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Characterisation of selective biodelignification of trembling aspen refiner mechanical pulp by Rigidoporus… Dawson-Andoh, Benjamin Ebo 1989

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C H A R A C T E R I S A T I O N OF S E L E C T I V E BIODELIGNIFICATION OF T R E M B L I N G A S P E N R E F I N E R M E C H A N I C A L P U L P B Y RIGIDOPORUS ULMARIUS By BENJAMIN EBO DAWSON-ANDOH B. Sc(Ed), B. Sc. University of Cape Coast M. Sc. University of Science and Technology A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S D E P A R T M E N T O F F O R E S T R Y We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A 1989 © BENJAMIN EBO DAWSON-ANDOH, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Forestry The University of British Columbia Vancouver, Canada Date: I X - O G> -A B S T R A C T Rigidoporus ulmarius (FT.) Imaz. has been reported briefly in the literature to selectively biodelignify wood both naturally in the field, and also under laboratory conditions. Selec-tive biodelignification of lignocellulosic materials by fungi is influenced to a great extent by the environmental or cultural factors employed. The effect ofthe following cultural factors: aeration; exogenous addition of hydrogen peroxide; concentration of mineral solution; addition of a surfactant (tween 80); and exogenous addition of veratryl alcohol on selective biodelignification of aspen (Populus tremuloides Michx.) refiner mechanical pulp (RMP) by R. ulmarius was studied. The objective was to optimise the depletion of lignin by R. ulmarius while maintaining the carbohydrate level, especially cellulose, at its original level. The quantitative success of this objective was defined as the selective biodelignification index (SBI), defined as the ratio of total carbohydrates to the total residual lignin. Fungal-treated pulps with the highest SBI and the greatest lignin depletion were compared with controls by physical means on handsheets. This study represents the first systematic attempt to determine the effect of combina-tions of several cultural factors on selective biodelignification of a lignocellulosic substrate by R. ulmarius. Also, a novel aspect was the exogenous addition of veratryl alcohol and hydrogen peroxide to the culture at the estimated beginning of the secondary metabolic phase of the fungus, because lignin biodegradation is hypothesized to commence during this phase, which is represented by the cessation of fungus growth. Optimal conditions for selective biodelignification of aspen RMP included addition of mineral solution (Kirk-Schultz mineral solution, eight-fold concentration) and oxygen flushing in the absence of n other chemical additives or nitrogen supplements. Pulps given this treatment were char-acterised by residual contents of 14.8% lignin, 51.5% glucose and 15.1% xylose. This was equivalent to a lignin reduction of 30.8%, and a xylan depletion of 23.7%, without proportional reduction in cellulose content. Consequently, the SBI was 4.5, versus 3.2 for the untreated controls. Other cultural conditions caused even greater lignin loss(up to 50.4 %), but these treatments were accompanied by severe cellulose and hemicellulose reduction, leading to pulps with impaired strength properties. In general, mineral solution (eight-fold concentration) enhanced selective biodeligni-fication and lignin biodegradation under oxygen flushing. Similarly, exogenous addition of veratryl alcohol to the culture also enhanced selective biodelignification. However, exogenous addition of hydrogen peroxide to treatments containing other factors had a common effect of limiting selective biodelignification. Under high oxygen flushing, both lignin and carbohydrate biodegradation was enhanced to the same extent by exogenous addition of tween 80 (0.05%), with the result that SBI was not much affected. The op-timum pH and incubating temperature for selective biodelignification were 4.5 and 28 0 C, respectively. Biodegradation of pulp was also attended by acidification of medium. Under optimal selective biodelignification conditions, significant changes in some opti-cal properties of pulps were noted. Brightness increased by 7.4%,while opacity decreased by 0.3%. Modest but significant improvement in some strength properties of handsheets made from this pulp was observed. Dry zero-span breaking length increased by 4.4%, while burst and tensile indices also increased by 16.8% and 28.9% respectively. The weighted average fiber length of pulps was reduced from 1.17 mm for the controls to 1.12 mm for pulps given the optimal treatment. This represented 4.2% reduction in weighted average fiber length. Fines content of such pulps were reduced by a negligible 3.2%. iu Table of Contents A B S T R A C T ii List of Tables viii List of Figures xvii A C K N O W L E D G E M E N T x x 1 I N T R O D U C T I O N 1 1.1 NOVELTY 11 1.2 HYPOTHESES . . 12 2 L I T E R A T U R E R E V I E W 13 2.1 SELECTIVE BIODELIGNIFICATION OF WOOD BY FUNGI 13 2.2 FACTORS INFLUENCING SELECTIVE BIODELIGNIFICATION . . . 16 2.2.1 STRUCTURE AND CHEMISTRY OF WOOD 17 2.2.1.1 WOOD SPECIES 17 2.2.1.2 WOOD CELL ELEMENTS 18 2.2.1.3 CELL WALL CHEMICAL COMPONENTS . . . . . . . 19 2.2.2 CULTURAL FACTORS 24 2.2.2.1 AERATION 26 2.2.2.2 HYDROGEN PEROXIDE 27 2.2.2.3 MINERAL (TRACE) SOLUTION 29 2.2.2.4 TWEEN 80 30 iv 2.2.2.5 VERATRYL ALCOHOL 32 2.2.2.6 pH 34 2.2.2.7 NITROGEN 34 2.2.2.8 AGITATION 36 2.3 PHYSICO-MECHANICAL PROPERTIES OF PULP AND HANDSHEETS MADE FROM WOOD CHIPS AND MECHANICAL PULP PRE-TREATED WITH FUNGI 39 3 M A T E R I A L S A N D M E T H O D S 51 3.1 MATERIALS 51 3.1.1 WOOD SPECIES . 51 3.1.2 PREPARATION OF ASPEN REFINER MECHANICAL PULP . 51 3.1.3 FUNGUS 52 3.1.4 BASAL MEDIUM 53 3.2 METHODS 54 3.2.1 MYCOLOGICAL AND PHYSIOLOGICAL METHODS 58 3.2.1.1 PREPARATION OF INOCULUM 58 3.2.1.2 DETERMINATION OF THE TIME REQUIRED FOR GROWTH TO BECOME LIMITING 59 3.2.1.3 SELECTIVE BIODELIGNIFICATION OF ASPEN RMP : OPTIMIZING THE EFFECT OF AERATION, HY-DROGEN PEROXIDE, MINERAL SOLUTION CON-CENTRATION, TWEEN 80 AND VERATRYL ALCO-HOL 60 v 3.2.1.4 SELECTIVE BIODELIGNIFICATION OF ASPEN RMP UNDER OPTIMAL CULTURAL CONDITIONS : EF-FECT OF P H , NITROGEN CONCENTRATION AND TEMPERATURE 63 3.2.2 CHEMICAL ANALYSIS OF PULP . . . 64 3.2.2.1 DETERMINATION OF LIGNIN CONTENT 64 3.2.2.2 ANALYSIS OF CARBOHYDRATES 65 3.2.2.3 DETERMINATION OF TOTAL NITROGEN CONTENT OF UNTREATED ASPEN REFINER MECHANICAL PULP 78 3.2.3 EVALUATION OF SOME PHYSICAL PROPERTIES OF CON-TROL AND FUNGAL TREATED ASPEN REFINER MECHAN-ICAL PULP 79 3.2.3.1 PHYSICO-MECHANICAL TESTING OF HANDSHEETS 80 4 RESULTS A N D DISCUSSION 82 4.1 DETERMINATION OF THE TIME REQUIRED FOR GROWTH TO BECOME LIMITING 82 4.2 CULTURAL FACTORS INFLUENCING SELECTIVE BIODELIGNIFI-CATION OF TREMBLING ASPEN REFINER MECHANICAL PULP . 84 4.2.1 AERATION 90 4.2.2 HYDROGEN PEROXIDE 93 4.2.3 MINERAL SOLUTION CONCENTRATION 97 4.2.4 TWEEN 80 100 4.2.5 VERATRYL ALCOHOL 102 4.2.6 GENERAL SUMMARY FROM FACTORIAL ANALYSIS . . . . 106 vi 4.3 S E L E C T I V E B I O D E L I G N I F I C A T I O N : E F F E C T O F P H , N I T R O G E N A N D T E M P E R A T U R E L E V E L S O N T R E M B L I N G A S P E N R M P UN-D E R O P T I M U M C U L T U R A L CONDITIONS 107 4.3.1 pH 107 4.3.2 N I T R O G E N I l l 4.3.3 T E M P E R A T U R E ... 113 4.4 E V A L U A T I O N O F S O M E P R O P E R T I E S O F P U L P A N D P A P E R M A D E F R O M F U N G A L T R E A T E D T R E M B L I N G A S P E N R M P 115 4.4.1 W E I G H T E D A V E R A G E F I B E R L E N G T H D I S T R I B U T I O N S . . 115 4.4.2 P H Y S I C A L P R O P E R T I E S O F H A N D S H E E T S 120 5 C O N C L U S I O N S 128 C I T E D B I B L I O G R A P H Y 1 3 3 Appendices 150 A 150 vii List of Tables T E X T 2.1 Polysaccharide composition of the cell wall layers of fibers and tracheids of a hardwood and a softwood 25 2.2 Nitrogen content of wood and lignin of some wood species 37 2.3 The effect of white-rot fungal treatment of mechanical pulp on pulp and handsheet properties 47 2.4 Properties of handsheets made from TMP treated with various fungi. . . 48 2.5 Physical properties of 60-g/m2 handsheets from unbleached aspen refiner mechanical pulps made from chips pre-treated with white-rot fungi. . . . 49 2.6 Bauer-McNett screen analysis of unbleached aspen refiner mechanical pulps made from chips pre-treated with white-rot fungi 50 4.7 Time required for growth to become limiting in media totally deficient in nitrogen. R. ulmarius 83 4.8 Changes in the concentration of glucose in the growth media. R. ulmarius. 83 4.9 Selective biodelignification of aspen RMP: Chemical composition of aspen refiner mechanical pulp given 32 different fungal treatments. Percentages are based on original oven-dry weight of pulp before fungal treatment. 87 4.10 Summary of anovas showing levels of significance of cultural factors and the effect of their interactions on the chemical composition of pulp. . . . 88 4.11 One-way analysis of variance for selective biodelignification index with respect to fungal-treated pulp : Duncan's multiple range test 89 vm 4.12 Selective biodelignification of aspen RMP: Effect of pH under optimum conditions 110 4.13 Selective biodelignification of aspen RMP: effect of exogenously added nitrogen under optimum conditions 110 4.14 Selective biodelignification of aspen RMP: Effect of temperature under optimum conditions I l l 4.15 Percentage population distribution of weighted average fiber length of con-trol trembling aspen refiner mechanical pulp 117 4.16 Percentage population distribution of weighted average fiber length of trembling aspen refiner mechanical pulp given optimum fungal Treatment 21 118 4.17 Percentage population distribution of weighted average fiber length of trembling aspen refiner mechanical pulp given fungal Treatment 24. . . . 119 4.18 Average physical properties of handsheets made from unbleached control and fungal-treated trembling aspen refiner mechanical pulp 127 A P P E N D I X A A.19 Refining of trembling aspen wood chips 151 A.20 Chemical composition of untreated trembling aspen wood chips 151 A.21 Various fungal-treatments used to evaluate the effect of some cultural fac-tors on selective biodelignification of trembling aspen RMP by Rigidoporus ulmarius 152 A.22 Analysis of variance for testing the effect of cultural factors: aeration, hydrogen peroxide, mineral solution, veratryl alcohol and tween 80 on lignin biodegradation as measured by the residual lignin content 153 ix A.23 Analysis of variance for testing the effect of cultural factors: aeration, hydrogen peroxide, mineral solution, veratryl alcohol and tween 80 on cellulose biodegradation as measured by the residual glucose content. . . 154 A.24 Analysis of variance for testing the effect of cultural factors: aeration, hydrogen peroxide, mineral solution, veratTyl alcohol and tween 80 on hemicellulose biodegradation as measured by the residual xylose content. 155 A.25 Analysis of variance for testing the effect of cultural factors: aeration, hydrogen peroxide, mineral solution, veratryl alcohol and tween 80 on selective biodelignification measured by SBI 156 A.26 Mean residual lignin content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: aeration, hydrogen per-oxide and veratryl alcohol 157 A.27 Mean residual lignin content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: aeration, mineral solution and veratryl alcohol 158 A.28 Mean residual lignin content of fungal-treated pulp under second order interaction involving cultural factors: hydrogen peroxide, veratryl alcohol and tween 80 159 A.29 Mean residual glucose content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: aeration, hydrogen per-oxide and mineral solution 160 A.30 Mean residual glucose content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: aeration, hydrogen per-oxide and tween 80 161 x A.31 Mean residual glucose content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: aeration, mineral solution and veratryl alcohol 162 A.32 Mean residual glucose content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: aeration, veratryl alcohol and tween 80 163 A.33 Mean residual glucose content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: aeration, mineral solution and tween 80 164 A.34 Mean residual glucose content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: hydrogen peroxide, min-eral solution and veratryl alcohol 165 A.35 Mean residual glucose content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: hydrogen peroxide, min-eral solution and tween 80 166 A.36 Mean residual glucose content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: hydrogen peroxide, vera-tryl alcohol and tween 80 167 A.37 Mean residual xylose content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: aeration, hydrogen per-oxide and Tween 80 168 A.38 Mean residual xylose content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: aeration, mineral solution and veratryl alcohol 169 xi A.39 Mean residual xylose content of fungal-treated pulp under significant sec-ond interaction involving cultural factors: aeration, mineral solution and tween 80 170 A.40 Mean residual xylose content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: hydrogen peroxide, min-eral solution and veratryl alcohol 171 A.41 Mean residual xylose content of fungal-treated pulp under significant sec-ond order interactions involving cultural factors: hydrogen peroxide, min-eral solution and tween 80 172 A.42 Mean residual xylose content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: mineral solution, veratryl alcohol and tween 80 173 A.43 Mean selective biodelignification index of fungal-treated pulp under signifi-cant second order interaction involving cultural factors: aeration, hydrogen peroxide and mineral solution. 174 A.44 Mean selective biodelignification index of fungal-treated pulp under signifi-cant second order interaction involving cultural factors: aeration, hydrogen peroxide and veratryl alcohol 175 A.45 Mean selective biodelignification index of fungal-treated pulp under signifi-cant second order interaction involving cultural factors: aeration, hydrogen peroxide and tween 80 176 A.46 Mean selective biodelignification index of fungal-treated pulp under sig-nificant second order interaction involving cultural factors: hydrogen per-oxide, veratryl alcohol and tween 80 177 A.47 Key to numbers used to designate levels of cultural factors in figures A.7 toA.30 178 xn A.48 Analysis of variance for initial pH of media on lignin biodegradation under optimum conditions as measured by the residual lignin content 191 A.49 Duncan's multiple range test for the effect of initial pH of media on lignin biodegradation under optimum conditions as measured by the residual lignin content. 191 A.50 Analysis of variance for initial pH of media on cellulose biodegradation under optimum conditions as measured by the residual glucose content. . 192 A.51 Duncan's multiple range test for the effect of initial pH of media on cellu-lose biodegradation under optimum conditions as measured by the residual glucose content 192 A.52 Analysis of variance for initial pH of media on hemicellulose biodegradation under optimum conditions as measured by the residual xylose content. . 193 A.53 Duncan's multiple range test for the effect of initial pH of media on hemi-cellulose biodegradation under optimum conditions as measured by the residual xylose content 193 A.54 Analysis of variance for initial pH of media on selective biodelignification under optimum conditions of fungal-treated aspen pulp 194 A.55 Duncan's multiple range test for the effect of initial pH of media on selec-tive biodelignification index under optimum conditions of fungal-treated pulp 194 A.56 Analysis of variance for exogenously added nitrogen on lignin biodegrada-tion under optimum conditions as measured by the residual lignin content. 195 A.57 Duncan's multiple range test for the effect of exogenously added nitrogen on lignin biodegradation under optimum conditions as measured by the residual lignin content 195 xm A.58 Analysis of variance for exogenously added nitrogen on cellulose biodegra-dation under optimum conditions as measured by the residual glucose content 196 A.59 Analysis of variance for exogenously added nitrogen on hemicellulose biodegra-dation under optimum conditions as measured by the residual xylose content. 196 A.60 Duncan's multiple range test for the effect of exogenously added nitrogen on hemicellulose biodegradation under optimum conditions as measured by the residual xylose content 197 A.61 Analysis of variance for exogenously added nitrogen on selective biodelig-nification index under optimum conditions of fungal-treated pulp 197 A.62 Duncan's multiple range test for the effect of exogenously added nitrogen on selective biodelignification index under optimum conditions of fungal-treated pulp. 198 A.63 Analysis of variance for temperature on lignin biodegradation under opti-mum conditions as measured by the residual lignin content 198 A.64 Duncan's multiple range test for the effect of temperature on lignin biodegra-dation under optimum conditions as measured by the residual lignin content. 199 A.65 Analysis of variance for temperature on cellulose biodegradation under optimum conditions as measured by the residual glucose content 199 A.66 Analysis of variance for temperature on hemicellulose biodegradation un-der optimum conditions as measured by the residual xylose content. . . . 200 A.67 Duncan's multiple range test for the effect of hemicellulose on hemicellulose biodegradation under optimum conditions as measured by the residual xylose content 200 A.68 Analysis of variance for temperature on selective biodelignification index under optimum conditions of fungal-treated pulp 201 xiv A.69 Duncan's multiple range test for the effect of temperature on selective biodelignification index under optimum conditions of fungal-treated pulp. 201 A.70 Analysis of variance for the effect of fungal treatment(treatments 21, 24 and control) on brightness of pulp handsheets 202 A.71 Duncan's multiple range test for the effect of fungal treatment(treatments 21, 24 and control) on brightness of pulp handsheets 202 A.72 Analysis of variance for the effect of fungal treatment(treatments 21, 24 and control) on (dry)zero-span breaking length of pulp handsheets. . . . 203 A.73 Duncan's multiple range test for the effect of fungal treatment(treatments 21, 24 and control) on (dry)zero-span breaking length of pulp handsheets. 203 A.74 Analysis of variance for the effect of fungal treatment(treatments 21, 24 and control) on (wet)zero-span breaking length of pulp handsheets. . . . 204 A.75 Duncan's multiple range test for the effect of fungal treatment(treatments 21, 24 and control) on (wet)zero-span breaking length of pulp handsheets. 204 A.76 Analysis of variance for the effect of fungal treatment(treatments 21, 24 and control) on tensile index of pulp handsheets 205 A.77 Duncan's multiple range test for the effect of fungal treatment(treatments 21, 24 and control) on tensile index of pulp handsheets 205 A.78 Analysis of variance for the effect of fungal treatment(treatments 21, 24 and control) on burst index of pulp handsheets 206 A.79 Duncan's multiple range test for the effect of fungal treatment(treatments 21, 24 and control) on burst index of pulp handsheets 206 A.80 Analysis of variance for the effect of fungal treatment(treatments 21, 24 and control) on tear index of pulp handsheets 207 A.81 Duncan's multiple range test for the effect of fungal treatment(treatments 21, 24 and control) on tear index of pulp handsheets 207 xv A.82 Analysis of variance for the effect of fungal treatment(treatments 21, 24 and control) on density of pulp handsheets 208 A.83 Duncan's multiple range test for the effect of fungal treatment(treatments 21, 24 and control) on density of pulp handsheets 208 A.84 Analysis of variance for the effect of fungal treatment(treatments 21, 24 and control) on opacity(Tappi) of pulp handsheets 209 A.85 Duncan's multiple range test for the effect of fungal treatment(treatments 21, 24 and control) on opacity of pulp handsheets 209 x v i List of Figures TEXT 1.1 Schematic representation of a section of spruce lignin. Source : Adler, 1977 4 1.2 Structural monomers of lignin : p-coumaryl alcohol(I), coniferyl alco-hol(II), sinapyl alcohol(III). Source : Crawford, 1981 5 1.3 Formation of phenoxy radicals, the immediate precursors for chemical polymerizations of lignin in woody plants, by peroxidase. Ri = R2 = H = p-coumaryl alcohol; Ri — H & R2 = OCi/3 = coniferyl alcohol; R1 = R2 = OCH3 — sinapyl alcohol. Source : Modified after Fengel and Wegener, 1984 6 3.4 Schematic representation of cuprous ion with 2,2 -bichinchoninate 67 3.5 Schematic representation of Varian HPLC 5000 71 3.6 Schematic representation of Packing Column system 72 A P P E N D I X A A.7 Plot of significant interaction of aeration and hydrogen peroxide on biodegra-dation of lignin by R. ulmarius as measured by residual lignin 179 A.8 Plot of significant interaction of aeration and mineral solution on biodegra-dation of lignin by R. ulmarius as measured by residual Hgnin 179 A.9 Plot of significant interaction of mineral solution and veratryl alcohol on biodegradation of hgnin by R. ulmarius as measured by residual hgnin. . 180 A. 10 Plot of significant interaction of hydrogen peroxide and mineral solution on biodegradation of lignin by R. ulmarius as measured by residual hgnin. 180 A. 11 Plot of significant interaction of hydrogen peroxide and veratryl alcohol 011 biodegradation of hgnin by R. ulmarius as measured by residual hgnin. 181 xvii A.12 Plot of significant interaction of hydrogen peroxide and tween 80 on biodegra-dation of lignin by R. ulmarius as measured by residual lignin 181 A. 13 Plot of significant interaction of aeration and hydrogen peroxide on biodegra-dation of cellulose by R. ulmarius as measured by residual glucose. . . . 182 A . H Plot of significant interaction of aeration and mineral solution on biodegra-dation of cellulose by R. ulmarius as measured by residual glucose 182 A. 15 Plot of significant interaction of aeration and veratryl alcohol on biodegra-dation of cellulose by R. ulmarius as measured by residual glucose. . . . 183 A.16 Plot of significant interaction of aeration and tween 80 on biodegradation of cellulose by R. ulmarius as measured by residual glucose 183 A. 17 Plot of significant interaction of hydrogen peroxide and veratryl alcohol on biodegradation of cellulose by R. ulmarius as measured by residual glucose. 184 A.18 Plot of significant interaction of hydrogen peroxide and tween 80 on biodegra-dation of cellulose by R. ulmarius as measured by residual glucose. . . . 184 A. 19 Plot of significant interaction of mineral solution and veratryl alcohol on biodegradation of cellulose by R. ulmarius as measured by residual glucose. 185 A.20 Plot of significant interaction of mineral solution and tween 80 on biodegra-dation of cellulose by R. ulmarius as measured by residual glucose. . . . 185 A.21 Plot of significant interaction of veratryl alcohol and tween 80 on biodegra-dation of cellulose by R. ulmarius as measured by residual glucose. . . . 186 A.22 Plot of significant interaction of aeration and hydrogen peroxide on biodegra-dation of hemicellulose by R. ulmarius as measured by residual xylose. . 186 A.23 Plot of significant interaction of aeration and mineral solution on biodegra-dation of hemicellulose by R. ulmarius as measured by residual xylose. . 187 A.24 Plot of significant interaction of aeration and tween 80 on biodegradation of hemicellulose by R. ulmarius as measured by residual xylose 187 xviii A.25 Plot of significant interaction of hydrogen peroxide and mineral solution on biodegradation of hemicellulose by R. ulmarius as measured by residual xylose 188 A.26 Plot of significant interaction of hydrogen peroxide and tween 80 on biodegra-dation of hemicellulose by R. ulmarius as measured by residual xylose. . 188 A.27 Plot of significant interaction of hydrogen peroxide and veratryl alcohol on biodegradation of hemicellulose by R. ulmarius as measured by residual xylose 189 A.28 Plot of significant interaction of mineral solution and tween 80 on biodegra-dation of hemicellulose by R. ulmarius as measured by residual xylose. . 189 A.29 Plot of significant interaction of veratryl alcohol and tween 80 on biodegra-dation of hemicellulose by R. ulmarius as measured by residual xylose. . 190 A.30 Plot of significant interaction of aeration and tween 80 on biodegradation of chemical components of aspen RMP by R. ulmarius as measured by SBI. 190 A.31 Chromatogram from the separation of standard solution of six common wood sugars and the internal standard, ribose 210 A.32 Chromatogram from the separation of hydrolysates of aspen refiner me-chanical pulp 211 xix A C K N O W L E D G E M E N T The author wishes to express his deepest gratitude to his research supervisor, Dr. R. W. Kennedy, Dean of the Faculty of Forestry, for his support, direction, inspiration and invaluable suggestions throughout the course of this work. I also wish to accord my sincere thanks to Drs B. J. van der Kamp and R. J. Bandoni, committee members, for their useful suggestions and crticisms. Special thanks also go to Dr. R. Smith and all the technical staff, notably Ms J. Clark, of the Protected Wood Department of Forintek for their considerable support, help and advice. I really enjoyed their companionship during the course of my studies here. The author wishes to express his deepest gratitude to Dr. J. W. Wilson, committee member and Director, Forestry graduate studies, for his support, care and guidance during the course of my studies here. I am grateful to Dr. L. Paszner who permitted the use of his HPLC for my sugar analysis, Dr. J. N. R. Ruddick for his encouragement and Mr. B. Daniels of Forintek and Mr. S. Yee of Food Science in the running of the Varian HPLC. I also acknowledge the considerable assistance accorded me by the late Mr. G. Boe-hemkamp of the Faculty of Forestry for his help and encouragement throughout the course of my studies. The author also wish to thank Dr. J. V. Hatton of Pulp and Paper Research Institute (PAPRICAN), Vancouver, for permiting me to use the pulp and paper making facilities at the institute. Sincere gratitude is also accorded to Dr. K. Hunt, Mr. B. Christie and Mr S. Johal, all of Paprican for their help in making making and testing the pulp. xx I would also want to thank Mr. B. Wong and Mr. D. McCarthy through whom I have acquired considerable computing skills. My thanks to fellow students and friends for their company and help during my stay in the Faculty of Forestry, the Canadian Commonwealth Scholarship and Fellowship Plan, Vandusen Graduate Fellowship in Forestry and the Faculty of Forestry for financial support. Finally, I would thank my wife, Araba and my children, Nana, Ebow and Aba for their patience, love and support during my studies here. D E O G RATI AS xxi Chapter 1 I N T R O D U C T I O N The bio-polymer lignin is the second most abundant organic compound on earth. Cellu-lose is the only organic substance present in the biosphere in larger quantity than hgnin. (Bellamy, 1974; Nimz, 1978; Crawford and Crawford, 1978; Ander and Eriksson, 1978). The distribution of lignin in the plant kingdom is wide but not universal. Lignin oc-curs in vascular plants, where it acts as a structural component of support and conducting tissues. Lignin has been identified in certain primitive plant groups such as ferns and club mosses, but seems to be absent in the bryophyta (true mosses) and lower plant groups (Erickson and Micksche, 1974a, 1974b; Micksche and Yasuda, 1978). Non-lignified lower plant groups such as the bryophyta (Erickson and Micksche, 1974a, 1974b) and even the green algae (Gunnison and Alexander, 1975) often synthesise non-lignin phenolic substances that may falsely analyse as lignin by standard assay procedures such as the Klason technique (Effland, 1977). Higher plants such as angiosperms and gymnosperms make up the bulk of organic matter in terrestrial enviroments and thereby dominate the total biomass of the earth. The stems of woody dicotyledonous angiosperms con-tain 18 - 25% lignin on a dry-weight basis. Corresponding values for gymnosperms and monocotyledons are 25-30% and 10-30% hgnin, respectively (Cowling, 1976). The biosphere's ability to produce vast quantities of lignocellulosic materials consti-tutes the driving force for life on earth. This capability has been exploited by mankind in the development of large lignocellulose-based industries. Prominent among these in-dustries are agriculture, lumbering and paper making. Large quantities of lignocellulosic 1 Chapter 1. INTRODUCTION 2 waste materials are produced directly or indirectly from these industries. Lignin (Fig. 1.1) is a complex aromatic polymer which is hydrophobic and has several types of non-hydrolyzable linkages. Lignin is also heterogenous and polydisperse. Lignins are generally classified into three major groups based on their structural monomer units (Fig. 1.2). Gymnosperm lignins contain mainly guaiacyl (coniferyl alcohol-derived) units. Angiosperm lignins contain both guaiacyl and syringyl (sinapyl alcohol-derived) units and grass lignin is composed of a mixture of coniferyl, sinapyl and p-coumaryl alcohol polymers. In grass hgnins, p-coumaric acid is esterified to the c7-hydroxyl group of the side chains in the lignin polymer. These precursors are synthesized from glucose via the shikimic acid pathway. The polymerization of lignin precursors proceeds by free radical condensation reac-tions, catalyzed by peroxidase and hydrogen peroxide (Fig. 1.3). The reaction is initiated by the abstraction of a hydrogen atom from the monomer. Extensive conjugation of the monomers occurs resulting in the formation of a number of mesomeric resonance forms which can react randomly with radicals from other monomeric units. These monomeric units react primarily with similar structures in the growing polymer to yield a three-dimensional macromolecule of indeterminate size. Some radical resonance forms are favoured over others resulting in the formation of certain preferred linkages notably the arylglycerol- /3-aryl ether or 8-0-4 hnkages. The arylglycerol- /3-aryl linkages predominate in lignin and constitute over 50% of the linkages in lignin. Lignin occurs in intimate as-sociation with cell wall polysaccharides. It has been shown that some hemicelluloses are linked by covalent bonds with lignin (Fengel and Wegener., 1984). Lignin preparations from wood contain besides carbohydrates, significant amounts of protein. Amino acid profiles of lignin preparations have suggested that the polymerising lignin links covalently with the cell wall glycoprotein and that these bonds may be formed preferentially with hydroxyproline (Whitmore, 1982). Chapter 1. INTRODUCTION 3 Lignin biodegradation occupies a central position in the earth's carbon cycle, because most renewable carbon is either in lignin or in compounds protected by lignin from enzy-matic degradation (Kirk, 1983). In natural decomposition, the lignin sheath is disrupted either mechanically by insects or marine borers, or biochemically by micro-organisms (Kirk and Cowling, 1984) or physically / chemically by atmospheric degradation. It has been suggested by Kirk (1983) that the biochemical decomposition of lignin is by far the more important process and constitutes the key to the recycling of most of the earth's car-bon. Alternatively, lignified tissues can be made susceptible to attack by non-degraders of lignin, provided that the lignin barrier is overcome by physical (eg, fine grinding of wood) or chemical (eg, sodium hydroxide pre-treatment) means. Thus, the importance of lignin as a rate-limiting component of the carbon cycle, becomes even more appar-ent since lignin acts as a barrier to slow the decomposition of the even more abundant polysaccharides. The importance of lignin as a barrier to microbial degradation of plant tissues is exemplified in the, observation that lignification is an important agent of disease resistance in plants (Vance et al, 1980). Microbial groups that can decompose lignin in axenic culture are essentially the same ones that can decay intact wood. This correlation is not surprising because in wood lignin physically protects the cellulose and hemicellulose from enzymatic attack. Only aerobes, primarily wood-decomposing members of the basidiomycetes, metabolize lignin efficiently. Bacteria and actinomycetes have been shown by 14C-radioisotopic lignin biodegradation studies to decompose lignin to 1 4 C 0 2 and water soluble catabolites (Crawford and Craw-ford, 1980; 1984). The most studied actinomycete is Streptomyces viridosporus. Strep-tomyces viridosporus and Streptomyces setonii have been shown to cause 32-44% loss in lignin content of spruce and maple as determined by chemical analysis of the insoluble residues (Antai et al, 1981). However, it has been argued by some workers that actino-mycetes cause only limited lignin degradation and grass lignin and lignin in phloem tissues Chapter 1. INTRODUCTION 4 C CH 2 0H C MCO-; HCOM H C ' O f c H , O M l c CH,OH ° M • J ' ^ O C H , ,, C H j O ^ V • „ ^ o H ^ CH 0 CM CH2OH HOH 2 C-C-C; HCOH M 0 C H ^ ^ O C H 3 C H 2 O H H < - T H VJ h 2coM H C 0 H V I 1 0 I CHjO-MC -0 H0H,C H O C H j J p M C O O j m C O h 0 -CM "<--HCOH C--0 C H j o X ^ O - O C H j OH OH[0-CJ Figure 1.1: Schematic representation of a section of spruce hgnin. Source : Adler, 1977 Chapter 1. INTRODUCTION 5 C H 2 O H C H 2 O H CHjOH I I I C H CH C H II II II C H CH C H O k j L O C H 3 0 C H 3 1 C H 3 0 » O H O H O H p - C O U M A T ^ L C O N I F E R Y L S I N A P Y L A L C O H O L A L C O H O L A L C O H O L Figure 1.2: Structural monomers of lignin : p-coumaryl alcohol(I), coniferyl alcohol(II), sinapyl alcohol(III). Source : Crawford, 1981. Chapter 1. INTRODUCTION 6 Figure 1.3: Formation of phenoxy radicals, the immediate precursors for chemical poly-merizations of hgnin in woody plants, by peroxidase. Ri = R2 = H = p-coumaryl alcohol; Ri = H & i?2 = OCH3 = coniferyl alcohol; R\ = R2 = OCH3 = sinapyl alcohol. Source : Modified after Fengel and Wegener, 1984. Chapter 1. INTRODUCTION 7 are more susceptible to degradation by these micro-organisms. Grasses are degraded by a number of actinomycetes to produce a water-soluble residue called 'acid-precipitable polymeric lignin' (APPL)(Crawford et al, 1983; McCarthy et al, 1986, Pettey et al, 1985). APPLs contain varying amounts of carbohydrates and might therefore result from carbohydrate rather than lignin degradation(McCarthy et al. 1986.) Bacteria shown by 1 4 C - assays to oxidise lignin to C02 include strains of Nocardia sp (Gradziel et al, 1978; Haider et al, 1978); a strain of Bacillus megaterium (Robinson et al, 1978); strains of Pseudomonas, Flavobacterium and Aeromonas (Kawakami, 1976a, 1976b; Odier et al, 1977). This is, however, disputed by Kirk et al. (1987) who contends that aerobic decay of intact wood by bacteria is unknown and they degrade only low molecular weight aromatics related to lignin. Nonetheless, Nilsson et al. (1981) have reported a new type of wood decay caused by bacteria belonging to the Myxobacteriales or Cytophagales. The major wood decay types are soft rots, brown rots and white rots. Soft rots of wood are caused mainly by Fungi Imperfecti and Ascomycetes. Soft rots of wood involve some lignin degradation but polysaccharides are preferentially removed (Eslyn et al, 1975). Both brown rots and white rots are caused primarily by fungi belonging to the basidiomycetes. Brown rots are characterised by limited degradation of lignin and greater degradation of cellulose and hemicellulose. Their major effect on lignin is demethylation of methoxyl groups (Kirk and Adler, 1970; Kirk, 1975). White rots are the major degraders of lignin, White rots degrade both lignin and carbohydrates. Despite the presence of a lignin barrier, there is a great potential for the use of micro-organisms to biologically transform plant materials and residues into industrially valuable products. Recent research on lignin biodeterioration has undoubtedly been stimulated by the several tantalizing potential applications of bio-ligninolytic systems (Kirk, 1983). Some of the potential applications of bio-ligninolytic systems are: Chapter 1. INTRODUCTION 8 1. Partial delignification to increase ruminant digestibility (Dekker and Richards, 1973; Kirk and Moore, 1972; Zadrazil, 1977); 2. Partial delignification as pre-treatment for enzymatic saccharification (Kirk, 1983); 3. Partial delignification and / or Hgnin modification to reduce energy requirements and improve pulp properties in mechanical pulping (Ander and Eriksson, 1975, 1978; Eriksson, 1976; Eriksson and Vallender, 1982); 4. Lignin modification of mechanical pulps to improve pulp properties (Fukuzumi et al, 1983; Yang et al, 1980): 5. Delignification to bleach pulps (Kirk, 1983); 6. Modification of natural or industrial by-product lignins to produce chemicals (Man-dels et al, 1971; Moo-Young et al, 1979, 1983; Crawford and Crawford, 1983; Glasser et al, 1984); and 7. Treatment of lignin-derived waste (Chang et al, 1983). Many wood-destroying fungi can degrade wood but only a relatively small number can remove hgnin without the attendant removal of appreciable amounts of cellulose and hemicellulose (Blanchette, 1980a, 1980b; Eriksson, 1981; Otjen and Blanchette, 1982). Fungi that selectively remove lignin without appreciable loss of carbohydrates are ex-tremely attractive for biological pulping; for improving digestibility of highly lignified plant residues, and for the bioconversion of lignocellulose into industrial products ( Craw-ford, 1981; Kirk et al, 1980). Cellulase-less mutants of some white-rot fungi have also been used to achieve selective delignification (Ander and Eriksson, 1977). With respect to Hgnin degradation, the most extensively studied white-rot fungus is Phanerochaete chrysosporium Burdsall ( Sporotnchum pruinosum Gilman et Abbot, Chapter 1. INTRODUCTION 9 Sporotrichum pulverulentum Novobranova and Chrysosporium lignorum Bergman et Nils-son (Burdsall and Eslyn, 1974). The ability of another white-rot fungus, Coriolus versi-color^, ex Fries) Quel[Polystictus versicolor (L. ex Fries) Sacc. and Polyporus versicolor L. ex Fries] to degrade lignin has been studied by some authors. Knowledge gained in the above studies is gradually being applied to studies on wood decay by other white-rot fungi. Mechanical pulps have been treated with a number of white-rot fungi notably Phane-rochaete chrysosporium with a view of improving some of their physical properties. Other fungi that have been employed in pre-treatment of mechanical pulps are Phlebia radiata Fr., Poria subacida (Peck) Sacc; Trametes gibbosa (Pers. ex Fr.) Fr.; Ganoderma sp and Dichomitus squalens (Polyporus anceps). In 1962, Kawase analysed wood decayed naturally by a number of white rot fungi including Rigidoporus ulmarius (Fr.) Imaz. (Fomes ulmarius (Sox. ex Fries) Gill). The decayed wood was collected from the forest of Hokkaido, Japan. The result of chemical analysis showed that several of the wood samples lacked lignin. Kirk and Moore (1972) using some of the fungi (including Rigidoporus ulmarius) reported by Kawase (1962), studied the decay of two hardwoods (aspen and birch) and three softwoods (southern pine, sitka spruce and Douglas-fir). In agreement with the observations of Kawase (1962), Rigidoporus ulmarius was reported by Kirk and Moore (1972) to remove lignin faster than carbohydrates, on relative basis, from aspen and birch. In pre-treatment of mechanical pulps with white-rot fungi, the general objective has been to modify and/or preferentially remove lignin from the mechanical pulps. Con-sequently, Rigidoporus ulmarius appeared to be a good candidate for pre-treatment of mechanical pulps to improve their properties. On this basis, Rigidoporus ulmarius was se-lected for use in the present study. Trembling aspen (Popolus tremuloides Michx) refiner mechanical pulp was selected for use in this study for the following reasons : (1) Aspen Chapter 1. INTRODUCTION 10 has been shown to be selectively delignified by Rigidoporus ulmarius (Kirk and Moore, 1972); and (2) Aspen is the most abundant hardwood in Canada and increasingly more pulp companies are considering its use. The experiments thus described in this thesis at-tempt to physiologically optimise conditions for the selective fungal biodelignification of aspen refiner mechanical pulp, and describe the resulting physico-mechanical properties of such fungal treated pulp. The specific objective of the research presented in this thesis was to determine the effect of the following cultural parameters on selective biodelignification of trembling aspen refiner mechanical pulp (RMP) by the white-rot fungus Rigidoporus ulmarius in shallow liquid cultures: 1." Type of aeration; 2. Exogenous addition of hydrogen peroxide; 3. Concentration of trace elements; 4. Use of sorbitan polyoxyethylene monooleate (tween 80); 5. Exogenous addition of veratryl alcohol; 6. Initial pH of medium; 7. Nitrogen content of medium; and 8. Temperature of incubation. On the basis of these results, optimum cultural conditions for selective biodelignifi-cation of aspen RMP were determined as those which gave the highest ratio of residual carbohydrates to residual hgnin in the treated pulp. The physical properties of pulp handsheets made from this RMP biodelignified under these optimum cultural conditions were subsequently evaluated to quantify changes in these physical properties. Chapter 1. INTRODUCTION 11 1.1 N O V E L T Y In all previous studies on selective biodelignification, the wood, thermomechanical pulp or the appropriate lignocellulosic substrates, were incubated with a fungus in liquid cultures characterised by low levels of nitrogen (1.2 mM nitrogen) in the liquid medium.. This study was based primarily on the premise that in nature, fungi thrive on the easily accessible carbon and nitrogen sources available in the lignocellulosic substrate until nitrogen becomes limiting. The fungus then switches on its ligninolytic system and attacks the lignin to enable it to gain access to the cellulose and hemicellulose screened by the lignin matrix. Thus, one of the novel ideas in this study was to add the fungal inoculum to media containing all the required nutrients including 1 % glucose and excluding nitrogen sup-plements. The media also contained sterilised aspen refiner mechanical pulp. The fungus used in the inoculum was first grown in a liquid medium containing all the required nu-trients including 24 mM nitrogen and 1 % glucose for two weeks. The mycelia were then filtered and blended under sterile conditions. The pre-treatment of mechanical pulp with white-rot fungi to selectively remove hgnin has been studied by only a few workers (Fukuzumi et al, 1983; Yang et al., 1980; Pilon et al, 1982a, 1982b). But all these previous studies generally employed either the white-rot fungi Phanerochaete chrysosprium, Polyporus giganteus Pers. ex Fr. or their respective mutants. The present study is the first instance in which Rigidoporus ulmarius has been used for this purpose. It has been shown that added veratryl alcohol enhances hgnin degradation, although it is also produced in vivo by a number of white-rot fungi ( Faison et al, 1986.; Leisola et al, 1985; Kirk et al, 1986; Kawai et al, 1987; Liwicki et al, 1985). Hydrogen peroxide is also required for Hgnin degradation by most white-rot fungi and it too is generated Chapter 1. INTRODUCTION 12 in vivo mainly by : (a) Oxidation of glucose by glucose-2-oxidase; and (b) Methanol oxidase oxidation of methanol, produced by demethoxylation of lignin. Consequently, both veratryl alcohol and hydrogen peroxide were added to the culture medium to see to what extent their addition in vivo might affect selective biodelignification by Rigidoporus ulmarius. 