for pulping of wood pre-treated with Stereum hirsutum. Pulping Active Alkali Liquor to Alkali Pulping Time Treatment Charge (%) Wood Ratio Activity (min)" 1 22 4,5 48,9 85 2 22 5,4 40,7 85 3 22 7,5 29,3 85 4 25 8,0 31,3 85 5 26 4,5 57,8 85 6 26 5,4 48,1 85 7 26 7,5 34,7 85 8 27 7,5 36,0 85 9 27 9,0 30,0 85 10 30 7,5 40,0 85 11 32 5,4 59,3 85 12 32 10,0 32,0 85 13 22 5,4 40,7 75 14 22 5,4 40,7 65 a Time at temperature (170°C) and 85 minutes to temperature Pulp evaluation The amount of delignification during pulping was determined as the reduction of kappa number using Tappi Test Method (T236). Pulp viscosity was determined with a capillary viscometer using Tappi Test Method (T230). Pulp yields were determined by weighing the pulp (oven dry) and applying a correction factor to account for weight loss during fungal pre-treatment. Alkali consumption was determined by analysis of black liquor according to Tappi Test Method (T625). 132 RESULTS AND DISCUSSION Lignin content The fungal pre-treatment of wood, pulped under all of the test conditions, resulted in reduction of lignin content, as reflected by kappa number (Table 2). The most successful pulping was done in Treatment 9, with fungal pre-treated wood that was pulped with 27 % active alkali at a liquor to wood ratio of 9 : 1 (Table 1). Under these conditions, pulp with a 30,6 % lower kappa number was obtained compared to untreated wood pulped under similar conditions (Table 2). Table 2. Influence of conditions of different pulping treatments on pulp quality and yield of wood, pre-treated with Stereum hirsutum compared to untreated wood. Pulping Kappa fl Kappa Pulp Yield fl Yield Viscosity fl Viscosity Treatment Number No. (%t (g/100 g)" (%t (crrr'zg) (%t 1 21,3 -12,3 41,5 -3,7 782 -12,6 2 23,1 -17,5 41,9 -4,8 873 -12,8 3 36,0 -15,3 42,7 -2,5 922 -17,9 4 25,4 -24,6 42,3 -3,2 891 -15,9 5 19,5 -3,0 40,5 -1,7 770 -2,0 6 16,3 -15,5 39,2 -7,5 697 -18,2 7 23,7 -22,3 40,3 -7,1 882 -13,3 8 22,3 -28,3 41,3 -6,3 853 -16,3 9 25,4 -30,6 42,3 -4,3 912 -15,5 10 18,9 -26,2 40,6 -3,1 806 -27,5 11 12,2 -23,3 38,8 -2,5 558 -15,3 12 21,1 -24,6 41,3 n.d. 857 -14,1 13 24,4 -23,3 42,7 -3,6 899 -12,3 14 30,2 -20,1 43,3 -3,8 978 -11,4 a Change compared to untreated woodpulped under similar condi.t.ions b Based on the oven dry weight of woodbefore treatment n.d. Not determined 133 As result of the varied conditions used for different pulping treatments, pulp with a range of kappa numbers was obtained. The pulping data could, therefore, be used to derive the following equation to calculate the effect of pulping conditions on kappa number: K = 71,8276 - 6,llF -0,299T - 1,531C + 3,219L (R2 = 88 %) (1) Where K is the kappa number, F is the pre-treatment (0 = untreated and 1 = fungal pre-treatment), T is the pulping time (min) at 170 oe, C is the active alkali charge (%) and L is the liquor to wood ratio. Equation 1 shows that fungal pre-treatment reduced the kappa number by 6,1 units in the experimental range. In comparison, kraft pulping of wood decayed by white-rot fungi, produced variable results. Western hemlock, decayed by Echinodontium tinctorium and Phellinus pini, produced pulp with a 2,5 % and a 2,4 % respective increase in permanganate number (~ kappa number/0,66) compared to sound wood (Hunt, 1978a). Another report showed a 3 % decrease in permanganate number of pulp from decayed softwood (Hunt, 1978c). These contradictory results make the mechanism of delignification in decayed wood more difficult to interpret. The reduction in lignin content of pulp can be ascribed to the degradation or modification of the lignin polymer (Scott et al., 1995). This results in improved penetration of cooking liquor and leads to more efficient pulping (Sjëstróm, 1981). 134 Modifications to cell walls and utilization of extractives can, therefore, also increase delignification during pulping (Wall et al., 1994). Pulp yield and degree of polymerization The ability of fungi to selectively degrade lignin has been an important criterion in the selection of fungi for use in biopulping processes (Blanchette et al., 1992b; Job- Cei et al,. 1991). The kappa number of pulp from fungal pre-treated wood is, therefore, often expressed in terms of yield and viscosity (Oriaran et al., 1990; 1991; Wall et al., 1996). Absolute selectivity does not appear to be possible in biopulping processes (Eriksson & Kirk, 1986) and in this study 1,1 % weight loss of wood occurred during fungal pre-treatment for nine weeks. This weight loss was included in the final yield calculation to maintain the mass balance (Table 2). Benefits in lignin reduction should be weighed-up against losses in yield. During this study, yield loss and reduction of viscosity accompanied reduction in kappa number (Table 2). Equations to describe linear relationships between pulp yield and kappa number have been proposed for kraft pulping of fungal pre-treated aspen and red oak (Oriaran et aI., 1990; 1991). However, from the results (Table 2) the following non-linear equations were the most effective for the estimation of yield at a specific kappa number (Figure 1): Ye = 37 + 8[1_e-O,1(K-12)] (R2=86%) (2) Yf= 37 + 7[1_e-O,1(K-12)] (R2=86%) (3) 135 Where Ye (g/100 g wood) is the pulp yield of untreated wood and Yf the yield of biopulped wood. 46 45 • 44 43 ..-, 't 42 '-' • "0 .].. 41 40 • •• ControlBiopulping 39 • -- Equation 238 -- Equation 3 37 11 16 21 26 31 36 41 46 Kappa number Figure 1. Relationship between pulp yield and kappa number for fungal pre-treated wood and untreated wood, pulped under different conditions. The influence of kappa number on cellulose viscosity in pulp from untreated and pre-treated wood (Figure 2) is described by: v = 1330(1 _ e-O,046K) (R2 = 86 %) (4) Where V is viscosity (cm'zg). 136 Figure 2. Relationship between cellulose viscosity and kappa number for fungal pre- treated wood and untreated wood, pulped under different conditions. Equations 2 and 3 can be used to show that the pulp yield of fungal pre-treated wood, compared to untreated wood is reduced by 0,84 glIOO g and 0,56 glIOO g wood when pulped to a kappa number of 30 and 20 respectively. These data show that biopulping reduces yield by less than 1 % in the experimental range for a given kappa number. The loss of yield also becomes smaller as wood is pulped to a lower kappa number. Viscosity, however, is a function only of kappa number and is not influenced by the fungal pre-treatment (equation 4, Figure 2). The loss of pulp yield observed during this study could be further reduced by using a shorter pre-treatment period, because it has been shown that selectivity of lignin degradation decreases with increased incubation time (Messner et al., 1992). 137 Alkali consumption An increase in consumption of cooking chemicals is a major concern when decayed wood is used for pulping (Hunt, 1978a; 1978b; 1978c). Results of this study showed that residual alkali can be calculated by using the equations (Figure 3): Re = 12 + 20 x e-O,II(K-15) (R2 = 92 %) (5) Rf= 10+ 17 x e-O,15(K-12) (R2 = 92 %) (6) Where Re is the residual alkali of the black liquor from the control treatment and Rf is the residual alkali in black liquor from the fungal pre-treatment. 