1.2 H Y P O T H E S E S For the fungus (Rigidoporus ulmarius)-host (trembling aspen RMP) combination : 1. Under conditions when nitrogen is growth-limiting, selective biodelignification of the aspen refiner mechanical pulp in shallow hquid culture is influenced by a com-bination of the following factors : type of aeration, presence of exogenously added hydrogen peroxide, concentration of mineral solution, use of tween 80, presence of exogenously added veratryl alcohol, pH and temperature of incubation. 2. Under optimum conditions for selective dehgnification of trembling aspen refiner mechanical pulp, the physical properties of the resultant pulp are significantly im-proved. C h a p t e r 2 L I T E R A T U R E R E V I E W 2.1 S E L E C T I V E B I O D E L I G N I F I C A T I O N O F W O O D B Y F U N G I Microbial conversion of wood has recently attracted considerable attention because of its immense potential biotechnological applications. Wood-decay fungi which have the capability to degrade all the major components of wood, namely hgnin and carbohydrates are called white-rot fungi (Liese, 1970; Cowling, 1961; Kirk and Highley, 1973). Liese (1970) made a distinction between two types of white-rot fungi. One type, such as Polyporus versicolor L. ex Fr. and Lentinus nigripes, removes lignin, cellulose and hemicellulose simultaneously from wood. This type of white-rot was described as 'simul-taneous rot' by Liese (1970). The second type described as 'white-rot' by Liese (1970) and exemplified by Polyporus berkeleyi Fr. and Rigidoporus ulmarius (Fr.) Imaz.(Fomes ulmarius (Sox. ex Fries) Gill.) (Kirk, 1973; Kirk and Moore, 1972) succesively degrades cell wall components commencing with preferential removal of lignin and hemicellulose, followed by the removal of cellulose at a later stage. Microscopic studies have shown that 'simultaneous rot' is characterised by a progres-sive thinning of the cell wall, proceeding from the S 3 layer of the secondary wall towards the compound middle lamella (Wilcox, 1970; Kaarik, 1974). The highly lignified middle lamella is relatively resistant to attack until the latter stages of wood decay (Cowling, 1961; Liese, 1970; Wilcox, 1968). However, 'white-rot' fungi which cause preferential removal of lignin from the cell wall leading to the exposure of the cellulose fibrils of the 13 Chapter 2. LITERATURE REVIEW 14 secondary wall result in the loss of the compound middle lamella (Blanchette, 1980a; Otjen and Blanchette, 1982). In 1874, histological examinations of white-rotted wood by Hartig led him to suggest that some white-rot fungi have the capability of selectively removing lignin from wood. Chemical analysis by Fahreus et al. (1949) of birch sawdust decayed by a number of wood decay fungi indicated that Polyporus abietinus (Dickinson) Fr. and Marasmius scorodonius (Fr.) Fr. preferentially degraded lignin, while Stereum rugosum (Pers. ex Fr.) Fr. attacked mostly cellulose. Panus stipticus (Bull, ex Fr.) Fr. which preferentially degrades lignin (Jurasek, 1955), commences its deterioration of the cell wall from the £3 layer to the middle lamella, finally weakening the bond between cells. The same organism has been reported (Necessany and Jurasek, 1956) to destroy the 63 layer early in decay, suggesting that some action on the wood other than strictly lignin deterioration was occuring. Necessany and Cetlova (1963) found a correlation between the amount of cell wall separation along the middle lamella and decrease in lignin content. Wilcox (1968) suggested that lignin-degrading enzymes penetrate and act upon layers within the cell wall in advance of the cellulolytic enzymes. Thus, lignin decomposition might precede cellulose decomposition in lignin-poor regions of the secondary wall. How-ever, in the lignin-rich compound middle lamella, lignin and cellulose decomposition might proceed at a similar rate. This behaviour of white-rot fungi has been ascribed to the requirement of an additional carbon source during degradation of lignin (Wilcox, 1968). Kirk and Moore (1972) have demonstrated that at 14% total weight loss of aspen, Polyporus resinosus Schrad. ex Fr. [Ischnoderma resinosum (Schrad. ex Fr.) Karst.] removed 47% of the hgnin and 13% of the carbohydrates. At 11% of total weight loss of birch, the same fungus removed 34% and 13% of hgnin and carbohydrates, respectively. Pleurotus ostreatus (Fr.) Quel, (examined only with birch), Polyporus berkeleyi Fr. Chapter 2. LITERATURE REVIEW 15 and Polyporus frondosus Dickinson ex Fr. (examined with the two hardwoods and three softwoods) also selectively removed lignin faster. However, Ganoderma applanatum (Pers. ex Wallr.) Pat. removed carbohydrates faster than lignin from aspen but removed lignin and carbohydrates at about the same rate from birch. A Phanerochaete spp called P-Bl (Henningsson et ai, 1972), at weight loss of 15% in birch, removed 50% hgnin but only 2% cellulose. However, cellulose was also attacked as decay proceeded. In order to find naturally occuring white-rot fungi which selectively degrade hgnin (at least in incipient decay), Ander and Eriksson (1977) screened 25 different species of fungi using pine blocks and kraft lignin agar plates. Pycnoporus cinnabannus (Jacquin ex Fr.) Karst. at total weight loss of 3.4% was found to degrade only lignin in the presence of malt extract. Ander and Eriksson (1977) thus concluded that selective degradation of lignin is to a large extent influenced by different nutrient sources. Selective degradation of wood has also been achieved using cellulase-less mutants of some white-rot fungi, notably Phanerochaete chrysosporium (Eriksson and Goodell, 1974). Cellulase-less mutants have also been produced from other white-rot fungi such as Phlebia radiata Fr. and Phlebia gigantea (Ander and Eriksson, 1978). Cell 44, a cellulase-less mutant of Phanerochaete chrysosporium degraded only lignin and xylan in birch wood (Ander and Eriksson, 1975). Although the xylanase producing power of the mutant Cell 44 is very low compared to the wild type, a great loss of xylan occurs simultaneously with the loss of lignin. This confirms the additional requirement of an extra carbon source during delignification (Ander and Eriksson, 1978). Using scanning electron microscopy as a screening procedure, Blanchette (1984c) demonstrated recently that 26 white-rot fungi selectively removed lignin from the wood of various softwood and hardwood tree species in nature. Selectively delignified wood tissues were characterised by lack of compound middle lamella; separation of cells (fibers) Chapter 2. LITERATURE REVIEW 16 from one another and the maintenance of the fibrillar appearance of the secondary wall layers (Blanchette, 1984b). Chemical analyses indicated that dehgnified tissues contained primarily cellulose and only small amounts of hemicellulose and hgnin. However, selec-tively dehgnified wood tissues were not uniformly distributed throughout the decayed wood samples and contained pockets of sound wood and simultaneously-white rotted wood. These various studies by Blanchette and his co-workers (Blanchette, 1979, 1980a, 1980b, 1984b, 1984c; Otjen and Blanchette, 1982; Blanchette et al, 1985) have shown that several white-rot fungi exhibit selective delignification in nature. Summarising, white-rot fungi can be divided into two groups as described by Liese (1970). One such group called 'simultaneous rot' removes hgnin, cellulose and hemicel-lulose simultaneously. The second group called 'white-rot' fungi preferentially degrades lignin. In the absence of an easily metabolizable carbon source, hemicelluloses are usually degraded simultaneously with hgnin even by some 'white-rot' fungi. Preferential degra-dation of lignin has also been achieved using cellulase-less white-rot fungi. In general, selectivity of lignin degradation is influenced largely by the interaction of fungal and wood species. 2.2 F A C T O R S I N F L U E N C I N G S E L E C T I V E B I O D E L I G N I F I C A T I O N Preferential delignification of wood in nature and in vitro is influenced (Highley and Kirk, 1979; Blanchette et al, 1985) by two main categories of factors: 1. Structure, and chemistry of wood. 2. Cultural factors that influence fungal physiology. Chapter 2. LITERATURE REVIEW 17 2.2.1 STRUCTURE AND CHEMISTRY OF WOOD Selective biodelignification of wood by 'white-rot' fungi (used in the narrow sense, Liese, 1970) has been reported to be .influenced by : Wood species; Type of wood cell; and The chemistry of the wood (Kawase, 1962; Kirk and Moore, 1972; Highley and Kirk, 1979; Yang et al, 1980; Blanchette, 1984a; Fries, 1973; Rayner and Hedges, 1982). 2.2.1.1 WOOD SPECIES Kawase (1962) analysed wood decayed naturally and showed that Polyporus resinosus selectively removed lignin from spruce wood but not from hardwood species. Rigido-porus ulmarius was studied for its ability to selectively remove lignin from the wood of three coniferous species and two hardwood species (Kirk and Moore, 1972). The rate of decay of all three conifers was slow compared with that of the hardwoods (Kirk and Moore, 1972). Yang et al. (1980) have shown that the rate of degradation of hgnin and hemicelluloses of thermomechanical (TMP) pulp of a hardwood (alder) was at least four times as fast as in a conifer (hemlock). These differences have been suggested to reflect neither morphological variations or differences in the hemicellulose content of the woods (Highley and Kirk, 1979), but instead have been attributed to the structural dif-ferences in the lignins (Kirk and Moore, 1972). Gymnosperms' hgnins consist mainly of guaiacyl units, whilst angiosperms' hgnins are made up of guaiacyl and syringyl units (Adler, 1977). Some white-rot fungi, such as Polyporus anceps, are usually found decay-ing conifers rather than hardwoods. Such fungi might degrade conifer lignin more rapidly than Phanerochaete chrysosporium which in nature is found, as most white-rot fungi, in angiospermous wood (Kirk and Moore, 1972). Differences have been observed also in the rate and selectivity of lignin removal and carbohydrates from different softwood or hardwood species. Polyporus gigantus removed Chapter 2. LITERATURE REVIEW 18 lignin faster than carbohydrates from aspen, but removed carbohydrates faster than lignin from birch (Kirk and Moore, 1972). Individual fungi also differed in the selectivity of hgnin removal. With species of wood and fungi studied, hgnin was more selectively removed in the initial stages of decay, when weight loss percentages were low (Kirk and Moore, 1972). Studies by Kirk and Highley (1973) indicated a greater effect of wood species than the fungal species on the relative rates of removal of the major structural components by white-rot fungi from the woods examined. In some species, such as white pine, the fungi used were generally similar in their relative rates of removal of glucan, mannan and lignin. In general, selective removal of lignin by white-rot fungi is influenced by wood species. Lignin is generally removed relatively faster from hardwoods than softwoods by most white-rot fungi. An exception to this observation is exhibited by Polyporus anceps which is usually found on decaying conifers rather than hardwoods. The observed differences in lignin degradation by white-rot fungi between softwoods and and hardwoods might be due to the difference in type of hgnin in these woods. 2.2.1.2 WOOD CELL ELEMENTS Some 'white-rot' fungi exhibit preference for certain wood cellular elements (Blanchette, 1984c; Otjen and Blanchette, 1982). In white-rotted wood in nature, wood cell elements not prefered by those fungi were left as borders between decayed areas. Occluding sub-stances, presumably decomposition products, appeared to be primarily responsible for delimiting pockets of degradation (Blanchette, 1984a). In nature, Dichomitus squalens appeared to dehgnify earlywood cells, whereas Phelli-nus pini selectively delignified latewood completely (Blanchette, 1979, 1984c).. Sound wood, bordering white-pocket areas, consisted of earlywood and resin-filled parenchyma. Chapter 2. LITERATURE REVIEW 19 Study of decay patterns in red oak caused by by Inonotus dryophilus (Otjen and Blanchette, 1982) indicated that this fungus selectively dehgnified earlywood fibers and axial parenchyma cells but not ray parenchyma cells or latewood. It has been also sug-gested that 'white-rot' fungi degrade less hgnin as nitrogen concentration increases (Kirk et al. 1976; Keyser et ai, 1978; Reid, 1979). Since the nitrogen content of parenchyma cells is often relatively high as compared to other cell types (Merrill and Cowling, 1966), lignin degradation may be subsequently inhibited. Thus by and large, some white-rot fungi preferentially degrade certain wood cell ele-ments. This phenomenon is not well understood although the relatively low degradation of parenchyma cells in some woods has been ascribed to their relatively high nitrogen content. 2.2.1.3 CELL WALL CHEMICAL COMPONENTS Wood is heterogenous not only in its structure but also in the distribution of its com-ponents within the cell walls (Fengel and Wegener, 1984). In the development of the wood cell, the first portions to appear are the middle lamella and the primary wall which are both rich in pectic materials (Timell, 1967; Harlow, 1958). During subsequent cell wall thickening, cellulose and hemicellulose are deposited to form the secondary wall. Formation of hgnin becomes noticeable at the end of this phase, beginning around the compound middle lamella at the cell corners, and extending from there into the secondary wall. 2.2.1.3.1 LIGNINS Using ultraviolet (UV) microscopy, the hgnin content of the compound middle lamella and secondary walls was determined in tracheids of black spruce and Douglas-fir as well as in fibers, vessels and ray cells of white birch (Fergus et al. 1969; Fergus and Goring, Chapter 2. LITERATURE REVIEW 20 1970). Similar studies with woods of the genera Pinus, Picea, Cryptomeria, Fagus and Abies were made by Fukuda and Haraguchi (1971) and Evdokimov (1974). Using scanning electron microscopy coupled with energy dispersive X-ray analysis (SEM-EDXA), the middle lamella at the cell corners was found to contain higher lignin content than middle lamella regions adjacent to the radial walls (Saka, 1980; Saka et al, 1979). Softwood hgnins are typically guaiacyl lignin with minor amounts of syringyl and p-hydroxyphenylpropane units. Although softwood lignins are said to be uniform (Sarke-nen et al. 1967; Erickson and Miksche, 1974c), no general guaiacyl: syringyl: p-hydroxyphenylpropane (G:S:H) ratio can be given for hardwoods and softwoods (Fengel and Wegener, 1984). The G:S:H ratios for spruce and pine hgnin are 94:1:5 (Erickson et al. 1973a) and 86:2:13 respectively. Hardwoods exhibit greater variability in their hgnin composition than softwoods (Fengel and Wegener, 1984). The syring}^  content of typical hardwood hgnin varies between 20 - 60% (Sarkenen et al, 1967; Erickson et al, 1973b). The G:S:H ratio for beech hgnin is reported to be 56:40:4 (Nimz, 1978). UV microscopy applied to ultra-thin sections of birch revealed that lignin in the secondary walls of the vessels and in the middle lamella is predominantly guaiacyl hgnin (Fergus and Goring, 1970). The secondary wall lignin of fibers and ray parenchyma however is mainly composed of syringyl units. Lignin from the middle lamella and cell corners of the fibers and ray cells is a typical mixed guaiacyl/syringyl hgnin. Birch hgnin has been reported by Tai et al. (1983) to be more susceptible to biodegra-dation by Phanerochaete chrysosporium than spruce lignin, and also syringylpropane units in birch hgnin were preferentially degraded over guaiacyl propane units. Earher on, Kirk et al. (1975) had also reported that syringyl units were degraded preferentially during the initial phase of decay of birch wood by the white-rot fungus, Coriolus versi-color. This phenomenon has been ascribed mainly to the relatively labile nature of the Chapter 2. LITERATURE REVIEW 21 chemical bonds in the syringyl units. In summary, both softwoods and hardwoods exhibit variation in their hgnin composi-tion and this could lead to differences in selective dehgnification. Studies have also shown that syringyl units in hgnin are preferentially degraded over guaiacyl units by white-rot fungi. 2.2.1.3.2 HEMICELLULOSES Hemicelluloses are wood polysaccharide that are characterised by shorter molecular chains (in comparison to cellulose), and by branching of the chain molecules and all are heteropolymers. Hemicellulose could be divided into two main groups: Xylan and Glucomannans. Xylans are made up of a homopolymer backbone of xylose units which are linked by f3 - (1 - 4)-glycosidic bonds. In hardwoods, 4-O-methylglucuronic acid units are linked at irregular intervals by a - (1 - 4)-glycosidic bonds with the xylan backbone. Many ofthe H O - groups at C-2 and C-3 ofthe xylose units are substituted by O-acetyl groups. Hardwoods have a ratio of about 10:1 (Xyl:Me-Glu). Softwood xylans differ from hardwood xylans by the lack of acetyl groups and by the presence of arabino-furanose units linked by a - (1 - 3) glycosidic bonds to the xylan backbone. In most softwoods, xylans exhibit a ratio of XyhMeGlu of 5-6:1 (Timell, 1967). Wood glucomannans are characterized by a heteropolymer backbone made up of glucose and mannose units. Hardwood glucomannans exhibit the simplest structure consisting of glucose and mannose units which are linked by (3 - (1 - 4) -glycosidic bonds to form chains which are slightly branched. The ratio of mannose and glucose units is about 1.5-2:1 in most hardwoods (Timell, 1967). In hardwoods, compared to xylans, glucomannans (3 - 5%) are of minor importance. Softwoods contain mannans made up of a glucomannan backbone to which acetyl Chapter 2. LITERATURE REVIEW 22 groups and galactose units are attached. Hence, softwood mannans are called O-acetyl-galactoglucomannan. The ratio of mannose and glucose units is about 3:1. Determination of hemicellulose distribution across the cell wall is more difficult than that of hgnin. The distribution of hemicellulose across the cell wall has been studied by only some few workers (Meier, 1961) in only some few wood species and could best be summarized in tabular form. Some of the results obtained by Fengel and Wegener (1984) are shown in Table 2.1. From this table, the variation of hemicelluloses across the cell wall is apparent. Cell wall thickness has also been shown to affect the distribution of hemicelluloses across the cell wall. In scots pine, Meier (1961) found that the thicker S2 layer in summerwood cells contains higher glucomannan (24.8%) in the tracheids than in the springwood tracheids which contained 20.3% of this hemicellulose. It followed that there was a lower glucurono-arabinoxylan content in the summerwood since the S3 makes up a smaller proportion of the total cell wall. Several authors (Ander and Eriksson, 1975, 1976; Eriksson, 1978) have shown that at least one of the polysaccharides in wood must be degraded simultaneously with hgnin. Chemical analysis of birch wood decayed by Cerrena umcolor (Fr.) Murr., Ganoderma applanatum, Polyporus resinosus and Poria medulla-panis (Jacq.:Fr.) Cke indicated that approximately equal amounts of ligniri, xylose and mannose were removed from the entire decay samples. Ganoderma tsugae did not remove large amounts of cellulose when hgnin was de-graded, but 76 - 86% of hemicellulose was lost (Blanchette, 1984b). Xylobolus frustulatus, selectively removed lignin and hemicellulose during the incipient (white-pocket) stage of oak decay. It has again been suggested (Kirk, 1980) that a carbohydrate source is necessary for delignification. Hemicellulose may be selectively removed with hgnin be-cause of the close spatial association that exist within the cell wall (Kerr and Goring, 1975; Parameswaran and Liese, 1981) and possibly because it is amorphous and more Chapter 2. LITERATURE REVIEW 23 accessible. The addition of an easily degradable carbon source to wood cultures inoculated with white-rot fungi generally represses cellulolytic activity, thus saving the carbohydrates in the wood. Studies by Tran et al. (1987) indicate that in the absence of an ex-ogenous co-metabolizable carbon subtrate, biodelignification of hardwood kraft pulp by P. chrysosporium was attended by considerable loss of carbohydrates. Leisola et al. (1984) have suggested that the fungus requires an easily metabolizable carbon source for abundant growth and, also, as energy source for its ligninolytic activity. However, high concentrations of a co-carbon substrate hke glucose might decrease ligninolytic activity in spite of apparent growth stimulation (Roch et al., 1983). This was similarly reflected in the biodelignification of alder thermomechanical pulp (Yang et al. 1980). Extensive biodelignification is usally attended by some loss of carbohydrates (especially hemicellu-lose) and this has been ascribed to the reported link between hgnin and hemicellulose. In conclusion, the availability of an easily metabolizable carbon source is required for the degradation of hgnin by most white-rot fungi. Thus, at least one of the polysaccha-rides usually hemicellulose must be degraded simultaneously. The exogenous addition of an easily metabolizable carbon source might prevent the degradation of carbohydrates especially cellulose during Hgnin loss by white-rot fungi. Chapter 2. LITERATURE REVIEW 24 2.2.2 CULTURAL FACTORS The selective degradation of lignin is greatly influenced by enviromental or cultural factors (Kirk, 1981; Reid, 1979; Ander and Eriksson, 1977; Plat et al, 1984; Highley and Kirk, 1979; Kirk, 1976). Studies with Phanerochaete chrysosporium have shown that hgnin metabolism oc-curs only during secondary metabolism (Kirk, 1981; Reid, 1979). In filamentous fungi (Bu lock, 1975), the primary growth phase (trophophase) continues until cultures become hmited for an essential nutrient. When a nutrient becomes limiting, fungi adapt to the limitation by complex regulatory controls that result in cessation of net replicative growth (ie, DNA increase) and shift to secondary ('idiophasic'or 'maintenance') metabolism that accomodates the limitation (Bu lock, 1975). The secondary phase is characterised in many organisms by the production of so-called 'secondary metabolites', among which certain antibiotics are the most studied. The nutrient limitation also often triggers a series of developmental changes that cul-minate in sporulation and eventual death of the mycelium (dechne phase). Studies with Phanerochaete chrysosporium have shown these phases and demonstrated that ligni-nolytic activity is associated with only the secondary phase. Since lignin metabolism does not occur during primary growth, the white-rot fungi do not use lignin as a sole source of carbon/energy (Jefferies et al, 1981, Kirk et al, 1976). Estimates indicate that lignin would only serve marginally as a carbon/energy source even for the secondary metabolic phase (Jefferies et al, 1981). Studies have also shown that a ligninolytic system appears in culture whether or not lignin is present, and that the activity is not increased when hgnin is present (Keyser et al, 1978). Therefore, it appears that lignin degradation by most white-rot fungi occurs only during secondary metabolism, triggered by a limited essential nutrient hke nitrogen. Chapter 2. LITERATURE REVIEW 25 Table 2.1: Polysaccharide composition of the cell wall layers of fibers and tracheids of a hardwood and a softwood. ^ Polysaccharide M + P(%) b Si{%) 52(%) outer part 52(%) inner part + T Betula verrucosa Galactan 16.9 1.2 0.7 0.0 Cellulose 41.4 49.8 48.0 60.0 Glucomannan 3.1 2.8 2.1 5.1 Arabinan 13.4 1.0 1.5 0.0 4-0-methyl -glucuronoxylan 25.2 44.1 47.7 35.1 Picea abies Galactan 20.1 5.2 1.6 3.2 Cellulose 33.4 55.2 64.3 63.6 Glucomannan 7.9 18.1 24.4 23.7 Arabinan 29.3 1.1 0.8 0.0 Arabino-4-0-methyl-glucuronoxylan 13.0 17.6 10.7 12.7 "Source: Fengel and Wegener, 1984. ''Compound middle lamella: also contains a high percentage of galacturonan. Chapter 2. LITERATURE REVIEW 26 Lignin does not serve as a carbon source or induce ligninolysis by its presence in the culture. 2.2.2.1 AERATION Heart-rot fungi grow in a nearly anaerobic atmosphere containing high levels of carbon dioxide. Concentrations of oxygen limited to little more than 1% of the volume of gases in tree trunks are common (Jensen, 1969). Concentrations of carbon dioxide as high as 20% have also been reported (Hintikka et al, 1970). In cultures growing in an atmosphere of 5% oxygen, synthetic hgnins were not de-graded (Kirk et al, 1978a). Lignin degradation of thermomechanical pulp of western hemlock in shallow cultures was at least 50% more rapid in cultures under 100% oxygen than in those under air (Yang et al., 1980). Degradation of Hgnin and carbohydrates in aspen wood was faster in oxygen than in air for seven (including Phanerochaete chrysospo-rium) out of nine sap-rot fungi (Reid and Seifert, 1982). Highley et a/.(1982), demonstrated that the decay in sapwood and heartwood caused by four heartrot fungi (Phellinus evarharti (Ell. et GaU.), Phellinus ignarius (L.:Fr.) Quel., Phellinus rugineofuscus (Karst.) Bourd. and Inonotus glomeratus (Pke.) Murr. and three sap-rot fungi (Phellinus ferreus (Pers.) Bourd.Galz., Phellinus ferrugineosus (Schrad.:Fr.) Cke. and Porta medulla-panis (Jacq.:Fr.) Cke.) was reduced by low oxygen (0.01 and 0.1 atm) and high carbon dioxide (0.1 and 0.2 atm). Although the lack of lignin decomposition at a low oxygen concentration (eg, 5%) at which growth still occured appeared to suggest that the Hgnin decomposing system has a high oxygen requirement ( Kirk et ai, 1978a), it stiU does not explain the cause of wood decay by heart-rot fungi in a nearly anaerobic atmosphere. It has been observed also that in hquid cultures, close hyphal-substrate contact and high oxygen tensions are required for significant hgnin degradation under submerged cultivation (Rosenberg and Chapter 2. LITERATURE REVIEW 27 Wilke, 1978). 2.2.2.2 HYDROGEN PEROXIDE The biochemical system of white-rot fungi that degrades the lignin polymer is said to be extra-cellular, oxidative and relatively non-specific (Kirk and Shimada, 1985). The fact that the hgninolytic activity of white-rot fungi is partly extracellular is ascribed to the fact that they degrade a polymeric substrate. Lignin is also a polydis-persed macromolecule and except for a very small fraction, it is too large to be taken up by microbial cells (Kirk and Shimada, 1985). Characterisation of fungal degradation products of hgnin and its model compounds indicates that its degradation is largely ox-idative. Degradation of lignin by white-rot fungi has been described as non-specific for the following reasons: 1. Lignin is heterogenous and several types of intermonomeric linkages in lignin lack stereo-regularity (Harken, 1967); 2. Industrially modified lignins including kraft lignin are readily degraded by white-rot fungi (Lundquist et ai, 1977); and 3. Stereo-specificity in fungal metabohsm of lignin model compounds is absent (Nakat-subo et al, 1982). 'Active oxygen species' have been speculated to be involved in hgnin degradation (Hall, 1980; Kirk and Shimada, 1985). Among these 'active oxygen species' are partially reduced or excited molecules derived from ground-state molecular oxygen. They include the following : 1. Hydrogen peroxide (H2O2)', Chapter 2. LITERATURE REVIEW 28 2. Superoxide radical (07); 3. Hydroxyl radical (HO - ) ; and 4. Singlet oxygen (102). All these species have been reported in biological systems (Kirk and Farrall, 1987). Only ^ 2 and H O - ' are reactive enough to be considered direct lignin oxidants (Kirk and Shimada, 1985). Initial studies by several authors suggested the involvement of both 102 and H O - ' ( Kirk and Shimada, 1985), but subsequent investigations have discounted the involvement of both 1 0 2 (Kirk et ai, 1983, Kutsuki et al., 1983); H O - ' (Kirk et al, 1985). The investigations of 'active oxygen species' in Phanerochaete chrysosporium have shown that hydrogen peroxide is involved in hgnin degradation (Forney et al, 1982b). These workers found a correlation between hydrogen peroxide production by broken-cell extracts and lignin degradation by intact cultures. Their later work (Forney et al, 1982a) suggested that peroxisome-like structures just inside the cytoplasmic membrane of P. chrysosporium are the site of hydrogen peroxide (H2O2) synthesis. Research by Faison and Kirk (1983) established the presence of extracellular hydrogen peroxide in ligninolytic cultures and demonstrated that specific scavengers of hydrogen peroxide, including catalase, strongly suppressed ligninolytic activity. Faison and Kirk (1983) have also shown that hydrogen peroxide production is temporarily and quantitatively correlated with ligninolytic activity. The knowledge that hydrogen peroxide is required for lignin degradation by P. chrysosporium has facilitated the discovery of a hgnin-degrading enzyme by Tien and Kirk (1983). This discovery followed investigations into the nature of the agent in ligni-nolytic cultures that is responsible for an oxidative cleavage reaction that has been well characterised in intact cultures using dimeric lignin model compounds (Enoki and Gold, Chapter 2. LITERATURE REVIEW 29 1982; Nakatsubo et al, 1982; Kirk et al, 1983). These workers detected cleavage activity in concentrated extracellular fluid of P. chrysosporium. This cleavage activity was found to be dependent on H202- Subsequent work showed that this agent was a peroxidase enzyme (Tien and Kirk, 1983). Hydrogen peroxide is produced by three main mechanisms. Forney et al. (1982b) suggested that one of its major sources is glucose-2-oxidase. This enzyme has been isolated and shown to oxidise glucose to 2-keto-gluconic acid and hydrogen peroxide. This enzyme, unlike glucose-1-oxidase, also oxidises xylose. Another mechanism involves the enzyme methanol oxidase, which has been shown to be produced during secondary metabolism by P. chrysosporium (Ander et al, 1985). Considerable amounts of methanol are produced during lignin degradation in liquid cul-tures by demethoxylation. Methanol is oxidized to formaldehyde and hydrogen peroxide. The latter is also produced through the action of alcohol oxidase which is produced during the secondary metabolic phase of the fungi. In summary, hgnin degradation by white-rot fungi is an oxidative process. Of the 'active oxygen' species predicted to be involved in lignin degradation, experimental evi-dence so far has implicated only hydrogen peroxide. During ligninolysis, white-rot fungi produce hydrogen peroxide in vivo by the enzymatic oxidation of glucose or methanol. 2.2.2.3 MINERAL (TRACE) SOLUTION Certain inorganics including manganese, iron and calcium have often been reported to be concentrated in zones of decay [Alcubilla et al, 1974; Ellis, 1959; 1965; Dawson-Andoh et al, 1985; Muhammad et al, 1984; Ruddick et al, 1987; Aho et al, 1979; Basham et al, 1976; Shortle et al, 1973; Safford et al, 1974]. Recently, the effect of trace element concentration on hgnin degradation has been investigated by a number of workers. Ligninase production has been shown to be increased by either copper or Chapter 2. LITERATURE REVIEW 30 manganese (Kirk ei al., 1986). There is growing evidence that manganese is important in lignin degradation. In the degradation of wood by several white-rot fungi in nature, manganese tends to concentrate as manganese dioxide deposits in the zones of decay (Blanchette, 1984a). Lignin degra-dation by Lentinus edodes is markedly influenced by manganese concentration (Leatham et ai, 1986). The importance of manganese, is attested to by the recent discovery of a Mn-dependent peroxidase enzyme in Phanerochaete chrysosporium Although relatively high concentrations of certain inorganic elements have been asso-ciated with wood decay in nature, the role of the trace (mineral) element solution in lignin degradation is not yet known, apart from the requirement of manganese by manganese dependent peroxidase enzyme. 2.2.2.4 TWEEN 80 The effect of non-ionic surfactants such as sorbitan polyoxyethylene monooleate(tween 80) on lignin degradation in liquid cultures has been studied by a number of reseachers. Studies by Jager et al. (1985) have shown that the addition to the culture medium of non-ionic surfactants such as tween 80, sorbitan polyox3rethylene monolaurate (Tween 20) or 3-(3-cholamido-propyldimethyl-ammonio 1 - propanesulphonate (CHAPS)) produced an increase in ligninase production even with agitation in cultures containing Phanerochaete chysosporium. The effect of incubation conditions on ligninase production by P. chysosporium INA-12 was examined in medium supplemented with 0.05% (W/V), tween 80 or 0.04% (W/V) oleic acid emulsified with tween 80. In stationary, as well as agitated cultures, supplemen-tation with these additives enhanced ligninase activity. Addition of oleic acid emulsified with tween 80 resulted in a reduction of incubation time for maximum activity of lign-inase. Specific activity of hgninase in the extra-cellular medium was stimulated in the Chapter 2. LITERATURE REVIEW 31 same way. Ligninase production in agitated cultures decreased slightly compared to stationary cultures in the same growth medium. The mechanism by which surfactants hke tween 80 enhance extracellular enzyme production in filamentous fungi has not been established. It has been suggested by several authors that tween 80 promotes both uptake and exit of compounds from the cell through modification of plasma membrane permeability (Reese et al, 1969). However, Tweens which, hke vegetable oils, contain ester groups, are hydrolysed by most fungi producing lipase activity. When grown in the presence of tween 80, P. chrysosporium IN A-12 has been found to be actively lipolytic, dissimilating fatty acids resulting from hydrolysis. Studies done by Asther et al. (1986) indicate that tween 80 provides both saturated and unsaturated fatty acids to the fungus. This is borne out by the fact that the exogenous addition of oleic acid to the culture medium results in higher production of ligninase and reduction of time required for maximum enzyme production. Asther et al. (1987) have also suggested that fatty acids provided exogenously are incorporated into the phosphohpids of the plasma membrane resulting in changes to the properties of the membrane. However, the mechanism by which surfactants increased enzyme activity in filamentous fungi might be more complex than only a change in lipid metabolism. The addition of CHAPS which lacked fatty acids also engendered increased ligninase activity comparable to that obtained with tween 80 ( Jager, 1985). Thus, the action of surfactants in extra-cellular enzymes production might vary. By and large, the addition of surfactants hke tween 80, Tween 20, oleic acid and tween 80 emulsified with oleic acid engendered an increase in hgninase activity and sub-sequently an increase in hgnin production, although the underlying mechanism is not well understood. Chapter 2. LITERATURE REVIEW 32 2.2.2.5 VERATRYL ALCOHOL Optimisation of the growth medium for ligninase production has been studied in several laboratories. Addition of veratryl alcohol has been shown by several workers (Faison et al, 1986; Kirk et al, 1986; Leisola et al, 1984, 1985; Kawai et al, 1987 and Liwicki et al, 1985) to markedly improve ligninase production. The transition from primary metabolism to secondary metabolism in the fungus P. chrysosporium is triggered by nitrogen starvation. In glucose-grown, nitrogen-starved cultures, the appearance of secondary metabolism is characterised by the de novo syn-thesis of the secondary metabolite veratryl (3,4 - dimethoxybenzyl) alcohol (Liwicki et al, 1985). In nitrogen-starved cultures of P. chrysosporium, veratryl alcohol is synthesized from phenylalanine via 3,4 - dimethoxycinnamyl alcohol. The latter is then oxidised to 1, - (3,4 - dimethyoxyphenyl) glycerol which is then oxidised by ligninase to veratryl alcohol (Shimada et al, 1981). Veratryl alcohol is oxidised by ligninase to veratryl aldehyde and other products. Veratryl alcohol may also stimulate the oxidation of other compounds by ligninase (Hammerli et al, 1986, Harvey et al., 1986, Faison et al, 1986, Leisola et al, 1984). In the abscence of added veratryl alcohol, Leisola et al. (1985) showed that the ligninolytic system and biosynthesized veratryl alcohol appeared simultaneously (Faison et al, 1986, Kirk et al, 1986, Leisola et al. 1985). Veratryl alcohol has also been shown to be synthesized de novo by the white-rot fungi Coriolus versicolor (Kawai et al, 1987), Pycnoporus cinnabarinus, Phlebia radiata (Hatakka, 1986) and Trametes species and by four other unindentified white-rot fungi (Kirk and Farrall, 1987). Kinetic studies by Tien et al. (1986) have shown that hydrogen peroxide first re-acts with the ligninase enzyme which then reacts with veratryl alcohol. Attempts to detect intermediate substrate-free, radicals failed. It has been suggested by Harvey et Chapter 2. LITERATURE REVIEW 33 al. (1986) that veratryl alcohol is oxidized to a cation radical which acts as a diffusible one-electron oxidant to interact with other substrates. However, Kirk and Farrall (1987) have suggested that veratryl alcohol might simply provide protection for the ligninase enzyme from deactivation or act as an electron relay at the enzyme active site or alter the enzyme conformation. Summarising, lignin degradation by some white-rot fungi is enhanced by the exoge-nous addition of veratryl alcohol. Veratryl alcohol is also produced de novo by white-rot fungi during ligninolysis and its synthesis coincides with commencement of lignin degra-dation. The mechanism by which veratryl alcohol enhances lignin degradation is not known, although several theories have been proposed. Chapter 2. LITERATURE REVIEW 34 2.2.2.6 pH The pH of the medium is critical to lignin decomposition (Kirk et al, 1978a). These authors observed that the optimum pH for the degradation of synthetic lignin (DHP) by P. chrysosporium was 4-4.5 with substantial suppression below pH 3.5 and above pH 5.5. The optimum pH for growth was somewhat higher than for lignin decomposition. Kirk et al. (1978a), also observed that the control of pH was difficult when concentration of nitrogen was high and when salts of carboxylic acids served as growth substrates. In several nitrogen-limitation and glucose replenishment experiments, pH has been controlled using 2,2' - dimethylsuccinate as a buffer (Reid et al, 1979). Studies by these authors indicate that 2,2 - dimethylsuccinate at concentrations up to 50 mM did not inhibit the growth of a P. chrysosporium isolate PRL 2750. Fenn and Kirk (1979) have shown that pthalate buffer inhibits ligninolysis by P. chrysosporium. 2.2.2.7 NITROGEN Ligninolytic activity is highest in cultures limited for nitrogen, with other nutrients being present in excess (Kirk, 1981). This is the most common situation in natural wood substrates. Although ligninolytic activity commences immediately upon depletion of supplied carbohydrates in carbohydrate-limited cultures, a considerably longer period occurs between depletion of supplied nutrient nitrogen and appearance of ligninolytic activity in cultures limited only by this nutrient (Kirk, 1981). It is possible to speed up hgnin degradation by using appropriate amounts of nitrogen in the medium (Ander and Eriksson, 1978). Thus, in the presence of about 1 mM asparagine, most lignin was degraded by P. chrysosporium (Ander and Eriksson, 1978). When the amount of asparagine was increased, weight loss occured proportionally more rapidly than hgnin depletion indicating that selectivity for lignin degradation decreased Chapter 2. LITERATURE REVIEW 35 when the amount of nitrogen increased. A similar effect of nitrogen on lignin degradation has also been reported (Kirk et al, 1976; Kirk et al, 1978). Degradation of hgnin in alder thermomechanical (TMP) pulp by P. chrysosporium was also inhibited by high nitrogen levels (Yang et al, 1980). The type of nitrogen (organic or inorganic) nutrient might also influence selectivity of lignin degradation. In the presence of asparagine, a Phanerochaete sp called P-Bl grew on birch wood and decomposed primarily the lignin component (Henningson et al, 1972). However, in the presence of an inorganic nitrogen source such as ammoniun nitrate, cellulose degradation was predominant. The carbon:nitrogen (C:N) ratio of most wood species is (350-500):l and sometimes as high as 1250:1 (Hagglund, 1951). Nitrogen in wood and its role in wood deterioration has been reviewed extensively by Cowling and Merrill (1966). These authors suggested that autolysis and re-use of nitrogen are mechanisms by which the wood-destroying fungi might conserve the meager resources of nitrogen in wood. Lignin preparations of woody material contain, besides carbohydrates, significant amounts of protein. The Klason hgnin preparations from the cell wall of Pinus elliottii, for example, were found to contain 9% protein (Whitmore, 1982). Evidence indicated that lignin formed covalent bonds with cell wall protein and ammonium-N of the nitrogen-containing compounds in the soil (Weichelt, 1981). Amino acid profiles of hgnin preparations have suggested that the polymerising lignin linked covalently with the cell wall glycoprotein, and that these bonds might be formed preferentially with hydroxyprohne (Whitmore, 1982). Using spruce, Westermark et al. (1986) showed that the compound middle lamella contained a considerably higher nitrogen content (2.4%) than whole wood (0.03%). Dill et al. (1984) have also demonstrated that in Fagus sylvatica, the nitrogen concentration on a dry-weight basis was more than two times higher in the lignin fraction than the whole wood. An analogous distribution of nitrogen was reported by the same workers Chapter 2. LITERATURE REVIEW 36 for other hardwoods, eg., Chilean Eucryphila cordifolia and Nothofagus dombegeyi. They also presented calculations to show that the lignin fraction always contained at least 50% of the total nitrogen in wood irrespective of the individual amounts of Hgnin and nitrogen in the wood (Table. 2.2). Thus, if about half of the extremely limited nitrogen source in wood is firmly linked to the lignin polymer as a lignoprotein complex, then a true white-rot fungus should be able to gain sufficient nitrogen in only two ways for its growth and development: Degradation of lignin; and Recychng of nitrogen from older parts of the mycelium (Dill et al, 1984). Consequently, Dill et al. (1984) have suggested that under field conditions, a white-rot fungus displays a 'quasi-stationary' metabohsm, with Hgninolytic activity being expressed either permanently or stepwise. In the latter case, degradation of Hgnin would yield the nitrogen required for a limited degradation of ceUulose or metabohsm of carbohydrates for a period of restricted growth, during which Hgninolytic activity might be suppressed. After the available nitrogen is exhausted, growth would stop and Hgnin degradation start again. This is supported by the results of work done by Ulmer et al. (1983) showing the occurence of intermittent growth cycles in P. chrysosporium under nitrogen-limited chemostat conditions. Thus, on the whole, lignin degradation by white-rot fungi is highest in cultures lim-ited for nitrogen, reflecting the general situation in nature. In some situations, the type of nitrogen (organic or inorganic) might have an effect on lignin degradation. The middle lamella and the primary wall contain the highest amounts of glycoproteins, which sub-sequently complex with Hgnin during Hgnification of the ceU waU to form Hgno-proteins. 2.2.2.8 AGITATION Agitation of submerged cultures has been shown by several authors to inhibit the degra-dation and metabolism of Hgnin and hgnin-related compounds by P. chrysosporium (Reid et al, 1984). Kirk et al. (1978a), found that P. chrysosporium degraded synthetic Hgnin Chapter 2. LITERATURE REVIEW 37 Table 2.2: Nitrogen content of wood and lignin of some wood species. Species Klason lignin Testing Nitrogen i(^g/mg) %Nitrogen (% dry wt) method Wood Lignin In hgnin Fagus sylvatica I 25 Kjeldahl 1.65 3.76 57.0 Amino acid 1.53 3.98 65.0 analysis Fagus sylvatica II 21 Kjeldahl 0.90 2.2 51.3 Eucryphila cordifolia 28 Kjeldahl 0.58 1.12 54.1 Nothofagus dombegeyi 17 Kjeldahl 0.42 1.26 51.0 "Source: Dil l et al., 1984. (dehydrogenative polymerizate, DHP) to carbon dioxide in shallow non-agitated cultures much faster than agitated cultures. Shaking inhibited DHP degradation more in an at-mosphere of oxygen than in air. Kirk et al. (1978a) also observed that in both agitated and non-agitated cultures, a substantial portion of the initially suspended hgnin became associated with the hyphae during the first few days of incubation. Binding of hgnin to fungal mycelia is a recognised phenomenon (Gottlieb and Pelczar, 1951; Tono et ai, 1968; Konishi and Inoue, 1971). It is speculated that in continously shaken cultures, most of the lignin bound by the hyphae became entrapped in the fungal pellets and the oxygen concentration became so low (Phillips, 1966) that hgnin decomposition is prevented on all but the outer surfaces. This is supported by the fact that pelleted cultures, when removed from the shaker, formed mats and then decomposed the hgnin at a rate and extent proportional to the amount of hgnin not already associated with the pellets. Culture agitation also gave different results under pure oxygen than under air. Lignin decomposition under pure Chapter 2. LITERATURE REVIEW 38 oxygen was inhibited by agitation whether or not an initial period without agitation was aUowed (Kirk et al, 1978a). The strong deleterious effect of culture agitation is probably influenced by aeration (Yang et al, 1980). In stationary cultures containing P. chrysosoporium and aspen or western hemlock thermomechanical pulp, the fungus formed a mycelial mat intermingled with the pulp over the bottom of the flask. However in agitated cultures, pulp and mycelium compacted together in a spherical form. It has been postulated, therefore, that some kind of interference with oxygen-requiring reactions in the compacted pellets was responsible for the adverse effect of agitation on lignin degradation. Some studies however indicate that agitation does not inhibit hgnin degradation. P. chrysosporium in shaken cultures has decolourized pulp bleaching plant effluents (Eaton et al, 1980). This discolouration probably involves the hgninolytic system ofthe fungus. In contrast to several previous reports, cultures of P. chrysosporium agitated on a rotary shaker degraded synthetic hgnin (DHP) to carbon dioxide as rapidly and extensively as static cultures (Reid et al, 1984). The hgnin in aspen wood was degraded to carbon dioxide and water-soluble products equally well in agitated and static cultures, if the wood particles became enmeshed in the mycelium. In summary, lignin degradation by white-rot fungi in submerged cultures was gener-ally inhibited by agitation, probably because the fungus tends to compact together to form pellets, thereby trapping the lignocellulosic material and inhibiting oxygen trans-fer. However, the cause of lignin degradation under conditions of agitation has not been explained. Chapter 2. LITERATURE REVIEW 39 2.3 P H Y S I C O - M E C H A N I C A L PROPERTIES OF P U L P A N D H A N D S H E E T S M A D E F R O M W O O D CHIPS A N D M E C H A N I C A L P U L P P R E - T R E A T E D W I T H F U N G I Two distinct types of lignocellulosic substrates have been used to produce pulp by biodelignification. These are wood chips and various types of mechanical pulps. The properties of pulp and paper produced by biodelignification using these two types of lignocellulosic substrates have been studied by a number of workers (Pilon, 1982a, 1982b; Samuelsson et al, 1980; Yang et ai, 1980; Eriksson and Vallender, 1982; Fukuzumi, 1983, Myers et al, 1988) and are reviewed together here. Most early biopulping studies focused primarily on energy savings derived from fungal treatment of wood chips, since energy consumption is one of the major considerations in the production of mechanical pulps. The effect of fungal treatments on the physico-mechanical properties of the resulting pulp has also been evaluated in some studies. One of the earlier studies in this catergory was done by Samuelsson et al. (1980). Samuelsson et al. (1980) investigated the relative effects of fungal treatment on pine wood chips which were later defibrated at 127° C and 170° C, respectively. Mechanical pulp produced by defibration of pine wood chips was also given the same fungal treatment. The white-rot fungi used in their study were Phlebia radiata Fr. L 12-41 (wild-type) and its cellulase-less mutant, Cel 26. For the same energy input, fungal treatment of pine wood chips with P. radiata (wild-type) gave pulp handsheets with the same tensile indices as those observed for the controls. However, treatment of pine mechanical pulp with the same wild-type fungus gave pulp handsheets with lower tensile values compared with the controls. Cel 26, whether applied to pine wood chips or mechanical pulp gave approximately the same tensile strength as the control handsheets. With respect to brightness, treatment of pine mechanical pulp, with P. radiata (wild-type) resulted in a Chapter 2. LITERATURE REVIEW 40 brightness loss of 8 units in comparison with the controls. Treatment of pine wood chips and pulp with the cellulase-less mutant resulted in even greater brightness reduction. The effect of exogenously added glucose on wood chips decayed by cellulase-less mu-tants of Phanerochaete chrysosporium and Phlebia gigantea ( Cel 44 and Cel 26, respec-tively) has been studied by Eriksson and Vallender (1982). Wood chips impregnated with glucose solution (1.7% on weight basis) in the presence of optimum quantities of urea and ammonium dihydrogen phosphate (NiJ4iJ2^04)(Eriksson et al, 1980) in 20 liter cylinders were incubated with both Cel 44 and Cel 26 for 14 days. In the absence of glucose, there was more intense fungal attack on the wood cell wall components. For glu-cose impregnated spruce wood, Cel 44-treated wood chips required 1250 kW h tonne-1 as compared to 1600 kW h tonne-1 for the controls to obtain a tensile index of 33 N m g-1 Johnsrud et al. (1986) have shown that even limited delignification (0.5% - 1.0%) in wood, gave rise to about 25% reduction in energy consumption in mechanical pulping. In co-operation with ISIDA (Cuban Research Institute for Sugar By-products), pre-treatment of bagasse with Ce/ - mutant of P. chrysosporium before the application ofthe Cuba - 9 - CTM pulping process (cold soda) gave rise to a considerable energy savings without altering the quality of pulp (Johnsrud et al., 1986). Yang et al. (1980) were some of the early workers to investigate the effect of pre-treatment of mechanical pulps with the fungus, P. chrysosporium in shallow hquid cul-tures. Using thermomechanical pulps (TMP) from western hemlock and red alder, Yang et al. (1980) demonstrated in hquid cultures that degradation of hgnin in these mechan-ical pulps in shallow Hquid cultures was ten times faster in stationary cultures than in agitated cultures. Under pure oxygen, lignin degradation in these mechanical pulps was also about 50% faster than under air. It was also observed that under the conditions employed in the studies, Hgnin degradation in the TMP pulps was greatly influenced by Chapter 2. LITERATURE REVIEW 41 nitrogen content ofthe media. For instance, the addition of 0.12% nutrient nitrogen (dry pulp basis) increased the rate of Hgnin degradation two to five fold. However, addition of 1.2% nitrogen at first suppressed, then stimulated lignin degradation. Lignin in alder TMP was degraded over five times more rapidly than that in hemlock TMP. Yang et al. (1980) also studied the effect of glucose added exogenously to the TMP on its degradation by the fungus employed in the study. Addition of glucose (35% of dry pulp) to both alder and hemlock TMP containing 0.12% added nitrogen completely inhibited polysaccharide deterioration during two weeks of incubation, but did not influence lignin degradation. The maximum lignin degradation in both pulps was 3% per day over the the two-week period of incubation or approximately 2.9 mg/mg fungal ceU protein/day. Another group of workers who have studied the effect of fungal treatment on the properties of mechanical pulps is Bar-Lev et al. (1981). In their study, alder TMP (50 g dry weight) treated with P. chrysosporium in the presence of glucose (2.5%), resulted in 25 - 30% reduction of energy requirements to refine the pulp to a given freeness when compared to the untreated control. Fungus-treated pulp samples after swelling in alkaH, required 50% less refining energy (PFI revolutions) than the control pulp. This was achieved without loss in strength properties of fungus-treated pulp when compared to the controls. The fungal treatment also reduced the lignin content by 19% with no loss in total carbohydrate. The added glucose evidently prevented degradation of cellulose and hemicellulose, probably by repression of polysaccharides' hydrolytic enzymes . However, when the alder TMP was treated with P. chrysosporium in the absence of glucose, there was no real difference in refining energy requirements between the fungus-treated and the control pulps after swelfing in alkah. These results were in accord with those of Samuelsson et al. (1980). Brightness was unaffected by the fungus/alkali treatment. Samuelsson et al. (1980) did not report any any decrease in brightness, although they they did not use alkah treatment. . Nonetheless, absence of glucose appeared to have Chapter 2. LITERATURE REVIEW 42 resulted in cellulose degradation with attendant loss in strength properties. In 1983, Fukuzumi et al. (1983) screened seven fungi for their potential use in fungal treatment of mechanical pulps. The fungi employed were Poria subacida, Phlebia gi-gantea, Ganoderma spp,Trametes gibbosa and three unidentified fungi namely Uh, Tas-2 and #21. In this study, 20 g (dry weight) of TMP produced from unspecified softwood chips by the Japan Pulp and Paper Research Institute, Inc. was incubated with the fungus in one liter of a medium containing ammonium hydrogen phosphate in addition to other mineral nutrients, trace elements and growth factors. Incubation was carried out on a rotary shaker at pH of 5.8 for one week. Evaluation of physico-meehanical properties (Table 2.4) of the resulting TMP handsheets showed that breaking length and burst factor were remarkedly decreased by Phlebia gigantea and Uh. Handsheets made from TMP pre-treated with the unidentified Fungus #21 exhibited an increase in only breaking length. TMP treated with all the other fungi showed a decrease in brightness, breaking length and burst factor. Water retention (Pilon et ai, 1982a) is said to be correlated with the mechanical properties of paper, i.e. is a measure of the swelling and flexibilitj' of fibers (Pilon et al., 1982a). It is related to the WO" groups available for inter-fibrillar and inter-fiber bonding. Increased swelling is said to produce greater contact between fibers during paper making, thus increasing the strength. However, water retention is also affected by the amount of fines in the pulp; the amount of beating and extreme pH values. Pilon et al. (1982a) treated mechanical pulp consisting of 10% pine, 30% balsam fir and 60% spruce with fungi belonging to three major classes of wood-inhabiting fungi, namely stains and molds, white-rot and brown-rot fungi. The white-rot fungi used were Schizophyllum commune, Pleurotus ostreatus, Polyporus versicolor, Phellinus pini and Phanerochaete chrysosporium,. The brown-rot fungi employed in this study were Fomes pinicola and Coniophora puteana. The two molds used were Aureobasidium pullulans and Chapter 2. LITERATURE REVIEW 43 Trichoderma reesei. They observed the greatest increase in water retention value of pulp treated with white-rot fungi (except P. chrysosporium). The two brown-rot fungi and the mold Aureobasidium pullulans produced only a slight increase in water retention value as compared to the controls. The mold T. reesei produced no effect on water retention value of the treated pulp. Overall, the highest increase (88%) in water retention value of the fungi-treated pulp as compared to the control pulp was produced by the white-rot fungus, Schizophyllum commune (Table 2.3). It was suggested by Pilon et al. (1982a) that one of the factors contributing to high water retention value was the relatively high endoglucanase activity and also the high adsorption of highly hydrated fungal polysaccharides. Schizophllum commune and Aureobasidium pullulans produce a polysaccharide called schizophyllan and pullulan, re-spectively. They also attributed the observed increase in water retention values of the white-rot treated pulps to partial hgnin degradation or removal. However, this failed to explain the low water retention values observed with pulp treated with Phanerochaete chrysosporium, since this fungus generally exhibits high lignin degradation. Perharps the incubation conditions employed tended to inhibit hgnin degradation by this fungus. The high endoglucanase activity observed would be expected to contribute to cellulose degradation. However, it was argued by Pilon et al. (1982a) that the observed increase in water retention values was caused by endoglucanase randomly cleaving the cellulose chains on the surfaces of the crystalline regions leaving loose cellulose tails to bind ad-ditional water. In a subsequent study, Pilon et al. (1982b) have shown that some of the white-rot fungi used in the previous study notably Phanerochaete chrysosporium, Polyporus versicolor and Pleurotus ostreatus improved some of the properties of hand-sheets made from mechanical pulp treated with these fungi. However, these changes in the paper properties were different for different fungi. The white-rot fungi also generally decreased brightness. The details of this study are given in Table 2.3 Chapter 2. LITERATURE REVIEW 44 Attempts have been made also to bleach chemical pulps by treatment of the pulp with white-rot fungi. In 1977, Lundquist et at. demonstrated that Phanerochaete chrysospo-rium metabolised kraft hgnins to carbon dioxide. This led Kirk et al. (1979) to investigate the possibility of bleaching kraft pulp with this and other white-rot fungi. Using southern pine kraft pulps (150 mg) in 125 ml culture flasks containing 10 ml of Kirk et al. (1979) basal medium; trace elements; vitamins, a nitrogen source(equimolar L-asparagine and ammonium nitrate, 0.2% nitrogen on a dry pulp basis) and glucose (33% of dry pulp), Kirk et al. (1977) reported a 50-75% reduction in kappa number in six to eight days. Fungal treatment periods longer than eight days resulted in even greater reductions. In the absence of exogenously added glucose, the cellulose content (50%) of the pulps was depleted in seven days. Addition of more nutrient nitrogen to the cultures suppressed delignification. For instance, in seven days, kappa number was only decreased from 28 to 24 in 2% nitrogen, but 28 to 14 in 0.2% nitrogen. In this study, delignification of the kraft pulp was also greatly enhanced by incubating cultures under pure oxygen instead of air. Kappa number of the southern pine kraft pulp was reduced from 28 to 7 in oxygen and from 24 to 13 under air in seven days. Other white-rot fungi namely Coriolus versicolor, Gloeoporus dichrous and Lentinus edodes reduced the original kappa number of this kraft pulp from 28 to 16, 21 and 24 respectively. Lignin metabolism has been shown (Keyser et al, 1978) to occur only after nutrient nitrogen has been depleted and a subsequent lag phase has passed. Addition of nutrient nitrogen also delayed the appearance of the hgninolytic system or suppressed it if it were already present (Keyser et al., 1978). The most recent and relevant work done on the effect of fungal treatment of wood chips or mechanical pulp on the physico-mechanical properties of the mechanical pulp derived from such treatment has been reported by Myers et al. (1988). These workers studied the effect of pre-treating aspen wood chips with two white-rot fungi, Phanerochaete Chapter 2. LITERATURE REVIEW 45 chrysosporium and Dichomitus squalens in a solid-substrate prior to refiner mechanical pulping. Pre-treatment of aspen wood chips in a rotating cylinder for 38 - 40 days at 39° C. with either fungus improved the strength properties of handsheets produced from the unbleached refiner mechanical pulp (Table 2.5). Burst, tear, tensile, and zero-span tensile indices were markedly increased, especially by D. squalens. At a comparable freeness of 85-95 ml CSF, the fungal pre-treatments improved burst by factors of 2.1 and 3.5, tear index by 1.3 and 2.5, tensile index by 1.4 and 1.7 and zero-span tensile by 1.2 for P. chrysosporium and D. squalens, respectively, when compared to controls. Opacity was not affected, but brightness was decreased from 51.0% (at 65 ml CSF freeness) for the controls to 39.8% and 41.0% respectively for D. squalens and P. chrysosporium treated wood chips. Scattering coefficient also decreased from 62.8 (m2/kg) for the controls to 40.7 (m2/kg) and 53.3 for handsheets made from wood chips treated with D. squalens and P. chrysosporium respectively. Using Bauer-McNett screen analysis of pulp produced from control aspen wood chips and aspen wood chips pre-treated with D. squalens and P. chrysosporium, Myers et al. (1988)(Table 2.6) observed that the fiber size distribution was greatly altered by D. squalens, whereas it was more or less unchanged with P. chrysosporium. Treatment with D. squalens decreased the pulp fines content [those fibers passing the 0.149 mm(100 mesh) screen] and maintained the longer fiber content [those fibers retained by the 0.149 mm screens]. At a nominal 90 ml CSF, the D. squalens pre-treatment decreased fines content from about 53 % to 33% and the fiber length index was higher with D. squalens. The pulp treated with P. chrysosporium was comparable to that of the controls. On the whole, D. squalens pre-treatment gave a greater improvement in handsheet properties than P. chrysosporium under solid state fermentation conditions employed in the study. In summary, white-rot fungi have been employed in the pre-treatment of wood chips Chapter 2. LITERATURE REVIEW 46 prior to conversion to mechanical pulps. Fungal bio-treatment of wood chips has been aimed mainly at the reduction of fiberizing energy and also the improvement of the physico-mechanical properties of paper from the bio-treated pulp. Treatment of mechan-ical pulps has been restricted to improving the physico-mechanical properties. Results of the relatively few studies done so far are somewhat contradictory owing to several factors, especially different culture conditions, fungal species, wood species and a general lack of complete understanding of the biochemical/physiological basis of delignification and bleaching. In spite of this , the following general observations could be made : 1. Treatment of wood chips with white-rot fungi resulted in reduction in defiberizing energy; 2. Exogenous addition of a co-carbon substrate like glucose tended to preclude the degradation of the carbohytrate content of chips or mechanical pulps; 3. In shallow hquid cultures, lignin degradation was enhanced by pure oxygen and inhibited by the presence of high nitrogen; 4. Under some cultural conditions, some physico-mechanical properties of handsheets were improved when wood chips were treated with white-rot fungi; and 5. Brightness generally tended to decrease by such treatments. Chapter 2. LITERATURE REVIEW 47 Table 2.3: The effect of white-rot fungal treatment of mechanical pulp on pulp and handsheet properties.*5"' Fungi Control P.b P.c P.d Pe s. * Tests3 versicolor chrysosporium ostreatus pini commune W R V (g/g) 1.00 1.53 (53) 1.46 (46) 1.32 (32) 1.68 (68) 1.74 (74) (normalized) Freeness 127 148 (18) 178 (42) 135 (8) 160 (28) 84 (-34) (CSF, ml) Bulk 3.28 3.06 (-7) 3.15 (-4) 3.06 (-7) 3.16 (-4) 3.20 (-2) (cm3/g) Burst index 1.03 1.39 (35) 1.24 (20) 1.23 (19) 1.02 (-1) 0.96 (-10) (kPa m V 1 ) Tear index 4.18 4.65 (32) 3.87 (-7) 3.72 (-11) 3.94 (-6) 4.08 (-4) (mN m V 1 ) B. L.{ 2220 2901 (31) 2749 (24) 2746 (24) 2378 (7) 2253 (-2) (m) Stretch 1.62 2.03 (25) 1.80 (11) 1.69 (4) 1.74 (7) 1.59 (-2) (%) Iso 52.80 34.80 (-38) 32.20 (-30) 38.60 (-56) 36.80 (-30) 52.00 (-2) brightness-7 Tappi 98.00 98.90 (1.0) 98.70 (-.7) 99.10 (1.0) 97.50 (-.5) opacity* Scattering 692 604 (-13) 560 (-19) 645 (-7) 606 (-12) 669 (-3) coefficient' Zero-span 8.58 8.90 (4) 8.24 (-4) 8.71 (2) 8.08 (-6) 8.34 (-3) B. L m (km) Incubation 3 3 3 3 2 period(wk) "Source: Pilon et al. 1982b. Polyporus '{Phanerochaete dPleurotus 'j Phellinus ' Schizophyllum 'Values in brackets indicate percentage over control. ''Water retention value. 'Breaking length. J8-457% abs. WS. fc10-FMY/C, %. W / g ) . '"Breaking length. Chapter 2. LITERATURE REVIEW 48 Table 2.4: Properties of handsheets made from TMP treated with various fungi. Wood-rotting fungus Brightness Breaking length km Burst factor Control TMP 46.8 1.42 0.947 Phlebia gigantea 41.8 0.58 0.476 Uh 38.2 0.72 0.469 Poria subacida 42.0 1.37 0.884 #21 44.0 1.63 0.941 Ganoderma sp 39.9 1.42 0.905 Trametes gibbosa 43.6 1.27 0.750 Tas-2 41.5 1.44 0.856 "Source: Fukuzumi ft al , 19»3 Chapter 2. LITERATURE REVIEW 49 Table 2.5: Physical properties of 60-g/m2 handsheets from unbleached aspen refiner mechanical pulps made from chips pre-treated with white-rot fungi. ^  Property measured Control Dichomitus squalens Phanerochaete chrysosporium Freeness, CSF 65 90 130 65 85 60 95 150 Burst index, kPa.m2/g 0.64 0.35 0.26 1.24 1.21 0.71 0.75 0.50 Tear index, mM.m2/g 1.66 1.69 1.74 4.24 4.30 2.00 2.14 2.52 Tensile index, Nm/g 25.4 21.3 19.1 36.0 36.6 30.0 30.6 27.2 Zero-span B.L.°, kPa/m 0.189 0.170 0.164 0.211 0.201 0.183 0.201 0.183 Density, kg/m3 422 408 368 388 382 419 405 370 Brightness,% 51.0 51.4 50.4 39.8 39.7 41.0 40.7 40.0 Opacity, % 97.9 96.8 97.1 95.2 97.1 97.7 98.5 97.8 Scattering coefficient, m2/kg 62.8 59.0 57.6 40.7 45.7 53.3 54.2 50.6 Drainage, s 54.2 16.5 10.9 14.4 13.0 17.0 13.3 8.1 "Breaking length. Source Myers et a! . 198B Chapter 2. LITERATURE REVIEW 50 Table 2.6: Bauer-McNett screen analysis of unbleached aspen refiner mechanical pulps made from chips pre-treated with white-rot fungi* Property measured Control Dichomitus squalens Phanerochaete chrysosporium Freeness, CSF 6 65 90 130 65 85 60 95 150 % Retained on 0.595 0.35 0.85 1.30 16.85 18.70 0.20 0.50 0.65 mm screen % Retained on 0.297 6.30 12.50 16.65 27.40 28.35 8.65 12.65 2.10 mm screen % Retained on 0.149 30.65 33.55 33.50 19.95 20.25 31.80 36.35 52.60 mm screen % Retained on 0.075 23.50 23.00 17.75 10.50 11.50 19.45 19.00 14.60 mm screen % Passing 0.075 39.20 30.10 30.80 25.30 21.20 39.90 31.50 30.05 mm screen Fiber length index 0.0696 0.0825 0.0838 0.1044 0.1162 0.0700 0.0815 0.0836 mm "Source: Myers et al, 1988. * Canadian standard freeness Chapter 3 M A T E R I A L S A N D M E T H O D S 3.1 M A T E R I A L S 3.1.1 WOOD SPECIES The lignocellulose material used in this study was refiner mechanical pulp (RMP) made from trembling aspen(Popu/us tremuloides Michx.). Trembhng aspen was selected for use in this study because it is the most abundant hardwood in Canada and it is severely underutilised (Rheade, 1983). The profile of this hardwood species as a pulp source in Canada is growing as more companies realize its value. Its main attractions are its relatively low lignin and high carbohydrate content, white colour and low density, making it ideal for mechanical pulping. 3.1.2 PREPARATION OF ASPEN REFINER MECHANICAL PULP A felled aspen tree, cut into bolts approximately 760 mm long x 300 mm diameter, was debarked with a machette. The tree was obtained near Williams Lake in the interior of British Columbia. The debarked logs were reduced to chips 2-5 mm thick x 4 - 5 cm wide x 4 - 5 cm long at Econo-Tech Services Ltd, New Westminster, B . C . Chips longer than 8 cm were ehminated using a Wennberg chip classifier at the Pulp and Paper Research Institute of Canada (Paprican), Vancouver. The aspen chips were refined in a Sprout Waldron 305 mm single-disc laboratory refiner ( Model 105-A, Plate pattern, D2A507) in two passes at Paprican. The plate gap, refining consistency, refining temperature and 51 Chapter 3. MATERIALS AND METHODS 52 wet power in each pass are given in Table A. 19. The moisture content of the pulp was reduced to about 35-45%(oven dry wt) by centrifuge-drying the pulp. The resulting pulp was stored in a cold room at 3° C until used. 3.1.3 FUNGUS Rigidoporus ulmarius (Fr.) Imaz. F 40a used in this study was obtained from the Center for Forest Mycolog3' Research, Forest Products Laboratory, U. S. D. A., Madison, Wisconsin. It was orignally isolated by Dr. Kiyowoo Aoshima in Japan. This isolate was selected for this study because of the reported capability of this fungus in Japan to completely delignify in the field (Kawase, 1962). The cultures were maintained on malt (1.5%)-agar(2%) slants at 3° C. The hyphal and growth characteristics of R. ulmarius have been described by Lombard et al. (1960). The hyphae of Rigidoporus ulmarius was 2.3 - 5 u, thin-walled, simple septate, with rare anastomoses (Lombard et al., 1960). These workers also noted that hyphae without contents are conspicuous in mounts and vesicular cells are present in moderate numbers; terminal with hyaline, thin walls; broadly ovoid to pear-shaped with the distal end being larger. Basiodiospores are produced in old cultures and are hyaline, globose to broadly ovoid, 4.5 - 7 X 4.5 - 5.5 p. (Lombard et al., 1960). R. ulmarius grows to form a white mat which is fine cobbwebby to downy; very thin to cleared and appressed around the inoculum. On malt agar, growth exhibited a temperature range of 18 - 34 0 C. Traces of growth was observed at 14 0 C and the optimum growth temperature was 28 0 C. The killing temperature range was 38 - 40 0 C (Lombard et al., 1960). According to Lombard et al. (1960), this fungus causes a 'soft white-rot of hardwoods'. The wood is reduced to a soft white condition rather quickly or at least there is no large amount of intermediate decay. An examination of a considerable number of herbarium specimens, collected mainly by Long (1914), indicated that the white-rot decay consisted mainly of Chapter 3. MATERIALS AND METHODS 53 wet stringy to soft woody tissue, which collapsed and dried to a parchment-like mass. This rapid disintegration apparantly accounts for the hollow condition associated with infections. The cultures employed by Lombard et ol. (1960) in their study included cultures of R. ulmarius received from Dr. K. Aoshima. 3.1.4 BASAL MEDIUM The basal medium used in this study was described by Kirk et al. (1978a). The compo-sition of the medium is given below : Macronutrients(per litre of distilled water). e Potassium dihydrogen phosphate(KH2P04) 0.2g o Magnesium sulfate(MgSO4.7H20) 0.05 g • Calcium chloride(CaCl2) 0.01 g o Mineral Solution containing the following per liter of distilled water) 1 ml 1. Nitriloacetate 1.5 g 2. Magnesium sulfate(MgSO4.7H20) 3.0 g 3. Manganese sulfate(MnSO4.H20) 0.5 g 4. Sodium chloride(NaCl) 1.0 g 5. Ferrous sulfate(FeSO4.7H20) 0.1 g 6. Cobaltous sulfate(CoS04) 0.1 g 7. Calcium chloride(CaCl2) 0.082 g 8. Zinc sulfate(ZnSO4.7H20) 0.1 g 9. Copper sulfate(CuSO4.5H20) 0.01 g Chapter 3. MATERIALS AND METHODS 54 10. Aluminium potassium sulfate(AlK(SO 4)2) 0.01 g 11. Boric acid(H3B03) O.Olg 12. Sodium molybdate(NaMO 4) O.Olg e Vitamin Solution per liter of distilled water 0.5 ml 1. Biotin 2 mg 2. Folic acid 2 mg 3. Thiamine hydrochloric acid 5 mg 4. Riboflavine 5 mg 5. Pyrodoxime hydrochloride 10 mg 6. Cyanocobalmine 0.1 mg 7. Nicotinic acid 5 mg 8. DL- Calcium panthonate 5 mg 9. p-Aminobenzoic acid 5 mg 10. Thiotic acid 5 mg o 2,2-dimethylsuccinate(10 mM) was used as a buffer to adjust the initial pH of the medium. 0 D-glucose 10 g 3.2 M E T H O D S The methods employed could be divided into three broad groups : 1. Mycological and physiological methods. Chapter 3. MATERIALS AND METHODS 55 2. Chemical analysis methods. 