40 35 • Control BiopuJping ,-.3.0 •-- Equation 5 ~.0_, -- Equation 6 :.:c; 25-; -; = 20".;C; ~ ~ 15 10 5 11 16 21 26 31 36 41 46 Kappa number Figure 3. Relationship between residual alkali in the black liquor of fungal pre-treated wood and untreated wood, pulped under different conditions and kappa number. 138 Alkali consumption is then calculated using: U=100(A-R)/A (7) Where A is the activity of the cooking liquor and R is the residual alkali in the black liquor. This model (equation 7) can be used to show that alkali consumption increase from 61,1 % to 72,7 %, when fungal pre-treated samples are pulped to a kappa number of 30 using conditions similar to those of a pulp mill (Table 3). These data are comparable with those reported by Hunt (1978b) for the pulping of decadent western red cedar. The increase in alkali consumption has been ascribed to the increase in products of the fungal degradation, such as low molecular weight polysaccharides (Hunt & Hatton, 1979). The reduction in yield is, therefore, correlated with the increased alkali consumption (Hunt & Hatton, 1979). The increased organic solids in the black liquor contribute additional material to the recovery boiler (Hunt, 1978b) that, frequently, has a limited capacity. However, expansion of the recovery capacity to increase the pulping capacity, would increase the fixed cost of pulp (Christie, 1979). 139 Table 3. Expected influence offungal pre-treatment of wood chips on kraft pulping parameters. Parameter Example No. Parameters 1 2 3 Pre-treatment Control Stereum hirsutum Stereum hirsutum Alkali charge (%) 22 22 22 I Liquor to wood ratio 5,4: 1 5,4: 1 5,4: 1 Kappa number 30 30 243 Time at 170°C (min.) 85,0 79,43 85,0 Yield (g/100g wood) 437,b 42,8c 41,9c Viscosity (ern' 19) 995d 995d 889d Alkali consumption (%) 61,1 e 72,t 686,f a Values calculated using model equation l. b Value calculated using model equation 2. c Values calculated using model equation 3. d Values calculated using model equation 4. e Value calculated using model equations 5 and 7. f Values calculated using model equations 6 and 7. Pulping time Variation of pulping times has shown that pre-treated wood can be pulped to a similar kappa number as untreated wood, in a shorter time (Table 3). The estimated reduction in pulping time of 5,6 min was calculated using the model equations (Table 3). Reduction of pulping time has also been reported for biokraft pulping of aspen (Oriaran et al., 1990) and for biosulphite pulping, where the pulping time was reduced from 5,75 h to 5,25 h (Scott eta!., 1995). The reduction of pulping time can be ascribed to modification of wood, which in turn allows quicker and more uniform impregnation by the cooking liquor (Wall 140 et aI., 1994). This is of specific importance in the k.raft process, where the initial phase of delignification is controlled by diffusion (Sjostrëm, 1981). CONCLUSIONS The fungal pre-treatment of softwood can cause a substantial reduction of the lignin content of pulp. Under optimal pulping conditions, fungal pre-treatment reduced kappa number by more than 30 %. An alternative benefit is a shorter pulping time. Pre-treated wood could be pulped to the same kappa number (kappa 30) as untreated wood in a shorter time at pulping temperature (6,6 % less than untreated wood). This saving in pulping time could be translated to increased output. Fungal pre-treatment of wood reduced the pulp yield, due to the non-selective delignification by S. hirsutum (WR 95). This yield reduction does, however, become less important when pulping is done to low kappa number. Selectivity of delignification is also influenced by many factors (Otjen et aI., 1987) and because solid substrate fermentation is used, these factors are often difficult to control. A reduction of pulping time from nine weeks to three weeks could, for instance, reduce the amount of cellulose that is degraded. The model (equation 4) shows that cellulose viscosity of pulp is a factor of kappa number. The degree of polymerization was, therefore, not negatively influenced by fungal action. 141 Alkali consumption was increased during kraft pulping of fungal pre-treated wood. It is, however, possible to reduce the use of bleaching chemicals due to the reduced lignin content of the pulp (Macleod, 1993). We do not expect that different results to those presented in this study, will be obtained by using other fungal strains for biokraft pulping. Our results have corroborated results of previous studies obtained with decayed wood (Hunt, 1978a; 1978b; 1978c; Hunt & Hatton, 1979). The benefits of biopulping, such as reduced kappa number and pulping time might be enhanced by the use of other fungal strains. Increased chemical consumption would, however, remain an important obstacle to a commercially viable process. REFERENCES AKHTAR,M., KIRK, T.K. & BLANCHETIE,RA. 1996. Biopulping: An overview of consortia research. In: Biotechnology in the pulp and paper industry: Recent advances in applied and fundamental research. Proceedings of the Sixth International Conference on Biotechnology in the Pulp and Paper Industry. Eds. E. Srebotnik & K. Messner, pp. 187-192. Facultas-Universitatsverlag, Vienna, Austria. BLANCHETIE,RA., LEATHAM,G.F., ATIRIDGE,M., AKHTAR,M. & MYERS, G.C. 1991. Biomechanical pulping with C. subvermispora. u.s. Pat. 5,055,159. 142 BLANCHETIE,RA., AKHTAR,M. & ATIRIDGE,M.C. 1992a. Using Simons stain to evaluate fiber characteristics of biomechanical pulps. Tappi 1. 75(11): 121- 124. BLANCHETTE,RA., BURNES, T.A., EERDMANS,M.M. & AKHTAR,M. 1992b. Evaluating isolates of Phanerochaete chrysosporium and Ceriporiopsis subvermispora for use in biological pulping processes. Holzforschung 46(2): 109-115. CHRISTIE,RD. 1979. The use of decayed wood in pulping process. In: Chip Quality Monograph. Ed. lV. Hatton, pp. 111-128. Tappi Press, Atlanta, USA. ERIKSSON,K. -E. & KIRK,T.K. 1986. Biopulping, biobleaching and treatment of kraft bleaching effluents with white-rot fungi. In: Comprehensive biotechnology in industry, agriculture and medicine. Ed. M. Moo-Young, pp. 271-294. Pergamon press, Oxford, UK. HUNT, K. 1978a. Pulping western hemlock decayed by white-rot fungi. Pulp Pap. Can. 79(6): 75-80. HUNT,K. 1978b. Kraft pulping of decayed wood: western red cedar and alpine fir. Pulp Pap. Can. 79(7): 30-35. HUNT, K. 1978c. How rot-decayed wood affects chipping and kraft pulping. Pulp Pap. Can. 79(8): 65-68. HUNT,K. & RATTON,LV. 1979. Predicting the kraft pulping behavior of decayed pulpwood. Pulp Pap. Can. 80(3): 55-57. JOB-CEl, C., KELLER,J. & JOB, D. 1991. Selective delignification of sulphite pulp paper: Assessment of 40 white-rot fungi.Mater. u. Organ. 26(3): 215-226. 143 MACLEOD,I.M. 1993. Extended delignification: a status report. Appita 46(6): 445- 451. MESSNER,K., MASEK, S., SREBOTNIKE, . & TEcm, G. 1992. Fungal pretreatment of wood chips for chemical pulping. In: Biotechnology in the pulp and paper industry. Proceedings of the 5th International Conference on Biotechnology in the Pulp and Paper Industry. Eds. M. Kuwahara and M. Shimada, pp. 9-13. Uni Publishers, Tokyo, Japan. MESSNER,K. & SREBOTNIKE, . 