3. Physico-mechanical methods for testing the properties of the pulp and handsheets. Biological methods used to prepare fungal inocula, and those employed in the selective biodelignification experiments are described under mycological / physiological methods. A major aspect of this study was to determine the influence of fungal treatment of aspen RMP on its three main chemical components : lignin, cellulose, and hemicellu-loses. The methods employed to determine the chemical composition of aspen RMP are described under chemical analyses. Physico-mechanical testing of handsheets describe methods used to evaluate the phys-ical properties of control and fungal treated aspen RMP. The overall strategy adopted in this study was (1) Optimise selective biodelignification of aspen RMP by R. ulmarius; (2) Determine the effect of pH, nitrogen and temperature on the optimal cultural factors obtained in the previous experiment; and (3) To determine the physico-mechanical properties of pulp and handsheets made from fungal treated pulp under optimal conditions for selective biodelignification. Items (1) and (2) are described in detail under mycological/physiological methods and chemical analysis and item (3) is described under physico-mechanical methods for testing the properties of pulp and handsheets. To determine the optimal cultural conditions for selective biodelignification, three separate steps were required. The first one involved preparing the fungal inocula used in this study. This technique of fungal inocula preparation was chosen because of its previous use by another worker (Reid, 1985) and its adaptabihty to facilities available in our laboratory. The second step was to determine when growth of the fungus in the media became nutrient-limited, as indicated by cessation of growth. Lignin degradation by white-rot fungi is a secondary metabolic process. These fungi switch from primary to Chapter 3. MATERIALS AND METHODS 56 secondary metabolism during growth when one of the growth nutrients Hke nitrogen be-come Hmiting in the media. Thus, the strategy adopted here was to vary certain cultural factors (step 3) when the fungus had switched from primary to secondary metabolism (i.e. when growth of the fungus became growth limiting). A review of literature indicated that selective biodelignification of wood by white-rot fungi is influenced by the following cultural factors: aeration, hydrogen peroxide, mineral solution, tween 80, veratryl alcohol, pH, nitrogen, carbon co-substrate, agitation and temperature. However, a factorial experimental design encompassing all these factors would be so large as to be impracticable to carry out. Thus, the approach adopted here was to keep some factors constant whilst varying the others. The factors that were kept constant were (1) pH, 4.5 (2) nitrogen, 0 mM (except that present in the pulp) (3) carbon co-substrate, 1% glucose (4) agitation, static incubation conditions (5) temperature, 28° C. The factors that were varied were : e Type of aeration (air or oxygen); « Hydrogen peroxide (0 mM or 0.2 mM); • Mineral solution concentration ( 1 ml or 8 ml); a Tween 80 (0% or 0.05%[W/V]); and o Veratryl alcohol (3,4-dimethoxy benzyl alcohol, 0 or 2 mM). The levels of aeration, hydrogen peroxide and veratryl alcohol used in this study were based on values reported in the literature (Faison, 1985). The pH was kept at 4.5 because the optimal pH for a number of white-rot fungi is between 4 - 4.5. Since the primary aim of this study was to mimic lignin degradation in nature, no nitrogen was added to the media. Thus, the only nitrogen present in the media was that present in the aspen Chapter 3. MATERIALS AND METHODS 57 RMP. At the end of each experiment, the total residual carbohydrates and lignin of the fungal treated pulp were determined. The degree of preferential removal of lignin from aspen refiner mechanical pulp was measured by the the selective biodelignification index (SBI). The selective biodelignification index (SBI) was defined as the ratio of the total resid-ual carbohydrates to the total residual lignin and computed as : S B I ' TR where : CR=% total residual carbohydrates; and LR=% total residual lignin. The cultural factors that gave the highest selective biodelignification index were de-fined as the optimum cultural conditions. Employing the optimal cultural conditions, the individual effect of pH, nitrogen and temperature on selective biodelignification was investigated. In three separate experi-ments, the effect of pH, nitrogen and temperature on selective biodelignification under optimal cultural conditions was structured as a completely randomised design with four treatments (3.5, 4.5, 5.5, 6.5) for the pH experiment; four treatments (0, 1.2, 2.4, 24 mM) for the nitrogen experiment and three treatments (22, 28 and 32° C) for the temperature experiment. The replications in each experiment were three. The culture flasks were kept stationary. In the final experiment, the physico-mechanical properties of pulp treated with R . ulmarius under two cultural conditions were determined. The cultural conditions used were those that gave the highest (1) SBI (i.e optimal cultural conditions), treatment 21; and (2) Lignin degradation, treatment 24. Chapter 3. MATERIALS AND METHODS 58 To produce enough fungal-treated pulp for handsheet testing(approximately 30 g oven-dry weight basis), aspen RMP (300 + 20 mg) given each fungal treatment under each cultural conditions was rephcated 100 times. This was done rather than scaling up the experiment because of possible changes in the effect of the cultural factors due to changes in mass/volume relationships. 3.2.1 MYCOLOGICAL AND PHYSIOLOGICAL METHODS 3.2.1.1 PREPARATION OF INOCULUM Rigidoporus ulmarius was grown in 250 ml liquid Delong culture flasks containing 50 ml of medium. Flasks stoppered with Marton culture tube closures were sterilised in an autoclave at 121° C(dry conditions) for five minutes. The growth medium was prepared by adding 24 mM nitrogen (equi-molar amounts of ammonium nitrate and asparagine, Kirk et al. 1978) to the basal medium described in 3.1.4. The growth medium was buffered with 2,2' -dimethylsuccinate (10 mM) to give an initial pH of 4.5 and filter-sterilised through a 0.2 pm membrane filter fitted into a millipore filtration apparatus. A plug of the fungus growing on malt (1.5%)-agar(2%) slants was transferred to malt (1.5%)-agar(2.0%) plates. After a week of growth, a mycelial plug of R. ulmarius was aseptically transferred to the culture medium in the Delong culture flasks. After two weeks of growth at 28° C, the contents of each flask were filtered through a sterilised Whatman #1 filter paper under vacuum. The filter apparatus, consisting of Buchner funnel on a 500 ml filter flask fitted with rubber adapter and cotton-plugged side con-necting spout, was sterihsed by autoclaving at 121° C for 15 minutes(dry conditions). The filtered fungal mycelia of R. ulmarius were washed with either sterile distilled water or 2,2' -dimethylsuccinate buffer (10 mM, pH 4.5). The washed myceha were then blended in a given volume of either sterile distilled water or 2,2 -dimethylsuccinate (10 Chapter 3. MATERIALS AND METHODS 59 mM, pH 4.5) in a sterile Waring Blender for 30 seconds. Appropriate volumes of the blended fungal mycelia were dispensed by the pipette to culture flasks employed in subsequent experiments. 3.2.1.2 DETERMINATION OF THE TIME REQUIRED FOR GROWTH TO BECOME LIMITING The basal medium described in 3.1.4 was filter-sterilised as described in section 3.2.1.1. and 10 ml portions dispensed by sterile pipette into sterile 125 ml Erlenmeyer flasks. To each flask was added 1 ml of fungal inocula as described in 3.2.1.1. The fungal cultures were incubated at 28° C under static conditions. The experiment was structured as a completely randomised design with five treatments(3, 6, 9, 12 and 20 days of incubation) and five rephcations. The index of growth of R. ulmarius at the conclusion of incubation was the increase in mycehal dry weight. The pH at the end of the incubation period was also determined. To determine mycehal dry weight, the contents of each flask were filtered through pre-washed, tared Whatman #1 paper under vacuum. The fungal mycelia were washed with 50 ml of distilled water, freeze-dried for 24 hours, and weighed. Chapter 3. MATERIALS AND METHODS 60 3.2.1.3 SELECTIVE BIODELIGNIFICATION OF ASPEN RMP : OPTIMIZING THE EFFECT OF AERATION, HYDROGEN PEROXIDE, MINERAL SOLUTION CONCENTRATION, TWEEN 80 AND VERATRYL ALCOHOL The aim of this study was to evaluate the effect of some cultural factors on selective biodelignification of trembling aspen RMP. The cultural factors evaluated were :-• Type of aeration (flushing of culture flask with air or oxygen on day 6 and every third day thereafter). e Exogenous addition of hydrogen peroxide (0 mM or 0.2 mM) to the medium at the onset of limited growth, day 6; o Mineral solution concentration in the medium (1 ml or 8 ml per liter of growth medium); o Tween 80 concentration in medium (0% or 0.05%[W/V]); and e Exogenous addition of veratryl alcohol (0 or 2 mM) to the medium at the onset of limited growth, day 6. The following factors were kept constant throughout this phase of the study: e Initial pH (4.5) of the medium; o Nitrogen concentration of culture medium (0 mM); and o Incubation temperature of 28° C. All culture media were buffered with 10 mM 2,2'-dimethylsuccinate to pH of 4.5. Chapter 3. MATERIALS AND METHODS 61 The experiment was structured as a completely randomised design with five cultural factors namely, aeration, hydrogen peroxide, mineral solution concentration, tween 80 and veratryl alcohol. Each factor was examined at two levels to give a 25 factorial design with three replicates. Approximately 300 mg (oven-dry weight) of aspen RMP was weighed to the nearest milligram into 125 ml Erlenmeyer flasks. This provided the original oven-dry weight basis for subsequent calculation of percentage of chemical constituents in treated and untreated pulps. The flasks were stoppered with 2-hole rubber bungs fitted with two cotton-plugged glass tubes for gas inlet and outlet. The inlet glass tube extended into the flask about 1 cm above the culture fluid. The outlet glass tube was made flush with the bottom of the rubber bung. Each culture flask and its contents were sterilised at 121°C for ten minutes in an autoclave. Inocula of R. ulmarius were prepared as described in section 3.2.1.1. On a sterile bench, 10 ml of the appropriate culture medium was dispensed by a sterile pipette into each culture flask containing the aspen RMP to provide the various treatment levels. To each culture flask, 5 ml of fungal inocula was aseptically added and the flask stoppered. All flasks were incubated at 28° C under static conditions for two weeks. In addition to each treatment, there were two types of controls. For one control of two replicates, the culture flask contained the required cultural medium and aspen RMP but no fungal mycehum. This was used to determine the baseline carbohydrate and hgnin composition of the RMP for comparison purposes. The second type of control, with two replicates, contained the necessary cultural medium and fungal mycehum, but no aspen RMP. This was employed to see if fungal growth would take place in the absence of lignocellulose source. On the sixth day of incubation, sterile veratryl alcohol and hydrogen peroxide were added to flasks requiring such treatment, respectively, and flasks requiring air or oxygen Chapter 3. MATERIALS AND METHODS 62 were flushed as described by Keyser et al. (1978) and Kirk et al. (1978). Veratryl alcohol and hydrogen peroxide were added to give a final concentration of 2 mM and 0.2 mM respectively. The other treatments were spiked with equivalent amounts of sterile distilled water. At the end of air or oxygen flushing, both the inlet and the outlet tubes were closed by tightening the rubber tubing attached to both the inlet and the outlet glass tubes. Subsequently, each flask requiring air and oxygen treatment was flushed with the corresponding gas on every third day until the end of the experiment. At the end of the experiment, the contents of the flask were quantitatively transferred onto a Whatman glass microfibre filter paper; washed with about 300 ml distilled water and dried. Each sample of treated and control aspen RMP was then analysed for lignin and carbohydrates as described in section 3.2.2. Chapter 3. MATERIALS AND METHODS 63 3.2.1.4 SELECTIVE BIODELIGNIFICATION OF ASPEN RMP UNDER OPTIMAL CULTURAL CONDITIONS : EFFECT OF pH, NITROGEN CONCENTRA-TION AND TEMPERATURE Under optimal cultural conditions obtained in the previous experiment, the effect of pH, nitrogen concentration and temperature on selective biodelignification were studied separately.' For each study, a completely randomised experimental design with three replicates was used. Approximately 300 mg of oven-dried aspen RMP was weighed into a 125 ml Erlenmeyer flask and the flask was stoppered as described in section 3.2.1.3. Each stoppered 125 ml Erlenmeyer flask containing aspen RMP was sterihsed as described in section 3.2.1.3. Inoculum of Rigidoporus ulmarius was prepared as described in 3.2.1.1. On a sterile bench, 10 ml of culture medium (filter sterilsed) was dispensed aseptically and randomly to each sterihsed flask. To each flask, 5 ml of the blended mycelia (suspended in ster-ile water) was added and the flask was stoppered. For each study, there were three replications and two controls as described in section 3.2.1.3. For each study, the culture flasks were incubated at the appropriate temperatures for six days. On the sixth day, each flask was flushed with air and oxygen as prescribed by Keyser et al. (1978) and Kirk et al. (1978). At the end of the oxygen flushing, both the inlet and outlet tubes were closed with clamps. After this, each flask was flushed with oxygen on every third day thereafter until the end of the experiment. At the end of 14 days, the pH of the contents of each flask was measured. The contents were filtered onto glass filter paper and washed with 300 - 500 ml of distilled water and dried. Chapter 3. MATERIALS AND METHODS 64 3.2.2 CHEMICAL ANALYSIS OF PULP The control and fungal treated pulps were subjected to alcohol-benzene extraction (Tappi T12 os-75), ground in a Wiley mill to pass 20 mesh screen, and analysed chemically (Effland, 1977; Kirk et ai, 1972). Two main chemical analytical methods were employed in the analyses ofthe pulp samples, namely a modified Klason Hgnin analysis and borate-anion exchange high performance liquid chromatography (HPLC) of the resulting wood sugars. These techniques permitted the determination of both carbohydrate and lignin con-tent of the same pulp sample. The number of replicates for analysis varied as described under each selective biodelignification experiment. AH results were expressed as a per-centage of the original oven-dry weight of pulp before fungal treatment. 3.2.2.1 DETERMINATION OF LIGNIN CONTENT To determine the acid-insoluble Hgnin (Klason) content of the extractive-free pulp sam-ples, a modified procedure of Klason (Effland, 1977) was followed. After the digestion of the wood pulp with 5.5 ml of 72% sulphuric acid at room temperature for two hours, the resulting solution was diluted to 3% sulphuric acid. The 3% sulphuric acid solution was then subjected to a modified secondary hydrolysis by autoclaving it at 121° C and steam pressure of 20 psi for one hour. After autoclaving, the resulting solution was allowed to stand overnight. The insoluble residue (Klason lignin) was collected on a tared, medium porosity, sintered-glass crucible by vacuum filtration and dried at 105 + 2°C. The filtrate was diluted to 500 ml in a volumetric flask. The diluted filtrate was used to determine the acid-soluble Hgnin and carbohydrate content, respectively. The acid-soluble Hgnin was determined according to the' TAPPI useful method 250 Chapter 3. MATERIALS AND METHODS 65 (TAPPI, 1976d) using the diluted filtrate solution saved in the determination of the insoluble lignin. The absorbance of the diluted filtrate was measured at 205 nm using a Unicam SP 800 spectrophotometer. Assuming an extinction coefficient of 110 g/ml, the percentage soluble hgnin was determined from the the total filtrate volume (500 ml), original oven-dry weight of the pulp and dilution factor using the equation shown below : T . . _ (B x V x 100) LigninYo = —777——TTTT- y (1000 x W) where: B = lignin content (g/1000 ml); V = total volume of solution; and W = original oven-dry weight of pulp. B is given by : AjzD 110 where : A = u.v. absorbance at 205 nm; D = dilution factor; and 110 = absorptivity of soluble hgnin (extinction coefficient). 3.2.2.2 ANALYSIS OF CARBOHYDRATES The total sugar content of lignocellulosic materials is very important in many silvichem-ical products. Thus, a rehable method for the separation and quantification of wood sugars is vital. In the past, descending paper chromatography has been routinely used to separate wood sugars (Jeffrey et ai, 1960). The major drawback of this method is the length of analysis time. Analysis of wood sugars by gas chromatography (GC) of Chapter 3. MATERIALS AND METHODS 66 trimethyl silyl ether derivatives (Sweeley et al, 1963) is a well-known procedure. This technique is suitable for analysis of wood sugars (Brower et al, 1966), as is the GC sep-aration of wood sugars as alditol acetates (Crowell et al, 1967). However, the extensive sample preparation procedure and the sensitive chromatographic separating parameters render it unsuitable for routine wood sugar analysis. Various modes of high performance chromatography have been applied to the separa-tion of wood sugars. A reverse-phase partition HPLC using silica based columns applied to the analysis of wood sugars failed to achieve separation of glucose from mannose and galactose acceptable for quantification. Using two resin-based Bio-rad laboratory's HPX-87P columns in series, separation of free glucose, mannose, galactose, arabinose, and xylose, present in very unequal proportions in spent sulphite liquor, was achieved in sixty minutes (Wentz et al, 1982). Borate-complex anion-exchange HPLC has been successfully applied to the complete separation of wood sugars. Although, this technique is characterised by long analysis time (90 to 180 minutes), it has several advantages. Aqueous solutions can be analysed without pretreatment and pre-derivatisation as is the case with GC. Contaminants are also tolerated to a greater extent, and the wood sug-ars present in acidic and enzymatic hydrolysates of wood sugars can be separated and quantitatively measured in one run (Sinner et al, 1975). The borate-complex anion-exchange HPLC technique was adopted partly for the above stipulated reasons and also for the fact that such facilities existed in our labo-ratory. However, chromatographic conditions had to be developed which would result in optimal separation of all the wood sugars. 3.2.2.2.1 EQUIPMENT The equipment used consisted of a Varian HPLC system Model 5000 fitted with the following : Chapter 3. MATERIALS AND METHODS Figure 3.4: Schematic representation of cuprous ion with 2,2-bichinchoninate. Chapter 3. MATERIALS AND METHODS 68 • Varian u. v./visible light detector model 100; • Varian 60 vial capacity autosampler Model 8055; • Varian data acquisition system fitted with a thermal printer, two disk drives and cathode ray tube (CRT) display model CDS 402; s Kratos post-column derivatization pump model URS-050; • Rheodyne 50 u\ capacity loop; • Water bath; • 4.6 mm (I.D) x 20 cm column slurry packed with DA-X8-11 anion exchange resin supplied by the Durrum Chemical Co; o A menu driven rom-based program which permited the programming of several pa-rameters including flow rates; column temperature; gradient or ternary conditions; attenuation; paper speed; wavelength of u.v/visible light detector and equilibration of column; and o A Bio-rad micro-guard fitted with anion HO~ refill cartridge (40 mm x 4.6 mm). 3.2.2.2.2 C H E M I C A L S Chromatographic grade water was used to prepare the buffers, sugar standards and sugar samples used in this study. All the sugar samples applied to the column were filtered through a 0.45 ^ m millipore membrane filter fitted into the bottom of 5 ml capacity vials fitted with a Varian septum and capped. The sample vials were loaded into the autosampler. Each sample vial was identified by its rack number and vial number. The autosampler was fitted with four racks and each rack could carry fifteen vials. Chapter 3. MATERIALS AND METHODS 69 3.2.2.2.2.1 PREPARATION OF BORATE BUFFERS Borate buffer solution A was prepared as follows : 1. 92.745 g of boric acid was dissolved in 2800 ml of chromatographic grade water; 2. Potassium hydroxide pellets were added to [1] until the pH was about 0.5 units below 9.0. Then, the pH was adjusted to 9.0 with 1.0 M potassium hydroxide solution; 3. The solution was made up to 3000 ml; and 4. Buffer solution A was then filtered through a 0.2 pm membrane filter using a milhpore filtration apparatus before use on the HPLC. Borate buffer solution B was prepared as follows : 1. 37.098 g of boric acid was dissolved in 2800 ml of chromatographic grade water; 2. Potassium hydroxide pellets were added to [1] until the pH of the resulting solution was about 0.5 units below 8.2. Then, the pH of the solution was adjusted to 8.2 with 1.0 M potassium hydroxide solution; 3. The solution was then made up to 3000 ml; and 4. Buffer solution B was then filtered through a 0.2 pm membrane filter using a milhpore filtration apparatus before use on the HPLC. The post column reagent was prepared as indicated below : 1. 414.0 g of potassium carbonate was dissolved in 1500 ml of chromatographic grade water; Chapter 3. MATERIALS AND METHODS 70 2. Potassium carbonate (5.0 g), aspartic acid (3.7 g) and copper sulfate (1.0 g) were dissolved in 100 ml of type 1 water; 3. Solution [2] was added to solution [1]; 4. 2.0 g of sodium bicinchoninate(4,4 -dicarboxy -2,2 - biquinoline, sodium salt) was dissolved in 100 ml of type 1 water. The resulting solution was added to [3]; and 5. . The solution was made up to 2000 ml with chromatographic grade water. All reducing sugars are detectable by this post column reagent. Reducing sugars reduce C u " ^ to Cu"*" which subsequently reacts with aspartate-bicinchoninate complex to produce an intense blue colour. The structure of Cu"*" bichinoninate is given in Fig. 3.4. The post-column reagent was stable for about a week without any cooling or protective atmosphere. The reagent was mixed with the eluate in the ratio of 2:1 by the post-column pump. The intensity of the colour was measured at 570 nm. 3.2.2.2.3 PACKING OF THE HPLC COLUMN The chromatographic resin used in the separation and quantitative determination of the wood sugars was DA-X8-11 supphed by Durrum Chemical Co. DA-X8-11 was a spherical resin co-polymer of divinyl benzene cross-hnked polystyrene, substituted with quaternary amine functional groups. The degree of cross-linking was 8% and the particle size was 11 pm. The resin (10 g) was supphed in the fully hydrated chloride form which was the most stable form of the resin for shipping and storage. Approximately 7 g of the resin was used for packing each 4.6 mm x 250 mm column. To convert the anion exchange resin to the borate form, 7 g of the resin was dispersed in about 250 ml of chromatographic grade water and poured into a Buchner funnel fitted with a No. 1 Whatman filter paper. The resin was then washed with chromatographic Chapter 3. MATERIALS AND METHODS 71 p r o p o r t i o n a t i n g p u n p v a l v e v 1 2 b o r a t e b u f f e r a u t o - s a a p l e r g u a r d COIUBO a n a l y t i c a l colvmn w , t e r d e t e c t o r b a t h ^ p o e t - c o l u & n r e a g e n t p O « t - C O l LOBE r e a c t i o n • y e t e a T w a a t e Vlata 401 Figure 3.5: Schematic representation of Varian HPLC 5000. Chapter 3. MATERIALS AND METHODS from HPLC pump s t i r r e d - s l u r r y column packer waste column zero dead-volume f i t t i n g r e s i n s l u r r y magnetic s t i r r i n g bar Figure 3.6: Schematic representation of Packing Column system. Chapter 3. MATERIALS AND METHODS 73 water until chloride ions could not be detected. To test for chloride ions, a sample of the filtrate was acidified with a few drops of nitric acid and 1% silver nitrate solution added. A white precipitate indicated the presence of chloride ions. The resin again was consecutively washed with 1 M sodium hydroxide solution, chromatographic grade water, 0.5 M borate buffer (pH 9.0) respectively and filter dried and transferred to a clean beaker containing about 100 ml of 0.5 M borate buffer (pH 9.0). Using a small magnetic bar, the suspension was stirred gently and continously overnight. This allowed the resin to swell and also be converted to the the borate form. The column was packed using a Micrometrics stirred-slurry column packer, model 705 (Fig. 3.6.). The swollen, converted (borate) resin was poured into the slurry packer. The inlet of the slurry packer was connected to Reservoir B (0.5M, pH 9.0) and its outlet to the top of the empty column which carried no frit. A frit was placed at the bottom' of the column to prevent the resin from flowing out of the column. The magnetic stir bar placed at the bottom of the slurry packer was set in motion to ensure a homogenous slurry with the resin particles of different sizes evenly distributed throughout the slurry. This produced a rapid and an even packing of the column. Packing was commenced by pumping the borate buffer in Reservoir B at 8 ml per minute. Back pressure, as read from the status screen on the Varian HPLC LC5000, increased rapidly but was kept below 340 atmospheres by stepwise reduction of flow rate until, at a flow rate of 2 ml per minute, the back pressure stabilized. Flow rate was maintained at 2 ml per minute for about 2 hours. The pump was stopped and the back pressure allowed to drop below 2 atmospheres. The slurry packer was then disconnected from the Reservoir and the column. A frit was then placed on top of the column and installed in the appropriate place(after the injection loop). A rheodyne injection loop of 50 /xl was used. The column compartment had heat-control panels that allowed the column temperature to be varied from 20 to 75° C. Buffer from Reservoir B (0.2M, pH 8.2) was then pumped through the Chapter 3. MATERIALS AND METHODS 74 column for 24 hours. A back pressure of 190 - 220 atmospheres indicated good packing. Back pressures exceeding 290 atmospheres indicated poor packing or a plugged column (resulting from contamination). In the latter situation, the resin was pumped out and repacked. 3.2.2.2.4 SEPARATION OF WOOD HYDROLYSATES The separation and quantitative determination of the composition of wood hydrolysates required the development of HPLC conditions which gave satisfactory separation of these sugars. To achieve this, separation of a sugar solution containing six monosaccharides (rhamnose, mannose, galactose, arabinose, xylose and glucose) was attempted xising the gradient HPLC conditions recommended by Sinner et al. (1975). By varying the flow rate of the buffer, length of analysis time, temperature of column and equilibration time, the HPLC conditions indicated below were found to be optimum for the separation of the wood sugars. The column temperature was kept at 68° C. TIME(MINUTES) EVENT VALUE 0.0 Flowrate l.Oml/min 0.0 Reservoir Buffers A &B 0.0 SignahNoise Ratio 2 0.0 %B 100 0.0 %A 0 100.0 %A 100 100.0 %B 0 101.0 %B 100 101.0 %A 0 Basehne-basehne separations were achieved between all the sugars apart from arabi-nose and galactose. Chapter 3. MATERIALS AND METHODS 75 The next step was to assign the six monosaccharides to each of the peaks. This was achieved by running successive samples containing one more additional sugar than the previous run. The new peak appearing in the chromatogram was assigned to the new sugar added. This procedure was followed until all of the six sugar peaks had been assigned; The relative position of the peak of each sugar in the chromatogram is called relative retention time. Initially, calibration curves for external standards for each of the six sugars were prepared. Considerable variation was observed between runs. Consequently, the method requiring the use of internal standards was chosen. In the use of the method of internal standards, results are not affected by sample size or detector sensitivity (Varian Vista 401/2, 1981). Generally, the internal standard method is used for large, numbers of the same type of analysis where an internal standard can easily be incorporated into the sample. Several reducing sugars were screened for their suitability as the internal standard. Ribose and lyxose were found to be suitable. Baseline-basehne separation of either of these sugars was achieved. D-ribose was however chosen as the internal standard. The use of internal standard required the determination of response factors for each of the seven sugars. The primary usefulness of the relative response factor was that it was needed to compensate for variations in detector response to different molecule and instrument conditions. The relative response factor (RR.Fi) is given by equation shown below (Varian Vista 401/402, 1981): j^j^p, Amount i x Area Peak (internal standard) 1 Areai x Amount (internal standard) Amount = Amount of Sugar; Area; = Peak area of Sugary Chapter 3. MATERIALS AND METHODS 76 The relative response factor for each sugar was determined by preparing a standard solution containing 200 pg per ml of the following sugars : rhamnose, ribose, mannose, arabinose, galactose, xylose and glucose. Samples of the standard solution were run in duphcate and the peak areas of each sugar was measured. The relative response of each sugar was then determined using the above equation. 3.2.2.2.5 A TYPICAL SUGAR SEPARATION BY BORATE ANION HPLC All the sugar samples, standard sugar solutions, and wood hydrolysates, used in this study were filtered through 0.45 disposable pm millipore membrane filter into 5 ml capacity sample vials and then loaded into the autosampler. The samples were filtered to remove particulate matter which might otherwise damage the column. The HPLC could be run in batch (Method Ready) or automatic mode. In the batch mode, a sample was run consecutively. The automatic mode permited all the loaded samples to be run using different methods if so desired. In this study, all the analyses were done in the automatic mode. The column was equilibrated for about 20 minutes by running buffer B (pH 8.2, 0.2 M) through the column. After equilibration, the autosampler was automatically switched on and 50 pA ofthe solution was automatically injected into the solvent stream just before the guard column by means of the rheodyne loop injector. The latter permitted equal volumes of sample to be injected with high repeatability. The program was initiated and the elution gradient commenced. The eluted sugars were mixed with the post-column derivatizing solution and pumped through a teflon tube (I.D. 0.8 mm, 30 m long) coiled around an immersed beaker in a water bath kept at 96° C to allow for the reaction of the separated sugars with the post-column derivatizing agent. The derivatized sugars were then pumped through a u.v./visible light detector where the Chapter 3. MATERIALS AND METHODS 77 absorbance was measured at 570 nm and reported as peak areas. The u.v./visible hght detector was fitted with a visible filter. The retention times, area, resolution and other data collected during each run was stored and processed by the Vista 402 microprocessor data acquisition and processing center. The peak areas were computed by a digital intergrator. Raw data for each run (chromatogram) was stored on one sided 360 K non-dos formatted diskette. A typical output for a run showing the results of separation of a mixture of seven sugars made up of six common wood sugars and the internal standard, ribose is shown in Fig. A.31. The recorded retention times included the period of time (about 10 minutes) spent by the eluant in the post column derivatization system before reaching the detector. Each run took 101 minutes. After each run, the column was automatically equili-brated with buffer B for 20 minutes. Chapter 3. MATERIALS AND METHODS 78 3.2.2.2.6 ANALYSIS OF HYDROLYSATES OF CONTROL AND FUNGAL-TREATED ASPEN REFINER MECHANICAL PULP The sample used in this analysis was the acid-soluble fraction obtained from modified Klason lignin analysis (section 3.2.2.1). To 8 ml of the hydrolysate of control or fungal treated aspen RMP pulp, 2 ml of ribose solution (to give a final concentration of 100 fig per ml) was added as an internal standard in a volumetric flask. The resulting solution was filtered through a 0.45 um milhpore filter into labelled autosampler vials. All the wood hydrolysates were loaded into the autosampler and analysed as described in section 3.2.2.2.5. A chromatogram from the separation of hydrolysates of aspen refiner mechanical pulp is given in Fig. A.32. From the peak area and response factor of each wood sugar in each sample and the response factor and concentration of the internal standard, the percentage concentration of each wood sugar in each pulp sample was determined on an oven-dry weight basis of the original pulp before fungal treatment. The amount of each sugar in the sample was given by the equation shown below(Varian Vista 401/2, 1981) :. Peak Areai x RRFi x Amount (internal standard) x Dilution Factor Amount i = —-— — — — Peak Areayinternal standard) 3.2.2.3 DETERMINATION OF TOTAL NITROGEN CONTENT OF UNTREATED ASPEN REFINER MECHANICAL PULP The quantitative determination of total nitrogen in the aspen refiner mechanical pulp (RMP) involved its digestion followed by quantitative measurement of the ammonia produced. The quantification of ammonia produced was effected by the utihsation of the Chapter 3. MATERIALS AND METHODS 79 Berthelot reaction in which the ammonia produced is reacted with sodium phenate, and then followed by the addition of sodium hypochlorite to form a blue indophenol complex. Approximately 200 + 20 mg (oven-dried) of untreated aspen RMP was weighed to the nearest milligram into a pyrex test tube . Employing the Kjeldahl procedure (Twine et al, 1971), the pulp was wet digested with 5 ml of the digestion mixture using a marble as a stopper. There were five rephcates. The digestion mixture was prepared by adding 100 g of potassium sulfate and 1 g of selenium to 1 litre of .concentrated sulfuric acid. The solution was then heated on a hot plate until it became clear. The pyrex test tubes containing the aspen RMP were heated in a block digester until their contents became clear and colourless. The pyrex test tubes were then removed from the block digester and allowed to cool to room temperature. The contents were diluted to 100 ml and analysed using the indophenol blue method on a Technicon Autoanalyser II (Keeney et ai, 1982) 3.2.3 EVALUATION OF SOME PHYSICAL PROPERTIES OF CONTROL AND FUN-GAL TREATED ASPEN REFINER MECHANICAL PULP The primary objective of this study was to assess the effect of fungal treatments on the ph3rsico-mechanical properties of the trembling aspen RMP. Trembhng aspen RMP was given two fungal treatments numbers, 21 and 24 as described in section 3.2. Fun-gal treatment 21 produced the optimal selective delignification. Fungal treatment 24 corresponded to the fungal treatment which resulted in both the highest hgnin and car-bohydrate degradation. The control pulp for analysis differed from treated pulps only by the absence of fungal myceha in their culture flasks. To obtain approximately 30 g (oven-dried) of fungal treated and control pulps respec-tively, each treatment consisted of 100 rephcates of culture flask containing 300 + 20 mg (oven-dry weight) of trembling aspen RMP. At the end of each treatment, the pulps were washed with distilled water, filtered in separate Buchner funnel and stored in plastic bags Chapter 3. MATERIALS AND METHODS 80 in darkness at 3° C until used to prepare the test handsheets. The handsheet properties reported for the control pulps were the average of the values for the respective controls for fungal treatments 21 and 24. 3.2.3.1 PHYSICO-MECHANICAL TESTING OF HANDSHEETS Handsheets were made from both control and fungal treated aspen refiner mechanical pulps (fungal treatments 21 and 24). All the handsheets were made and tested at the Pulp and Paper Research Institute of Canada (Paprican), Vancouver, British Columbia. 3.2.3.1.1 FORMING HANDSHEETS FOR THE PHYSICAL TESTING OF PULP Samples of both treated and control pulps were oven-dried to determine their consis-tency. All the pulp samples were diluted in hot water and disintegrated to remove latency associated with dried mechanical pulps. After disintegration, the stock was screened to remove shives using the Pulmanac Shive Analyser. The pulp stock was diluted to 0.3% consistency and 400 ml of this stock slurry used to make each handsheet in a British sheet machine according to Tappi standard T 205 om-81 (TAPPI, 1981). The back water was recirculated. The handsheets were pressed under a constant pressure of 345 kPa in a standard British Sheet Press. The sheets so formed were conditioned according to Tappi standard T 402 os-70 (TAPPI, 1970) at 23+ 2° C and 50+ 2% relative humidity. 3.2.3.1.2 PHYSICAL TESTING OF HANDSHEETS Handsheets were tested in a controlled temperature (23+ 2° C) and humidity (50+ 2° relative humidity)(CTH) room according to TAPPI standard T 402 (TAPPI, 1970). The following physical properties of the handsheets were measured according to the Tappi Chapter 3. MATERIALS AND METHODS 81 standard T 220 os-71 (TAPPI, 1971) : o Brightness and Opacity were measured using the Technidyne Technibrite Micro TB-1C; • Freeness was determined using the Canadian freeness tester (TAPPI, 1985). • Zero-span tensile strength (both wet and dry) was measured using the Pulmanac Trouble Shooter zero-span Tester (Model V-S-167); • Tensile strength was determined using an Instron Tester (Model 4202)(TAPPI, 1976b); o Burst was determined using the Mullen Tester (TAPPI, 1976a); o Tear was evaluated using the TMI Elmendorf Tear Tester with 800 g pendulum (Model 83-10)(TAPPI, 1965); o Basis weight was determined using Mettler Gram-atic Balance Model 55 H26 (TAPPI, 1968); and o Caliper was measured using a TMI Precision Micrometer Series 400 Tester (Model # 49-60)(TAPPI, 1976e). Brightness of the handsheets was determined within 24 hours after conditioning the test handsheets according to TAPPI standard T 220 os - 71 (TAPPI, 1971). The pulp handsheets were were stored in the dark in the CTH room. Chapter 4 RESULTS A N D DISCUSSION 4.1 D E T E R M I N A T I O N OF T H E T I M E R E Q U I R E D F O R G R O W T H T O B E C O M E LIMITING Mycelia of Rigidoporus ulmarius F 40a increased rapidly in weight reaching a maximum on Day 6 (Table 4.7). After Day 6, mycelia weight remained appreciably constant, so it was considered as the day growth became limiting in the system. Day 6 was also considered to represent the begining of the secondary phase of growth, whereas days 1 to 5 represented primary growth. Since hgnin biodegradation is reported to commence at the onset of the secondary (idiophasic) phase, exogenous addition of hydrogen peroxide and veratryl alcohol to the growth media was made on Day 6. The concentration of glucose in the growth media was also monitored. Results shown in Table 4.8 indicate that the initial concentration of 1% glucose in the media fell rapidly to 0.65 % on the second day and then a little less rapidly to 0.48% on Day 6. After Day 6, the concentration of glucose decreased gradually to 0.32% on Day 20 . Since the maximum duration for all the other experiments was 14 days, the concentration of glucose in the growth media did not constitute a limiting factor throughout the course of the study. 