1994. Biopulping: An overview of developments in an environmentally safe paper-making technology. FEMS Microb. Rev. 13: 351-364. ORIARAN, T.P., LABOSKY, P. & BLANKENHORN,P.R. 1990. Kraft pulp and papermaking properties of Phanerochaete chrysosporium-degraded aspen. Tappi J. 73(7): 147-152. ORIARAN, T.P., LABOSKY, P. & BLANKENHORN,P.R. 1991. Kraft pulp and papermaking properties of Phanerochaete chrysosporium-degraded red oak. Wood Fiber Sci. 23(3): 316-327. OTJEN,L., BLANCHETIE,R., EFFLAND,M. & LEATHAM,G. 1987. Assessment of 30 white rot Basidiomycetes for selective lignin degradation. Holzforschung 41(6): 343-349. REID, I.D. 1989. Optimization of solid-state fermentation for selective delignification of aspen wood with Phlebia tremellosa. Enzyme Microb. Techno!. Il: 804- 809. REID, I.D. 1991. Biological pulping in paper manufacture. Tibtech 9(8): 262-265. 144 SCOTI, G.M., AKHTAR, M. & LENTZ, M. 1995. Fungal pretreatment of wood chips for sulfite pulping. Tappi Pulping Conference, p. 355-361. Tappi Press, Atlanta, USA. SJOSTROM, E. 1981. Wood chemistry: Fundamentals and Applications, 2nd ed. Academic Press, San Diego, USA. WALL, M.B., BRECKER, 1., FRITZ, A., IVERSON, S. & NOEL, Y 1994. Cartapip® treatment of wood chips to improve chemical pulping efficiency. Tappi Biological Sciences Symposium, pp. 67-76. Tappi Press, Atlanta, USA. WALL, M.B., STAFFORD, G., NOEL, Y, FRITZ, A., IVERSON, S. & FARRELL, R.L. 1996. Treatment with Ophiostoma piliferum improves chemical pulping efficiency. In: Biotechnology in the pulp and paper industry: Recent advances in applied and fundamental research. Proceedings of the 6th International Conference on Biotechnology in the Pulp and Paper Industry. Eds. E. Srebotnik & K. Messner, pp. 205-210. Facultas-Universitatsverlag, Vienna, Austria. \ 145 SUMMARY KEY WORDS: Basidiomycetes, Biodiversity, Biopulping, Biotechnology, Fungi, Inhibition, Kraft, Pulping, White-rot, Wood. The forest products industry is one of the most important earners of foreign exchange for South Africa. The major focus of the industry is the production of pulp with an annual capacity of 2,4 million tons. Wood from plantations of exotic trees is the most important source of fibre, but other fibre sources are also used. Biotechnology can play a significant role in the industry to produce high value products at lower cost and could reduce the environmental impact of conventional processes. Biopulping is potentially the most important of these biotechnological processes, because it can influence all downstream operations. The aim of this study was, therefore, to develop a biopulping process for the treatment of softwood at the Sappi Ngodwana kraft mill in Mpumalanga. Initially, 278 strains of wood-decay fungi were collected from various natural habitats. This collection represents a diversity of fungal families and included species that have not previously been recorded from South Africa. The first step in selecting suitable fungal strains for biopulping was to characterize different groups on the basis 146 of the enzymes that they produce and their oxidase reactions. The suitability of these strains for the pre-treatment of softwood chips prior to kraft pulping was subsequently assessed by evaluating their influence on kappa number, yield and strength properties of pulp. Seven of the strains tested were able to reduce the kappa number of pulp significantly, without having a significant influence on the pulp yield. These strains were more efficient than strains of Phanerochaete chrysosporium and Ceriporiopsis subvermispora that have been patented for other biopulping applications. Treatment of wood with strains of Peniophora sp., an unidentified specie as well as two strains of Stereum hirsutum resulted in pulp with improved strength properties. The envisaged biopulping process aimed at treating wood chips in outside chip storage with a biopulping fungus. The aim of one study was to investigate conditions such as temperature, moisture, CO2 and microbial populations that develop in a chip pile, and to determine the suitability of the chip pile for colonization by biopulping fungi. High temperatures and high moisture levels were observed in some areas of the chip pile, which suggested that part of the pile was unsuitable for colonization by mesophilic fungi. It will, therefore, be necessary to manage the chip pile to maintain a suitable environment for biopulping. Problems were experienced with poor colonization of freshly chipped softwood by biopulping fungi. The effect of contaminating microbes and inhibitory compounds present in wood was, therefore, studied. It was found that the inhibition of biopulping fungi by a-pinene and by contaminating microbes were both very important. The inhibition by microbes, as well as by extractives, was mitigated by a 147 short steam treatment of wood chips. Steaming for ten minutes under atmospheric pressure could be an economical method to improve colonization by biopulping fungi. Alternatively biopulping fungi with good competitive ability and tolerance to monoterpenes could be selected. Pinus patula wood chips were pre-treated with a selected strain of Stereum hirsutum to determine the optimal conditions for the kraft pulping of pre-treated softwood and to do an economic evaluation of the process. Chips were pulped on a small scale under various pulping conditions. Lignin content, yield, and viscosity of the pulp as well as alkali consumption were evaluated. The results were used to develop models for biokraft pulping. This study showed that biopulping can reduce the kappa number of pulp or reduce the pulping time. Pulp yield losses were relatively small when pulps with low kappa number were produced. Increased alkali consumption was, however, an important factor in the economic evaluation. 148 OPSOMMING SLEUTELWOORDE: Basidiomisete, Biodiversiteit, Biotegnologie, Bioverpulping, Fungi, Hout, Inhibisie, Kraft, Verpulping, Witvrot,. Die bosbou-industrie is een van die belangrikste bronne van buitelandse valuta vir Suid-Afrika. Die industrie fokus hoofsaaklik op die produksie van pulp met 'n jaarlikse produksiekapasiteit van 2,4 miljoen ton. Die belangrikste roumateriaal vir die industrie is hout uit plantasies van uitheemse bome, maar ander bronne word ook benut. Biotegnologie kan 'n belangrike rol in die industrie speel om hoë-waarde produkte te vervaardig teen 'n laer koste en om die omgewingsimpak van konvensionele prosesse verminder. Bioverpulping is potensieël die belangrikste metode, omdat alle verdere vervaardigingsprosesse daardeur beïnvloed word. Die doel van hierdie studie was dus om 'n bioverpulpingsproses te ontwikkel vir die behandeling van sagtehout by die Sappi Ngodwana kraft-meule in Mpumalanga. Aanvanklik is 278 isolate van witvrot fungi versamel uit verskillende natuurlike habitatte. Hierdie versameling het 'n verskeidenheid fungus families verteenwoordig en het ook spesies ingesluit wat nie voorheen in Suid-Afrika beskryf 149 IS me. Die eerste stap om geskikte isolate vir bioverpulping te kies, was om verskillende groepe te karakteriseer in terme van hulle produksie van ensieme en oksidase-reaksies. Die geskiktheid van hierdie isolate vir die behandeling van houtskerfies voor kraft-verpulping is vervolgens beslis deur hulle invloed op kappagetal, opbrengs en sterkte van pulp te bepaal. Sewe van die isolate wat getoets is, het die kappagetal van pulp betekenisvol verlaag sonder om pulpopbrengs betekenisvol te verlaag. Hierdie isolate was meer doeltreffend as isolate van Phanerachaete chrysasparium en Ceripariapsis sub vermispara, wat vir ander toepassings van bioverpulping gepatenteer is. Behandeling van die hout met 'n isolaat van Peniophora sp., 'n isolaat wat nie geidentifiseer is nie en twee isolate van Stereum hirsutum, het pulp met verbeterde sterkte-eienskappe gelewer. Die voorgestelde bioverpulpingsproses het ten doel gehad om houtskerfies met die bioverpulpingsfungus te behandel waar dit in die buitelug gestoor word. Die doel van een studie was om toestande soos temperatuur, houtvog, C02 en populasies van mikrobe te ondersoek, sodat die geskiktheid van die hope vir kolonisasie deur bioverpulpingsfungi bepaal kon word. Hoë temperature en houtvog wat in sommige areas van die houthope waargeneem is, het laat blyk dat gedeeltes van die hope ongeskik sal wees vir kolonisasie deur mesofiele fungi. Dit sou dus nodig wees om bestuurspraktyke toe te pas om 'n geskikte omgewing vir bioverpulping te handhaaf. Probleme IS ondervind met swak kolonisasie van varsgekapte sagtehoutskerfies deur bioverpulpingsfungi. Die invloed van kontaminerende mikrobes en inhiberende verbindings in hout is daarom ondersoek. Dit is gevind dat 150 inhibisie van bioverpulpingsfungi deur a-pineen en deur kontaminerende mikrobes beide baie belangrik is. Die inhibisie deur mikrobe sowel as vlugtige bestanddele is verlig deur die houtskerfies vir 'n kort periode met stoom te behandel. Stoom vir tien minute by atmosferiese druk kan 'n ekonomiese oplossing wees om kolonisasie van bioverpulpingsfungi te verbeter. Alternatiewelik kan isolate vir bioverpulping met goeie kompeterende vermoë en verdraagsaamheid vir monoterpene geselekteer word. Pinus patula-skerfies is met 'n geselekteerde isolaat van Stereum hirsutum behandel om die optimale toestande vir die kraft-verpulping van fungusbehandelde hout te bepaal en 'n ekonomiese evaluasie van die bioverpulpingsproses te doen. Skerfies is op 'n klein skaalonder verskillende toestande verpulp en die lignien inhoud, opbrengs en viskositeit van die pulp asook alkali-verbruik bepaal. Die data is gebruik om modelle te ontwikkel vir biokraft-verpulping. Die studie het getoon dat die kappagetal of verpulpingstyd deur bioverpulping verminder kan word. Verlies aan pulpopbrengs was relatief laag waar pulp met lae kappagetalle geproduseer is. Die verhoogde alkali-verbruik was egter 'n belangrike faktor by die ekonomiese evaluasie. 151 APPEND liC]ES 152 APPENDIX A: ORIGIN OF FUNGAL STRAINS IN CULTURE COLLECTION. Substrates or hosts, and locality from where different strains of wood-inhabiting fungi were collected. Strain no." Species Substrate / host Area Locality \VR ~.4 IPycnop~rus sanguineus . ".................. .. . .. " . .. .. ':... . WR 17 Lenzites eie ans " " " ·~.·.X.~·.·.·.·.·.·.·.·.·.·.·.·.·.·.·.I.c:~.~i~Fé.ii~q.iy.i.o.~.~·.·.·.·.·.·.·.·.·.·.·.··.··.·.··.·..·.·..·..·.··.··.·.·.·.·.·.·.·.·.·.·.ii.·.·.·.·.•·.•.•.•.•.•.•.•.•.•.•...•.•.•.•.•••.•.•.•.•...•.•.•.•.•.•.•••••.•.•.'•0.'.,.•.................................................................................. :.: ..................•...................... WR 19 Stereum ostrea " " " :~:?)::::::::::::::]p.y~~:~p9.F~~:iq~g~jd.i~~::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::':':::::::::::::::::::::1::::::::::::::::::::':':::::::::::::::::::: .W....R.....2...2...................I.S...t.e..r..e..u...m......h..i..r..s.u...t.u...m.....................................................".............................................."..I.."..................................................... .W.....R....•.2..3.....•••••••••••......L...e..n...z..i.t.•e..s.•••••b••e•••t•u•••l•i•.n.•a••..••..................••.•.••••.•••••••••••••.•••.••••••"...•.....••..•.................•.....••••••••••...".....••••.••..........•.•••.••...•......... " ,~,~,~" ..,""" ,..f.f!.'!. .~. ~'!..L!!f.-?_~l:!.f?f:1:!§,,,,'''''''''''''''''''''',.." ..- ,,,,,,,,.'.:,,,, ,,,,,,,,,,,, ..,, "",,,,,,,,,,,,,,,,,:.',,,,,,,,,,,,,,, __ """" """,,,::,,,,,,, .,.,,,, ,,,.. WR...2...5................ Stereum hirslltum" " . . ·~.·.g·.·.·.·.·.·.·.·.·.·.·.·.·.·.·.·r$i.:.~;.;.~i:.·.·~.;~i;;·.·.·.·.·.·".·""""""".·""".·.·.·.·.·.·.·".·".·.· .. ~.~.~.~ ~.~.~.·~;.;m=.~:.~.·~~·.$.1:.~:..~~.~7·.J~~. .~~ WR 30 Stereum rimosum t.~p.~ ~y"~M9p.u4m::a:l:a:n::g:a::::: Sudwala:~.j:I:::::::::::::]fiq~;~:t~i.:é~g~/~!~:::::::::::::::::::::: Qa.~j~~g:::::::::::::::::::::::::]~i~t9.~~:::::::::::::.::::::::::: ~}~ Stereum ~ir.s.lI.tll.'!I A~.4 Mpumalanga Chrissierneer . .W.....R.....3...3.....................G....a..n...o..d...e..r..m....a.......l.u..c...i.d...u..m...........................A....c..a...c..i.a......t.r..e..e................................................"................................................ ~J~ f(!.'!.~!.t(!.s..b..e.t.u.(!.'!..a. :E:1l.~~w.~.'.Y~<.>.(}.~ " Y!.yc_'!..opo_rYs..s.l1_'!.glf.!I1.~lf.S. P'.in.:~.:".:().o.c:I: ~9.~9,1.~~.9p~ ~~.qg~!}~.~~ . .~.P.o. ?YC_f1.O_PCJ.~l._.sl.sc.:.'!.~u.!.'!.~.l!.s. '.' :: ~.()~~~Y.i~ . ~ ..~.3..l fJ.1..c.Y1.O'Po.r..u.s..s.l1_Y18Y.in..e.lf.S. :' '.' J:3.~~~():n. . .~.1~.2. ?y'C_f1.o.Po.0S..c_yc_'!..C!PO_0.S..S.l1_'!.glf.!I1.~lf.S. .I.':lc:l:ige.l1.o.l!:~.~~: '.' :' . .~ ..1.~.? §~~~~I.I11!..~_i~S.I~.tlf.11! :E:~~~}Y.P.~.~.wood :: " . ·WWRR"'i1'33g7"··"··"··' sCoi~rio;/.u;s~;;;v'ejr1siic;o.t;o~r t~;'"''''''''''''''''''.''.,'..,'.','""""""""';;"'".','.,'''''''''''",,,,,,,,,,,",,,,,,,.,;.,.,,,,",,",".,",",,""",.,."";;.,.,'''.,.,''''.,_. _.., ........................................................................................................................................................................................................................... .~ ..~.3..? !!y.P~O_.c!.O'.'!.!.!l1_.~p· ~~.':l.~.~Y.O'9.q :: ~: . ~ ..~~~ J>Y..c.'!..C!Po_r.!Is..s.l1_Ylg':'.!I1.~~'.S. IndigenousHw, " " . .W......R.. 1•....41 '.'.'.'.'.C'.' oriolus versicotor Pine wood " "' ' '.' - ' ' '.' '.'.' '.' '.'.' '.'.'.'.' ' '.'.' '.'.'.'.'.'.'.'.'.'.'.'.'.' ' ' '.' ' ' ' '.'.'.' '.' ' '.'.' '.'.'.' '..' ' '.' ' ' '.'.' ' '.' ..' '.'.'.'.'.' ..'.'.'.' ..,.,., ...•.•., .~ ..~.~.2. .9.!o_.e.O_P..~y./!u.'!I.S.(!Pi.Clr.i.l_l'.n. '.' :: " . ~}~~ q/q_~o.P~y.!/IJ_J!_I..s.epil1_~!!!.11! :: '.' :' . .~ ..~.~.s. ?y.c_~?PCJ.0.S. ..S.Cl'!.~l!.!.rI~.u.s. A~~.c:i~ ..~.()<.>d. " " . ~ ..,~,~().q...................... " :' . .~ ..~.??. Bjerkandera ..Cl.c!.lf.S.~l1_ '.' :: ~: . W., R 164 Corio/us vers..i.colo, " , , ......r............••••••.•••••••.•......................•......•"•.......................••..••.•.••••.........".......••.•......•••...........•.•.••••••••••"••....••... WR 165 Skeletocutis sp. Pine wood " " S!!EI~§·~·.·~E~.·~.~0·.·/.~iiá_!.iJ![q,~[áq,~iJ!i_~~.·Q.~~i·i.l~i·..·~.·.~~·?~(~·~.·.·.~~·.~·.~~~~·~~·~~~~~~~:i.~·,.~.~·~..~..~:~·~.~~·~·~·~.~~~~~..~...:~.::~.·~.:~~.~: W.....R......1..6...7...............C....o..r..i.o...l.u...s.....v..e...r.s..i..c..o...t.o..r.....................................................".............................................."