82 Chapter 4. RESULTS AND DISCUSSION 83 Table 4.7: Time required for growth to become limiting in media totally deficient in nitrogen. R. ulmarius. Day Mycehal weight, mga 2 6.5b 4 9.1 0 11.2 8 10.4 20 8.5 "average of 5 replicates, ''standard deviation=+1.2. Table 4.8: Changes in the concentration of glucose in the growth media. R. ulmarius. Day % Concentration of glucose0 0 1.00 6 2 0.65 4 0.59 6 0.48 8 0.41 20 0.32 "average of 5 replicates, ^standard deviation=+-22. Chapter 4. RESULTS AND DISCUSSION 84 4.2 C U L T U R A L F A C T O R S I N F L U E N C I N G S E L E C T I V E BIODELIGNI-F I C A T I O N OF T R E M B L I N G A S P E N R E F I N E R M E C H A N I C A L P U L P Several studies (Kirk et al, 1987) have demonstrated that lignin biodegradation by a number of white-rot fungi, notably Phanerochaete chrysosporium and including Coriolus versicolor, is influenced by a number of nutritional and cultural factors including the fol-lowing: (a) Presence of co-metabohzable substrate; (b) High oxygen tension; (c.) Growth as mycehal mats rather than as submerged pellets in agitated cultures; (d) Buffer t3^ pe and pH of buffer; (e) Levels of mineral (trace) elements; (f) Veratryl alcohol; and (g) Tween 80. In this study, the following cultural factors were studied: (a) Aeration; (b) Hydrogen peroxide; (c) Mineral solution; (d) Veratryl alcohol; and (e) Tween 80. The results of this study (Tables 4.9) indicated that biodegradation of lignin, cellulose and hemicellulose, as measured by their residual contents, was influenced in a number of ways by all the cultural factors studied. This is borne out by many significant interactions observed in the statistical analysis of the results (Tables A.22 - A.25). These interactions obviously tend to make the interpretation of the results somewhat complex. In general, interpretation of third and higher order interactions are highly complex and, therefore, difficult to interpret. Thus, only the first and second order interactions were interpreted for their effects on biodelignification. Lignin, glucose and hemicellulose content of the control and fungal-treated pulps are given in Table 4.9. Their sums total to less than one hundred percent, and this could be ascribed to several possible sources. In this study, only the hgnin, glucose and xylose contents of both untreated controls and fungal-treated pulps were determined based on the unextracted original (O.D.) weights of these pulps. Certain extractive components were lost through the alcohol/benzene extraction. Other components simply were not determined, such as uronic acids, acetyl content, ash, pectin, and hemicelluloses other Chapter 4. RESULTS AND DISCUSSION 85 than xylan. In the case ofthe fungal-treated pulps, some ofthe carbohydrate components may have become oxidised by the fungus, and therefore not measured. In addition to the hydrolytic enzymes involved in biodegradation of carbohydrates, an oxidating enzyme has been detected which produces aldonic acids from cellobiose and cellodextrin (Fengel and Wegener, 1984). Finally, the fungal-treated pulps also suffered some weight reduction owing to metabolism of carbohydrate and hgnin components. However, weight loss in the system was not determined. Usually between 75 and 90% of the weight of original dry weight of the pulp was accounted for by the three major components, but this figure dropped as low as 52.4% in the case of treatment number 24. The sum of the three major components in the control pulp totalled 90.8%. This compares with Myers (1988) who reported the basic composition of unextracted trembling aspen as 19.6% hgnin, 52.3% glucose, and 18.1% xylose, for a total of 90.0%. In addition, he also reported 2.2% mannose. The summative chemical composition of trembling aspen, based on extractive-free wood, has been reported by Timmel (1969). This researcher, in addition to the lignin, glucose and xylose contents totalling 86.5% also reported ash, acetyl content and uronic acid contents as well as non-xylans. By doing so, he was able to account for the entire wood substance. The total lignin content reported in this thesis was the sum of Klason lignin and acid-soluble hgnin (2 - 3 %). With respect to the different fungal treatments, since no pattern could be discerned for the acid-soluble hgnin content, it was considered prudent to report only the total residual lignin content. The optimum treatment was defined in section 3.3.3 as the treatment which gave the highest selective biodelignification index (SBI). SBI referes to the ratio of the total residual carbohydrate content to residual lignin. Pulps given Treatment 21 (aeration[oxygen flushing]-hydrogen peroxide[0 mM]-mineral solution[8 X concentration]-veratryl alcohol[0 mM]-tween 80[0 %]) (Table A.21) Chapter 4. RESULTS AND DISCUSSION 86 exhibited the highest SBI value of 4.5 (Table 4.9). This pulp was characterised by residual lignin, glucose and xylose content of 14.8% (21.4%)*, 51.5% (50.8%) and 15.1 (18.6%)(Ta-ble 4.9), respectively. To select the optimum treatment, one-way analysis(fixed) of vari-ance with 32 levels (treatments) was done and Duncan's multiple range test was used to distinguish between levels (Tables 4.11). Thus Treatment 21 was chosen as the op-timum condition for the selective biodelignification of trembling aspen refiner mechani-cal pulp. The pulp given Treatment 24 (aeration[oxygen fiushingj-hydrogen peroxide[0 mM]-mineral solution[8 X concentration]-veratryl alcohol[2 mM]-tween 80[0.05 %])(Table A.21) exhibited the lowest residual lignin content of 10.6% (21.4%), the lowest resid-ual glucose content of 38.5% (50.8%) and the lowest residual xylose content of 3.3% (18.6%)(Table 4.9). Factorial analyses of the selective biodelignification index of the fungal treated pulp with respect to the five cultural factors are summarized in Table 4.10, showing the sig-nificant main and interacting effects at the 0.001, 0.01 and 0.05 levels of probabihty. The significant first order interacting effects are graphically depicted in Figs. A.7 - A.30. Similarly with respect to the five cultural factors, Table 4.10 also summarizes the factorial results of the biodegradation of lignin, cellulose and hemicellulose of the fungal-treated pulp as measured by their respective residual total lignin, glucose and xylose contents, by showing significant effects at 0.001, 0.01 and 0.05 levels of probabihty 1 Values in parenthesis represent mean residual values of controls Chapter 4. RESULTS AND DISCUSSION 87 Table 4.9: Selective biodelignification of aspen RMP: Chemical composition of aspen refiner mechanical pulp given 32 different fungal treatments. Percentages are based on original oven-dry weight of pulp before fungal treatment.0' Treatment SBI Total Glucose Xylose (see Table A.21) hgnin, % % % 1 3.2+1 21.2+.8 50.3+.5 18.3+.5 2 3.5+.2 20.0+.9 51.2+.4 19.8+.6 . 3 3.4+.4 18.4+2.1 45.6+1.1 15.2+.9 4 3.6+.3 16.2+1.9 45.7+.9 13.4+.8 5 3.5+.1 18.3+.6 47.7+.4 16.4+.6 6 3.8+.1 16.4+.7 47.6+.4 14.9+.8 7 3.2+.1 20.3+.6 50.0+1.1 15.7+1.9 8 3.9+.1 15.1 +.3 45.7+1.5 13.8+.8 9 3.1+.3 20.7+.8 48.6+1.1 14.7 +.4 10 3.1+.2 19.7+1.3 47.3+2.1 14.3+.6 11 3.2+.2 18.3+1.9 42.8+1.6 15.3+1.2 12 3.7+.1 18.5+1.1 51.5+1.7 17.1 + .3 13 3.5+.2 19.9+1.1 52.2+.4 17.1 + .4 14 3.4+.2 17.4+.6 39.9+1.5 19.5+1.7 15 3.9+.4 18.3+1.9 51.3+.7 20.1+1.6 16 3.2+.3 19.6+1.5 49.5+1.1 15.3+.5 17 3.4+.1 19.3+1.1 49.3+.9 15.0+.7 18 3.4+.1 17.5+.5 44.3+2.1 14.7+.9 19 3.8+.1 16.7+.3 48.3+2.1 15.1 + 1.7 20 3.8+.1 15.3+.8 43.7+2.2 16.8+.8 21 4.5+.3 14.8+1.0 51.5+1.5 15.1 + .6 22 3.5+.1 16.3+.6 44.3+1.2 13.6+1.1 23 4.2+.3 13.2+.9 42.4+1.5 13.2+1.1 24 4.0+.6 10.6+2.0 38.5+1.1 3.3+.8 25 3.5+.2 19.2+1.0 51.1+.6 16.4+.8 26 3.9+.3 17.3+1.1 50.6+1.7 17.0+.8 27 3.8+.2 17.5+1.0 50.2+.6 17.7+.6 28 3.3+.2 19.4+1.1 47.8+.8 15.9+.8 29 3.3+.4 18.2+2.0 45.2+1.9 14.9+.9 30 4.0+.2 15.4+.9 46.2+.6 15.2+.6 31 3.9+.5 16.4+1.9 46.5+.9 18.2+.5 32 3.5+.2 18.3+1.2 47.3+.4 18.5+1.0 Treated control6 3.2+.3 21.4+1.1 50.8+1.1 18.6+.8 "Values for all fungal treated pulp=average of 3 replicates. The notation, +SD, where the value of SD represents standard deviation will be used throughout this thesis. ^Average of controls for all treated pulps. Chapter 4. RESULTS AND DISCUSSION 88 Table 4.10: Summary of anovas showing levels of significance of cultural factors and the effect of their interactions on the chemical composition of pulp. Sources of Degrees Mean Mean Mean S B I b variation of residual residual residual freedom lignin glucose xylose Aeration 1 *** ^ ^ ^ ^: ^: ^ Hydrogen peroxide0 1 ^ ^ *** % ^ ^ ** Mineral solutiond 1 *** *** Veratryl alcohol6 1 ^ ^ tween 80/ 1 *** % ^ ^ Aeration x Peroxide 1 * ^ ^ ^ Aeration x Minsol 1 *** Aeration x Veralcoh 1 * * * Aeration x Tween 80 1 ** Peroxide x Minsol 1 * * ^ ^ Peroxide x Veralcoh 1 * * * ^ ^ ^ ^ ^ Peroxide x Tween 80 1 ^ % *** Minsol x Veralcoh 1 Minsol x Tween 80 1 *** *** Veralcoh x Tween 80 1 *** ^ ^ ^ Aeration x Peroxide x Minsol 1 * * Aeration x Peroxide x Veralcoh 1 * * Aeration x Peroxide x Tween 80 1 *** *** Aeration x Minsol x Veralcoh 1 *** Aeration x Minsol x Tween 80 1 *** Aeration x Veralcoh x Tween 80 1 Peroxide x Minsol x Veralcoh 1 * Peroxide x Minsol x Tween 80 1 * ** Peroxide x Veralcoh x Tween 80 1 *** ^ %^ Minsol x Veralcoh x Tween 80 1 *** Aeration x Peroxide x Minsol x Veralcoh 1 *** Aeration x Peroxide x Minsol x Tween 80 1 ^ Aeration x Peroxide x Veralcoh x Tween 80 1 *** * ** Aeration x Minsol x Veralcoh x Tween 80 1 ** Peroxide x Minsol x Veralcoh x Tween 80 1 * Aeration x Per x Minsol x Veralcoh x T80 1 Error 62 Corrected total 93 a * indicates significance at the 0.05 level of probability; ** indicates significance at the 0.01 level of probability; *** indicates significance at the 0.001 level of probability. ^Selective biodelignification index. cPeroxide, Per is equivalent to hydrogen peroxide. d Minsol is equivalent to mineral solution. eVeralcoh is equivalent to veratryl alcohol, •'tween 80 is equivalent is to T80. Chapter 4. RESULTS AND DISCUSSION Table 4.11: One-way analysis of variance for selective biodelignification index with respect to fungal-treated pulp : Duncan's multiple range test.0-10 Treatment number Mean selective biodelignification index 21 4.5 A 23 4.2 A B 24 4.0 B C 30 . 4.0 B C 31 4.0 B C D 8 3.9 B C D 15 3.9 B C D 26 3.9 B C D E 27 3.9 B C D E F 6 • 3.8 B C D E F G 20 3.8 B C D E F G 19 3.8 B C D E F G H 12 3.7 B C D E F G H I 4 3.7 C D E F G H I J 32 3.6 C D E F G H I J K 2 3.6 C D E F G H I J K 22 3.6 C D E F G H I J K 25 3.5 C D E F G H I J K 5 3.5 C D E F G H I J K 13 3.5 C D E F G H I J K 3 3.4 D E F G H I J K 14 3.4 E F G H I J K 17 3.4 E F G H I J K 18 3.4 F G H I J K 29 3.3 F G H I J K 28 3.3 F G H I J K 16 3.3 G H I J K 1 3.3 H I J K 7 3.2 H I J K 11 3.2 I J K Control 3.2 I J K 10 3.1 J K 9 3.0 K "Means with the same letter are not significantly different at 0.05 level of probability. hOne way anova : Treatment was significant at 0.001 level of probability. F=4.64; Error mean square=0.074; Treatment df=31 Chapter 4. RESULTS AND DISCUSSION 90 4.2.1 AERATION Aeration was a significant main factor affecting selective biodelignification index and the biodegradation of lignin, cellulose and hemicellulose. However, significant first and higher order interactions were observed between aeration and the other cultural factors, hydrogen peroxide, mineral solution, veratryl alcohol and tween 80. As result of these significant interaction effects, no interpretation could be made of the significant main aeration effect. The effect of aeration on lignin biodegradation is depicted in the significant second order interactions: aeration*hydrogen peroxide*veratryl alcohol and aeration*mineral so-lution*veratryl alcohol (Tables A.26 and A.27). The aeration*hydrogen peroxide*veratryl alcohol interaction (Table A.26), showed that (1) In the absence of exogenously added hydrogen peroxide, the higher level of exogenously added veratryl alcohol (2 mM) en-hanced hgnin biodegradation at both levels of aeration, especially at the higher level of aeration (oxygen flushing); and (2) In the presence of exogenously added hydrogen per-oxide (0.2 mM), addition of of veratryl alcohol produced greater hgnin biodegradation under oxygen flushing than under air flushing. This apparent inhibitory effect of exoge-nously added hydrogen peroxide and the positive moderation effect by oxygen flushing were also evident in the significant first order interaction: aeration*hydrogen peroxide (Fig. A.7). With respect to aeration>;:mineral solution*veratryl alcohol (Table A.27), the higher level of veratryl alcohol at both levels of mineral solution produced greater hgnin biodegra-dation under oxygen flushing than under air flushing. Similarly, mineral solution in the absence of exogenously added veratryl alcohol also produced greater biodegradation un-der oxygen flushing than under air flushing. The enhancement effect of the higher level of mineral solution on Hgnin biodegradation under oxygen flushing was also evident in Chapter 4. RESULTS AND DISCUSSION 91 the significant first order interaction, aeration*mineral solution (Fig. A.8). For carbohydrate biodegradation as measured by the respective residual glucose and xylose contents, aeration exhibited significant first to fourth order interactions. With respect to cellulose biodegradation as measured by glucose loss, five significant second order interactions were observed (Tables A.29 - A.33). The trend that could be discerned from the significant second order interactions: aeration*hydrogen perox-ide*mineral solution (Table A.29) and aeration*hydrogen peroxide*tween 80 (Table A.30) was that in the absence of exogenously added hydrogen peroxide and both levels of tween 80 and mineral solution, oxygen flushing produced greater glucose (cellulose) biodegrada-tion. The significant second order interaction, aeration*mineral solution*tween 80 (Table A.33), exhibited at both levels of mineral solution and at the higher level of tween 80 greater cellulose biodegradation under oxygen flushing. Similarly, for the significant sec-ond order interaction, aeration*mineral solution*veratryl alcohol (Table A.31), cellulose biodegradation was greatest at the higher level of mineral solution and the higher level of veratryl alcohol under oxygen flushing. Likewise, the greatest cellulose biodegrada-tion for aeration*veratryl alcohol*tween 80 (Table A.32) occured at the higher levels of both tween 80 and veratryl alcohol under oxygen flushing. All but one first order in-teraction was significant. With the addition of exogenous chemicals (except hydrogen peroxide), greater cellulose degradation was observed under oxygen flushing conditions for the first order significant interactions as shown in Figs. A.13 - A.16. Appreciable cel-lulose biodegradation also occured under air flushing for the interaction, aeration*tween 80 (Fig. A.16). For hemicellulose biodegradation as measured by xylose loss, the significant sec-ond order interactions involving aeration and mineral solution: aeration*mineral solu-tion*veratryl alcohol (Table A.38) and aeration*mineral solution*tween 80 (Table A.39) exhibited a trend whereby enhanced hemicellulose biodegradation was noted for pulps Chapter 4. RESULTS AND DISCUSSION 92 given treatments involving oxygen aeration and the higher level of mineral solution and tween 80 or veratryl alcohol. For the significant second order interaction: aera-tion*hydrogen peroxide*tween 80 (Table A.37), enhanced hemicellulose biodegradation was observed in the absence of hydrogen peroxide tween 80 under oxygen flushing. The lowest residual hemicellulose biodegradation 11.4%, was noted for the treatment com-bination: Aeration [oxygen flushing]-hydrogen peroxide [0 mM]-Tween 80[0.05%](Table A.37). The significant first order interactions observed were aeration*hydrogen peroxide, aeration*mineral solution, aeration*tween 80 (Figs. A.22 - A.24). These interactions demonstrated that except for the the exogenous addition of hydrogen peroxide, oxygen flushing in the presence of chemical additions also enhanced hemicellulose biodegradation as measured by the residual xylose contents. With respect to selective biodelignification index, three significant second order interactions: aeration*hydrogen peroxide*mineral solution, aeration*hydrogen perox-ide*veratryl alcohol and aeration*hydrogen peroxide*tween 80 (Tables A.43 - A.45) were observed. For first two of these interactions, the highest selective biodelignification in-dex was associated with the absence of exogenously added hydrogen peroxide and the higher level of mineral solution or veratryl alcohol under high oxygen tension. On the other hand, tween 80 did not appear to be an important factor in combination with aeration and hydrogen peroxide (Table A.45). This could be ascribed to greater carbo-hydrate biodegradation in relation to lignin produced by 0.05% of tween 80 under these conditions. Several previous studies have shown that bio-degradation of hgnin and cellulose is enhanced by enriched oxygen atmosphere. Exposure to higher oxygen levels cause an increase in the levels of partially reduced oxygen species (Freeman et al, 1981; Turvens et al, 1982). These reduced oxygen species have been implicated in lignin and cellulose Chapter 4. RESULTS AND DISCUSSION 93 degradation. Yang et al. (1980) have demonstrated that lignin biodegradation of ther-momechanical pulp in shallow liquid cultures was greatly enhanced under high oxygen tension. Enhanced biodegradation of both hgnin and cellulose under high oxygen tension has also been reported by Reid and Seifert (1980). The results emanating from this study showed that but for the presence of exogenously added hydrogen peroxide (0.2 mM), biodegradation of hgnin relative to the carbohydrate component was greater under high oxygen tension at the higher level of either veratryl alcohol or mineral solution. 4.2.2 HYDROGEN PEROXIDE The results of the factorial analysis indicated a significant main hydrogen peroxide effect for selective biodelignification index and biodegradation of lignin, cellulose and hemicel-lulose. However owing to interactions observed between hydrogen peroxide and the other cultural factors, no conclusions could be drawn on the main effects. The significant second order interaction, aeration*hydrogen peroxide*veratryl alcohol (Table A.26), showed that in the absence of exogenously added hydrogen peroxide, the higher level of veratryl alcohol enhanced lignin biodegradation at both levels of aeration, but especially at the higher level of aeration (oxygen flushing). For the second order inter-action, hydrogen peroxide*veratryl alcohol*tween 80 (Table A.28), lignin biodegradation was again generally greater in the absence of hydrogen peroxide, especially when veratryl alcohol and tween 80 was present. The significant first order interactions observed were aeration*hydrogen peroxide, hydrogen peroxide*mineral solution concentration; hydro-gen peroxide*veratryl alcohol and hydrogen peroxide*tween 80 (Figs. A.7, A.10, A.11 and A.12). In all these interactions, exogenous addition of hydrogen peroxide to treat-ments containing the other cultural factors respectively had a common effect of limiting hgnin biodegradation to a greater extent than the absence of hydrogen peroxide. Chapter 4. RESULTS AND DISCUSSION 94 For the biodegradation of cellulose, all five second order interactions (Tables A.29, 30, 34, 35 and 36) showed that maximum cellulose loss occured in the absence of hydrogen peroxide, while the other two cultural factors were at their higher levels. The three significant first order interactions: aeration*hydrogen peroxide, hydrogen peroxide*veratryl alcohol and hydrogen peroxide*tween 80 (Figs. A.13, A.17 and A.18) indicated that with the exception of the hydrogen peroxide*tween 80 interaction (Fig. A.18), exogenous addition of hydrogen peroxide under these significant first order inter-actions produced a smaller cellulose biodegradation when compared to similar treatments which lacked exogenously added hydrogen peroxide. An opposite effect was observed with hydrogen peroxide*tween 80 interaction, where cellulose biodegradation was enhanced by the higher level of tween 80 at both levels of hydrogen peroxide. However, under these conditions, cellulose biodegradation was greater at the lower level of hydrogen peroxide. With respect to hemicellulose biodegradation, the significant second order interactions were: hydrogen peroxide*mineral solution*tween 80 (Table A.41) and hydrogen perox-ide*mineral solution*veratryl alcohol (Table A.40). For these interactions, the lowest residual xylose contents for both interactions were given by the higher levels of the other two factors respectively in the absence of hydrogen peroxide (Tables A.40 and A.41). In this respect, these interactions gave results similar to the analysis of residue glucose. For the significant second order interaction: aeration*hydrogen peroxide*tween 80 (Table A.37), less hemicellulose biodegradation occured at both levels of aeration in the pres-ence of exogenously added hydrogen peroxide. Except for the hydrogen peroxide*mineral solution interaction (Fig. A.25), the trend of significant first order interactions observed for hemicellulose biodegradation(Figs. A.26 and A.27) was similar to that of cellulose biodegradation (Figs. A.13, A.17 and A.18). For selective biodelignification index, the significant second order interactions: Chapter 4. RESULTS AND DISCUSSION 95 aeration*hydrogen peroxide*mineral solution (Table A.43), aeration*hydrogen perox-ide*veratryl alcohol (Table A.44) and aeration*hydrogen peroxide*tween 80 (Table A.45) showed that at both levels of aeration, selective biodelignification index was in most cases reduced slightly by the exogenous addition of hydrogen peroxide. Similarly, for the hy-drogen peroxide*veratryl alcohol*tween 80 interaction (Table A.46), selective biodeligni-fication index in the absence of veratryl alcohol and tween 80 was generally reduced by the exogenous addition of hydrogen peroxide. One of the objectives of this study was to investigate whether the exogenous addition of hydrogen peroxide (0.2 mM) would effect an increase in hgnin biodegradation under both air and oxygen flushing. Several recent studies have shown that hydrogen peroxide is produced in ligninolytic fungal cultures and is required for the activity of the fungal ligninases. Hydrogen peroxide is produced by three main mechanisms. One of the primary mechanisms is the oxidation of glucose to 2-keto-gluconic acid and hydrogen peroxide by glucose-2-oxidase (Forney et al., 1982). This enzyme also oxidises xylose and 6-gluconolactone. A considerable amount of methanol is produced during lignin biodegradation in ligninolytic cultures by demethoxylation. Methanol oxidase then oxidises the methanol to formaldehyde and hydrogen peroxide. Hydrogen peroxide is also produced through the action of alcohol oxidase which is produced during the secondary metabolic phase of fungi. Exogenously added hydrogen peroxide seemed to depress both hgnin and carbohy-drate biodegradation especially when other cultural factors were at their lower level, lead-ing to important difference in SBI. The results presented here indicate that exogenous addition of hydrogen peroxide (0.2 mM) was attended by a general decrease in selective biodelignification index especially under air flushing. In the presence of enriched oxygen atmosphere, glucose is oxidised by glucose oxidase to hydrogen peroxide and gluconic acid. The apparent inhibitory action of exogenously added hydrogen peroxide, notably Chapter 4. RESULTS AND DISCUSSION 96 under air flushing, could be ascribed plausibly to some feed-back mechanism whereby the in-situ generation of hydrogen peroxide and other reactive species by the action of glucose oxidase on glucose or other mechanisms is inhibited by the exogenously added hydrogen peroxide. Similarly, Asther et al. (1986) have reported that concentrations of tween 80 higher than 0.1% completely inhibited ligninase production in ligninolytic cultures of P. chrysosporium. Ligninase has been reported to be easily overoxidised by excess hydrogen peroxide to an inactive form (Leisola et al., 1988). The apparent moder-ation of the inhibitory effect of exogenously added hydrogen peroxide by oxygen flushing could be ascribed to the possible involvement of 'active oxygen species' although some experimental evidence in support of this is contradictory. Although Faison and Kirk (1983) have shown that hydrogen peroxide production is temporarily and quantitatively correlated with ligninolytic activity of P. chrysosporium in shallow hquid cultures, recent studies by Tran et al. (1987) ofthe culture conditions that affected delignification of unbleached hardwood kraft pulp by P. chrysosporium reported a negative correlation between the extent of delignification (as measured by Klason hgnin analysis) and residual extracellular hydrogen peroxide, such that the lowest residual hydrogen peroxide concentration in the media corresponded to the highest delignification. Consequently, Tran at al. (1987) concluded that the residual hydrogen peroxide in the media could serve as an indicator of the extent of delignification by P. chrysosporium This is in agreement with a report made previously by Glen et al. (1983) that high Hgninolytic activity occured in low hydrogen peroxide concentration in media where hydrogen peroxide is produced continously in-situ by a hydrogen peroxide generating system. This might explain the observed apparent inhibitory effect of exogenously added hydrogen peroxide on biodegradation of lignin and carbohydrate components ofthe aspen RMP. Chapter 4. RESULTS AND DISCUSSION 97 4.2.3 MINERAL SOLUTION CONCENTRATION Factorial analysis results similarly indicated significant main effect and significant first to third order interactions and this obscured any interpretation on the main effect. With respect to lignin biodegradation, the significant second order interaction: aera-tion ^ 'mineral solution*veratryl alcohol (Table A.27), showed that the higher level of min-eral solution with one exception produced greater lignin biodegradation at both levels of aeration and veratryl alcohol. The significant first order interaction observed were, aera-tion*mineral solution; mineral solution*hydrogen peroxide and mineral solution*veratryl alcohol. These interactions are shown in Figs. A.8, A. 10 and A.9 respectively. The trend observed in all the interactions was similar and indicated that the addition of mineral so-lution (8 X concentration) to treatments containing the lower level of hydrogen peroxide and the higher level of veratryl alcohol (2 mM) or aeration (oxygen flushing) produced greater lignin biodegradation. Although lignin biodegradation was greater in the absence of hydrogen peroxide, lignin biodegradation was also enhanced in the presence of exoge-nously added hydrogen peroxide when mineral solution (8 X concentration) was added to the treatment (Fig. A. 10). For the biodegradation of cellulose, significant second order interactions: aeration*mineral solution*tween 80 (Table A.33) and aeration*mineral solution*veratryl alcohol (Table A.31) showed that at the higher level of aeration and both levels of tween 80 or veratryl alcohol, the higher level of mineral solution produced greater cellulose biodegradation. Also for the second order interactions: hydrogen peroxide*mineral solution*tween 80 (Table A.35) and hydrogen peroxide*mineral solution*veratryl alcohol (Table A.34), the greatest cellulose biodegradation was produced by the higher level of mineral solution in the absence of hydrogen peroxide and presence of veratryl alcohol or tween 80. The Chapter 4. RESULTS AND DISCUSSION 98 observed significant first order interactions were aeration*mineral solution; mineral solu-tion* veratryl. alcohol and mineral solution*tween 80 (Figs. A.14, A.19 and A.20). In the presence of mineral solution (8 X concentration), cellulose biodegradation was greatly enhanced by the higher level of aeration (oxygen flushing) or tween 80 (0.05%) but to a lesser extent by the exogenous addition of veratryl alcohol' (2 mM). With respect to hemicellulose biodegradation, the significant interactions: aera-tion*mineral solution*tween 80 (Table A.39) and aeration*mineral solution*veratryl al-cohol (Table A.38), the higher level of mineral solution produced greater hemicellulose biodegradation at either level of tween 80 or veratryl alcohol under oxygen flushing. For the significant hydrogen peroxide*mineral solution*veratryl alcohol (Table A.40)and hy-drogen peroxide*mineral solution*tween 80 interaction (Table A.41), the higher level of mineral solution produced the greatest hemicellulose biodegradation in the absence of hydrogen peroxide, but in the presence of either veratryl alcohol or tween 80. It was also evident from the significant second order interaction, mineral solution*veratryl alco-hol*tween 80 (Table A.42), that the greatest hemicellulose biodegradation was produced by the treatment representing higher levels of all cultural factors. The observed signif-icant first order interactions were mineral solution*aeration, mineral solution*hydrogen peroxide and mineral solution*tween80 (Figs. A.23, A.25 and A.28). Similar to cellulose biodegradation, hemicellulose biodegradation was greatly enhanced by aeration (oxygen flushing) or tween 80 (0.05%) in the presence of the higher level of mineral solution. An opposite effect was produced by the addition of hydrogen peroxide (0.2 mM). For selective biodelignification index, the significant second order interaction of aeration*hydrogen peroxide*mineral solution (Table A.43), indicated that at both levels of aeration and hydrogen peroxide, the higher level of mineral solution produced higher selective biodegradation index than its lower level. The highest selective biodelignification index was associated with the treatment: aeration [oxygen flushingj-hydrogen peroxide[0 Chapter 4. RESULTS AND DISCUSSION 99 mM]-mineral solution [8 X concentration] (Table A.43). The mineral (trace element) solution contained twelve different compounds (Section 3.1.4), the most notable trace elements being manganese (II), iron (II), aluminium (III) and calcium (II) cations. Wood decayed by several white-rot fungi have been shown to ac-cumulate manganese as manganese dioxide (Blanchette, 1984). X-ray fluorescence spec-troscopic analysis of sound, incipient and advanced decay in trembling aspen wood has also demonstrated the concentration of calcium, iron, and manganese (Dawson-Andoh et al., 1985) in zones of decay. Copper and managanese have also been reported to engender an increase in Hgnin biodegradation in hgninolytic cultures (Blanchette, 1984; Kirk et al, 1986). The concentration of manganese has a marked influence on Hgnin biodegradation by Lentmus edodes (Leatham, 1986). Manganese (II) and hydrogen peroxide-requiring-peroxidase have recently been reported by Kuwahara et a/.(1984). Several mechanisms for the the activity of this enzyme have been proposed (Glen et al, 1986). Glen et al. ( 1985, 1987) have suggested that the manganese peroxidases oxidize Mn (II) to Mn (III), which in turn oxidizes the organic substances. However, Kirk et al. (1987) have suggested that manganese peroxidases appear to function as phenol-oxidizing enzymes and perharps participate in hydrogen peroxide production. In Hgninolytic cultures of P. chrysosporium containing veratryl alcohol, the presence or absence of a sevenfold increased concentration of trace element solution (Kirk-Schultz medium [1979] containing the same amounts of magnesium, manganese, cobalt, copper, iron, aluminium, zinc, and molybdenum salts used in this study) gave a 4.1 and 1.9-fold increase in lignin degradation respectively. The results of this study have similarly demonstrated that the addition of eight-fold increased concentration of mineral solution (Kirk-Schultz) also increased selective biodehgnification of Hgnin under oxygen flushing in the absence of hydrogen peroxide and tween 80. Chapter 4. RESULTS AND DISCUSSION 100 4.2.4 TWEEN 80 Tween 80 exhibited significant main effects and interactions with respect to biodegrada-tion of hgnin, cellulose and hemicellulose. As result of these interactions, no interpreta-tion could be made on the significant main effects. For lignin biodegradation, the significant second order interaction, hydrogen per-oxide*veratryl alcohol*tween 80 (Table A.28), showed that in general the higher level of tween 80 at both levels of hydrogen peroxide and veratryl alcohol enhanced lignin biodegradation. The first order interaction between tween 80 and hydrogen peroxide (Fig. A.12) was significant at 0.01 level of probability and showed that the higher level of tween 80 enhanced the biodegradation of hgnin at both levels of hydrogen peroxide but to a greater extent in the absence of hydrogen peroxide. Factorial analysis (Table 4.9) for cellulose biodegradation indicated that for the sig-nificant second order interactions, aeration*veratryl alcohol*tween 80 (Table A.32) and aeration*mineral solution*tween 80 (Table A.33), the higher level of tween 80 enhanced cellulose biodegradation at both levels of veratryl alcohol or mineral solution under oxy-gen flushing. In the second order interaction, aeration*hydrogen peroxide*tween 80 (Ta-ble A.30) only the single combination of oxygen flushing and tween 80 in the absence of hydrogen peroxide was effective in biodegrading cellulose. For the significant second order interaction, hydrogen peroxide*mineral solution*tween 80 (Table A.35) and hy-drogen peroxide*veratryl alcohol*tween 80 (Table A.36), cellulose biodegradation was maximized in the absence of hydrogen peroxide by presence of tween 80 combined with the level of either veratryl alcohol or mineral solution. Significant first order interac-tions: aeration*tween 80 (Fig. A.16), hydrogen peroxide*tween 80 (Fig. A.18), mineral solution*tween 80 (Fig. A.20), and tween 80*veratryl alcohol (Fig. A.21) were observed. The general pattern that could be discerned from these first order interactions indicated Chapter 4. RESULTS AND DISCUSSION 101 that the higher level of tween 80 produced greater cellulose biodegradation at the higher level of the other cultural factors (Fig. A.20). However for the hydrogen peroxide*tween 80 interaction (Fig. A. 18), the higher level of tween 80 enhanced cellulose biodegradation in the absence of exogenously added of hydrogen peroxide. With respect to hemicellulose biodegradation, the significant second order interac-tions, aeration * hydrogen peroxide*tween 80 (Table A.37) and aeration* mineral solu-tion*tween 80 (Table A.39), showed that hemicellulose biodegradation was particularly enhanced by the presence of tween 80, and the absence of hydrogen peroxide under oxy-gen flushing, or by the combination of tween 80 and high mineral content under oxygen flushing. Similarly, the significant second order interaction, hydrogen peroxide* min-eral solution*tween 80 (Table A.41) showed that in the absence of hydrogen peroxide, hemicellulose biodegradation was enhanced by the higher level of both tween 80 and mineral solution. The significant second order interaction, mineral solution*veratryl al-cohol*tween 80 (Table A.42) did not show an effect of tween 80's presence, except when both the other cultural factors were at their higher level. Factorial analysis also revealed significant first order interactions between tween 80 and other cultural factors, aera-tion, hydrogen peroxide, mineral solution and veratryl alcohol (Fig. A.24, A.26, A.28 and A.29). Except for hydrogen peroxide*tween 80 interaction (Fig. A.26), treatments containing tween 80 (0.05%) and the higher level of other interacting cultural factors produced greater hemicellulose biodegradation. With respect to selective biodelignification index, factorial analysis showed that for the significant second order interactions: aeration*hydrogen peroxide*tween 80 (Table A.45) and hydrogen peroxide*veratryl alcohol*tween 80 (Table A.46), no clear pattern of the effect of tween 80 on selective biodelignification index could be discerned.The significant first order interaction, aeration*tween 80 (Fig. A.30), showed that under air flushing, selective biodelignification index was enhanced by addition of tween 80 (0.05%). Chapter 4. RESULTS AND DISCUSSION 102 Under oxygen flushing, the opposite effect was observed. The addition of non-ionic surfactants such as sorbitan polyoxyethylene monooleate (tween 80) to ligninolytic cultures has recently been shown (Jager et al., 1987) to effect an increase in lignin degradation although the underlying mechanism is poorly understood. Tween 80 is said to effect increase in lignin biodegradation in ligninolytic cultures by promoting both uptake and exit of compounds for the cell through modification of plasma membrane permeability (Reese et al, 1969; Takao et al, 1984). Several mechanisms involving changes in rapid metabolism have been proposed (Ashter et al, 1987). However, the mechanism by which tween 80 effected an increase in hgnin degradation might to be more complex than this. It has been demonstrated that non-fatty acid containing a surfactant like chaps [ 3-(colamidopropyl)-dimethyl-aminonio 1-propane-sulphamate] also engenders increase in hgnin biodegradation in hgninolytic cultures. The effect of tween 80 on cellulose and hemicellulose degradation is not reported in the hterature. The results emanating from this study indicated that tween 80 enhanced selective biodelignification of lignin under air flushing. However, the opposite effect was observed under oxygen flushing. The latter effect could be attributed to increased biodegrada-tion of carbohydrates under these conditions. Tween 80 seemed to enhance lignin and carbohydrate reduction at the same rate, with the result that its effect on selective biodelignification index was not radically different than control samples. 