............................................."... ~Hi I~~~~;:::!i?,~::;I;~~=:~j :::::1 i::: :~)fï.::::::::::~........ .'.' .. . .. .W....R.......1..8...5...............C...o..r.i.o../.u..s..........v..e.r..s.i.c..o..t.o..r....................................O....a..k.....w....o...o..d.................................................."..............................................". ~ ..~.~() fJ.~.~l.li'!.IJ_s..~i!.".u.~ .. " " . ..W........R 188 Corio/us hirsutus A'.' '.'.'.' ..'. . ' ' '.' ' '.,.' '... .'.'.'.' '.' '.'.' '.. '.' ..•...•.c•. acia wood ",.•...•.• ' '.'.'.'.' ' ' '.'.'.'.'.'.'. .'.' ' •.......... "'.'.' '.' . WR 189 Skeletocutis sp. Pine wood " 155 Strain no. Species Substrate / host Area Locality ~.}.?9 9.o_:.i.o.!ll:.5.~~:':5i.~~_I~.r:. Acacia ..~().'?9. \Y~.~~~~.9,lp~ ~.~~il?.()f.l.~.F..: . .~}.?} ?Y..t?rI_~pC!.r_!~:..5~c:.rI_~U:!!!.~.ll:5 9.~.~~:'?()~ :: ': . ~ ..l..?~ P..yc_YI.°P.o_r_.ll~.~q_YlS.'!/'!.~'!.~ :: '.' :' . WWRR'1"199'43 .. "P(h~ell~in'u;sjoi~g;i~l;vu;s;i~;i;r "",," ·"ii····· . ~~ll!~~".?~.f .. .~ .{..~.~ ~ji~j1%t~i1:Yq········································ ;.; :.: ···························r·························· ~: ...............•....................... ·iY!L~§Ï,·.~·.·.·.·.·§.~i·i.·;~i~~i~~!á~!~~!~~~.~~·.~=~~.:~:~::~"::~:::~·~~~~~~~·~.~·~.~:::~·.:·:::.:~:i.~·::.:·:::.::··::~:~~:·:I::::::.:.::::.::.~:.::::::::::::: ~.}.?? P..e_.n.i.op~.o_:.q.~P: I'.if.l~.~v.'?()~ '.' ~~~~':l~.r':ls..~F.:. .~.?9.q [jJ~:.~~.~. i!:.~'!.~1:f.'!! J;:~~~Y.P.~~..~().'?~ :: ': . ~..2.9.J. lfy.ph.()_cl_O_rI_t!c.:..s.P: I'.if.l~.~v.().()~ '.' ~~~~.~.~.r.g§F.: . WR 202 Hyphodontia sp. " " " :~.::~~}:::::.:.:.:.:::::.::%.1~~;X;j~il::.;~~;i.;;:iil.:.:.:.:.:.:::.::.:::::::::.:.:.:::::::::::::::::::::::::::::::.::::::: :' ::.::::::::::. ~.?9.S. S.~.h.iz_o_p~Y..l!ll'(!.I.~o_rn..'!!.ll'!.~ " .. " .. .~.~Q§ " "..§.~~'-e_t..O_EZ.'.t..!~"~P,:, ., " .. , .. . ~~.2.9.7I. H P..e_'!.i.op~.o_:.a...~P: ..~Z;z~~tZ:pïq;.;·..~.·.··;.;·~,.·.=· ~;~••·;.·•.L••·•·~.•.·••"~••••. " .I•~••'~••.~.•~.f~.•.•v...~..•.:•.•:•.•...•.••• il!! g;~~~~-~~~tl:!~-;::-] .-.::-~ .W.~~R 216 Stereum hirsutum Acaci~a ~w~oo~d~~~~~~~~~ " ~~~~~ ~~~~" ~ . ~ ..2..~7. 13j~r.~.a_YI.d..e!.{l.qcJ_ll:5!.a. .. " " . .W.......R........2....1...8...... B..'i1erkandera adusta " " 1 " . WWRii..2ï'250'ï ····uC··foi{rCioïlëunstilvi'eërds·i·csopto: r ···· ·iOiaarkdwwooooCdïtrë·ë··· ·Noïth'ë~'P"ro~incë" Entab·ëIïi's"F:· · · ~l~F:·:·:::·:·:·:l~~W:~jt.{~:.:.·~:·~..·.~~t~j~1i.;t::.·i:~: ·t·~.:..:.!:.·.:.::.::.·;:..;....:..:..:..:..::...:...:...:.:.:.;.:;.'.::.::.:.:,.:.: WR 255 Coriolus versicotor " " " ~..2..5.~ §.t~r..~ll'.1~ ..S.P: :: " :: .. WR257 Schizopora paradoxa Hardwoodtree u .. " il!l J~fL~~;;!~~:f:~::~~'~;~;~I·- r: ~2.,?~. Crepidotus sp. . ., , '.' ",... . ., , .. ~ ..2.?~ ::::: ;;i~t;~:;;;::sp:::.:::::::·::::::::::::::::::::::::: r.:~.C.élly.p.~s..~'?()4 '.' tI~?gIyG.'.!opO''!J.s..S:C!.n.gll.!!'!.e.If.s. " ".................... " . .~.~.~.? f(!.'.!t.!r1.II~..S.t'!p.~':'~ Q~.~~.~~ :: " . WR 386 Stereum hirsutum " " " :~:·n~·:·:·:·:·::u:u:.~f~~t~~ïf7.~~w;~:.t.t:u:.:u:.:.:.:.:.:.:.: ·~·i:~~:<:~·~:~:·.u:.:.:.:.:.:.:.:.:.:.:.:..:'.::.:.:.:.:.~:::.:.:.:.:.:u ;; u.:..:.:.:.:.:.:.: W.....R......3...9..3................P...h..a...e..o.../.u...s.....s..c..h..w....e..i..n..i..t.z..i.i................................................."..............................................."................................................ .~.~.9..s. ?.h.(!_I/_i'.!.I~..~ l?: J:<;~~~.lyp~.~.~9.~~ ': Y!..i~p..~r.:: . .~}.~? , , <:;.9.!!~(!P!!.()f.q"q!.i.y,q~.~_q" ,', .., " ~!.~!.,!!!!,~,~,,"""", ,,, ::,.,,., ,.,. ,,,,"." "..,.".::"""".,",",, WR 398 f>y~n..opo.r..u_s..S:qflgll_i!'!.e.II.s. I_Il~g~~.()~s..I.f.~: '.' :~ . .~.~g.9. !!..e.~qgq_'.!q.~p. J:<;~~~.~yp~..~~9.~~ :: :: . W.....R......4..0...1...............C....o..r..i.o.../.u...s....h...i.r..s..u...t.u..s........................................................."...............................................".............................................."..... \W.V'RR·44"O0·24··········LLeenntzinit~ess·sbï~e'tPu.l·i·n·ëa~s······················· " " "····················ï,····················· ·················jj···················l···········jj· . II ~~t~ff::~~~~~~t~~:-~:;;~]~:-;~:_::-- WR 409 Lentinus stupeus Eucalyptus wood " lSwartfontein SF. a Refers to the reference numbers of strains that are maintained in the culture collection of the CSIR. wood Refers to dead wood as opposed to "tree" that refers to living trees. Hw Hardwood. Sw Softwood. SF. State Forest. 158 APPENDIXB: PHYSIOLOGICAL CHARACTERISTICS OF CULTURES Results of physiological tests on cultures and grouping of different strains of fungi according to the reactions of cultures. Culture reactions Gallic acid Tannic acid Enzymes" medium medium C,) C,) "Cl ::l""- o § ~ ~ " ë~ " f~ " C J. C,) " ~ Q=...= C -£ ~ ~ .~ 0 .C~ C,)C,.cC,) C,)C,.cC,)~.S - ~ ~.S t:.e 'C0ol 0 ". _0 "c0= c..J J0... 0J... ."_0 -c.J ~0..5......."._0 -c.J 0'" 5 Q.. J....!:: ...... ~ c= C,) ..... ~ ~ J... 5 ~ c= J... 5 00 c.!l 00 U 0 ~ ~ Eo-; 0 J... c.!l_ 0 t c.!l_ 1IA-n-t-r-o.d.,i,a--v-a-r-i-i-fo-r-m--i-s--;-----._-:~V. II :W--R--~46 -.-+--._-i_~.: +_.-: + .- .-.°.-..-.-.:;.-.-1-,.5 -- 1.:-..-.----0',-0-- Bjerkandera adusta : la :WR 101 +: + : + : + 2 : 0,0 2: 0,0 i iWR 181 +: + i + : + 2 i 0,0 2 i 9,5 i VII :WR 70 + i + : + i + °1 : 2,0 1: 2,0i iWR 71 +: + i + : + ° : 8,0 ° 0,0. :WR 74 +: + : + : + 2,5 °° 0,0iWR 159 + i + i + i + 1 2,5 0,0 :WR 195 +: + : + : + ° 1,0 ° 0,0 :WR217 + i + : + i ° 2,0 1 3,0 f-=--:---------~: :WR 218 + i. + :. + i. + ---°-i--!..:..'4 °--I-"':__°-i-____':'1--,-5I Chondrostereum purpureum i la iWR 66 + ::;++:.:++ :::++ 2 0,0 2 0,0 f-=-----,------'-: _V_I_I---!i_WR-=--:-78,,-::-+-_+--<"~.: 0,0 1 2,5 Coniophora o/ivacea VI :WR 397 : + i ° 6,0 1 0,0 Coriolopsis po/yzon--a---- la :WR 1 +: + : + : + 2 0,0 2 0,0 iWR 6 +: + i + i + 2 0,0 2 0,0 iWR 18 + i + : + i + 2 0,0 2 1,0 :WR 308 + i + i + i + 2 0,0 2 1,0 iWR 326 : + : + : + 2 . 0,0 2. 0,0 Thri%p~isstrumosa ---'-r--Ia:-1WR 361 + :--+--i +-!- -'--'i--'-'r-O;o -2 ---r 0,0 _ .._--_._------;._---:~VI:I :W-_R._37-3---..+..:;_ ..+..._~:: _+ ..;:._+._-- ._-_._ ...1_--_._-;:..._°--°~_._.0_:..._-'--°'-°-- Corio/us hirsutus : la :WR 2 +: + : + : 2' 0,0 2: 0,0 i !WR 94 +: + ! + ! + 2 0,0 2: 0,0 iWR 105 +: + i + i + 2 0,0 2 0,0 iWR 188 + i + i + i + 2 0,0 2 4,5 !WR 196 + i + ! + ! + 2 0,0 2 11,5 iWR 379 ! + i + i 2 0,0 2 3,5 !WR 401 +: + i + : 2 0,0 2 0,0 iWR 407 + i + i + : 2 0,0 2 0,0 !WR 408 +: + : + : 2: 0,0 2 2,5 :..VII iWR 100 +: + i. + .i + ---1_:--:_0.,_0 '----'-1: 0,0: Corio/us pubeseens : la iWR 62 +: + : + : + 2 i 0,0 2 i 0,0 159 Culture reactions Gallic acid Tannic acid Enzymes" medium medium c.=..ee Corio/us versicotor la iWR 37 + i + i + + 2 0,0 2 0,0 iWR 44 +: + I + + 2 0,0 2 1,0 !WR 61 +: + : + + 2 0,0 2 0,0 'E ï~ +L.'i: +~+·:::+1~+ ~ ~ ~:~ ~ !:~ !WR81 + 2 0,0 2 1,5 .~:; + I:·:: ; ~:~; ~:~ iWR 86 +: + i + + 2 0,0 2 2,0 !WR 137 +: + ! + + 2 0,0 2 7,0 iWR 141 +: + : + + 2 0,0 2 3,0 !WR 164 + I + ! + + 2 0,0 2 4,0 iWR 167 +: + : + + 2 0,0 2 6,5 !WR 169 + I + ! + + 2 0,0 2 9,0 iWR 177 +: + i + + 2 0,0 2 9,5 !WR 179 +! + i + + 2 0,0 2 9,0 iWR 184 +! + I + + 2 0,0 2 5,0 !WR 185 +: + : + + 2 0,0 2 9,5 lEm :++.: :++':++: ~ H ~ r~ !WR 255 + 2 0,0 2 9,0 iWR 283 + i + i + + 2 0,0 2 9,5 . --'- __ ;.1WR....:...:..:..+:3:..:2: :.+:.3_: 1+ + 2 0,0 2 L~ Corio/us zonata la 1WR 73 + i + 1 + i + 2 0,0 2 1 4,5 <.;'.:::.yL'P_to_tr_a_m_a_a--'sp,_r_a_..t-a;:._V_II_ :WR 252 + i + L!__i_ 0,0 1 j~_. Qaec/a/ea quercina i VIII jWR 63 Fomitopsis lilacino-gilva 1VIIITWR65 --+::--·1.i.--~ °° 18,0 °° 13,0 °° L. 23,5 _. i 16,5 . IX )WR 166 8,5 i 6,5 f...,G-a-n-od-'-e-r-m-a-a-pp-/-a-na-t-um---i--la-jWR 150 + i + 1 + i + 2 0,0 2 10,5 iWR 182 +! + ! + : + 2 0,0 2 2,5 __._ . 1: Ic.__:iWR 253 ~:-1_+ _i;_;i-f--,...2-:-'-i-_1-5.;-._22'~ 1 25 Ganoderma curtisii i la iWR 349 + ~i -+-+i + i + 2 : 0,0 2 i 0,0'G;;:;;;(i~'rma /ucid-;;m--'-~-IV\VlfD"--+ :-+--:--"--2--G~5----'O-"r-'-0,0 - i VII iWR 372 + i + i + i + 1 i 0 0 1 .- ---:--, :- .. : --:°-'----_ ..,i .0_°'-G/oeophyllum abietinum XI 1WR 155 i 3,5 0 1 0,0 160 Culture reactions Gallic acid Tannic acid Enzymes" medium medium Gloeophy/lum sepiarium III iWR 151 3,0 1 0,0 IV iWR259 2 0,0 1 0,0 XI jWR67 : 1+ ° 2,5 ° 0,0iWR 133 + ~ 2,5 ° 0,0 jWR 142 + 2,5 ° 0,0 iWR 144 + : °° 2,5 °° 0,0iWR 149 ° 2,0 0,0iWR 176 ° 2,0 °° 0,0jWR204 2,5 0,0 iWR 210 °° 2,5 °° 0,0jWR292 + 2,5 0,0 iWR295 °° 1,5 ° 0,0jWR299 ° 1,0iWR 381 2,0 °1 0,00,0 jWR390 ° iWR406 ° 1,5 ° 0,01,0 ° 0,0 Gloeophy/lum trabeum ~,E~r :++' +:::::::~_ L + ~ H ~ ~:~ ~WR68 iWR 171 °° 4,58,5 °° 0,00,0 He~go;;;;rigida-----;IV!W:WRR331680--+1+! ° 1,5 ° 0,0+:--+ 2 _ 0,0 1 -":-0,-0- Hypholomafascieulare : la ]WR 85 - --+-r+-r-+-'-i-+-1--2-]0,0 -2--T3,O -axl-t-extun;bieolor ;_'x _iWR2W1R739 ": ;_:_~i_:----: :: + °2 ~~ -0-,108,5-02--1~ 00,,0L 0 Lentinus stupeus la ~WR 11 +; + : + ; + 2 0,0 2 ~ 7,5 ~WR24 + i + i + ~ 2 0,0 2 i 3,5 ~WR 385 + .: i : i 2 0,0 2 ~ 2,0 iWR404 + 2 0,0 2 ~ 0,0 jWR 409 + j + j + : 2 0,0 2 ~ 2,0 Lentin-u-s--V-ill-O-SU-s-----'---la-:~ ~~9 -: i: I : i: -Y-' ~:~ ~ ~:~ Lenzites betulina la iWR i()--+1+j--+-_'-: -+-1-~2-;_':0:2.,0:_1-":::-2-;--":4::.!.,5-:.-J jWR 23 + ~ + ~ + i + 2 0,0 2 3,0 IWR 118 + j + I + j + 2 0,0 2 8,5 IWR402 + i + I + j 2 0,0 2 2,5 II IWR 34 + i + I + i + 2 0,0 2 9,5 161 Culture reactions Gallic acid Tannic acid Enzymes" medium medium Lenzites elegans : Ic :WR 5 + .:.+ ~:+: +::~+ 22 0,0 2: 0,0 I-:-c:------~- .-.-.----: 1~__lWR 17 :: i-- ;___:.0!..:.,0-j.__:0_L._0_,_0_ /!._igroponlsvinosus : __!~_j_WR_27_8_~L~)_+_.;_+ .?_.~.0,0 2:_ 0,0 Phaeo/us schweinitzii ~ Ic ~WR393 +: ~+ : + 2 ~ 3,0 2 ~ 1,0 ________________ l----l -----~--~-- --- .-.-~--- --:----- Phellinus gilvus : la ~WR104 +: + + + 2 : 0,0 2 ~ 0,0 : :WR 180 + ~+ + + 2 : 0,0 2: 10,5 ~WR186 ++:: + + 2· 0,0 2 ~ 4,0~WR 211 + + + 2 0,0 2· 2,0 ~WR175 +:+ + + 2 0,0 2 10,5 :WR 193 _+:_ + + + 2 : 0,0 2 7,5----_-_._._---_._._. _ :_,._:-W-R-~26-0--_+.: ._+~+.---+--_2,--i--0,_0 .....2.:.,_.0-,0- Pu/cherricium caeru/eum ~ la ~WR273 +: + ~+ : + 2 ~ 1,5 2 ~ 9,0 Pycnoporuscoccin~us ---·j---Ïa··-·lWR58-~: + :-+[+-i- 0,0 2: 7,0 ~:: ~WR102 +: + ~: + :~+ 2 °'° 2 6,0: 1-- . . ._:; ;:WR 132 .----+.-<-.-~--+'-: :--+-;-.~-+~ .-.--2----~--0.,.0:--I__2-.-' -_3:_,5--l Pycnoporus sanguineus : la :WR 8 + ~+ : + ~ 2 2,0 2 0,0 ~ ~WR12 + i + ~+ i + 2 0,0 2 5,5 ~ :WR 14 + ~+ : + : + 2 0,0 2 2,0 ~ ~WR21 + ~+ ~+ ~ 2 0,0 2 0,0 iWR 89 + i + i + ~+ 2 4,0 2 3,5 :iWR 93 + :~+ :i + : 2 °'° 2 1,5: ::WR 97 +: + :: + : + 2 °: : '° 2 4,0 ~WR103 + ~+ ~+ ~+ 2 1,0 2 2,0 :WR 112 + i + : + i + 2 0,0 2 3,5 ~WR113 + ~+ ~+ i + 2 0,0 2 6,5 :WR 114 + i + : + i + 2 0,0 2 1,0 ~WR123 + ~+ ~+ ~+ 2 0,0 2 2,5 iWR 124 + i + ~+ : 2 1,0 2 1,5 I~!~~: i:·: 2 1,0I : ~ !:~ 2 1,0 ~WR130 + ~+ ~+ ~+ 2 0,0 2 3,5 iWR131 + i+:+i+ 2 0,0 2 5,5 :WR 135 + ~+ i + i + 2 0,0 2 9,0 ::WR 140 + +: : + ~+ 2 00' 2 3,5. : ~WR145 + ~+ ~+ ~ 2 0,0 2 5,0 l~ 2 2,5m : .: i : ~ ~:~ 2 2,02 4,5 iWR 173 + ~+ : + ~+ 2 0,0 2 5,5 ;iWR 191 + .i + i: + i. + 2 00, 2 7,0 162 Culture reactions Gallic acid Tannic acid Enzymes" medium medium c. e.=. t.-' Pycnoporus sanguineus la iWR 192 + + ~ + ~ 2 0,0 2 9,0 iWR 212 +: + Ii + I: + 2 0,0 2 9,5iWR 213 + i + + + 2 0,0 2 4,5 IiWR:214 +: + : + : + 2 0,0 2 9,0I:m : I:,: I : ~ ~:~ ~ ~::H: : I: I: I : ~ ~:~ ~ i:: ': ;;~ :;:' : ' : ~ ~:~ ~ H iWR 338 + i + i + i + 2 0,0 2 2,5 iWR 343 +: + ~ + : + 2 0,0 2 4,0 I: ;:~ : I: 1: 1: ~ ~:~ ~ ~:~ I: ;:~ : I: 1: 1: ~ ~:~ ~ ~:~ lÊm : i ~ i ~ i : ~ g ~ I:~ I:~:+~I: I : I ~ ~:~ ~ !:~ III iWR 365 + i + i + : + 2 0,0 2 1,0 IV iWR 4 + i + i + : + 2 ° ° 1 2 ° :WR 99 + ~ + i + ~ + 2 0:0 1 0:0 Schi~phyli~;;l commune i r5CjWR 47- ----r--1-~:--o--rTs- ----6--·-:---l,O __._._.__._._._..._. ._--_._---.--:,-----i_W.R 205 ---+-:-:-,-+_:.:_>---- -_.0_: ---.,_. -6,5 -_.-.0.:_.._.,.-..-2.',-5-- Schizopora paradoxa : III :WR 257 ::~ 2· 1,0 2: 7,0 Stereum fulvum i VI rW'R 277 --- i + : + i ---1--· 0,0 ° 0,0 i VII iWR 376 +: + i + : 1 0,0 ° 0,0 163 Culture reactions Gallic acid Tannic acid Enzymes" medium medium : o= 1=.....~ 00 Stereum hirsutum la iWR 9 +: + : + : + 2 5,5 2 8,0 iWR 22 + i + i + i + 2 6,5 2 10,5 :WR 25 +: + +: + 2 7,0 2 9,0 iWR 90 i + + i 2 3,0 2 7,5 :WR 136 +: + +: + 2 4,0 2 7,0 iWR 200 +: + + i + 2 0,0 2 10,5 :WR 216 + ~ + + ~ + 2 0,0 2 10,5 iWR313 + i+i+:+ 2 1,0 2 6,5 Th I:~~6 : I: I + I+ ~ ~:~ ~ ::~ II :WR 3 +: + : + : + 2 10,5 2 14,0 iWR 64 +: + :i + : + 2 9,5 1 17,5:WR 87 + i + + i + 2 9,0 1 15,5 :WR 91 +: + : + : + 2 14,5 1 17,5 lE:h : I:':I + ~ :3:~: ; :H iWR 197 +: + ~ + : + 2 8,0 2 14,0 iWR 297 +: + i + : 2 11,5 2 15,0 __.._._.__.._... . ._. . .__. _:_._.. __.l~310_... + i + ~ + : + 2 . 7,5 2. 13,0 Stereum il/udens : la iWR 38 ····--:t·--i-+-·j-..;-T+- ·--·--2·T-W ---2-T···-6,O iWR 324 +. + i + i 2: 1,0 2 i 4,5 Ic iWR 96 :: + : 2: 1,5 2 i 1,5 --~ .-.:---l----"-----~-----:-- Stereum astrea la ~WR 19 ~ + : + : + 2 : 6 ° 2: 10 ° iWR 27 ~ + ~ + ~ + 2 . 0:0 2 ~ 9,0 :WR 261 : + : + : + 2 0,5 2: 0,5 : Th :WR 7 : +: j + 2 1,5 2 j 9,0 -------_ ...._._._---_._---_._.--_._ .._ ...~.-..V_._ .I._.I_._.- .~.._WR-2-8- -__+':--'-_+._-:-:+-._i-_+. --_._--~ 1,0 _.__1:.._ .._ ...•---1",-5- Stereum rimosum : la ~WR30 + ~ + ~ + ~ + 2 : 3,0 2 ~ 8 5 ---------.----- .i. ----.-; . .;.--.~-._----.' .-: ' Stereum sanguinolentum ~ Th ~WR45 + ~ + ~ ~ 2. 0,0 Tram,',s cingu/aia la lE~t:I:,: I: ~ ~:~- ~2 ~ ~3,0 :~ jWR 340 +: + ~ + j 2 0,0 2 0,0 :: ~:!: I: : I : :I+ ~ ~:~ ~ ~:~: ._.. . : jIWR:3~75 ~+:: i~+:1i +~: :--2.f--01,0 f·-2:~-~3-,0Trametesglabrescens . la j:~ 164 Culture reactions Gallic acid Tannic acid Enzymes" medium medium Trametes nivosa v WR337 + + + 1 3,0 oo 3,5WR342 + + + + 1 3,0 3,5 WR367 1 1,5 o 3,0 VI WR311 + + o 4,0 o1 3,0VII WR355 + + + + o 6,5 5,0 WR366 + + + + o 7,5 o 5,0 VIII WR 307 o 10,0 o 11,5 WR309 + o 12,5 o 15,5 WR315 o 9,5 o 13,0 WR316 o 11,5 o 16,5 WR329 o 8,0 o 13,5 IX WR303 + o 6,5 o 6,5 XI WR327 o 8,5 o 9,0 WR344 + + o 4,5 o 4,0 WR352 + o 4,5 o 3,0 WR356 o 2,0 o 2,5 WR364 i I I o 1,5 o 4,5 a A total of 42 strains were not Identified to species level and were, therefore, excluded from this table. b Qualitative tests + = present. c 0 = negative reaction; 1 = weak positive reaction; 2 = strong positive reaction. d Growth rate was determined by measuring the distance from the inoculum plug to the colony margin in four directions after incubation for one week. 165 APPENDIX C: RESULTS OF THE FIRST SCREENING STEP TO SELECT STRAINS FOR KRAFT BIOPULPING. Change in kappa number (%) caused by treatment with different strains of fungi compared to control treatments after three or eight weeks. Treatment time Strain no. Species Three weeks Eight weeks .