4.2.5 VERATRYL ALCOHOL The results of the factorial analysis for veratryl alcohol like the previous cultural factors exhibited both significant main effects and first and higher order interactions, for selective biodelignification index. As result of these interactions, no clear conclusions could be drawn on the main effects. Chapter 4. RESULTS AND DISCUSSION 103 With respect to hgnin biodegradation, the significant second order interaction: aera-tion*mineral solution*veratryl alcohol (Table A.27), showed that the exogenous addition of veratryl alcohol at both levels of mineral solution enhanced lignin biodegradation under oxygen flushing. However, for the significant second order interaction, aeration*hydrogen peroxide*veratryl alcohol (Table A.26), hgnin biodegradation was enhanced by the addi-tion of veratryl alcohol at lower level of hydrogen peroxide and at both levels of aeration. This effect was more pronounced under oxygen flushing. For the significant second order interaction, hydrogen peroxide*veratryl alcohol*tween 80 (Table A.28), lignin biodegra-dation was enhanced (with one exception) by the addition of veratryl alcohol at both levels of hydrogen peroxide and tween 80. As shown in Figs. A.11 and A.9, two significant first order interactions, veratryl alcohol*hydrogen peroxide and veratryl alcohol*mineral solution were observed. The addition of veratryl alcohol (2 mM) produced greater lignin biodegradation at both concentrations of mineral solution but this effect was greater for the higher level of mineral solution (8 X concentration)(Fig. A.9). In presence of exoge-nously added hydrogen peroxide, lignin degradation was only slightly enhanced by the addition of veratryl alcohol, but definitely enhanced when the hydrogen peroxide was absent. With respect to cellulose degradation, significant first to third order interactions were observed. The significant second order interactions are depicted in Tables A.31, A.32, A.34 and A.36. The interactions, aeration*mineral solution*veratryl alcohol (Table A.31) and aeration*veratryl alcohol*tween 80 (Table A.32), indicate that under high oxygen tension, the effect of veratryl alcohol on cellulose biodegradation was enhanced by the addition of tween 80 (0.05%) or mineral solution (8 X concentration). Under the latter conditions, the highest cellulose biodegradation was given by the treatment combination: aeration [oxygen flushingj-mineral solution[8 X concentrationj-veratryl alcohol[2 mM]. For the significant second order interactions, hydrogen peroxide*mineral solution*veratryl Chapter 4. RESULTS AND DISCUSSION 104 alcohol (Table A.34) and hydrogen peroxide*veratryl alcohol*tween 80 (Table A.36), cellulose biodegradation was enhanced by the addition of veratryl alcohol at both levels of either tween 80 or mineral solution at the lower level of hydrogen peroxide. The significant first order interactions observed included veratryl alcohol*aeration (Fig. A.15), veratryl alcohol*hydrogen peroxide (Fig. A.17), veratryl alcohol*mineral solution (Fig. A.19) and veratryl alcohol*tween 80 (Fig. A.21). At both levels of the cultural factors(except the higher level of hydrogen peroxide) involved in the first order interactions, addition of veratryl alcohol produced greater cellulose degradation. The opposite effect was observed in the presence of exogenously added hydrogen peroxide. In the case of hemicellulose biodegradation, the second order interaction, aeration*mineral solution*veratryl alcohol (Table A.38), hemicellulose biodegradation was enhanced by the addition of veratryl alcohol (2 mM) to treatments containing the higher level of mineral solution under oxygen flushing.For the hydrogen peroxide*mineral solution*veratryl al-cohol (Table A.40), the higher level of veratryl alcohol at both levels of mineral solution in the absence of hydrogen peroxide enhanced hemicellulose biodegradation. With re-spect to the interaction, mineral solution*veratryl alcohol*tween 80 (Table A.42), only the higher levels of all the cultural factors resulted in a particularly greater hemicellulose biodegradation. For the first order interaction (Fig. A.29), addition of veratryl alco-hol (2 mM) to tween 80 (0.05%) enhanced hemicellulose biodegradation. Exogenously added hydrogen peroxide to treatments containing veratryl alcohol (2 mM) produced the opposite effect. For selective biodelignification index, the significant second order interactions ob-served were aeration*hydrogen peroxide*veratryl alcohol and hydrogen peroxdde*veratryl alcohol*tween 80 (Tables A.44 and A.46). Under the aeration*hydrogen peroxide*veratryl alcohol interaction (Fig. A.44), the presence of veratryl alcohol at both levels of aera-tion in the absence of hydrogen peroxide increased the selective biodelignification index. Chapter 4. RESULTS AND DISCUSSION 105 For this interaction, the highest selective biodelignification index was produced by the treatment combination: aeration [oxygen flushingj-hydrogen peroxide[0 mM]-veratryl al-cohol[2 mM]. Similarly, for the hydrogen peroxide*veratryl alcohol*tween 80 interaction (Table A.46), selective biodelignification index, was enhanced by the presence of veratryl alcohol and tween 80, in the absence of hydrogen peroxide. The association of veratryl alcohol with hgnin biodegradation has been observed in nitrogen limited cultures of P. chrysosporium (Faison et al, 1986; Kirk et al, 1986 and Liwicki et al, 1987), Coriolus versicolor (Kawai et al, 1987), et al; 1987), Pycnoporus cinnabarius, Phlebia radiata (Hatakka et al, 1986) and a Trametes spp. In P. chrysospo-rium, veratryl alcohol is synthesized from phenylalanine via 3,4-dimethoxycinnamyl al-cohol. The latter is then oxidized to l-(3,4-dimethoxyphenyl) glycerol which is then oxidised to veratrylaldehyde (Shimada et al, 1981). However, in vivo, those oxidations are catalyzed by ligninase. Veratrylaldehyde is also reduced to veratryl alcohol in vivo (Shimada et al, 1981). Veratryl alcohol also stimulates the oxidation of other compounds by ligninase (Habe et al, 1985; Harvey et al, 1986). Exogenously added veratryl alcohol hastens the appearance of the ligninolytic system ([14C] lignin —> uCOi) as part of secondary metabolism. In the absence of exogenously added veratryl alcohol, the hgninolytic system and biosynthesized veratryl alcohol ap-peared simultaneously in cultures of P. chrysosporium. In the latter, exogenously added veratryl alcohol appeared to increase the titer of ligninase (Faison et al, 1985; Leisola et al, 1985) probably via induction (Faison et al., 1985). It has been suggested by Leisola et a/(1984) that veratryl alcohol is the normal inducer of the hgninolytic system. Several studies (Tien et al, 1986; Harvey et al, 1986) indicate that the oxidation of veratryl alcohol by ligninase is involved in lignin biodegradation. Results of studies done by Tien et al, (1986) indicate that hydrogen peroxide first reacts with the enzyme. The oxidized enzyme then in turn reacts with veratryl alcohol. Veratryl alcohol was Chapter 4. RESULTS AND DISCUSSION 106 also found to be oxidized at the active site by direct oxygenation or via two rapid one-electron oxidations. However. Harvey et al, (1986) have suggested that veratryl alcohol is oxidized to a radical which then acts as a diffusible one-electron oxidant to interact with other substrates. Kirk et al, (1987) have proposed that veratryl alcohol acts to protect the enzyme from inactivation or as a electron relay at the active site or alter the enzyme conformation. The results of this study indicate that the addition of 2 mM veratryl alcohol at both levels of aeration in the absence of exogenously added hydrogen peroxide was associ-ated with enhanced selective biodelignification of lignin. Thus, the results obtained in this study are by and large in agreement with the reported effect of veratryl alcohol in ligninolytic cultures of other white-rot fungi. 4.2.6 GENERAL SUMMARY FROM FACTORIAL ANALYSIS The primary objective of this study was to maximize lignin biodegradation and minimize carbohydrate biodegradation. Consequently, one would' select from our current study the treatment associated with the maximum selective biodelignification index. One would expect with respect to maximization of selective biodelignification index the following factors: oxygen flushing, 8 X mineral solution, exogenously added hydrogen peroxide would be very important. Exogenously added tween 80 appeared to affect equally hgnin and carbohydrate biodegradation. The latter in some situations seemed greater. This was reflected by Treatments 21 - 24 although the observed interactions tended to obscure this interpretation. Chapter 4. RESULTS AND DISCUSSION 107 4.3 S E L E C T I V E BIODELIGNIFICATION: E F F E C T OF p H , N I T R O G E N A N D T E M P E R A T U R E L E V E L S O N T R E M B L I N G A S P E N R M P U N -D E R O P T I M U M C U L T U R A L CONDITIONS 4.3.1 pH The effect of four levels of initial pH : 3.5, 4.5, 5.5 and 6.5 on the biodegradation of lignin, cellulose, xylose and selective biodelignification index under the optimum conditions ob-tained in section 3.3.3 was studied. With respect to lignin biodegradation, the effect of pH was found to be significant at 0.001 level of probability (Table A.48). Duncan's multiple range test showed that hgnin biodegradation at the four different pH levels was significantly different from each other (Table A.49). Initial pH of 4.5 exhibited the lowest residual lignin content (15.2%) and therefore the highest lignin biodegradation (Table 4.12). The least lignin biodegradation was observed at initial pH of 6.5 (Table 4.12). Cellulose biodegradation at the four different pHs used was significantly different at 0.05 level of probability (Table A.50). Duncan's multiple range test showed cellulose biodegradation was characterised by three groups (Table A.51). Cellulose biodegradation at pH of 6.5 was significantly less than from that at pH 3.5, but not significantly different from that at pH levels of of 4.5 and 5.5 (Table A.51). Hemicellulose biodegradation as measured by residual xylose content at the four dif-ferent pH levels was also significant at 0.001 level of probability(Table A.52). Duncan's multiple range test showed that residual xylose content at pH of 3.5 was not significantly different from that at pH of 4.5, but significantly less than that at pH 5.5 and 6.5 re-spectively (Table A.53). Hemicellulose biodegradation at pH 5.5 was not significantly different from that at the pH level of 6.5. The greatest hemicellulose biodegradation occured at pH of 4.5 (16.0% residual hemicellulose content, Table 4.12). Chapter 4. RESULTS AND DISCUSSION 108 Selective biodelignification index was found to be significant at 0.001 level of prob-ability (Table A.54). The highest SBI, 4.4, occured at pH of 4.5 (Table 4.12). Duncan's multiple range test showed that SBI at each pH level was significantly different from each other (Tabel A.55). The pH of the growth medium has been shown to be critical for lignin biodegradation by Phanerochaete chrysosporium (Kirk et al, 1978). On the contrary, Reid (1985) has demonstrated that the biodelignification of aspen by Merulius iremellosus is affected only to a small extent by the initial pH of the growth media over a long period (42 days). The results indicated that lignin degradation at the four levels of pH used was sig-nificantly different. The greatest hgnin biodegradation (residual lignin content of 15.2%) occurred at pH 4.5 and the least at pH 6.5. Biodegradation of the carbohydrates indi-cated that for cellulose, the lowest glucose residual content (48.6%, Table 4.12) occurred at initial pH of 3.5. In the case of the hemicelluloses, the lowest residual xylose content (16.0%) was observed at pH of 4.5. For each of hgnin, cellulose and hemiceUulose, the least biodegradation was observed at pH of 6.5. By the conclusion of the incubation period, the pH was reduced by about one unit , except in the highest case, where the reduction was only from 6.5 to 6.1. (Table 4.12). The effect of the initial pH of the culture media on biodelignification of aspen wood by M. tremellosus has been shown to be related to timing of lignin biodegradation as demonstrated by the kinetics of 1 4 C 0 2 release (Reid, 1985). Thus initial commence-ment of biodelignification for initial pH of 3.5 and 5.5 might have been delayed with respect to that of pH 4.5. These results also show that pH of 6.5 was unfavourable to hgnin biodegradation. Reid (1985) similarly reported that pH values above 6 were un-favourable to growth and lignin biodegradation by M. tremellosus. Acidification of the wood and media as indicated by the low final pH values was also observed by Reid (1985) during the biodelignification of aspen wood by M. tremellosus. Attempts by Reid (1985) Chapter 4. RESULTS AND DISCUSSION 109 to increase biodelignification by counteracting acidification were unsuccessful. Addition of sobd calcium carbonate to wood before inoculation caused a long delay before delig-nification began. Similarly periodic addition of sodium bicarbonate solution in amounts calculated to keep the pH of the cultures between 4 - 5 did not produce any increase in hgnin biodegradation. Reid (1985) also reported that M. tremellosus has a strong tendency to acidify the wood in which it is growing. It was unclear whether this acidi-fication resulted from the excretion of organic acids or the uptake of basic molecules or the generation of lignin biodegradation products (Kirk et al, 1975). M. tremellosus has also been shown to produce L-malic acid in high yield from glucose and xylose (Sasaki, 1967). In the study of biodegradation of birch and spruce by P. chrysosporium, Tai et al. (1983) identified about thirty seven carbonylic acids from the low molecular weight fraction. For both spruce and birch, the acidic fraction of methanol fractions were about 62% and 70% respectively. By and large, lignin and carbohydrate biodegradation of aspen RMP was generally favoured by acidic media. In this study, the optimum pH for selective lignin biodegra-dation was observed at pH of 4.5. Biodegradation of the aspen refiner mechanical pulp was attended by acidification of the media. Chapter 4. RESULTS AND DISCUSSION 110 Table 4.12: Selective biodelignification of aspen RMP: Effect of pH under optimum conditions. P H Q Lignin % Glucose % Xylose % SBI Final pH 3.5 16.2+.5 48.6+.6 16.6+.6 4.0+.1 3.0+.1 4.5 15.2+.4 49.5+.8 16.0+.9 4.4+.2 3.4+.2 5.5 17.8+.5 49.2+.5 18.1+.3 3.7+.1 4.5+.4 6.5 20.8+.3 50.2+.4 18.4+.4 3.3+.2 6.1+.2 Treated controlsb 21.4+.4 50.4+.8 18.8+.6 3.2+.3 c "For each p H level, values of lignin, glucose, xylose, SBI and final pH represent an average of triplicate determinations. ^Values represent average of eight measurements(2 replicates at each pH level) c Final pH values for controls: (a) for pH=3.5, final pH=3.3+.l; for pH=4.5, final pH=4.3+.2; for pH=5.5, final pH=5.3+.l; for pH=6.5. final pH=6.4+.l Table 4.13: Selective biodelignification of aspen RMP: effect of exogenously added nitro-gen under optimum conditions. Nitrogen Level" mM Lignin % Glucose % Xylose % SBI Final pH 0 14.9+.6 49.8+.4 15.7+.4 4.4+.2 3.3+.1 1.2 15.1+.3 49.8+.2 15.8+.8 4.3+.1 3.5+.2 2.4 18.3+.4 49.9+.6 16.4+.6 3.6+.2 3.9+.1 24 19.7+.9 50.0+1.2 17.2 +.5 3.4+.1 4.1+.2 Treated controls6 20.9+.9 50.4+1.1 18.5+.7 3.3+.3 4.4+.1 "For each nitrogen level, values of lignin, glucose, xylose, SBI and final pH represent an average of triplicate determinations. ''Values represent average of eight measurments(2 replicates at each nitrogen level) Chapter 4. RESULTS AND DISCUSSION 111 Table 4.14: Selective biodelignification of aspen RMP: Effect of temperature under opti-mum conditions. Temperature" °c Lignin % Glucose % Xylose % SBI Final pH 22 19.1+.5 49.8+.7 17.8 +.6 3.4+.1 4.0+.2 28 15.1+.6 49.5+.4 15.6+.9 4.3+.2 3.4+.2 32 20.2+.5 50.1+.7 18.1+.3 3.6+.1 4.2+.3 Treated controls6 20.8+1.1 50.5+.6 18.8+.7 3.3+.2 4.4+. 1 a For each temperature level, values of lignin, glucose, xylose, SBI and final pH represent an average of triplicate determinations. ^Values represent average of six measurements(2 replicates at each temperature level) 4.3.2 NITROGEN Four different levels of nitrogen concentration were used in this study. With respect to hgnin biodegradation, analysis of variance results showed the effect of exogenously added nitrogen to be significant at 0.001 level of probabihty (Table A.56). The highest lignin biodegradation was obtained at two nitrogen levels, namely 0 and 1.2 mM (Table 4.13). Duncan's multiple range test showed that the means of residual lignin contents at 0 and 1.2 mM nitrogen levels were not significantly different from each other (Table A.57). However, their means were significantly lower than those at nitrogen levels of 2.4 and 24 mM respectively. Lignin biodegradation at the nitrogen level of 2.4 mM was also significantly greater than that at the nitrogen level of 24 mM. Cellulose biodegradation, based on residual glucose content, was not significantly different at the four levels of nitrogen concentration used in this study (Table A.58). Hemicellulose biodegradation was significantly different at 0.05 level of probability (Table A.59). Hemicellulose residuals at 0, 1.2 and 2.4 mM levels of exogenously added nitrogen Chapter 4. RESULTS AND DISCUSSION 112 were not significantly different, but these values collectively were less than that at the nitrogen level of 24 mM (Table A.60). The effect of nitrogen concentration of the medium on selective biodelignification index was found to be significant at 0.001 level of probability (Table A.61). Analysis of their means by Duncan's multiple range test showed two significantly different groups. SBI at 0 mM and 1.2 mM nitrogen levels were not significantly different from each other, but were significantly greater than the two higher levels (Table A.62). In this experiment, initial pH was held at 4.5, nitrogen content was varied. The lowest final pH of 3.3 occured in 0 mM nitrogen media. The highest final pH of the growth media was 4.1 for media containing 24 mM of nitrogen. The maximum Hgnin biodegradation was obtained under conditions of 0 mM or 1.2 mM nitrogen in media (plus 0.14% in pulp). The lowest lignin biodegradation occurred in the culture that contained the highest nitrogen level, 24 mM. With the exception of only a few reported white-rot fungi, Hgnin is biodegraded only during the secondary (id-iophasic) metabohsm, which is triggered by limiting growth cultures for nutrient nitrogen (Jeffries et al, 1981; Keyser et al, 1978; Kirk et al, 1978; Reid, 1983). Nitrogen-limited conditions are natural for wood decay fungi, because wood is poor in nitrogen content (Cowling et al, 1966). Reid (1985) has demonstrated that Merulius tremellosus degrades Hgnin in aspen wood (Populus tremuloides) under conditions of solid-state fermentation where no exogenous nitrogen is added to the aspen wood. Under these conditions, both simple and complex nitrogen supplements inhibited delignification of the aspen wood by this fungus. Reid (1983) had shown earlier that biodegradation of aspen wood Hgnin and carbohydrate components by P. chrysosporium was strongly inhibited by simple nitrogen supplements like asparagine, casein hydrolysates and urea supplements (1% added N). However, complex nitrogen sources like peptone and yeast extract sfightly stimulated Chapter 4. RESULTS AND DISCUSSION 113 lignin biodegradation. The highest lignin biodegradation occurred during the biodegra-dation of unsupplernented wood. The latter might explain the inability to duplicate lignin degradation of wood in nitrogen-supplemented sources. However, Kirk et al. (1979) using shallow liquid cultures containing 10 ml of Kirk-Schultz medium, trace elements, vita-mins, and a nitrogen source (equimolar amounts of L-asparagine and ammoniun nitrate, 0.2% nitrogen on pulp basis), reported a 50 - 75% reduction in kappa number of south-ern pine kraft pulps in 6 - 8 days. Addition of more nitrogen to the cultures suppressed biodelignification. For instance, kappa number was only decreased from 28 to 24 in 2% nitrogen, but from 28 to 14% in 0.2% nitrogen. Keyser et al. (1978) have also shown that lignin biodegradation occurs only after nutrient nitrogen has been depleted and a subsequent lag phase has passed. Addition of nitrogen also delayed the appearance of the ligninolytic system or suppressed it if it were already present (Keyser et al., 1978). The data presented in this study study indicated that extensive hgnin biodegradation is favoured by very low nitrogen content. However, degradation of the carbohydrate content (cellulose and hemicellulose) was not significantly influenced by the nitrogen content as reported by Reid (1983, 1985). The latter observation could stem from the provision of an easily metabolizable, simple carbon source, 1% D-glucose, in the media. The optimum conditions for selective biodelignification of trembling aspen refiner me-chanical pulp by Rigidoporus ulmarius, unsupplernented with nitrogen or supplemented with 1.2 mM nitrogen, reflects conditions that occur naturally in wood decayed by most fungi. Reid (1985) pointed out that restricted nitrogen supply has a double benefit: it increases both the extent and selectivity of biodelignification. 4.3.3 TEMPERATURE Employing the optimum conditions obtained for selective biodehgnication in section 3.3.3, the effect of three growth temperatures on selective biodelignification index and the Chapter 4. RESULTS AND DISCUSSION 114 biodegradation of the three major chemical components (lignin, cellulose and hemicellu-lose) were studied. With respect to lignin biodegradation, the effect of temperature was found to be significant at 0.001 level of probability (Table A.63). Duncan's multiple range test showed that a growth temperature of 28° C was significantly superior to either 22° C or 32° C (Table A.64) which were also significantly different from each other. In the case of cellulose biodegradation, the F value of 1.08 (Table A.65) indicated no significant effect of temperature. Biodegradation of hemicellulose was found to be significant at 0.01 level of probability (Table A.66). Using Duncan's multiple range test, an incubating temperature of 28° C gave the highest xylose degradation (Table A.67). Also, the effect ofthe lower and higher temperatures, 22 and 32° C, was not significantly different from each other. With respect to selective biodelignification index, temperatures was found to be significant at 0.001 level of probability (Table A.68). Duncan's multiple range test indi-cated that SBI at 22° C and 32° C was not significantly different, but a growth temper-ature of 28° C yielded significantly greater values (Table A.69). Generally, temperature exerts some influence on wood decay by fungi. Tran (1987) showed that delignification of unbleached hardwood kraft pulp by P. chrysosporium ex-hibited a maximum at 38° C. In contrast, the optimum temperature for the biodelig-nification of aspen wood in the solid state by M. tremellosus appeared between 28 -30° C. The results of this study indicated that the optimum temperature for selective biodelignification of trembling aspen RMP by R. ulmarius was 28° C. In summary, the indicated imposition of pH 4.5, temperature 28 0 C, with no exoge-nous nitrogen, on the cultural conditions previously determined (section 3.3) as optimal would appear to maximize selective biodelignification. These conditions were employed Chapter 4. RESULTS AND DISCUSSION 115 to treat aspen RMP prior to evaluation of its handsheet properties. Earlier tests indi-cated that interactions were critical and the absence of tests for interactions in this phase is an acknowledged weakness of this phase of the study. 4.4 E V A L U A T I O N OF S O M E PROPERTIES OF P U L P A N D P A P E R M A D E F R O M F U N G A L T R E A T E D T R E M B L I N G A S P E N R M P 4.4.1 WEIGHTED AVERAGE FIBER LENGTH DISTRIBUTIONS The weighted average fiber length distributions of the control and fungal-treated pulps as measured by the Kaajani FS 200 Fiber Analyser are shown in Tables 4.15, 4.16 and 4.17. The control pulp exhibited the highest weighted average fiber length of 1.17 mm and pulps given Treatment 24 were characterised by the lowest weighted average fiber length of 1.02 mm. The weighted average fiber length of pulps given Treatment 21 (optimum treatment) was 1.12 mm. The latter weighted average fiber length thus exhibited a 4.2% decrease when compared to that.of the control pulp. The modal value occured at about the same distribution (0.60 - 0.70) in all cases. Pulps with weighted average fiber lengths in the 0.00 - 0.20 mm range were considered as fines. On that basis, there was a difference in the fines content of the three different pulps. In this range of fines, the control pulp exhibited the highest percentage content of 6.20% (Table 4.15). Pulps given Treatment 24 gave the lowest percentage content (4.77%) in this range (Table 4.17). However, the weighted percentage fines content in this range for pulps given Treatment 21 (optimum treatment) was 6.00% (Table 4.16). Thus, the difference in fines content in this range between the control pulp and the pulp given the optimum Treatment (21) was only 3.2% in contrast to 23.1% for pulps given Treatment 24. The strength properties of handsheets made from mechanical pulps depend on two Chapter 4. RESULTS AND DISCUSSION 116 main factors: Weighted length distribution factors; and a Shape factor as measured by specific surface area ofthe fibers and fines (Forgacs, 1963). Specific surface area is defined as the external surface area per unit weight. The shape factor provides an index of the bonding potential of the whole pulps from which such fractions are taken. Thus, the na-ture of particles'external surface are of importance in inter-fiber bonding. In mechanical pulps, long fibers are rigid and characterised by little external fibrillation. These fibers therefore contribute Uttle to inter-fiber bonding, but function like steel reinforcement in concrete. However, any treatment of fibers which improves their bonding potential also contributes to the strength properties of the paper. Fines of high specific area also act as bridges between fibers, leading to better cohesion and bonding between fibers during sheet consohdation and consequently higher strength properties. In summary, pulps given fungal Treatments 21 and 24 resulted in a reduction of 4.2% and 12.8% respectively in weighted average fiber lengths, accompanied by a reduction in fines. The greater reduction in fiber length owing to Treatment 24 could be ascribed to greater biodegradation of cellulose and hemicellulose as measured by the residual glucose and xylose contents, causing breakage. Fines have greater surface area than the higher pulp fractions. Consequently, fines might be subjected to greater enzymatic action. This appears to be in agreement with the fact that Treatment 24, characterised by the greatest enzymatic action (as indicated by lowest lignin, cellulose and hemicellulose), produced pulp with the smallest percentage of fines. Fines constitute an important part of mechanical pulp and aid in good formation, improved smoothness, outstanding opacity and good printabihty. Chapter 4. RESULTS AND DISCUSSION 117 Table 4.15: Percentage population distribution of weighted average fiber length of control trembling aspen refiner mechanical pulp. Weighted average fiber length 1.17 mm Total fibers counted 42896 Weighted average fiber length (mm) Percentage distribution Weighted average fiber length (mm) Percentage distribution 0.00 - 0.10 1.62 1.80 - 1.90 1.13 0.10 - 0.20 4.58 1.90 - 2.00 0.87 0.20 - 0.30 5.93 2.00 - 2.10 0.69 0.30 - 0.40 6.98 2.10 - 2.20 0.64 0.40 - 0.50 7.68 2.20 - 2.30 0.61 0.50 - 0.60 9.07 2.30 - 2.40 0.50 0.60 - 0.70 9.50 2.40 - 2.50 0.52 0.70 - 0.80 8.92 2.50 - 2.60 0.48 0.80 - 0.90 9.25 2.60 - 2.70 0.36 0.90 - 1.00 7.84 2.70 - 2.80 0.33 1.00 - 1.10 6.17 2.80 - 2.90 0.28 1.10 - 1.20 4.28 2.90 - 3.00 0.22 1.20 - 1.30 3.20 3.00 - 3.10 0.00 1.30 - 1.40 2.37 3.10 - 3.20 0.00 1.40 - 1.50 2.08 3.20 - 3.30 0.00 1.50 - 1.60 1.40 3.30 - 3.40 0.00 1.60 - 1.70 1.38 3.40 - 3.50 0.00 1.70 - 1.80 1.12 3.50 - 3.60 0.00 Chapter 4. RESULTS AND DISCUSSION 118 Table 4.16: Percentage population distribution of weighted average fiber length of trem-bling aspen refiner mechanical pulp given optimum fungal Treatment 21. Weighted average fiber length 1.12 mm Total fibers counted 35509 Weighted average fiber length (mm) Percentage distribution Weighted average fiber length (mm) Percentage distribution 0.00 - 0.10 1.52 1.80 - 1.90 0.80 0.10 - 0.20 4.48 1.90 - 2.00 0.85 0.20 - 0.30 6.17 2.00 - 2.10 0.78 0.30 - 0.40 7.56 2.10 - 2.20 0.62 0.40 - 0.50 7.94 2.20 - 2.30 0.42 0.50 - 0.60 9.53 2.30 - 2.40 0.29 0.60 - 0.70 9.67 2.40 - 2.50 0.28 0.70 - 0.80 9.35 2.50 - 2.60 0.33 0.80 - 0.90 8.87 2.60 - 2.70 0.26 0.90 - 1.00 7.23 2.70 - 2.80 0.24 1.00 - 1.10 5.87 2.80 - 2.90 0.23 1.10 - 1.20 4.65 2.90 - 3.00 0.09 1.20 - 1.30 3.30 3.00 - 3.10 0.00 1.30 - 1.40 2.37 3.10 - 3.20 0.00 1.40 - 1.50 2.08 3.20 - 3.30 0.00 1.50-1.60 1.40 3.30 - 3.40 0.00 1.60 - 1.70 1.38 3.40 - 3.50 0.00 1.70 - 1.80 1.12 3.50 - 3.60 0.00 Chapter 4. RESULTS AND DISCUSSION 119 Table 4.17: Percentage population distribution of weighted average fiber length of trem-bling aspen refiner mechanical pulp given fungal Treatment 24. Weighted average fiber length 1.02 mm Total fibers counted 42896 Weighted average fiber length (mm) Percentage distribution Weighted average fiber length (mm) Percentage distribution 0.00 - 0.10 1.02 1.80 - 1.90 0.72 0.10 - 0.20 3.75 1.90 - 2.00 0.61 0.20 - 0.30 6.12 2.00 - 2.10 0.69 0.30 - 0.40 7.60 2.10 - 2.20 0.56 0.40 - 0.50 8.88 2.20 - 2.30 0.50 0.50 - 0.60 9.86 2.30 - 2.40 0.56 0.60 - 0.70 9.98 2.40 - 2.50 0.31 0.70 - 0.80 9.22 2.50 - 2.60 0.34 0.80 - 0.90 8.87 2.60 - 2.70 0.27 0.90 - 1.00 7.28 2.70 - 2.80 0.20 1.00 - 1.10 6.25 2.80 - 2.90 0.10 1.10 - 1.20 4.35 2.90 - 3.00 0.00 1.20 - 1.30 3.18 3.00 - 3.10 0.00 1.30 - 1.40 2.23 3.10 - 3.20 0.00 1.40 - 1.50 1.97 3.20 - 3.30 0.00 1.50 - 1.60 1.88 3.30 - 3.40 0.00 1.60 - 1.70 1.52 3.40 - 3.50 0.00 1.70 - 1.80 1.20 3.50 - 3.60 0.00 Chapter 4. RESULTS AND DISCUSSION 120 4.4.2 PHYSICAL PROPERTIES OF HANDSHEETS The aspen RMP at the end of the fungal treatment exhibited a slight reddish brown colouration. This was more prominent in the pulp given Treatment 24, although pulps given fungal Treatment 21 also showed this colouration to some extent. However, this colouration disappeared from the pulp when it was disintegrated in hot water to remove latency and shives. This action immediately gave noticeably brighter pulp than that of the control. This colouration phenomenon was not observed with the control pulps. Merulius tremellosus (Schrad.:Fr), a white-rot fungus which causes selective removal of hgnin when grown on chipped aspen wood, also imparts a reddish-brown colour to the wood during biodelignification. In this case, only a portion of this colouration is removed by hot water extraction (Reid, 1985). Since this colouration was more prominent in pulp exhibiting considerable biodelignification, it could be ascribed largely to lignin degradation products and secondary metabolites. The average physical properties of handsheets made from unbleached, fungal-treated trembling aspen refiner mechanical pulp are given in Table 4.18. Brightness of paper is a measure of reflectance of light of wavelength 457 nm. Brightness of handsheets made from pulps given Treatments 21 and 24 was significantly higher than that of the control pulp (Tables A.70 and A.71). Brightness of handsheets made from pulps given Treatment 21 and 24 were also significantly different from each other (Tables A.70 and A.71). The highest brightness, 54.1%, was recorded for pulps given Treatment 24. This treatment was also characterised by the lowest residual hgnin content of 10.6% (Table 4.9). The brightness of the control pulp was 49.8% while pulp given the optimum selective biodelignification treatment (21) had a brightness of 53.5% (Table 4.18). Brightness is a reflection of hgnin content. A reduction in hgnin and/ or chromophore content is attended by increase in brightness. This is reflected in pulps given the optimum selective Chapter 4. RESULTS AND DISCUSSION 121 biodelignification Treatment 21, and Treatment 24. Freeness, as measured according to the Canadian standard freeness varied from 700 ml for pulps given fungal treatments to 650 ml for the control pulp (Table 4.18). The observed increase is apparently due to fungal treatment and its attendant decrease in fines content. The slight difference in freeness should not affect comparisons of strength made among treatments. ' The dry zero span-breaking lengths for pulps given different fungal treatment were significantly different from each other (Tables A.72 and A.73). The dry zero-span break-ing length, 7.1 km, was highest for the pulp given the optimum Treatment 21 (Table 4.18). Pulps given Treatment 24 were associated with the lowest value of 5.8 km. The dry zero-span breaking length for the control pulp was 6.8 km. However, paper made from the control pulp exhibited the highest wet zero-span breaking length (5.7 km), while pulp given Treatment 24 continued to give significantly the lowest value, 4.2 km (Tables A.74 and A.75). The wet zero-span breaking length for pulp given Treatment 21 was 5.6 km. This was not significantly different from that of the control pulp (Table A.74 and A.75). As could be discerned from above, there are two types of zero-span tensile strength, namely dry and wet. Although both are employed as an index of average ultimate strength of the randomly oriented fibers in paper, dry zero-span tensile strength also depends to a degree on the extent of inter-fiber bonding which is absent in the wet zero-span tensile test. Zero-span tensile strength is not a fundamental test. However, it is a useful index for measuring the loss in individual fiber strength resulting from some degrading effects of treatment (Casey, 1961). With respect to dry zero-span breaking length, the significantly higher value for pulps given Treatment 21 could be ascribed to enhanced inter-fiber bonding, resulting from both selective delignification of the fibers and improvement in the quality of fines. Fines fill the void spaces between fibers, and Chapter 4. RESULTS AND DISCUSSION 122 good quality fines serve as bridges between the fibers. Since inter-fiber bonding in water saturated paper is absent, wet zero-span breaking length is a better measure of the intrinsic fiber strength. The results of this study indicate that no significant difference in wet zero-span breaking length was observed between the control pulp and pulp given the optimum fungal treatment. For handsheets made from pulp given fungal Treatment 24, the significantly lower values for both wet and dry zero-span breaking length was indicative of loss of both intrinsic tensile strength and inter-fiber bonding potential. This was borne out by the appreciable loss of carbohydrates, notably the cellulose component for this pulp. Tensile index showed a trend for both the control and fungal-treated pulps similar to dry zero-span breaking length (Table 4.18). Values for the control, Treatment 21 and Treatment 24 were significantly different from each other (Tables A.76 and A.77). Handsheets made from pulp given Treatment 21 exhibited the highest tensile index (2.2 Nm/g) . The lowest tensile index (0.7 Nm/g) was shown by pulp given fungal Treatment 24. The tensile index for the control pulp was 1.7 Nm/g. Tensile strength is a measure of the resistance of paper to direct tension and is defined as the force required to break a strip of paper of specified length (usually 100 mm) and width (15 mm) at a prescribed loading rate. In mechanical pulps, the factors affecting tensile strength are weighted average fiber length and most importantly, the quantity and quahty of inter-fiber bonding. The latter is a function of the specific surface area of the fines and is usually defined by the quahty of the fines. The observed significant increase in the tensile strength of pulp given the optimum fungal Treatment 21, as indicated by the tensile index (Table A.77), demonstrates the fact that notwithstanding the small decrease in weighted average fiber length, improvement in bonding potential of fibers and the quality of fines produced greater inter-fiber bonding and consequently a higher tensile strength. Casey (1961) noted that the most important factor affecting tensile strength Chapter 4. RESULTS AND DISCUSSION 123 was the amount and quality of fiber bonding. The significantly lower value of tensile index reported for pulps given treatment 24 (Table A.77) is a reflection of the degradation of the carbohydrate component of the fibers notably the cellulose component. The burst indices ofthe control pulp (0.6 kPa. m2/g) and pulp given Treatment 21 (0.7 kPa. m2/g) were significantly different from each other (Tables A. 78 and A.79) and also significantly greater than that of Treatment 24 (0.2 kPa. m2/g) (Table 4.18). Bursting strength of paper is a highly empirical test which is defined as the hydrostatic pressure required to rupture paper when deformed in an approximate sphere, 3.05 mm (1.20 ins), in diameter at a controlled rate of loading. Two factors are responsible for bursting strength, measured as burst index in this study : Fiber length; and Inter-fiber bonding (Casey, 1961). Thus, the trend observed here is very similar to that observed for the tensile index. The observed significant increase in burst index for pulps given fungal Treatment 21 (Table A.79), notwithstanding the slight decrease in weighted average fiber length, could be ascribed to increase in inter-fiber bonding engendered by selective removal of lignin and quality of fines as measured by their specific surface area. The significant reduction in burst index for pulps given Treatment 24 is a reflection of reduced weighted average fiber length, degradation of carbohydrate contents, and a reduction in fines content. Tear index was much less for pulps given Treatment 24 (0.2 mN. m2/g) in comparison to either the control pulp or Treatment 21, both of which exhibited a comparable tear index of 1.0 mN.m2/g. The internal tearing resistance reported in this study as tear index measures the amount of work done in tearing the paper through a fixed distance after a tear has been started by means of a cutter attached to the instrument. The internal tearing resistance is usually measured on the Elmendorf tear tester, a pendulum type instrument. Chapter 4. RESULTS AND DISCUSSION 124 Tear index is dependent upon three properties: (1) Total number of fibers participat-ing in the sheet; (2) Fiber length; and (3) The number and strength of the inter-fiber bonding (Casey, 1961). This author points out that the work involved in tearing consists of two components, work involved in pulling fibers out of the paper and work involved in rupturing the fibers. For mechanical pulps, tear strength is initially proportional to the specific surface area which depends on weighted average fiber length; eventually tear index departs from linearity and flattens out (Shallhorn et al., 1979). Tear index increases with fiber length, because longer fibers mean an increase in frictional drag work per fiber (Casey, 1961). The latter in turn is greatly influenced by specific surface area of mechanical pulps. On this basis, pulps given Treatment 21 exhibiting a somewhat decreased weighted average fiber length were expected to show a decrease in tear index. However, this expected reduction in tear index might have been compensated by improvement in inter-fiber bonding, originating from an increase in specific surface area due to selective delignification. The significant reduction in tear index for pulps given Treatment 24 (Table A.80 and A.81) is a direct consequence of degraded carbohydrates. The greatest sheet density was exhibited by pulp given Treatment 24 (293.2 kg/m3) and was significantly greater than that of the other pulps.. The sheet densities for the control and pulp given optimum fungal Treatment (21), 255.5 and 241.0 kg/m3 respectively, were also significantly different from each other (Tables A.82 and A.83). The highest sheet density observed for Treatment 24 reflects low residual hgnin content and reduced weighted average fiber length and improved conformability. Opacity (Tappi) of the fungal treated pulps was signficantly different from each other (Tables A.84 and A.85). The highest opacity, 93.2%, was exhibited by handsheets made from control pulp