~·.·l'·'·'·'·'·'·'·'·'·'·'·'·'·'I·g~i.i~!f:;fi.;;;;{~i.~~~ .1[ ·.·.~l.~.:·}.·.·.·.·.·.·.·.·.·.·.·.·..· WR 3 Stereum hirsutum -26,1 if],f~J!~f~~=~~i_:l{L~_ WR 7 Stereum ostrea -6,8 ~····*··················:I?y.~·~~p·éF.~:~.·[.~q~~_i~~:~~· : :.?.~. .. WR 9 Stereum hirsutum -29,1 :~.:·:.:·:•·. :·.:·.:·:·.:·.:·.ïO.;.: ..:..:.: ..:.: ..:.:·:·:·.It.}~.1:~.t~~.:·.~.:·.~.:·.;.··:·:~.!7.:.::.;.:.:..:·....:.::...:..:.:.::.:.. :.:':'.:':'.:':'.:'.:'.:.:.:1-.:'.:'.:':':':'.:'.:'.:':'.:'.--.-.:':'.:'.:'.:':':'.:'.:'.:':'.:'.:.:.:.:.:.: ..:·:·:·.I.:·:·.:·:·.:·.:·.:·.:·:·.:·:·:·:·: ... ~}~::·i.:·.:':'.:'.:'.:'.:'.:'.:'.:':':'.:':'.:'.:'.:'.' WR 12 IPycnoporus sanguineus 1 -0,9 WR 22 IStereum hirsutum 1 -17,0 ~iE=[ff:~~1~~:~-t:~:~-22,0==]~-= WR 25 Stereum hirsutum -16,4 .~·"?f"""".·"""". $.(ii.~.~i9ii.~.~.~q ·· · 1 ~.?.;j_ .. WR 28 Stereum ostrea -4,6 WR 38 Stereum il/udens -8,4 ········ ······· ··.. ····.. ·············1··········..· . WR 39 Trametes cingulata 1 -2,2 :;~ ]~~:1~:,;~;;~:I1~; :~I WR 45 IStereum sanguinolentum I -0,0 \ 166 Treatment time Strain no. Species Three weeks Eight weeks WR 60 Unidentified ..S.P: I1.A- . ~ ..?,! "",,..9.g.~t9.!.1!X.~.~!.~!~9.!.q_"'":",'.", ,'"''',,".,,,.,',,.,,.,~,!,~,1,!.,,,,.",."' ", .,.' "" .".",."" ,"',,,,,,,,,,,.,,.,,,,,,,, .~.?~ C;C!r._iC?!u..~p.~~.~.e.~c..e.~~ :.~.!.~ . ~ ..~~ IP.q~.cjq!.e.q..q~~.e.r.c._if!q [ :.1.?? . ·~·?1.···········ISJe.r.e..~~.f.~!!i.~~.u..t.u..'!!. ;~\.?.jg~~?.~ ..~ ~.~ S6.;/;~~i~i:~g·;:~~:..~.~.·.·.·J·.·.·.·.·.·.·.·.·.·.·.·· . .:~.~~--~~~Jg%.;~~~-.:~:~.;·...L}~;::=~.=;:;=:;~=~=:~.=~~;..~.~:~.= .~ .7.9 l}je.r.k.Cf!.lcje.':q_..qcjlf§~Cf... . . ~}.?'.? ~ ..7..1 §j~.~~Cl_f!cI:.~r..q.q_cl:.u..~t.q ~.I.~.?_g . WR 73 Corio/us zonatus -12,2 ·~.·.j4.·.·.· ·.·[ijj~?fp~4q~4iq~~·~q· [ +I.;..~ . WR 75 Corio/us versicotor -4,2 ·~···j~.·.·····1·c;o.do.ï.~~i.·.y.~át.c..o.ï.o..~··.·.· ··.···· ·.·1· ··.·· · ·· ·..·· ..···· · ~.~.; ..8. . WWRR·.7778······C··o·r·i'·o·/·u··slchve~r;s;dic;ootsotr~;~~~;·p;;p;;;;;;;";·,··,·.,, ",.."."".·"··············"n·=.i·d1:.·o······..·,·,,,··, .W~R .~8. 4.s. Phel/inus sp. .." ::!,~?~ ,.."lj)-'P~.C?!C!'!!q!q~cJC.u..!Cf'..e. P.A: . W~R 86 i[!Corio/lIs versicotor -17,1l~~~~~~;neur s~~_~:i~~Im~_:;.;:: ~~]~~~t/%~~=~3=;:c-:J_~~::~ ~iij~F!!~;JEkrJ~~:~;i; ~~ :~~:~ WR 96 lS'tereum il/udens T -11,8 167 Treatment time Strain no. Species Three weeks Eieht weeks ~:~:~:'I~~J~~J~f.{:~::=~!..~~:::z.:.:.::.:·:~:.::::::::,:,:::::.::.,:::::::':::.:~::~:::]:::.:' :":":" WwiR'i051"0"4"'I'CPoh;jeolïl~in;u~s"hgj;ilsv~u~s t;;s""'"'''''''''''''''''.''.,'',','' ,,.,,,, ,,,,,,,,,, ,,,,,,,"-:211'8,:0'0""""""'" ~Itr ]I~i~~t~:~;~~~:~:~!!::f~l~ WR 118 Lenzites betulina -17,0 Biif~!'illl~~j!~l~ê~l~~::~-~::i1: i~ ~J~~ lr.y~n..()Pf!r__ll.s..sanguineus I .l ~~9.?9. . .w.:R.:: ..1..3..9 f.YC!'.19P.C!..r.l./~.~qn..~i.n..~.1!s. ::~.s..~9. . ~WRm131 IPycnoporus sanguineus -30,01~~;%~fjk~f1-k~?:~~£~;;:~~ -~~~~,~:- .~·.·.g·.~.···.···.·.I~:;;fg.6;;s0.~~·~~·~.~J '.~.·.·.·~..·;..·:.} . ~ H~·11~~~f;;~%~:%~~C!.·Ir. ~~:.~ . ~ill]~f!{3j~~~;~~-:~~~~-[3:~~}-~:~:-~~:~~~ ~!i!jij;~~~4;%~i-~j:;~f,~78] ~ ~ }~? ?YE'!9P9.t.Z.'.S..s.Cl.rl_8!!t'!.~.z.t.§, ,.,'" " "~.!,s..!::!",, ,,""" ..' '.'.---- " . .~~ 1~.? [9!f!~9PhY..!t.ll_r'.1..sepianum [ :.}9.?~ . ~}~.9 Ganoderma ..ClPp!a..'!Cl_t.lIr.rz [ I1.:~: . .w.:R.::.}..s..~ 9Jf!~9P~Y..!t.~/.'!!..sepiarium ::~.~.?~ .. \\'R 152 ISkeletoc.utis sp.. .. 1.......... ~6,5................. .. .. ~.1..s.} PyC!nopo!z./~.~an_~i.n..~.1!s. ~9~S. . ·WWRR·1i5554·····IlSGkjeol~etoopchuytiïsï~;s;pn.··abi~ïi;;~;········I······r·· n.d.-·40,2 . 168 Treatment time Strain no. Species Three weeks Ei~ht weeks WR 156 Stereum hirsutum -18,5 ".".,""'''".'"-' , , ,.., ,..""""""", ..,,,,,,,,,,,,,,,"w.,,,,,, "" ..,." "", ,,"", ..,,"""""", ..,,, ..,,""w., ..,,"""""""·.w."w.w.",·.·.",,, WR 157 Skeletocutis sp. -7,9 ~.·).~§.: ·r§j~.~k.iij4.~iq.·q4.~i~Tq·..·.·.· · ~?;.4..·..·.·..·.· ·..·.··: : :.. :.:. : : . WR 164 Coriolus versicolor -10,4 :~·:}~:~:·:·:·:·:·:·:I~~·;~;~:;s.tt~~;~~;.i:i.;:~.:~.:·:.:·:.:·:.:·:.:·:.:.:.:.:.:.:..:.:..:.:..:.:..:.:..:.:..~:.::~.::.:{.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:. ~j:~~~:,~:~if~,~:~::·~~~~~~~::::~:~~~,,~·~~,~~~~~~~~,~~~~~~~~,;~~~~~;,~~,~,~~I~~,~~,.~,~·~,~,~~~::,~~.,~~,~.~:,::~~~~,,~~~,~~~~~~ .~ ..~.?.?. 9.9.r._i.? !~q'!!.f:r.~.~ ..1]/.'!C!s..q ::~.~~ . .~·.}~.~.·.·.·.·.·.·.·.l~~Jï.~~~7~;.:~~~~.~·~.~~.~·~·.·.·.~·.·.·.·.~·.·.·.·.·..·.··.·.·..·~:·.4·:.·.·.·.·.·.·.· . WR372. IGanodernalucidum 1....... .. . nA . . . ·~·j·~.·.i···I~f~~:~~;7.·.d·. ~··~··;·;··;-!:·~:·5;8· J . WR 3~? ?..~~'-!i.'-?1.~.!:~P: " " ,," " " " ":.!. }.1...2"..""" ,, " " "" "" .. WR 397 . Coniophora olivacea . n.d. WR 407 Corio/us hirsutus -13,0 :~·:·;~:~:·:·:·:·:·:·]z.:;i~i::.;·:;:t;;.·~t:·:·:·:·:·:·:·:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.[.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:~:~::.~:.:.:.:.:.:.:.:.:.:':':':':':':':'[:':':':':':':':':':':':':':':':':':':.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.' .A...T..C....C...3...2..6...2..9...:·1Phanerochaete chrysosporium -48? -r . CZ-3 TCeriporiopsis subvermispora -12,3 I n.d. Not determined, because of insufficient growth. 172 APPENDIXD: TEMPERATURES OBSERVED IN A COMMERCIAL CHIP PILE Temperature (OC) at different positions, times and at different ages of the experimental chip pile. Age Position Time of pile 08:00 10:00 12:00 14:00 16:00 18:00 20:00 22:00 00:00 02:00 04:00 06:00 08:00 1 21,1 21,2 21,4 21,4 21,4 21,4 21,6 21,7 21,9 22,1 2 19,8 20,0 20,2 20,6 20,7 20,8 21,0 21,2 21,6 21,9 22,1 3 19,4 19,6 19,8 20,1 20,3 20,2 20,4 20,5 20,7 21,3 21,4 4 19,5 19,5 19,5 19,5 20,5 21,3 21,9 22,7 23,5 24,5 25,4 26,3 1 Day 5 20,2 20,4 20,7 20,7 20,7 20,9 21,0 21,2 21,5 21,7 6 21,3 21,4 21,5 21,6 21,6 21,6 21,6 21,6 21,6 21,6 21,6 7 19,6 19,8 20,0 20,5 20,5 20,5 20,7 20,9 21,2 21,6 21,9 8 20,3 20,6 20,6 20,7 20,9 21,1 21,3 21,6 21,8 9 20,0 20,0 19,9 19,9 19,9 19,9 20,0 19,6 10 19,7 19,0 16,7 15,9 15,4 15,0 14,9 15,5 1 51,7 51,6 52,0 51,9 52,1 52,0 51,6 51,6 51,7 51,9 51,9 52,0 2 52,1 52,6 52,5 52,4 52,5 52,5 51,9 51,9 51,8 51,9 51,8 51,8 3 51,4 51,6 51,6 51,6 51,6 51,6 51,5 51,6 51,6 51,6 51,6 51,6 4 42,7 43,5 43,6 43,9 44,4 44,7 44,6 45,1 45,4 45,8 46,2 46,7 1Week 5 41,2 40,6 40,7 40,4 40,4 40,4 39,8 39,6 39,6 39,5' 39,2 39,1 6 42,0 42,0 42,0 41,6 41,5 41,2 40,7 40,4 40,0 39,9 39,5 39,2 7 32,7 32,9 32,7 32,6 32,5 32,5 32,3 32,4 32,5 32,8 32,9 33,1 8 20,3 20,6 20,9 20,7 20,7 20,9 20,7 20,9 21,0 21,2 21,3 21,4 9 14,5 14,9 16,1 14,9 13,4 13,4 13,2 13,3 13,2 12,9 12,3 11,3 10 12,4 16,1 15,6 15,7 15,8 16,5 13,7 10,9 8,3 6,7 6,2 4,8 1 55,2 54,8 57,1 56,0 56,4 56,3 56,0 55,7 55,6 55,5 55,4 55,2 2 54,0 52,9 55,9 54,0 53,8 53,9 53,8 53,7 53,8 53,7 53,8 53,8 3 55,3 54,1 56,1 54,9 54,7 54,9 54,8 54,7 54,9 54,9 55,0 55,2 4 55,1 54,0 55,6 54,8 54,8 54,9 54,8 54,7 55,0 55,1 55,2 55,3 2 Weeks 5 35,6 36,3 38,9 37,3 37,1 37,1 36,7 36,3 36,3 36,1 35,8 35,6 6 35,2 35,4 38,3 36,5 36,3 36,1 35,9 35,7 35,7 35,5 35,4 35,4 7 30,2 29,1 31,8 30,6 30,7 30,9 31,0 31,0 31,1 30,8 30,7 30,5 8 19,9 21,7 23,1 22,2 zz.: 21,9 21,6 21,3 21,2 21,0 20,5 20,4 9 11,3 10,3 11,5 10,8 10,2 10,4 10,8 11,2 11,5 11,6 11,7 11,4 10 10,4 23,3 29,2 24,3 22,4 17,9 14,6 11,3 9,6 8,0 7,3 6,8 1 48,9 49,7 50,4 51,0 52,4 53,1 52,5 52,1 51,6 51,3 51,0 50,8 2 51,3 52,4 51,6 50,7 50,1 50,0 49,9 49,8 49,7 49,5 49,2 49,3 3 54,6 55,7 54,9 54,0 53,5 53,3 53,3 53,3 53,3 53,2 53,2 53,2 4 52,8 53,2 53,3 52,8 52,7 52,5 52,4 52,2 52,0 51,9 51,9 51,9 3 Weeks 5 31,3 32,3 32,3 31,6 31,3 31,3 30,9 30,6 30,2 29,9 29,5 29,4 6 31,2 32,4 32,2 31,3 31,1 31,0 30,8 30,6 30,3 30,1 29,8 29,8 7 27,4 28,8 28,4 27,9 27,9 27,9 27,8 27,6 27,3 27,1 26,8 26,7 8 15,8 16,0 16,4 15,7 15,5 15,4 15,2 15,0 14,7 14,5 14,3 14,2 9 4,3 4,8 6,2 6,1 6,2 6,1 5,5 5,0 4,1 3,3 2,5 2,0 10 6,3 8,8 18,5 18,7 17,5 10,7 6,5 3,1 0,6 -0,2 -1,1 -1,3 173 APPENDIXE: LEVELS OF CO2 OBSERVED IN A COMMERCIAL CHIP PILE Concentration of CO2 (%) at different positions, times and at different ages of the experimental chip pile. Age Position Time of pile 08:00 10:00 12:00 14:00 16:00 18:00 w:oon:oo 00:00 m:oo M:OO M:OO 08:00 1 0,4 0,4 0,6 0,1 0,8 0,8 0,8 0,3 0,6 1,0 2 0,0 0,0 0,2 0,7 0,9 1,5 2,3 3,0 3,5 3,7 3,7 3 0,1 0,1 0,6 1,3 1,8 2,0 2,6 3,2 3,9 4,2 5,1 4 0,1 1,1 1,1 2,0 3,0 4,2 4,6 5,9 6,7 8,4 8,6 9,7 1 Day 5 1,1 1,1 0,9 1,5 2,0 0,9 1,2 1,4 1,6 1,7 6 1,6 1,7 1,7 1,6 2,0 2,7 1,7 1,5 2,0 2,1 2,7 7 1,3 1,7 2,1 2,6 3,1 3,6 3,9 3,4 4,6 4,2 6,5 8 0,8 0,5 1,3 0,7 0,4 0,3 0,4 0,5 0,6 9 0,1 0,0 0,2 0,1 0,1 0,2 0,2 0,2 10 0,0 0,0 0,1 0,1 0,1 0,1 0,1 0,1 1 1,3 1,5 1,8 1,8 1,6 1,3 1,2 1,4 1,5 1,3 1,3 1,3 2 2,3 2,0 2,4 2,5 2,2 2,1 1,9 1,9 1,9 1,8 1,7 1,7 3 6,0 5,4 6,2 6,0 5,4 6,2 3,8 4,2 3,6 3,4 3,2 3,0 4 7,3 8,5 112,7110,8 9,4 8,9 7,1 5,5 4,7 4,1 3,6 3,7 1 Week 5 0,7 0,8 0,8 0,8 0,8 0,8 0,7 0,9 0,8 0,8 0,8 0,8 6 0,9 1,0 1,2 1,2 1,2 1,1 1,1 1,0 0,9 0,8 0,8 0,8 7 0,7 0,8 0,9 1,0 0,9 0,9 0,8 0,8 0,7 0,7 0,7 0,6 8 0,3 0,3 0,3 0,4 0,3 0,4 0,4 0,4 0,4 0,3 0,3 0,3 9 0,0 0,2 0,2 0,2 0,2 0,5 0,3 0,3 0,2 0,2 0,2 0,2 10 0,0 0,1 0,1 0,2 0,1 0,2 0,2 0,2 0,2 0,1 0,2 0,2 1 1,2 1,2 1,4 1,4 1,4 1,6 1,4 1,1 0,9 1,0 1,2 1,4 2 1,8 1,5 2,1 1,8 2,2 2,0 1,9 1,8 1,6 1,7 1,6 1,6 3 2,4 2,1 2,8 2,7 2,7 2,8 2,6 2,4 2,3 2,3 2,3 2,4 4 3,1 2,8 2,9 2,8 3,6 3,2 3,1 3,0 2,9 2,9 2,9 3,0 2 Weeks 5 0,6 0,8 0,8 0,8 0,8 0,8 0,8 0,7 0,6 0,6 0,6 0,6 6 0,6 0,7 0,8 0,8 0,8 0,8 0,7 0,6 0,6 0,6 0,6 0,5 7 0,4 0,5 0,5 0,5 0,5 0,5 0,5 0,4 0,4 0,4 0,4 0,4 8 0,2 0,2 0,2 0,2 0,2 0,2 0,2 0,2 0,2 0,2 0,2 0,2 9 0,1 0,1 0,1 0,1 0,1 0,1 0,1 0,1 0,1 0,1 0,1 0,1 10 0,1 0,1 0,1 0,1 0,1 0,1 0,1 0,1 0,1 0,1 0,1 0,1 1 1,1 0,7 1,0 0,9 0,9 0,9 0,8 0,7 0,6 0,5 0,5 0,9 2 1,2 1,5 1,0 1,0 1,0 1,2 1,1 1,0 1,0 1,0 1,0 1,2 3 1,9 1,9 1,1 1,2 1,1 1,6 1,5 1,5 1,5 1,6 1,6 1,6 4 1,9 1,4 2,9 2,9 2,3 2,1 1,7 1,7 1,7 1,6 1,5 1,5 3 Weeks 5 0,5 0,4 0,5 0,5 0,5 0,3 0,3 0,2 0,2 0,2 0,1 0,1 6 0,5 0,5 0,6 0,6 0,6 0,4 0,3 0,2 0,2 0,2 0,1 0,2 7 0,4 0,3 0,4 0,4 0,3 0,2 0,1 0,1 0,1 0,0 0,0 0,0 8 0,3 0,1 0,2 0,2 0,2 0,0 0,0 0,0 0,0 0,0 0,0 0,0 9 0,2 0,1 0,2 0,1 0,1 0,0 0,0 0,0 0,0 0,0 0,0 0,0 10 0,2 0,1 0,1 0,1 0,1 0,0 0,0 0,0 0,0 0,0 0,0 0,0 174 APPENDIXF INFLUENCE OF CO2 ON THE BIOPULPING EFFICIENCY OF SELECTED STRAINS OF WHITE- ROTFUNGI. Methods Inoculum and wood chips were prepared as described previously (Chapter 3). The wood chips were inoculated with Corio/us hirsutus (WR 83), Stereum hirsutum (WR 95), Pycnoporus sanguineus (WR 124) and sterile growth medium as a control. Treatments were incubated for three weeks at 28°C under atmospheric CO2 concentration for comparison with treatments incubated at 10 %, 15 % and 21,5 % CO2. Only one CO2 incubator was available, therefore, three factorial experiments with completely randomized designs were. conducted where each experiment consisted of four fungal treatments and two CO2 treatments. Treated chips were harvested, dried and pulped according to the methods described for screening (Chapter 3). Kappa numbers of treated chip samples were determined and the data subjected to one-way analysis of variance. Means for the fungal treatments were compared with Tukey's test. Results All three fungal strains were able to reduce kappa number significantly at 10 % CO2 when compared to the control inoculation. Furthermore, the mean kappa number for all three strains and the control did not differ significantly between the incubation at atmospheric C02 and incubation at 10 % C02 (Table 1). All three fungal strains were also able to reduce kappa number significantly at 15 % CO2 when compared to the control treatment. No significant difference was found between the mean kappa number of any treatments incubated at atmospheric CO2 and the treatments incubated 175 at 15 % CO2 (Table 2). At 21,5 % CO2, all three fungal strains were again able to reduce the kappa number significantly. However, the treatments incubated at 21,5 % C02, resulted in a significantly higher kappa number when compared to the treatments incubated at atmospheric CO2 (Table 3). Table 1. Effect of different fungal strains on kappa number in an environment with 10 % CO2. Treatment Control 10 % CO2 Control 26,8a 28,Oa Coriolus hirsutus WR83 23,9c 23,6c Stereum hirsutum WR95 25,Ob 25,8b Pycnoporus sanguineus WR124 22,3c 22,3c Treatment means 24,5z 24,9z a, b, c Means of three replications. Means ill the same column followed by the same letter do not differ significantly (p s 0,05; Tukey's test) z Means of four treatments. Means in the same row followed by the same letter do not differ significantly (p s 0,05; Tukey's test) Table 2. Effect of different fungal strains on kappa number in an environment with 15 % CO2. Treatment Control 15 % CO2 Control 30,7a 30,6a Coriolus hirsutus WR83 28,4b 28,3b Stereum hirsutum WR95 27,5bc 27,4bc Pycnoporus sanguineus WR124 27,lc 25,5c Treatment means 28,4z 28,Oz a, b, c Means of three replications. Means ill the same column followed by the same letter do not differ significantly (p s 0,05; Tukey's test) z Means of four treatments. Means in the same row followed by the same letter do not differ significantly (p s 0,05; Tukey's test) 176 Table 3. Effect of different fungal strains on kappa number in an environment with 21,5 % CO2. Treatment Control 21,5 % CO2 Control 31,5a 32,3a Coriolus hirsutus WR83 29,4b 31,3b Stereum hirsutum WR9S 27,6c 28,7c lYcnoporussan~uineus WR124 25,8d 26,9d Treatment means 28,6y 29,8z a, b, c, d Means of three replications. Means ill the same column followed by the same letter do not differ significantly (p s 0,05; Tukey's test) y, z Means of four treatments. Means in the same row followed by the same letter do not differ significantly (p s 0,05; Tukey's test) Conclusion All tree fungal strains were able to degrade lignin, as reflected by kappa number, at concentrations of CO2 as high as 15 %. 177 APPENDIXG: INFLUENCE OF a-PINENE ON THE GROWTH OF FUNGAL STRAINS Growth of different fungal strains in environments saturated with a-pinene in comparison with growth under atmospheric conditions. .c8_.I c -c<:.o- .".:c!o ~ c<: § ~.c I-'1E"O Colony diameter (mm) Relative Group ~c<:§ Strain growth (%)ao-pinene Control means Botryosphaeria dothidea CHS52 41,9 50,0 0,84 _"CI,I,'0""0 '{:eratocystis vireseens CMW3276 18,4 40,8 0,45 CI,I ~ c....I.".O '(:ylindrocladium candelabrum CMW3911 30,6 42,3 0,728 ,...c<: O,.Q Ipphiostoma piliferum CMW2524 17,3 22,1 0,78