and the lowest, 90.8%, opacity was associated with pulpsgiven the fungal Treatment 24 (Table 4.18). Opacity is a property of paper which is determined by Chapter 4. RESULTS AND DISCUSSION 125 the total amount of transmitted light (diffuse and non-diffuse). It is thus defined as the reciprocal ofthe amount of light transmitted through paper (Casey, 1961). Consequently, a perfectly opaque paper is one which is absolutely impervious to the passage of all visible light. Fines constitute an important part of mechanical pulps and are known to be respon-sible for enhancement of some optical properties such as opacity and good printability. Thus, the observed significant decrease in opacity for handsheets made from pulps given Treatment 24 (Table A.85) could be ascribed to the reduction in fines content of the pulps. Results of previous studies on the effect of fungal treatment of mechanical pulps and wood chips have been mixed (Samuelsson et ai, 1980; Fukuzumi et al, 1983; Pilon et al, 1982b; Myers et al, 1988). Decrease in strength properties of handsheets made from fungal-treated mechanical pulps have been reported by Samuelsson ei al, 1980 and Fukuzumi et al, 1983 (Tables 2.4). However, some workers (Pilon et al, 1982b; Myers et al, 1988) have demonstrated that under certain conditions, pulp handsheets made from from fungal-treated mechanical pulps and wood chips were characterised by improved strength properties (Tables 2.3 and 2.5). In general, the results of this study indicated some improvements in most strength properties of handsheets made from pulp given the optimum Treatment 21. The most notable was the increase in tensile index and brightness of the optimally treated pulp when compared to the controls. Several previous studies (Samuelsson et al, 1980; Myers et al, 1988) have shown that pre-treatment of wood chips and mechanical pulps with several white-rot fungi including Phanerochaete chrysosporium, Dichomitus squalens and Coriolus versicolor was attended by a considerble loss(10%) in brightness. Recently, Meyers ei al. (1988) reported that pre-treatment of aspen wood chips with both P. chrysosporium and D. squalens resulted in brightness loss of 22.8% and 20.8% respectively. In this study, treatment of the aspen Chapter 4. RESULTS AND DISCUSSION 126 RMP with optimum Treatment 21, and Treatment 24 produced 7.4% and 8.6% respective increase in brightness when compared to the controls. At comparable CSF freeness of 650 - 700 ml, optimum Treatment 21 improved tensile strength by 29%, dry zero-span breaking length (km) by 4.4% and burst index by 16.8%. Relative to the controls, opacity (Tappi) of pulps given Treatment 21 decreased by a relatively low 0.3%. Pulps given Treatment 24 were characterised by considerable biodegradation of hgnin, cellulose and hemicellulose as shown by their respective residual hgnin (10.6%), glucose (38.5%) and xylose (3.4%) contents. The considerable loss in cellulose and hemicellulose contents is reflected in poor strength properties of the resulting handsheets. At compa-rable freeness of 650 - 700 ml, dry zero-span breaking length (km) decreased by 14.7%, burst index by 67.7%, tear index by 80:0% and tensile index by 58.8%. Opacity was also 2.6% lower than that of the control. However, brightness increased by 8.6%, a reflection of enhanced hgnin biodegradation and reduction in chromophore content. In summary, aspen refiner mechanical pulps given maximum hgnin and carbohydrate removal (Treatment 24) and the optimal selective biodelignification (Treatment 21) were characterised by a significant increase in brightness and decrease in opacity. Modest im-provement in strength properties was observed for the pulp given the optimum Treatment 21, dry zero-span breaking length, tensile index and burst index being particularly signif-icant.As stated previously, pulps given Treatment 21 were characterised by low residual hgnin content (14.8%) coupled with a high retention of cellulose (residual glucose con-tent, 51.5%) and high hemicellulose (residual xylose content, 15.1%) and consequently higher bonding potential. This coupled with reasonably high retention of fines and ade-quate fiber length undoubtedly contributed to some of the improved handsheet properties observed. Conversely, pulps given Treatment 24 were characterised by low cellulose and hemi-cellulose, and a significant reduction both in fines and fiber length. In spite of maximum Chapter 4. RESULTS AND DISCUSSION 127 lignin reduction in this pulp, it manifested poor strength properties owing to excessive carbohydrate deterioration. Table 4.18: Average physical properties of handsheets made from unbleached control and fungal-treated trembling aspen refiner mechanical pulp. Property measured Control 21 24 Brightness (%) 49.8 53.5 54.1 Freeness(ml, CSF) 650 700 700 (Dry) Zero-span breaking length(km) 6.8 7.1 5.8 (Wet) Zero-span breaking length(km) 5.7 5.6 4.2 Tensile index(Nm/g) 1.7 2.2 0.7 Burst index(kPa. m2/g) 0.6 0.7 0.2 Tear index(mN. m2/g) 1.0 1.0 0.2 Density(kg/m3) 255.5 241.0 293.2 Opacity(%, Tappi) 93.2 92.9 90.8 Type of pulp treatment Chapter 5 C O N C L U S I O N S Cultural factors that enhance selective biodelignification of lignocellulosic materials by micro-organisms, notably white-rot fungi, are of vital importance in attempts to utilise non-chemical methods to enhance wood processing. This study examined the effects of some cultural factors on selective biodelignification of trembling aspen refiner mechanical pulp (RMP) by the white-rot fungus Rigidoporus ulmarius. The ability of this fungus to selectively delignify wood, especially hardwoods in the field was first reported by Kawase (1962). Later on, Kirk and Moore (1972) also demonstrated the ability of R. ulmarius to selectively delignify wood, notably hardwoods including aspen, under laboratory con-ditions. This study takes the previous studies further and tries to optimize the selective biodelignification of aspen RMP, using this fungus and combinations of the following cultural factors: aeration, hydrogen peroxide, mineral solution, a surfactant (tween 80), and veratryl alcohol. A novel aspect was the exogenous addition of veratryl alcohol and hydrogen peroxide to the culture at the estimated commencement of the secondary metabolic phase of the fungus, because hgnin biodegradation was hypothesized to begin during this phase represented by the cessation of fungus growth. The following conclusions were drawn from the present study: • Addition of a higher level of mineral solution (Kirk-Schultz mineral solution, eight-fold concentration) enhanced selective biodelignification, especially under condi-tions of oxygen flushing. This was reflected in enhanced hgnin degradation when 128 Chapter 5. CONCLUSIONS 129 compared to carbohydrate degradation and therefore increased selective biodelig-nification index under these conditions. « Similarly, exogenous addition of 2 m M veratryl alcohol in general enhanced selective biodehgnification(SBI) under conditions of oxygen flushing. o Generally, exogenous addition of hydrogen peroxide to treatments containing the other cultural factors commonly had the effect of limiting selective biodelignification (SBI). • Addition of tween 80 (0.05%) enhanced selective biodelignification under air flush-ing. Under oxygen flushing, selective biodelignification (SBI) was enhanced, but to a smaller extent when compared to treatments in which tween 80 (0.05%) was ab-sent. This observation was attributed to increased carbohydrate degradation under oxygen flushing conditions. A similar trend was observed in interactions between tween 80 and mineral solution and veratryl alcohol respectively. • In this study, the optimal conditions for selective biodelignification of aspen refiner mechanical pulp by R. ulmarius were mineral solution (eight-fold concentration)-aeration (oxygen flushing) in the absence of nitrogen supplements or hydrogen peroxide. This condition reflects the situation that occur naturally in wood decayed by most fungi. The aspen pulp produced by this treatment was characterised by residual lignin content of 14.8%, residual glucose content of 51.1% and residual xylose content of 15.1%. This was equivalent to a loss in hgnin content of 30.8%, and a depletion in xylan of 23.7%, with no significant change in cellulose content. The resulting SBI was 4.5, compared with a value of 3.2 for the untreated controls. • The optimum pH and incubating temperature for selective biodelignification were 4.5 and 28° C respectively. Exogenous addition of nitrogen beyond 1.2 m M was Chapter 5. CONCLUSIONS 130 not effective in enhancing selective biodelignification of the pulp. Biodegradation of pulp was attended by acidification of media. • Considerable biodegradation of both hgnin and carbohydrates was observed for pulp given treatment number 24: mineral solution (eight-fold concentration)-veratryl al-cohol (2 mM)-tween 80 (0.05%)-aeration (oxygen flushing). The residual lignin, glucose and xylose contents of pulps given this treatment were 10.6%, 38.5% and 3.3% representing loss of 50.4%, 24.9% and 83.3% lignin, cellulose and xylan re-spectively. The resulting SBI of this treatment was 4.0. Although handsheets made from pulp given this treatment exhibited 8.6% increase in brightness, they had very poor strength properties reflecting the considerable loss in carbohydrate content. • Treatment of pulp with the optimum selective biodelignification treatment number 21 produced a 7.4% increase in brightness. Opacity (Tappi) ofthe pulp given this treatments was reduced by 0.3 %. • Modest but significant improvement in some strength properties of handsheets made from pulps given the optimum selective biodelignification Treatment 21 was observed. At comparable CSF freeness of 650 - 700 ml, the optimum Treatment 21 significantly improved dry zero-span breaking length by 4.4 %, tensile index by 28.9 % and burst index by 16.8%. • The weighted average fiber length of pulp was reduced from 1.17 mm for the con-trols to 1.12 mm and 1.02 mm for pulps given Treatments 21 and 24 respectively. This represented a 4.2% and 12.8% reduction in respective weighted average fiber lengths. Similarly, there was corresponding decrease in the fines content, i.e fibers found in the 0.00 - 0.02 mm range. In this range, the fines content were reduced Chapter 5. CONCLUSIONS 131 by 3.2% and 23.1% respectively for pulps given Treatments 21 and 24. The reduc-tion of fines content could be attributed to higher extracellular enzymatic activity because of the relatively high surface area of the fines. Fungal-treated pulps exhibiting extensive lignin biodegradation were characterised by a reddish-brown colouration, which disappeared when they were disintegrated in hot water to remove latency and shives. Evaluation of the nature of this colouration is a subject for future studies designed to elucidate the process by which lignin is biodegraded by R. ulmarius. This study represents the first systematic attempt to determine the effect of combi-nation of several factors : aeration, hydrogen peroxide, mineral solution, tween 80, and veratryl alcohol on selective biodelignification of a ligniceUulosic substrate by R. ulmar-ius. Results of several previous studies on the effect of fungal treatment on the chemical components of lignocellulose have been conflicting. This confusion has been ascribed to the varying cultural conditions employed (Meyers, 1988), and is borne out by the results of this present study, which clearly demonstrate a strong interaction effect of cultural fac-tors on selective biodelignification. For instance, differences in cultural conditions alone caused the experimental organism R. ulmarius to behave in one instance like a typical white-rot fungus (optimal Treatment 21) and in another case hke a 'simultaneous rot' (Treatment 24). Although it is difficult to compare the effectiveness of R. ulmarius as an agent of biodelignification with fungi that have been studied in other laboratories because of differences in starting materials and methods used to evaluate the products (Reid, 1985), the observed significant increase in brightness, dry zero-span breaking length, burst index, and tensile index of pulps given the optimum selective biodelignification is indicative of the potential of this fungus in bio-treatment of mechanical pulps. Chapter 5. CONCLUSIONS 132 Future extension of this work might address the question of further improvement in pulp properties. For example, the optimal treatment (21), involving a 14-day incubation period, left cellulose completely intact, whereas hgnin and hemicellulose were significantly depleted. It is possible that a longer incubation period under these optimum conditions might further reduce lignin without significant impact on carbohydrates, especially cel-lulose. In this case, one would expect an even better quality of pulp as evaluated by handsheet tests. CITED B I B L I O G R A P H Y Adler, E. 1977. Lignin chemistry - past, present and future. Wood. Sci. Technol. 1 : 169 - 218. Aho, P. E., Seidler, R. J., Evans, H. J. and P. N. Raju. 1974. Distribution, enumeration and identification of nitrogen-fixing bacteria associated with decay in living white fir trees. Phytopathology 64 :1413 - 1420. Alcubilla, M., Aufsess, A. V., Cerny G. and K. E. Rehfuess. 1974. Untersuchungen uber pilzhemmwirkung des Fichlenholzes(Picea abies Karst. In 'Proc. 4th Int. Conf. on Fomes annosus'. Athens, G. A., U.S. Ed : E. G. Kulman. pp 139 - 162. Ander, P and K.-E. Eriksson. 1975. Influence of carbohydrates on lignin degradation by the white-rot fungus Sporotrichum pulverulentum. Sven. Papperstid. 5(18) : 643 - 652. Ander, P. and K.-E. Eriksson. 1976a. The importance of phenol oxidase activity in lignin degradation by the white-rot fungus, Sporotrichum pulverulentum. Arch. Microbiol. 109 : 1 - 8. Ander, P. and K.-E. Eriksson. 1976a. Methanol formation during lignin degradation by Phanerochaete chrysosporium- Appl. Microbiol. 21 : 96 - 102. Ander, P. and K.-E. Eriksson. 1977. Selective degradation of wood components by white-rot fungi. Physiol. Plant. 41 : 239 - 248. Ander, P. and K.-E. Eriksson. 1978. Lignin degradation and the utilisation by micro-organisms. In 'Progress in Industrial Microbiology'. Ed: Bull, M. J. Else-vier. Armsterdam. 14 : 1-58. Ander, P., K.-E. Ericksson. 1985. Methanol formation during lignin degradation by Phanerochaete chrysosporium. Appl. Microbiol. Biotechnol. 21 : 96 - 102. Antai, S. P. and D. L. Crawford. 1981. Degradation of softwood, hardwood and grass lignocelluloses by two Streptomyces strains. Appl. Environ. Microbiol. 4%'-378 - 380. Asther, M., Corrieu, G. and E. Odier. 1986. Production of hgninolytic enzymes of Phanerochaete chrysosporium INA-12 in submerged agitated cultures. In 'Biotechnology in the pulp and paper industry'. The third international conference. Stockholm June, 16 - 19, 1986. Sponsored by STFI and SPCI. pp 152 - 153. 133 CITED BIBLIOGRAPHY 134 Asther, M., Corrieu, G., Drapron, R. and E. Odier. 1987. Effect of tween 80 and oleic acid on ligninase production by Phanerochaete chrysosporium INA-12. Enzyme Microbiol. Technol. 9 : 245 - 249. Bar-Lev, S. S. and T. K. Kirk. 1981. Effects of molecular oxygen on lignin biodegra-dation by Phanerochate chrysosporium. Biochem. Biophys. Res. Commun. 99(22) : 373 - 378. Basham, H. G. and E. B. Cowling. 1976. Distribution of essential elements in forest trees and their role in wood deterioration. In 'Organism und Holz'. Interna-tional Symposium. Berlin-Dahlem. 1975. Duncker und Humboldt/Berlin. Ed: W. G. Baecker and W. Liese. pp 155 - 165. Bellamy, W. D. 1974. Single cell protein from cellulosic wastes. Biotechnol. Bioeng. 16(7) : 869 - 880. Blanchette, R. A. 1979. Wood decomposition by Phellinus(Fomes) pini : A scanning electron microscopy study. Can. J. Bot. 58 : 1496 - 2503. Blanchette, R. A. 1980a. A submicroscopic view. J. Fores. 75(12) : 734 - 737. Blanchette, R. A. 1980b. Wood decomposition by Phellinus(Fomes) pini. a scanning electron microscopy study. Can. J. Bot. 58 : 1496 - 1503. Blanchette, R. A. 1984a. Manganese accumulation in wood decayed by white-rot fungi. Phytopathology 74(6) : 725 - 730. Blanchette, R. A. 1984b. Selective delignification of eastern hemlock by Ganoderma tsugae. Phytopathology 74(2) : 153 - 160. Blanchette, R. A. 1984c. Screening wood decayed by white-rot fungi for preferential lignin degradation. Appl. Environ. Microbiol. 5^(3) : 647 - 653. Blanchette, R. A., L. Otjen, M. J. Effland and W. E. Eslyn. 1985. Changes in struc-tural and chemical components of wood dehgnified by fungi. Wood. Sci. Technol. 19(1) : 35 - 46. Brower, H. E., J. E. Jeffery and M. N. Folson. 1966. Gas chromatographic sugar analysis in hydrolyzates of wood constituents. Anal. Chem. 38: 362 - 364. Bu lock, J. D. 1975. Secondary metabolism in fungi and its relationship to growth and development. In 'Filamentous fungi'. Eds: Smith, J. E and D. R. Berry. Industrial Mycology. Vol 1. John Wiley, New York pp 33 - 58. Burdsall, H. H. Jr and W. E. Eslyn. 1974. A new Phanerochaete with a chrysosporium imperfect state. Mycotaxon. 1 : 123 - 133. Chang, H.-m., J. W. Joyce, A. G. Campbell, E. D. Gerrard, V.-B. Huynh and T. K. Kirk. 1983. Fungal decolorisation of bleach plant effluents. In 'Recent advances in lignin biodegradation'. Eds: Higuchi,T; H.-m and T. K. Kirk. Uni Publishers, Tokyo, Japan pp 257 - 268. CITED BIBLIOGRAPHY 135 Casey, J. P. 1961. Properties of paper. In 'Pulp and Paper. Chemistry and chemical Technology'. Second edition. Interscience publishers, Inc. New York pp 1251 - 1519. Cowling, E. B. 1961. Comparative biochemistry of the decay of sweetgum sapwood by white-rot and brown-rot fungi. U . S. Dept. Agric. Techn. Bull. No. 1258 pp 1 - 79. Cowling, E. B. and W. Merrill. 1966. Nitrogen in wood and its role in wood deterio-ration. Can. J. Bot. 44 : 1539 - 1554. Cowhng, E. B. 1976. Properties of cellulose and lignocellulose materials as substrates for enzymatic conversion processes. Biotechnol. Bioeng. Symp. 6 : 95 - 123. Crawford, D. L. 1978. Lignocellulose decomposition by selected Streptomyces strains. Appl. Environ. Microbiol. 35 : 1041 - 1045. Crawford, R. L. 1981. Lignin biodegradation and transformation. John Wiley and Sons, Inc. New York 154 pp. Crawford, D. L. and R. L. Crawford. 1980. Microbial degradation of lignin. Enz. Microbial. Technol. 2 : 11 - 22. Crawford, D. L., A. L. Pometto III and R. L. Crawford. 1983. Lignin degradation by Streptomyces viridosporus: Isolation and characterisation of a new poly-meric hgnin degradation intermediate. Appl. Environ. Microbiol. ^5: 898 -904. Crawford, R. L. and D. L. Crawford. 1984. Recent advances in studies of the mech-anisms of microbial degradation of lignins. Enz. Microbiol. Technol. 2(1) : 11 - 22. Crawford, R. L. and D. L Crawford. 1978. Radioisotopic methods for the study of hgnin biodegradation. Dev. Ind. Microbiol. 19: 35 - 49. Crowell, E. P. and B. B. Burnett. 1967. Determinations of the carbohydrate compo-sition of wood pulps by gas chromatography of the alditol acetates. Anal. Chem. 39: 121 - 124. Dawson-Andoh, B. E. and J. W. Wilson. 1985. Distribution of inorganic elements in sound, discoloured and decayed wood of trembhng aspen. Unpubhshed work 15 pp Dekker, F. H. and G. N. Richards. 1973. Effect of delignification on in vitro digestion of polysaccharides of bagasses. J. Sci. Food Agri. 24 '• 45 - 51. Dill, I. J. Salnikow and G. Kraepelin. 1984. Hydroxyproline-rich protein material in wood and hgnin of Fagus sylvatica. Appl. Environ. Microbiol pp 1259 -1261. CITED BIBLIOGRAPHY 136 Eaton, D., H.-m. Chang and T. K. Kirk. 1980. Fungal decolorisation of kraft bleach effluents. Tappi 63(10) : 103 - 106. Effland, M. J. 1977. Modified procedure to determine acid-insoluble lignin in wood and pulp. Tappi 60(10) : 143 - 144. Enoki, A. and M. H. Gold. 1982. Arch. Microbiol. 133 : 132 - 130. Quoted by Kirk, T. K and M. Shimada. 1985. Lignin biodegradation : The micro-organisms involved and the physiology and biochemistry of degradation by white-rot fungi. In " Biosynthesis and biodegradation of wood components." Ed : Higuchi, T. Academic Press, New York pp 579 - 605. Ellis, E. L. 1965. Inorganic elements in wood. In ' Cellular ultrastructure of woody plants'. Syracuse. Univ. Ed: W. A. Cote, Jr pp 181 - 189. Erickson, M., G. E. Miksche and I. Somfai. 1973a. Holzforschung. Quoted by Kirk, T. K and M. Shimada. 1985. Lignin biodegradation : The microorganisms involved and the physiology and biochemistry of degradation by white-rot fungi. In 'Biosynthesis and biodegradation of wood components'. Ed: Higuchi, T. Academic Press, N. York pp 579 - 605. Erickson, M., S. Larsson and G. Miksche. 1973b. Gas chromatographishe analyse von lignoxydationsproduken VII . Acta. Chem. Scand. 27 : 127 -140. Erickson, M. and G.E. Miksche. 1974a. On the occurence of lignin or polyphenols in some mosses and liverworts. Phytochemistry 13 : 2295 - 2299. Erickson, M. and G. E. Miksche. 1974b. Two dibenzofurans obtained on oxidative degradation of the moss Polytrichum communa Hedw. Acta. Chem. Scan. B. 28 : 109 - 113. Erickson, M. and G. E. Miksche. 1974c. Characterisation of gymnosperm hgnins by oxidative degradation. Holzforschung 28(A) : 135 - 138. Eriksson, K.-E. 1976. Delignification of wood chips by mutant white-rot fungi. In 'Biodelignification : Present status and future directions'. Ed: Abson, D. Weyerhauser Co, Seattle. Washington pp 5 - 30. Eriksson, K.-E. 1978. Enzyme mechanisms involved in cellulose hydrolysis by the rot fungus Phanerochaete chrysosporium. Biotechnol. Bioeng. 20(2): 317 - 376. Ericksson, K.-E and Vallender, L. 1980. Biomechanical pulping. In : 'Lignin biodegra-dation : microbiology, chemistry and application'. Eds : Kirk T. K., T. Higuchi, H.-m. Chang. Vol II. Boca Raton. CRC Press Inc pp 213 - 224. Eriksson, K.-E. 1981. Fungal degradation of wood components. Pure. Appl. Chem. 55(1): 33 - 43. CITED BIBLIOGRAPHY 137 Eriksson, K.-E. and E. W. Goodell. 1974. Pleiotropic mutants of the wood-rotting fungus, Phanerochaete chrysosporium. Biotechnol. Bioeng. Symp. 20 : 317 - 332. Eriksson, K.-E. and L. Vallender. 1982. Properties of pulps from thermomechanical pulping of chips pre-treated with fungi. Svensk. Papperstid. 55(6): 33 - 38. Eslyn, W. E., T. K. Kirk and M. J. Effland. 1975. Changes in the chemical composition of wood caused by six soft-rot fungi. Phytopathology 65(1): 473 - 476. Evdokimov, A. M. 1974. Khim. Drev. No. 1 : 26 - 30. Quoted by Kirk,T.K. and M. Shimada. 1985. Lignin biodegradation and white-rot fungi. In 'Biosynthesis and biodegradation of wood components'. Ed: Higuchi, T. Academic Press, New York pp 579 - 605. Forgacs, 0. L. 1963. Characterisation of mechanical pulps. Pulp and paper magazine of Canada 64 : T89 - T115. Fahreus, G., R. Nilsson and G. Nilsson. 1949. Studies in the decomposition of wood by means of three white-rot fungi. Svensk. Botanisk. Tidskrift. ^5(2-3): 1140 - 1145. Faison, B. D. and T. K. Kirk. 1983. Relationship between lignin degradation and production of reduced oxygen species by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 46(5): 1140 - 1145. Faison, B. D. and T. K. Kirk. 1985. Factors involved in the regulation of a ligninase activity in Phanerochaete chrysosporium. Appl. Environ. Microbiol. 49 '• 299 - 304. Faison, B. D., T. K. Kirk and R. L. Farrall. 1986. Role of veratryl alcohol in reg-ulating ligninase activity in Phanerochaete chrysosporium. Appl. Environ. Microbiol. 52: 251 - 254. Fengel, D. and G. Wegner. 1984. Distribution of the components within the wood cell wall. In 'Wood: chemistry, ultrastructure, reactions'. Walter de Gruyter. Berlin, New York pp 227 - 238. Ferin, P. and T. K. Kirk. 1979. Ligninolytic system of Phanerochaete chrysosporium : inhibition by o-pthalate. Arch. Microbiol. 123 : 307 - 309. Fergus, B. J., J. Procter, A. N. Scott and D. A. I. Goring. 1969. The distribution of hgnin in spruce wood as determined by ultraviolet microscopy. Wood. Sci. Technol. 3: 117 - 138. Fergus, B. J. and D. A. I. Goring. 1970. The location of guaiacyl and syringyl lignins in birch xylem tissue. Holzforshung 24 '• 113 - 117. CITED BIBLIOGRAPHY 138 Forney, L. J., C. A. Reddy and H. S. Pankratz. 1982a. Ultrastructural localization of hydrogen peroxide production in ligninolytic Phanerochaete chrysosporium cells. Appl. Environ. Microbiol. 44(3): 732 - 736. Forney, L. J., C. A. Reddy, T. Tien and S. D. Aust. 1982b. The involvement of hy-droxyl radical derived from hydrogen peroxide in hgnin degradation by the white-rot fungus Phanerochaete chrysosporium J. Biol. Chem. 257: 1145 -1162. Freeman, B. A., M. K. Topolosky and J. D. Crapo. 1982. Hyperoxia increase oxygen radical production in rat lung homogenates. Arch. Bicochem. Biophys. 216 : 477 - 484. Fries, N. 1973. The effects of volatile organic compounds on the growth and develop-ment of fungi. Trans. Bri. Mycol. Soc. 66: 1 - 21. Fukuda, K. and T. Haraguchi. 1971. Bull. Exp. For. Tokyo Univ. Agri. Technol. No. 9 : 25 - 29. Quoted by Kirk, T. K and M. Shimada. 1985. Lignin biodegradation and white-rot fungi. In 'Biosynthesis and biodegradation of wood components'. Ed: Higuchi, T. Academic Press, New York pp 579 - 605. Fukuzumi, T., A. Yotsukra and Y. Hayashi. 1983. Screening of fungi for biological pulping. In 'Recent advances in lignin biodegradation'. Eds: Higuchi, T, H.-m Chang and T. K. Kirk. Uni Press. Tokyo, Japan: 246 - 256. Glasser, W. G., C. A. Barnett, J. G. Rials and V. P. Sarat. 1984. Engineering plastics from hgnin. II. Characterisation of hydroxyalkyl lignin derivatives. J. Appl. Polymer Sci. 29 : 1815 - 1830. Glenn, J. K., M. A. Morgan, M. B. Mayheld, M. Kuwahara and M. H. Gold. 1983. An extracellular H202-requiring enzyme preparation involved on hgnin biodegradation by white-rot basidiomycete Phanerochaete chrysosporium. Biochem. Biophys. Res. Commun. 114 '• 1077 - 1083. Glenn, J. K. and M. H. Gold. 1985. Purification and characterisation of an extracellu-lar Mn(II)-dependent peroxidase from the hgnin-degrading basidiomycete Phanerochaete chrysosporium. Arch. Biochem. Biophys. 242 : 329 - 341. Glenn, J. K., L. Akileswaran and M. H. Gold. 1987. Mn(II) oxidation is the principal function of the extracellular Mn-peroxidase from Phanerochaete chrysospo-rium. Arch. Biochem. Biophys. 251 : 688 - 696. Gottheb, S. and M. J. Pelzcar Jr. 1951. Microbiological aspect of hgnin degradation. Bacteriol. Rev. 15: 55 - 76. Gradziel, K., K. Haider, J. Kochmanska, E. Malarczyk and J. Trojanowski. 1978. Bac-terial decomposition of synthetic 1 4 C - labelled hgnin and hgnin monomer derivatives. Acta. Microbiol. Pol. 27 : 103 - 109. CITED BIBLIOGRAPHY 139 Gunnison, D. and M. Alexander. 1975. Basis for the resistance of several algae to microbial decomposition. Appl. Microbiol. 29: 729 - 738. Habe, T., M. Shimada, T. Okamoto, B. Panijpan and T. Higuchi. 1985. Incorporation of dioxygen into hydroxylated product during the C - C single bond cleav-age of 1,2 - bis(p-methoxyphenyl)propane-l,3-diol catalysed by hemin. A novel model system for the hemoprotein ligninase. J. Chem. Soc, Chem. Commun. : 1323 - 1324. Hagglund, E. 1951. Chemistry of Wood. Academic Press, New York pp 351 Hall, P. 1980. Enzymatic transformation of lignin: 2. Enz. Microbiol. Technol. 2(2>): 170 - 176. Haider, K., J. Trojanowski and V. Sundman. 1978. Screening for lignin degrading bacteria by means of 1 4 C - labelled lignins. Arch. Microbiol. 119. : 103 -106. Arch. Microbiol. 119: 103 - 106. Hammerh, S. D., M. S. A. Leisola., D. Sangland and A. Fiechter. 1986. Oxidation of benzo(a)pyrene by extracellular ligninases of Phanerochaete chrysospo-rium. J. Biol. Chem. 261: 6900 - 6903. Hatakka, A. 1986. Degradation and conversion of hgnin, lignin-related aromatic com-pounds and lignocellulose by selected white-rot fungi. Ph. D. Thesis. Univ. of Helsinki, Finland 97pp. Harvey, P. J., H. E. Schoemaker and J. M. Palmer. 1986. Veratryl alcohol as a media-tor and the role of radical cations in lignin biodegradation by Phanerochaete chrysosporium. FEBS Letter 195: 242 - 246. Harken, J. M. 1967. Lignin - a natural polymeric product of phenol oxidation. In 'Oxidative coupling of phenols'. Eds: Taylor, W. I. and A. R. Battersby. Dekker, New York pp 243 - 321. Harlow, W. M. 1958. The chemistry of the cell walls of wood. In 'Wood Chemistry'. Wise, L. E and E. C. Jahn. Rheinhold Publishing Co. Vol 1 pp 99 - 116. Hartig, R. 1874. Wichtige Krankheiten der Waldbame. Phytopathological classic No. 12. The American Phytopathological Society, St. Paul, M. N(Translated by Merril, W., D. H. Lambert and W. Liese. 1972). Henningson, B., M. Henningson and T. Nilsson. 1972. Deliberation of wood by means of some white-rot fungi. Dept. For. Prod. Royal. Coll. Forest. Stockholm, Res. Notes. NR78. Highley, T. L. and T. K. Kirk. 1979. Mechanisms of wood decay and the heart-rots. Phytopathol. 69(10) : 1151 - 1157. CITED BIBLIOGRAPHY 140 Highley, T. L., S. S. Bar-Lev, T. K. Kirk and M. J. Larsen. 1982. Influence of oxygen and carbon dioxide on wood decay by heartrot and saprot fungi. Phy-topathology 76: 630 - 633. Hintikka, V. and K. Korhonen. 1970. Effects of carbon dioxide, on the growth of lignocolous and soil inhabiting Hymenomycetes. Commun. Ints. For. Fenn. 69: 1 - 29. Jager, A., S. Croan and T. K. Kirk. 1985. Production of ligninases and degradation of hgnin in agitated submerged cultures, of P. chrysosporium. Appl. Environ. Microbiol. 50: 1274 - 1278. Jeffery, J. E., E. V. Partlow and W. J. Polglase. 1960. Chromatographic estimation of sugars in wood cellulose hydrolyzates. Anal. Chem. 32: 1774 - 1777. Jeffries, J. W., S. Choi and T. K. Kirk. 1981. Nutritional regulation of hgnin degra-dation by Phanerochaete chrysosporium. Appl. Microbiol. \2: 290 - 296. Johnsrud, S. C. and K.-E. Eriksson. 1986. Pulp production from different fungal pre-treated materials. In 'Biotechnology in pulp and paper industry'. The third international conference, Stockholm June, 16 - 19, 1986. Sponsored by STFI and SPCI: 53. Jensen, K. K. 1969. Oxygen and carbon dioxide concentrations in sound and decaying red oak trees. For. Sci. 15: 246 - 251. Jurasek, L. 1955. Zmeny v mikrostructure zdrevanatele bunecene blany pri rozkladu drevokaznymi houbami. Biologia(Bratislava). 10: 569 - 579. Translation Joint Pub. Res. Serv. U.S. Dept. Commerce FPL - 635. Kaarik, A. 1974. Decomposition of wood. In 'Biology of plant litter decomposition'. Vol. 1. Eds: Dickinson, C. H and G. H. F.Pugh. Acad. Press, London, New York pp 129 - 174. Kawai, S., T. Umezewa, T. Higuchi. 1987. De novo synthesis of veratryl alcohol by Coriolus versicolor. Wood Res. 73: 18 - 21. Kawakami, H. 1976a. Bacterial degradation of hgnin model compounds. IV. On the aromatic ring cleavage of vanillic acid. Mokuzai Gakkaishi 22 : 246 - 251. Kawakami, H. 1976b. Bacterial degradation of hgnin. I. Degradation of MWL by Pseudomonas ovalis. Mokuzai Gakkaishi 22 : 252 - 257. Kawase, K. 1962. Chemical components of wood decayed under natural conditions and their properties. Hokkaido Daigaku Sapporo. J. Fac. Agric. 52: 186 -245. Keeney, D. R. and J. L Nelson. 1982. Nitrogen-inorganic forms. In ' Methods of soil analysis. Part 2,. Agron. No. 9. Second Edition.' Eds : Page, A. L., Miller, R. H. and D. R. Keeney pp 643 - 693. CITED BIBLIOGRAPHY 141 Kerr, A. J. and D. A. I. Goring. 1975. The ultrastructural arrangement of the wood ceU wall. Cellul. Chem. Technol. 9: 563 - 573. Keyser, P., Kirk, T. K. and J. G. Zeikus. 1978. Ligninolytic enzyme system oi Phane-rochaete chrysosporium: synthesised in the absence of lignin in response to nitrogen starvation. J. Bacteriol. 155(3): 790 - 797. Kirk, T. K. 1973. The chemistry and biochemistry of decay. In 'Wood deterioration and its prevention by preservative treatments'. Eds: Nicholas, D. D and W. E. Loos. Syracuse University Press, Syracuse. New York pp 149 - 181. Kirk, T. K. 1975. Effects of a brown-rot fungus, Lenzites trabea on lignin spruce wood. Holzforschung 29(3): 99 - 107. Kirk, T. K. 1976. Biodelignification research at the U. S Forest Products Laborar-tory . In 'Biological delignification: Present status-future directions'. Eds : Abson, D. Weyerhayser Symp. Aug, 30, 1976 pp 31 - 54. Kirk, T. K. 1980. Studies on the physiology of hgnin metabolism white-rot fungi. In 'Lignin biodegradation: microbiology, chemistry and potential applica-tions'. Eds: Kirk, T. K, Higuchi, T. and H.-m. Chang. Vol 1. CRC Press, Boca Raton, Flo pp 51 - 63. Kirk, T. K. 1981. Toward elucidating the mechanism of action of the ligninolytic systems in basidiomycetes. In 'Trends in the biology of fermentatons for fuels and chemicals'. Ed: Hollaender, E. Plenum Press, New York pp 131 - 149. Kirk, T. K. 1983. Lignin biodegradation : importance and historical research per-spectives . In 'Recent advances in lignin biodegradation research'. Eds : Higuchi, T, Chang, H. -m. and T. K. Kirk. Uni Publishers, Tokyo pp 1 -11. Kirk, T. K. and Adler, E. 1970. Methoxyl-deficient structural elements in hgnin of sweetgum decayed by a brown-rot fungus. Acta. Chem. Scand. 24'- 3379 -3390. Kirk, T. K. and W. E. Moore. 1972. Removing lignin from wood with white-rot fungi and digestibility of the resulting wood. Wood and Fiber (^2): 72 - 79. Kirk, T. K. and T. L. Highley. 1973. Quantitative changes in structural components of conifer woods during decay by white- and brown-rot fungi. Phytopathology 63: 1338 - 1342. Kirk, T. K and H.-m. Chang. 1975. Decomposition of lignin by whie-rot fungi.II. Characterisation of heavily degraded lignins from degraded spruce. Holz-forschung 29 : 56 - 64. CITED BIBLIOGRAPHY 142 Kirk, T. K., Connors, W. J. and J. G. Zeikus. 1976. Requirement for growth during hgnin decomposition by two wood-rotting fungi. Appl. Environ. Microbiol. 32(1): 192 - 194. Kirk, T. K., Connors, W. J. and J. G. Zeikus. 1977. Advances in understanding the microbial degradation of hgnin. In 'The structure, biosynthesis and degradation of wood. Recent advances in phytochemistry'. Vol. 11. Eds: Loewus F. A. and Runeckles. Plenum Press, New York pp 369 - 394. Kirk, T. K., Schultz, E., Connors, E., Lorenz, W. J. and J. G. Zeikus. 1978. Influence of cultural parameters on lignin metabohsm by Phanerochaete chrysospo-rium. Arch. Microbiol. 117. 277 - 285. Kirk, T. K., H. Yang and P. Keyser. 1978. The chemistry and physiology ofthe fungal degradation of lignin. Develop. Ind. Microbiol. 19: 51 - 61. Kirk, T. K. and H. H. Yang. 1979. Partial delignification of unbleached kraft pulp with lignolytic fungi. Biotechnol Lett. 1: 347 - 325. Kirk, T. K., T. Higuchi and H.-m. Chang. 1980. Lignin biodegradation: summary and perspectives. In 'Lignin biodegradation: microbiology, chemistry and potential applications'. Eds: Kirk, T. K and H.-m. Chang. CRC Press, Boca Raton, Flo pp 235 - 244. Kirk, T. K. and H.-m. Chang. 1981. Potential applications of bioligninolytic systems. Enz. Microbiol. Technol. 5(3): 189 - 196. Kirk, T. K. and M. Shimada. 1985. Lignin biodegradation and white-rot fungi. In 'Biosynthesis and biodegradation of wood components'. Ed: Higuchi, T. Academic Press, New York pp 579 - 605. Kirk, T. K., Nakatsubo, F. and I. D. Reid. 1983. Biochem. Biophys. Commun. 104: 320 - 327. Quoted by Kirk, T. K and M. Shimada. 1985. Lignin biodegrada-tion : The microorganisms involved and the physiology and biochemistry of degradation by white-rot fungi. In 'Biosynthesis and biodegradation of wood components'. Ed: Higuchi, T. Academic Press, N. York pp 579 - 605. Kirk, T. K. and E. B. Cowhng. 1984. The chemistry of solid wood. In 'Advances in Chemistry Series'. Ed : Rowell, R. M. Vol 27. American Chemical Society, Washington, D. C pp 435 - 487. Kirk, T. K., Croan, S., Tien, M., Murtagh, K. E. and R. L. Farrall. 1986. Production of multiple ligninases hy Phanerochaete chrysosporium. Effect of selected growth studies and use of mutant strain. Enzyme Microbiol. Technol. 8: 27 - 32. Kirk, T. K. and R. L. Farrall. 1987. Enzymatic 'combustion': the microbial degrada-tion of hgnin. Ann. Rev. Microbiol. J^l: 465 - 506. CITED BIBLIOGRAPHY 143 Konishi, K. and Y. Inou. 1971. Decomposition of lignin by Coriolus versicolor. II : Mode of action of laccase-type enzyme on lignin. J. Japan. Wood. Res. Soc. 17: 255 - 262. Kutsuki, II. and M. H. Gold. 1983. Generation of hydroxyl radical and its involvement in lignin degradation by Phanerochaete chrysosporium. Biochem. Biophys. Res. Commun. 109: 320 - 328. Leatham, G. F. and T. K. Kirk. 1983. Regulation of ligninolytic activity by nutrient nitrogen in white-rot basidiomycetes. Feins. Microbiol. Lett. 16 : 65 - 67. Leatham. G. F. 1986. The ligninolytic activities of Lentinus edodes and Phanerochaete chrysosporium. Appl. Microbiol. Biotechnol. 24 '• 51 - 58. Leisola, M. S. A., Ulmer, D. C , Waldner, R. and A. Fiechter. 1984. Role of veratryl alcohol in lignin degradation by Phanerochaete chrysosporium. J. Biotech-nol. 1: 331 - 339. Leisola, M. S. A., Thanei-Wyss, U. and A. Fiechter. 1985. Strategies for production of high ligninase activity by Phanerochaete chrysosporium. J. Biotechnol. 3: 92 - 107. Leisola, M. and R. W. Waldner. 1986. Production, characterisation and mechanisms of hgnin peroxidases. In 'Treatment of lignocellulosics with white-rot fungi'. Eds: Zadrazil, F. and P. Reiniger. Elsevier Applied Science. London pp 37 - 42. Liese, W. 1970. Ultrastructural aspects of woody tissue disintegration. Ann. Rev. Phytopathology 8: 231 - 258. Liwicki, R., Paterson, A., MacDonald, M- J- and P. Broda. 1985. Phenotypic classes of phenol-oxidase negative mutants of the hgnin degrading fungus Phane-rochaete chrysosporium. J. Bacteriol. 162: 641 - 644. Lombard, F. F., Ross, W. D. and J. L. Lowe. 1960. Cultural characteristics of Fomes ulmarius and Poria ambigu. Mycologia 52 :280 - 294. Long, W. H. 1914. A preHminary note on the cause of'pecky' cypress. Phytopathology. 4. 39 pp. Lundquist, K., Kirk, T. K. and Connors, W. J. 1977. Fungal degradation of kraft Hgnin and lignin sulufonates prepared from synthetic UC hgnins. Arch. Microbiol. 112: 291 - 296. McCarthy, A. J., Paterson, A. and P. Broda. 1986. Lignin solubilisation by Ther-momonospora mesophila, Appl. Microbiol. Biotechnol. 24- 347 - 352. Mandels, M., Kostich, J. and R. Darizek. 1971. The use of adsorbed cellulose in the continous conversion of ceUulose to glucose. J. Polym. Sci. Part. C 36: 445 - 459. CITED BIBLIOGRAPHY 144 Meier, H. 1961. The distribution of polysaccharides in wood fiber. J. Polym. Sci. S.l: 11 - 18. Meier, H. 1962. Pure. Appl. Chem. 5: 37 Quoted by Timmel, T. 1967. Recent progress in the chemistry of wood hemicelluloses. Wood. Sci. Technol. 1: 45 - 70. Merril, W. and E. B. Cowling. 1966. Role of nitrogen in wood deterioration: Amount and distribution of nitrogen in fungi. Phytopathology 56(7): 1083 - 1090. Myers, G. C , Leatham, G. F. and T. H. Wegner. 1988. Fungal pretreatment of aspen chips improves strength of refiner mechanical pulp. Tappi pp 105 - 108. Miksche, G. E. and S. Yasuda. 1978. Lignin of 'giant' mosses and some related species. Phytochemistry 17(3): 503 - 504. Moo-Young, M. A., Daugliss, A. J. D., Chahal, S. and D. G. Macdonales. 1979. The Waterloo Process for SCP from waste biomass. Proc. Biochem. 14'- 38 - 40: Moo-Young, M., Lamptey, J. and P. Girard. 1983. Paper pulpmill sludge utilisation: Technoeconomic potential for fuel ethanol,methane and SCP production. In 'Biotechnology in pulp and paper industry'. Paper and Board Division, Pira, Surrey, England pp 382 - 435. Muhammed, A. F. and M. M. Micko. 1984. Accumulation of calcium crystals in decayed wood of aspen attacked by Fomes igniarius. Inst. Wood Anat. Bull. ? - ?. Nakatsubo, F., Reid, I. D. and T. K. Kirk. 1982. Incorporation of 1 8 0 2 and abscence of streospecincity in primary product formation during fungal metabolism of a hgnin model compound. Biochimica et Biophysica Acta. 719: 284 -291. Necessany, V. and J. Cetlova. 1963. Rozkland bunecnych bian bukoveho dreva houbou Stereum purpureum Pers. Drevarsky Vyskum. 4'- 195 - 202. Necessany, V. and L. Jurasek. 1956. Zmeny submikroskopicke struktury dreva na-padeneho bilon hnildoon. Lesnicjy Casopis. 2: 43 - 52. Nilsson, T. and G. Daniel. 1986. Ligninolytic activity of wood-degrading bacteria. In 'Biotechnology in the pulp and paper industry. The third international conference, Stockhlom. June, 16 - 19, 1986. Sponsored by STFI and SPCI pp 54 - 57. Nimz, H. 1978. Beech hgnin-proposal of a constitutional scheme. Angew. Chem. 86: 336 - 344. Odier, E. and B. Monties. 1977. Activite ligninolytique in vitro de Bacteria isolees depaille de Ble en decomposition. C. A. Acad. Sci. Paris, Serie D. 284- 2175 - 2178. CITED BIBLIOGRAPHY 145 Otjen, J. and R. A. Blanchette. 1982. Patterns of decay caused by Inonotus dryophilus (Aphyllophorales: Hymenochaetaceae), a white-pocket rot of red oaks. Can. J. Bot. 60{11): 2270 - 2279. Parameswaran, N. and W. Liese. 1981. Ultrastructural localisation of wall components in wood cells. In 'Ekman Days'. Int. Symp. Wood, and Pulping Chemistry . Stockholm, June, 9 - 12. Vol 1 pp 16 - 131. Pettey, T. M. and D. L. Crawford, D. L. 1985. Characterisation of acid-precipitable, polymeric lignin(APPL) produced by Streptomyces viridosporus and proto-plast fusion recombinant Streptomyces strains. Biotechnol. Bioeng. Symp. 15: 179 - 190. Phillips, D. H. 1966. Oxygen transfer into mycelial pellets. Biotech. Bioeng. Symp. 8: 456 - 460. Pilon, L., Derochers, M., Jurasek, L. and P. J. Neumann. 1982a. Increasing water retention of mechanical pulp by biological treatmnets. Tappi £5(6) : 93 -Pilon, L., Barbe, M. C , Desrochers, M. and L. Jurasek. 1982b. Fungal treatment of mechanical pulps-its effect on paper properties. Biotechnol. Bioeng. 2^ (9): 2063 - 2076. Piatt, M. N., Haddar, Y. and I. diet. 1984. Fungal actitvites involved in lignocellulose degradation by Pleurotus. Appl. Microbiol. Biotechnol. 20: 150 - 154. Rayner, A. D. and M. J. Hedges. 1982. Observation in the specificity and ecological role of basidiomycetes colonizing dead elm wood. Trans. Bri. Mycol. Soc. 78: 370 - 373. Reid, I. D. 1979. The influence of nutrient balance on lignin degradation by the white-rot fungus Phanerochaete chrysosporium. Can. J. Bot. 57: 2050 - 2058. Reid, I. D. 1983. Effects of nitrogen supplements on degradation of aspen wood hgnin and carbohydtrate components by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 45: 830 - 837. Reid, I. D. 1985. Biological delignification of aspen wood by sohd-state fermentaion with the white-rot fungus Merulius tremellosus. Appl. Environ. Microbiol. 50{1): 133 - 139. Reid, I. D. and K. A. Seifert. 1982. Effect of an atmospere of oxygen on growth, respiration and lignin degradation by white-rot fungi. Can. J. Bot. 60: 2357 - 2364. Reid, I. D., Chao, E. E. and P. S. S. Dawson. 1984. Lignin degradation by Phane-rochaete chrysosporium in agitated cultures. Can. J. Microbiol. 31: 88 -90. CITED BIBLIOGRAPHY 146 Reese, E. T. and A. Maguire. 1969. Surfactants as stimulants of enzyme production by micro-organisms. Appl. Microbiol. 17: 242 - 245. Rheade, A. E. and R. E. McQueen. 1983. Investigation of white-rot fungi for the con-version of poplar into potential feedstuff for ruminants. Can. J . Microbiol. 29(3): 456 - 460. Robinson, L. E. and R. L. Crawford. 1978. Degradation of 1 4 C - labelled lignins by Bacillus megaterium. Ferns. Microbiol. Lett. 4'- 301 - 302. Roch, P., Odier, E., Monties, B. and K.-E. Eriksson. 1983. The influence of simple sugar supplementation on lignin degradation by different white-rot fungi. In 'Biotechnology in the pulp and paper industry'. London, 12 - 14 Sept, Pira pp 323 - 332. Rosenberg. S. L. and C. R. Wilke. 1978. Ligin biodegradation on the production of ethyl alcohol from cellulose. In 'Lignin biodegradation: microbiology, chemistry and potential applications'. Vol II. Eds: Kirk, T.K., T. Higuchi and H.-m. Chang. CRC Press, Boca Raton, Florida pp 199 - 212. Ruel, K., Barnoud, F. and D. A. I. Goring. 1978. Lamellation in the 52 layer of softwood tracheids as demonstrated by scanning transmission electron mi-croscopy. Wood. Sci. Technol. 12: 287 - 291. Ruel, K. and B. Barnoud. 1984. Ultrastructural aspects of wood degradation by Phanerochaete chrysosporium: observations on spruce wood impregnated with glucose. Holzforshung 38(2): 61 - 68. Ruddick, J . N. R. and P. I. Morris. 1987. The movement of iron into field test stakes. International Research Group on Wood Preservation Document IRG/WP/2286 14 pp. Safford, L. O., Shigo, A. and M. Ashley. 1974. Gradients of cation concentration in discoloured and decayed wood in sugar maple. Can. J . For. Res. 4'. 435 -440. Saka, S. 1980. Lignin distribution as determined by energy dispersive X-ray analysis. Ph.D. Thesis. North Carolina State University; Raleigh, North Carohna. Saka, S., Thomas, R. J. and J. S. Gratzl. 1979. Lignin distribution and determination by energy dispersive analysis of X-rays. Tappi 61 (1): 73 - 76. Samuelsson, L., Mjoberg, P. J., Harley, N., VaUender, L. and K.-E. Eriksson . 1980. Influence of fungal treatment in the strength versus energy relationship in mechanical pulping. Sven. Papperstidn. 83: 221 - 225. Sasaki, Y. and S. Takao. 1967. Organic acid production by basidiomycetes. III. Cul-tural conditions for L-malic acid production. Appl. Microbiol. 15 : 373 -377. CITED BIBLIOGRAPHY 147 Shallhorn, P. M. and A. Karnis. 1979. The tear and tensile strength of mechanical pulps. 1979 international mechanical pulping conference. Sponsored by the technical section, CPPA and TAPPI. The Sheraton Centre, Toronto, Ont. June, 11 - 14 pp 25 -36. Sarkenen, K. V., Chang, H. -m. and G. G. Allan. 1967. Species variation in hgnins. II .conifer hgnins. Tappi 50: 583 - 587. Shortle, W. C. and A. L. Shigo. 1973. Concentration of manganese and microorgan-isms in discoloured and decayed wood in sugar maple. Can. J. For. Res. : 354 - 358. Shimada, M., Nakatsubo, F., Kirk, T. K. and T. Higuchi. 1981. Biosynthesis of the secondary metabolite veratryl alcohol in relation to lignin degradation in Phanerochaete chrysosporium. Arch. Microbiol. 129 : 321 - 324. Shimada, M. and M. H. Gold. 1983. Direct cleavage of the vicinal diol hnkage of the lignin model compound dihydroanisoin by the basidiomycete Phane-rochaete chrysosporium. Arch. Microbiol. 13^: 299 - 302. Sinner, M., Simatupang, M. H. and H. H. Dietrichs. 1975. Automated quantitative analysis of wood carbohydrates by borate complex ion exchange chromatog-raphy. Wood Sci. Technol. 9 : 307 - 322. Sweeley,'C. C , Bentley, R., Makita, M. and W. W. Wells. 1963. Gas-liquid chro-matography of trimethylsilyl derivatives of sugars and reliable substances. J. Am. Chem. Soc. 85: 2497 - 2507. Tai, D., Teraasawa, M., Chen, C. -L., Chang, H. -m. and T. K. Kirk. 1983. Biodegra-dation of guaicyl and guaiacyl-syringyl lignins in wood by Phanerochaete chrysosporium. In ' Recent advances in lignin biodegradation research'. Rds : T. Higuchi, H.-m. Chang and T. K. Kirk. Uni publishers Co. Ltd. Chuo-ku, Tokyo 103 pp 44 - 63. T.A.P.P.I. 1958. Freeness of pulp. T 227 os - 58, TAPPI standard , Tech. Assoc. Pulp Paper Industry, New York. T.P.P.I. 1965. Internal resistance of paper. T 414 os - 65, TAPPI standard , Tech. Assoc. Pulp Paper Industry, New York. T.A.P.P.I. 1968. Weight per unit area(basis weight or substance) of paper and paper-board, T 410 os - 68, TAPPI Standard, Tech. Assoc. Pulp Paper Industry, New York. T.P.P.I. 1970. Standar conditioning and testing atmospheres for paper board, pulp sheets and related products. T 402 - 70, TAPPI standard, Tech, Assoc. Pulp Paper Industry, New York. CITED BIBLIOGRAPHY 148 T.A.P.P.I. 1971. Physical testing of pulp handsheets. T 220 os - 71, TAPPI standard, Tech. Assoc. Pulp Paper Industry, New York. T.A.P.P.I. 1976a. Bursting strength of paper. T403 os - 76, TAPPI Standard, Tech. Assoc. Pulp. Paper Industry, New York. T.A.P.P.I. 1976b. Tensile breaking strength and elongation of paper and paper boards(using pendulum-type tester). T404 os - 76, TAPPI Standard, Tech. Assoc. Pulp. Paper Industry, New York. T.P.P.I. 1976c. Alcohol-benzene and dichloromethane solubles in wood and pulp. T 204 Os - 76. TAPPI standard, Tech. Assoc. Pulp Paper Industry, New York. T.P.P.I. 1976d. Acid soluble hgnin in wood and pulp. TAPPI standard, Tech. Assoc. Pulp Paper Industry, New York. T.A.P.P.I. 1981. Forming hansheets for physical tests of pulp. T 205 om - 81, TAPPI Standard, Tech. Assoc.Pulp. Paper. Industry, New York. T.A.P.P.I. 1976. Thickness(Caliper) of paper and paperboard. T 411 os - 76, TAPPI Standard, Tech. Assoc. Pulp Paper Industry, New York. T.A.P.P.I. 1985. Freeness of pulp. T 227 om - 85, TAPPI Standard, Tech. Assoc. Pulp Paper Industry, New York. Turrens, J. F., Freeman, B. A. and J. D. Crapo. Hyperoxia increases H 2 0 2 release by lung mitochondria and microsomes. Arch. Biochem. Biophys. 217 : 411 -412. Tien, M. and T. K. Kirk. 1983. Lignin-degrading enzyme from the Hymenomycete Phanerochaete chrysosporium Burds. Science. 221: 661 - 663. Tien, M., Kirk, T. K., Bull, C. and J. A. Free. 1986. Steady state and transient-state kinetic studies on the oxidation of 3,4-dimethylbenzyl alcohol catalyzed by the ligninase of Phanerochaete chrysosporium 261: 1657 - 1693. Timmel, T. E. 1967. Recent progress in the chemistry of wood hemicelluloses. Wood. Sci. Technol. 1: 45 - 70. Timmel, T. E. 1969. The chemical composition of tension wood. Svensk. Papperstid-ning. 72 (6) ; 173 - 181. Tono, T., Y. Tani and K. Ono. 1968. Microbial degradation of agricultural wastes. I: Adsorption of lignin and clarification of lignin containing liquor by molds. J. Ferment. Technol. 9: 569 - 576. Tran, A. V. and R. P. Chalmers. 1987. Delignification of an unbleached hardwood kraft pulp by Phanerochaete chrysosporium. Appl. Microbiol. Biotechnol. 25 : 484 - 490. CITED BIBLIOGRAPHY 149 Twine, J. R and C. H. Williams. 1971. The determination of phosphorus of plant material by automatic analysis. Comm. Soil. Sci. Plant Anal. 2(6). : 485 -489. Ulmer, D., Leisola, M., Puhakka, J. and A. Fiechter. 1983. Phanerochaete chrysospo-rium and hgnin degradadtion: growth pattern and lignin degradation. Eur. J. Appl. Microbiol. Biotechnol. 18: 153 - 157. Vance, C. P., Kirk, T. K. and R. T. Sherwood. 1980. Lignification as a mechanism of disease resistance. Ann. Rev. Phtopathology 18: 259 - 288. Varian Vista 401/2. 1981. Chromatography data system. Operators system manual. Varian assocites Inc.pp 5-1 - 5-78. Wentz, F. E., Marcy, A. D. and M. J. Gray. 1982. Analysis of wood sugars in pulp and paper industry samples by HPLC. J. Chromatogr. Sci. 20: 349 - 352. Westermark, U., Hardell, H. -L. and T. Iversen. 1986. The content of protein and pectin in the lignified middle lamella/primary wall from spruce fibers. Holz-forschung 40(2) : 65 - 68. Whitmore, F. N. 1982. Lignin-protein complex in cell walls of Pinus elliottii: amino acid constituents. Phytochemistry 21: 315 - 318. Weichelt, J. 1981. Reaktionsprodukt aus stickstoff und hgnin sowie dessen oxidative. Abbau. Z. Acker. Pflanzenb. 150: 480 - 488. Wilcox, W. W. 1968. Changes in wood microstructure through progressive stages of decay. U.S. For. Serv. Res. Paper. FPL No.70: 45 pp. Wilcox, W. W. 1970. Anatomical changes in wood cell walls attacked by fungi and bacteria. Bot. Rev. 36(1): 1 - 27. Yang, H. H., Effland, M. J. and T. K. Kirk. 1980. Factors influencing fungal degra-dation of hgnin in representative lignocellulosic thermomechanical pulp. Biotech. Bioeng. 22: 65 - 67. Zadrazil, F. 1977. The conversion of straw into feed by basidiomycetes. save J. Appl. Microbiol. 4: 273 - 281. Appendix 150 Appendix A. 151 Table A.19: Refining of trembling aspen wood chips. First Pass Second Pass Plate gap (inches) 0.020 0.003 Refining consistency(%) Refining temperature( 0 C) 21.6 15.0 90.0 90.0 Net power(kwh/t) 455 1061 Net power(Hpd/t) 25.4 59.3 Net power(MJ/kg) 1.6 3.8 Canadian standard freeness N. D. a "Note determined. Table A.20: Chemical composition of untreated trembling aspen wood chips. Wood component Percentage composition" Lignin 21.5+.3 Glucose 50.8+1.2 ' Xylose 19.8+.8 Nitrogen 0.14+.2 "average of 5 replicates. Appendix A. 152 Table A.21: Various fungal-treatments used to evaluate the effect of some cultural factors on selective biodehgnification of trembling aspen RMP by Rigidoporus ulmarius. Fungal-treatmenta Fungal-treatment type number Aeration Hydrogen Mineral Veratryl Tween peroxide, mM solution, ml alcohol, mM 80, % 1 Air 0 1 0 0 2 Air 0 1 0 0.05 3 Air 0 1 2 0 4 Air 0 1 2 0.05 5 Air 0 ' 8 0 0 6 Air 0 8 0 0.05 7 Air 0 8 2 0 8 Air 0 8 2 0.05 9 Air 0.2 1 0 0 10 Air 0.2 1 0 0.05 11 Air 0.2 1 2 0 12 Air 0.2 1 2 0.05 13 Air 0.2 8 0 0 14 Air 0.2 8 0 0.05 15 Air 0.2 8 2 0 16 Air 0.2 8 2 0.05 17 Oxygen 0 1 0 0 18 Oxygen 0 1 0 0.05 19 Oxygen 0 1 2 0 20 Oxygen 0 1 2 0.05 21 Oxygen 0 8 0 0 22 Oxygen 0 8 0 0.05 23 Oxygen 0 8 2 0 24 Oxygen 0 8 2 0.05 25 Oxygen 0.2 1 0 0 26 Oxygen 0.2 1 0 0.05 27 Oxygen 0.2 1 2 0 28 Oxygen 0.2 1 2 0.05 29 Oxygen 0.2 8 0 0 30 Oxygen 0.2 8 0 0.05 31 Oxygen 0.2 8 2 0 32 Oxygen 0.2 8 2 0.05 "Used throughout this thesis as reference to fungal-treatment type. Appendix A. 153 Table A.22: Analysis of variance for testing the effect of cultural factors: aeration, hydro-gen peroxide, mineral solution, veratryl alcohol and tween 80 on hgnin biodegradation as measured by the residual hgnin content. Source Degree of Sum of F Freedom squares ratio0 Aeration 1 105.26 69.96*** Hydrogen peroxide6 1 57.45 38.18*** Mineral solution0 1 68.73 45.68*** Veratryl alcohold 1 36.74 24.41*** Tween 80 1 31.34 20.83*** Aeration x Peroxide 1 8.66 5.75* Aeration x Minsol 1 14.34 9.53** Aeration x Veralcoh 1 3.55 2.36 Aeration x Tween 80 1 3.92 2.60 Peroxide x Minsol 1 13.25 8.81** Peroxide x Veralcoh 1 23.12 15.37*** Peroxide x Tween 80 1 11.75 7.81** Minsol x Veralcoh 1 7.55 5.02* Minsol x Tween 80 1 2.87 1.91 Veralcoh x Tween 80 1 2.38 1.58 Aeration x Peroxide x Minsol 1 0.90 0.60 Aeration x Peroxide x Veralcoh 1 7.90 5.25* Aeration x Peroxide x Tween 80 1 3.03 2.01 Aeration x Minsol x Veralcoh 1 18.51 12.30*** Aeration x Minsol x Tween 80 1 2.34 1.56 Aeration x Veralcoh x Tween 80 1 1.37 0.91 Peroxide x Minsol x Veralcoh 1 0.46 0.31 Peroxide x Minsol x Tween 80 1 0.00 0.00 Peroxide x Veralcoh x Tween 80 1 45.16 30.01*** Minsol x Veralcoh x Tween 80 1 0.71 0.47 Aeration x Peroxide x Minsol x Veralcoh 1 2.34 1.55 Aeration x Peroxide x Minsol x Tween 80 1 4.65 3.09 Aeration x Peroxide x Veralcoh x Tween 80 1 1.78 1.18 Aeration x Minsol x Veralcoh x Tween 80 1 3.03 2.01 Peroxide x Minsol x Veralcoh x Tween 80 1 9.54 6.34* Aeration x Peroxide x Minsol x Veralcoh x Tween 80 1 0.00 0.00 ° * * * indicates significance at the 0.001 level of probability; ** indicates significance at 0.01 level of probability; * indicates significance at 0.05 level of probability. ^Peroxide is equivalent to hydrogen peroxide. c Minsol is equivalent to mineral solution. rfVeralcoh is equivalent veratryl alcohol. Appendix A. 154 Table A.23: Analysis of variance for testing the effect of cultural factors: aeration, hydro-gen peroxide, mineral solution, veratryl alcohol and tween 80 on cellulose biodegradation as measured by the residual glucose content. Source Degree of Sum of F Freedom squares ratioa Aeration 1 40.66 24.10*** Hydrogen peroxideb 1 43.04 25.51*** Mineral solution0 1 43.85 25.99*** Veratryl alcohold 1 43.33 25.69*** Tween 80 1 91.22 54.07*** Aeration x Peroxide 1 44.95 26.64*** Aeration x Minsol 1 54.82 32.49*** Aeration x Veralcoh 1 28.93 17.15*** Aeration x Tween 80 1 12.57 7.45** Peroxide x Minsol 1 0.04 0.02 Peroxide x Veralcoh 1 89.45 53.04*** Peroxide x Tween 80 1 25.17 14.92*** Minsol x Veralcoh 1 20.45 12.12*** Minsol x Tween 80 1 61.05 36.19*** Veralcoh x Tween 80 1 28.81 17.08*** Aeration x Peroxide x Minsol 1 10.07 5.97* Aeration x Peroxide x Veralcoh 1 0.00 0.00 Aeration x Peroxide x Tween 80 1 50.18 29.74*** Aeration x Minsol x Veralcoh 1 71.95 42.65*** Aeration x Minsol x Tween 80 1 81.42 48.26*** Aeration x Veralcoh x Tween 80 1 16.39 9.71** Peroxide x Minsol x Veralcoh 1 37.26 22.09*** Peroxide x Minsol x Tween 80 1 7.54 4.47* Peroxide x Veralcoh x Tween 80 1 41.97 24.88*** Minsol x Veralcoh x Tween 80 1 0.00 0.00 Aeration x Peroxide x Minsol x Veralcoh 1 33.54 19.88*** Aeration x Peroxide x Minsol x Tween 80 1 41.15 24.39*** Aeration x Peroxide x Veralcoh x Tween 80 1 83.07 49.24*** Aeration x Minsol x Veralcoh x Tween 80 1 4.42 2.62 Peroxide x Minsol x Veralcoh x Tween 80 1 0.00 0.00 Aeration x Peroxide x Minsol x Veralcoh x Tween 80 1 4.28 2.54 " * * * indicates significance at the 0.001 level of probability; ** indicates significance at 0.01 level of probability; * indicates significance at 0.05 level of probability. ''Peroxide is equivalent to hydrogen peroxide. c Minsol is equivalent to mineral solution. ^Veralcoh is equivalent veratryl alcohol. Appendix A. 155 Table A.24: Analysis of variance for testing the effect of cultural factors: aeration, hydro-gen peroxide, mineral solution, veratryl alcohol and tween 80 on hemicellulose biodegra-dation as measured by the residual xylose content. Source Degree of Sum of F Freedom squares ratio" Aeration 1 46.39 50.67*** Hydrogen peroxide6 1 96.45 105.34*** Mineral solution0 1 14.14 15.45*** Veratryl alcohol^  1 20.80 22.72*** Tween 80 1 24.57 26.84*** Aeration x Peroxide 1 42.81 46.75*** Aeration x Minsol 1 43.69 Aeration x Veralcoh 1 2.00 2.19 Aeration x Tween 80 1 7.24 7.91** Peroxide x Minsol 1 97.15 106.11*** Peroxide x Veralcoh 1 86.77 94.77*** Peroxide x Tween 80 1 17.26 18.85*** Minsol x Veralcoh 1 1.95 2.13 Minsol x Tween 80 1 37.54 40.99*** Veralcoh x Tween 80 1 35.60 38.88*** Aeration x Peroxide x Minsol 1 0.00 0.00 Aeration x Peroxide x Veralcoh 1 1.03 1.12 Aeration x Peroxide x Tween 80 1 13.89 15.18*** Aeration x Minsol x Veralcoh 1 12.80 13.98*** Aeration x Minsol x Tween80 1 5.73 6.25* Aeration x Veralcoh x Tween 80 1 1.50 1.64 Peroxide x Minsol x Veralcoh 1 3.94 4.30* Peroxide x Minsol x Tween 80 1 6.30 6.88** Peroxide x Veralcoh x Tween 80 1 3.19 3.49 Minsol x Veralcoh x Tween 80 1 26.15 28.56*** Aeration x Peroxide x Minsol x Veralcoh 1 92.75 101.21*** Aeration x Peroxide x Minsol x Tween 80 1 1.93 2.11 Aeration x Peroxide x Veralcoh x Tween 80 1 3.95 4.31* Aeration x Minsol x Veralcohx Tween 80 1 8.45 9.23** Peroxide x Minsol x Veralcoh x Tween 80 1 0.00 0.00 Aeration x Peroxide x Minsol x Veralcoh x Tween 80 1 41.28 45.49*** ° * * * indicates significance at the 0.001 level of probability; ** indicates significance at 0.001 level of probability; * indicates significance at 0.05 level of probability. ^Peroxide is equivalent to hydrogen peroxide. c Minsol is equivalent to mineral solution. dVeralcoh is equivalent veratryl alcohol. Appendix A. 156 Table A.25: Analysis of variance for testing the effect of cultural factors: aeration, hy-drogen peroxide, mineral solution, veratryl alcohol and tween 80 on selective biodeligni-fication measured by SBI. Source Degree of Sum of F Freedom squares ratio" Aeration 1 2.00 26.95*** Hydrogen peroxide6 1 0.52 7.08** Mineral solution0 1 1.52 20.56*** Veratryl alcohol"" 1 0.41 5.59* Tween 80 1 0.03 0.41 Aeration x Peroxide 1 0.00 0.02 Aeration x Minsol 1 0.00 0.00 Aeration x Veralcoh 1 0.05 0.63 Aeration x Tween 80 1 0.51 6.95** Peroxide x Minsol 1 0.09 1.20 Peroxide x Veralcoh 1 0.00 0.00 Peroxide x Tween 80 1 0.02 0.20 Minsol x Veralcoh 1 0.05 0.68 Minsol x Tween 80 1 0.25 3.32 Veralcoh x Tween 80 1 0.10 1.34 Aeration x Peroxide x Minsol 1 0.34 4.55* Aeration x Peroxide x Veralcoh 1 0.32 4.36* Aeration x Peroxide x Tween 80 1 0.78 10.56*** Aeration x Minsol x Veralcoh 1 0.07 0.96 Aeration x Minsol x Tween 80 1 0.00 0.00 Aeration x Veralcoh x Tween 80 1 0.24 3.21 Peroxide x Minsol x Veralcoh 1 0.18 2.38 Peroxide x Minsol x Tween 80 1 0.02 0.20 Peroxide x Veralcoh x Tween 80 1 0.91 12.31*** Minsol x Veralcoh x Tween 80 1 0:00 0.00 Aeration x Peroxide x Minsol x Veralcoh 1 0.12 1.64 Aeration x Peroxide x Minsol x Tween 80 1 1.12 15.13*** Aeration x Peroxide x Veralcoh x Tween 80 1 0.53 7.17** Aeration x Minsol x Veralcoh x Tween 80 1 0.15 2.06 Peroxide x Minsol x Veralcoh x Tween 80 1 0.54 7.28** Aeration x Peroxide x Minsol x Veralcoh x Tween 80 1 0.00 0.00 a*** ; n (Ji c ates significance at the 0.001 level of probability; ** indicates significance at 0.001 level of probability; * indicates significance at 0.05 level of probability. bPeroxide is equivalent to hydrogen peroxide. c Minsol is equivalent to mineral solution. ""Veralcoh is equivalent veratryl alcohol. Appendix A. 157 Table A.26: Mean residual lignin content of fungal-treated pulp under significant second order interaction involving cultural factors: aeration, hydrogen peroxide and veratryl alcohol. Treatments Mean residual hgnin content % of original oven-dry weight of pulp Aeration Hydrogen peroxide Veratryl alcohol 0a 0b 0C 18.9 0 0 ld 17.5 0 l e 0 19.4 0 1 1 18.7 1> 0 0 16.9 . 1 0 1 13.9 1 1 0 17.5 1 1 1 17.9 "air flushing. 60 m M . <;0 m M . d2 m M . c0.2 m M . •'oxygen flushing. Appendix A. 158 Table A.27: Mean residual lignin content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: aeration, mineral solution and veratryl alcohol. Treatments Mean residual lignin content % of original oven-dry weight pulp Aeration Mineral solution Veratryl alcohol oa l b 0C 20.4 0 1 ld . 17.8 0 2 0 . 18.0 0 2e 1 18.3 If 1 0. 18.3 1 1 1 17.2 1 2 0 16.2 1 2 1 14.3 aair flushing. bl X concentration of mineral solution. " i O m M . d2 m M . e8 X concentration of mineral solution, 'oxygen flushing. Appendix A. 159 Table A.28: Mean residual lignin content of fungal-treated pulp under second order interaction involving cultural factors: hydrogen peroxide, veratryl alcohol and tween 80. Treatments Hydrogen peroxide Veratryl alcohol Tween 80 % of original oven-dry weight of pulp 0° 0b 0C 18.4 0 0 ld 17.5 0 l e 0 17.1 0 1 1 14.3 1* 0 0 19.5 1 0 1 17.4 1 1 0 17.6 1 1 1 19.1 Mean residual hgnin content "0 m M . b0 m M . c0 %. "0.05%. r-2 m M . '0.2 m M Appendix A. 160 Table A.29: Mean residual glucose content of fungal-treated pulp under significant second order interaction involving cultural factors: aeration, hydrogen peroxide and mineral solution. Treatments Mean residual glucose content % of original oven-dry weight of pulp Aeration Hydrogen peroxide Mineral solution 0° 0 B r 48.2 0 0 47.8 0 l e I 47.6 0 1 2 48.3 If 0 1 46.4 1 0 2 44.2 1 1 1 49.9 1 1 2 46.2 ttair flushing. '0 m M . '1 X concentration of mineral solution. 8 X concentration of mineral solution. "0.2 m M . •'oxygen flushing. Appendix A. 161 Table A.30: Mean residual glucose content of fungal-treated pulp under significant second order interaction involving cultural factors: aeration, hydrogen peroxide and tween 80. Treatments Mean residual glucose content % of original oven-dry weight of pulp Aeration Hydrogen peroxide Tween 80 0° 0b 0C 48.4 0 0 ld 47.5 0 l e 0 48.7 0 1 1 47.1 1' 0 0 47.8 1 0 1 42.7 1 1 0 48.1 1 1 1 48.0 "air flushing. h0 mM. c0 %. d0.05%. c0.2 mM. •'oxygen flushing. Appendix A. 162 Table A.31: Mean residual glucose content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: aeration, mineral solution and veratryl alcohol. Treatments Mean residual glucose content % of original oven-dry weight Aeration Mineral solution Veratryl alcohol 0° l b 0C 49.4 0 1 ld 46.4 0 2e 0 46.8 0 2 1 49.2 \f 1 0 48.8 1 1 1 47.3 1 2 0 46.7 1 2 1 43.3 a air flushing. ' ' I X concentration of mineral solution. °0 m M . doxygen flushing. X concentration of mineral solution, •'oxygen flushing. Appendix A. 163 Table A.32: Mean residual glucose content of fungal-treated pulp under significant second order interaction involving cultural factors: aeration, veratryl alcohol and tween 80. Treatment Mean residual glucose content % of original oven-dry of pulp Aeration Veratryl alcohol Tween 80 0° 0b 0C 49.7 0 0 ld 46.5 0 l e 0 47.4 .0 1 1 48.1 1' 0 0 49.2 1 0 1 46.4 1 1 0 46.6 1 1 1 44.1 "air flushing. ''0 m M . c 0 %. d0.05%. e2 m M . 'oxygen flushing. Appendix A. 164 Table A.33: Mean residual glucose content of fungal-treated pulp under significant second order interaction involving cultural factors: aeration, mineral solution and tween 80. Treatment Mean residual glucose content % of original oven-dry weight of pulp Aeration Mineral solution Tween 80 0a l b . 0C 46.8 0 1 ld 48.9 0 2e 0 50.3 0 2 1 45.7 \f 1 0 49.7 1 -< 1 1 46.6 1 2 0 46.4 1 2 1 43.7 aair flushing. ' ' I X concentration of mineral solution. '0 %. 0.05%. X concentration of mineral solution, 'oxygen flushing. Appendix A. 165 Table A.34: Mean residual glucose content of fungal-treated pulp under significant second order interaction involving cultural factors: hydrogen peroxide, mineral solution and veratryl alcohol. Treatments Mean residual glucose content % of original oven-dry weight of pulp Hydrogen peroxide Mineral solution Veratryl alcohol 0 a l f c 0 C 48.8 0 1 ld 45.8 0 2e 0 47.8 0 2 1 44.2 l ' 1 0 49.4 1 1 1 47.9 1 2 0 45.9 1 2 1 48.8 "0 m M . I X concentration of mineral solution. c 0 m M . d2 m M "8 X concentration of mineral solution. '0.2 m M . Appendix A. 166 Table A.35: Mean residual glucose content of fungal-treated pulp under significant second order interaction involving cultural factors: hydrogen peroxide, mineral solution and tween 80. Treatments Hydrogen peroxide Mineral solution Tween 80 % of original oven-dry weight of pulp 0° lb o c 48.4 0 1 ld 46.2 0 2 e 0 47.9 0 2 1 44.0 1> • 1 0 48.1 1 1 1 49.3 1 2 0 48.8 1 2 1 45.6 Mean residual glucose content "0 m M . I X concentration c0%. d0.05%. e8 X concentration '0.2 m M . of mineral solution, of mineral solution. Appendix A. 167 Table A.36: Mean residual glucose content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: hydrogen peroxide, veratryl alcohol and tween 80. Treatments Mean residual glucose content % of original oven-dry weight of pulp Hydrogen peroxide Veratryl alcohol Tween 80 0a 0b 0C 49.7 0 0 ld 46.8 0 l e 0 46.5 0 1 1 43.4 1' 0 0 49.3 1 0 1 46.1 1 1 0 47.5 1 1 1 49.2 a 0 m M . ''0 m M . c0%. d0.05%. c2 m M . '0.2 m M Appendix A. 168 Table A.37: Mean residual xylose content of fungal-treated pulp under significant second order interaction involving cultural factors: aeration, hydrogen peroxide and Tween 80. Treatments Mean residual xylose content % of original oven-dry weight of pulp Aeration Hydrogen peroxide Tween 80 Qa 0" 0C 16.4 0 0 ld 15.5 0 l e 0 16.8 0 1 1 16.6 If 0 0 14.6 1 0 1 11.4 1 1 0 16.8 1 1 1 16.5 a air flushing. b0 m M . "0 %. d0.05%. '0.2 m M . 'oxygen flushing. Appendix A. 169 Table A.38: Mean residual xylose content of fungal-treated pulp under significant sec-ond order interaction involving cultural factors: aeration, mineral solution and veratryl alcohol. Treatments Mean residual xylose content % of original oven-dry weight of pulp Aeration Mineral solution Veratryl alcohol 0° lb 0C 16.8 0 1 ld 15.3 0 2e 0 16.9 0 2 1 16.2 If 1 0 15.9 1 1 1 16.2 1 2 0 14.7 1 2 1 12.9 "air flushing. bl X concentration of mineral solution. c 0 m M . 2 m M . °8 X concentration of mineral solution, 'oxygen flushing. Appendix A. 170 Table A.39: Mean residual xylose content of fungal-treated pulp under significant second interaction involving cultural factors: aeration, mineral solution and tween 80. Treatment s Mean residual xylose content % of original oven-dry weight of pulp Aeration Mineral solution Tween 80 0Q l b 0C 15.9 0 1 l d 16.2 0 2e 0 17.3 0 2 1 15.9 •1' 1 0 15.9 1 1 1 16.2 1 2 0 15.4 1 2 1 12.8 "air flushing. ' ' I X concentration of mineral solution. ';0 %. d0.05%. "8 X concentration of mineral solution, 'oxygen flushing. Appendix A. 171 Table A.40: Mean residual xylose content of fungal-treated pulp under significant second order interaction involving cultural factors: hydrogen peroxide, mineral solution and veratryl alcohol. Treatments Mean residual xylose content % of original oven-dry weight of pulp Hydrogen peroxide Mineral solution Veratryl alcohol 0° l b 0C 17.1 0 1 ld 14.9 0 T 0 15.1 0 2 1 11.5 1> 1 0 15.6 1 1 1 16.4 1 2 0 16.7 1 2. 1 18.1 "0 m M . I X concentration of mineral solution. c0 m M . d2 m M . c8 X concentration of mineral solution. '0.2 m M . Appendix A. 172 Table A.41: Mean residual xylose content of fungal-treated pulp under significant second order interactions involving cultural factors: hydrogen peroxide, mineral solution and tween 80. Treatments Hydrogen peroxide Mineral solution Tween 80 % of original oven-dry weight of pulp Q a l b 0C 15.9 0 1 ld 16.3 0 2e 0 15.2 0 2 1 11.4 1 0 15.9 1 1 1 16.1 1 2 0 17.6 1 2 1 17.1 Mean residual xylose content ° 0 m M . I X concentration of mineral solution. c 0 %. d0.05%. = 8 X concentration of mineral solution. '0.2 m M . Appendix A. 173 Table A.42: Mean residual xylose content of fungal-treated pulp under significant second order interaction involving cultural factors: mineral solution, veratryl alcohol and tween 80. . Treatment s Mean residual xylose content % of original oven-dry weight of pulp Mineral solution Veratryl alcohol Tween 80 1° 0b 0C 16.1 1 0 ld 16.6 1 r 0 15.6 1 I 1 15.7 2' 0 0 15.9 2 0 1 15.8 2 l 0 16.8 2 I 1 12.2 " I X concentration of mineral solution. h0 m M . c0 %. d0.05%. ef2 m M . ' 8 X concentration of mineral solution. Appendix A. 174 Table A.43: Mean selective biodelignification index of fungal-treated pulp under signifi-cant second order interaction involving cultural factors: aeration, hydrogen peroxide and mineral solution. Treatments Mean selective biodelignification index Aeration Hydrogen peroxide Mineral solution Qa 0b l c 3.5 0 0 2d 3.6 0 r 1 3.3 0 l 2 3.5 \* 0 1 3.6 1 0 2 4.1 1 I 1 3.6 1 l 2 3.7 "air flushing. ''0 m M . *1 X concentration of mineral solution. 8 X concentration of mineral solution. e0.2 m M . 'oxygen flushing. Appendix A. 175 Table A.44: Mean selective biodelignification index of fungal-treated pulp under signifi-cant second order interaction involving cultural factors: aeration, hydrogen peroxide and veratryl alcohol. Treatments Mean selective biodelignification index Aeration Hydrogen peroxide Veratryl alcohol 0a 0b 0C 3.5 0 0 ld 3.8 0 l e 0 3.3 0 1 1 3.5 1' 0 0 3.7 1 0 1 4.0 1 1 0 3.7 1 1 1 3.7 "air flushing. bQ m M . c 0 m M . d2 m M . e0.2 m M . 'oxygen flushing. Appendix A. 176 Table A.45: Mean selective biodelignification index of fungal-treated pulp under signifi-cant second order interaction involving cultural factors: aeration, hydrogen peroxide and tween 80. Treatments Mean selective biodelignification index Aeration Hydrogen peroxide Tween 80 0a 0b 0C 3.4 0 0 ld 3.8 0 l e 0 3.4 0 1 1 3.4 1' 0 0 4.0 1 0 1 3.7 1 1 0 3.6 1 1 1 3.7 "air flushing. '0 mM. c0 %. d0.O5%. e0.2 mM. 'oxygen flushing. Appendix A. 177 Table A.46: Mean selective biodelignification index of fungal-treated pulp under signif-icant second order interaction involving cultural factors: hydrogen peroxide, veratryl alcohol and tween 80. Treatments Mean selective biodelignification index Hydrogen peroxide Veratryl alcohol Tween 80 0b 0C 3.7 0 0 ld 3.6 0 l e 0 3.7 0 1 1 3.9 l ' 0 0 3.4 1 0 1 3.6 1 1 0 3.7 1 1 1 3.5 ?0 m M . b0 m M . ';0 %. d0.05%. c2 m M . '0.2 mM Appendix A. 178 Table A.47: Key to numbers used to designate levels of cultural factors in figures A.7 to A.30. Cultural Aeration factor Number Corresponding level of aeration 0 Air flushing 1 Oxygen flushing Cultural Hydrogen peroxide factor Number Corresponding level of hydrogen peroxide 0 0 mM 1 0.2 mM Cultural Mineral solution factor Number Corresponding level of mineral solution 1 1 X concentration 2 8 X concentration Cultural Tween 80 factor Number Corresponding level of tween 80 0 0 % 1 0.05% Cultural Veratryl alcohol factor Number Corresponding level of veratryl alcohol 0 0 mM 1 2 mM Appendix A. 179 • ACB*TIQN=AIB HUSHING O ACRATIQH=QITGCN flUSHINO HYDROGEN PEROXIDE Figure A.7: Plot of significant interaction of aeration and hydrogen peroxide on biodegra-dation of lignin by R. ulmarius as measured by residual hgnin. 20 • ACRATIQN=AIB TLUSHINC O AERATI0N=0XYCCN HUSHING 18 z ^ 16 14 MINERAL SOLUTION Figure A.8: Plot of significant interaction of aeration and mineral solution on biodegra-dation of hgnin by R. ulmarius as measured by residual hgnin. Appendix A. 180 14 VERATRYL ALCOHOL Figure A.9: Plot of significant interaction of mineral solution and veratryl alcohol on biodegradation of hgnin by R. ulmarius as measured by residual hgnin. 20 • 1 Figure A.10: Plot of significant interaction of hydrogen peroxide and mineral solution on biodegradation of lignin by R. ulmarius as measured by residual hgnin. Appendix A. 181 • WTQ»QCCM t»t»0»IDI <PPtP=HQ 0 HTQUQCtN POOIIPI «00CD=T^S ^ 10 VERATRYL ALCOHOL Figure A . l l : Plot of significant interaction of hydrogen peroxide and veratryl alcohol on biodegradation of hgnin by R. ulmarius as measured by residual hgnin. 20 • HVQBQGtM P(B0»ID( AOPtO-MO Q HTQBOOCH PtBQXtQt «00C0=T 18 ° TWEEN 80 ' Figure A. 12: Plot of significant interaction of hydrogen peroxide and tween 80 on biodegradation of hgnin by R. ulmarius as measured by residual hgnin. Appendix A. 182 60 • Af RATION-AIR flUSHING LU 48-| CO O O so 0 s 44 42 O AC RATIO N=QXYCCN HUSHING H Y D R O G E N P E R O X I D E Figure A. 13: Plot of significant interaction of aeration and hydrogen peroxide on biodegradation of cellulose by R. ulmarius as measured by residual glucose. • AEWATION=AI» HUSHING O AERATION= OXYGEN H.USHINC M I N E R A L S O L U T I O N Figure A. 14: Plot of significant interaction of aeration and mineral solution on biodegra-dation of cellulose by R. ulmarius as measured by residual glucose. © Appendix A. 183 • ACRATIONrAlB fLUSHING o AcaATiON=qxYGCn r iusHtNo V E R A T R Y L A L C O H O L Figure A. 15: Plot of significant interaction of aeration and veratryl alcohol on biodegra-dation of cellulose by R. ulmarius as measured by residual glucose. 60 • ACRATlON=AIR RUSHING O AERATION=OXYGCN FLUSHING LU «M CO o CJ 46 -44 42 ° T W E E N 8 0 1 Figure A.16: Plot of significant interaction of aeration and tween 80 on biodegradation of cellulose by R. ulmarius as measured by residual glucose. Appendix A. 184 • MTQ«octN >C»0«iOt *OOCD'WQ Q HTQBQCtN y[>OtlDC AQOtO-TtS O U I . ' 1 ' '"• LU 48 -CO o o - J sp 0 s 4 4 A 42 V E R A T R Y L A L C O H O L Figure A. 17: Plot of significant interaction of hydrogen peroxide and veratryl alcohol on biodegradation of cellulose by R. ulmarius as measured by residual glucose. • MTD8QCIW PIBOXIOf «00tP«MO O MTDBQCCW Pt»0«iPt *DDtQrTtS LU ««-CO o o _j O so 44 H 42 T W E E N 8 0 Figure A. 18: Plot of significant interaction of hydrogen peroxide and tween 80 on biodegradation of cellulose by R. ulmarius as measured by residual glucose. Appendix A. 185 50 • w<yi i f t u t i o ^ . n f ^ f . ' t i t u i r> Q J 48 CO o CJ D 48 _J V P 0 s 44 42 V E R A T R Y L A L C O H O L Figure A.19: Plot of significant interaction of mineral solution and veratryl alcohol on biodegradation of cellulose by R. ulmarius as measured by residual glucose. 5Q • |t»i»u »itf|.o«-tity«titflt«flfl| o LU 4 8 CO O C J - j C> 44 42 ° T W E E N 8 0 ' Figure A.20: Plot of significant interaction of mineral solution and tween 80 on biodegra-dation of cellulose by R. ulmarius as measured by residual glucose. Appendix A. 186 • VERATRYL ALCOHOL=0 mM O VERATRYL ALC0H0L=2 mM ° T W E E N 8 0 ' Figure A.21: Plot of significant interaction of veratryl alcohol and tween 80 on biodegra-dation of cellulose by R. ulmarius as measured by residual glucose. • ACRATIQN=AIB HUSHING O ACRATION=OXYCCN HUSHING U J C O O > X H Y D R O G E N P E R O X I D E Figure A.22: Plot of significant interaction of aeration and hydrogen peroxide on biodegradation of hemicellulose by R. ulmarius as measured by residual xylose. Appendix A. 187 • ACBATIQN = AIR FLUSHING O ACRATIQN=OXYGCN TLUSHING MINERAL SOLUTION Figure A.23: Plot of significant interaction of aeration and mineral solution on biodegra-dation of hemicellulose by R. ulmarius as measured by residual xylose. • ACRATION=AIR HUSHING O ACRATlQN=OXYGCN FLUSHING TWEEN 80 Figure A.24: Plot of significant interaction of aeration and tween 80 on biodegradation of hemicellulose by R. ulmarius as measured by residual xylose. Appendix A. 188 • HTQBQCtM PtHQUQf «OOCP'NQ O HTPBQOtM HBQmQt APOtP ' U S MINERAL SOLUTION Figure A.25: Plot of significant interaction of hydrogen peroxide and mineral solution on biodegradation of hemicellulose by R. ulmarius as measured by residual xylose. ^mj^QOQQlM P(»0«iPt AQPtPsHQ O HTPBQGIN P^BQXiPt «PP{P=Tt3 ° TWEEN 80 1 Figure A.26: Plot of significant interaction of hydrogen peroxide and tween 80 on biodegradation of hemicellulose by R. ulmarius as measured by residual xylose. Appendix A. 189 • HYQBQCtH HHOIIOf «0PCD'MQ Q HTPtQGtN H,t>QItP( «P0{P«Tt? V E R A T R Y L A L C O H O L Figure A.27: Plot of significant interaction of hydrogen peroxide and veratryl alcohol on biodegradation of hemicellulose by R. ulmarius as measured by residual xylose. UJ CO 1 8 2 >-X vp 14 12 T W E E N 8 0 Figure A.28: Plot of significant interaction of mineral solution and tween 80 on biodegra-dation of hemicellulose by R. ulmarius as measured by residual xylose. Appendix A. 190 18 9 VC»*T»Tl AlCQHQl AQDCQ.MQ O VtBATHTl ALCQHQl A Q O C P ^ S 1 2 J J T TWEEN 80 Figure A.29: Plot of significant interaction of veratryl alcohol and tween 80 on biodegra-dation of hemicellulose by R. ulmarius as measured by residual xylose. x • AC8ATION=AI» riUSHINC O ACRATION=QXYGCN r i U S H I N C UJ * ] -1 O z ° TWEEN 80 ' Figure A.30: Plot of significant interaction of aeration and tween 80 on biodegradation of chemical components of aspen RMP by R. ulmarius as measured by SBI. Appendix A. 191 Table A.48: Analysis of variance for initial pH of media on lignin biodegradation under optimum conditions as measured by the residual lignin content. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 3 58.22 19.41 73.93***" Error 8 2.10 0.26 Corrected total 11 60.32 "indicates significance at 0.001 level of probability. Table A.49: Duncan's multiple range test for the effect of initial pH of media on hgnin biodegradation under optimum conditions as measured by the residual lignin content. Duncan grouping" Mean hgnin content, % of original oven-dry weight of pulp Initial pH A 20.8 6.5 B 17.8 5.5 C 16.2 3.5 D 15.2 4.5 "Means with the same letter are not significantly different at 0.05 level of probability. Appendix A. 192 Table A.50: Analysis of variance for initial pH of media on cellulose biodegradation under optimum conditions as measured' by the residual glucose content. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 3 3.82 1.27 4.23*° Error 8 2.41 0.30 Corrected total 11 6.23 "indicates significance at 0.05 level of probability. Table A.51: Duncan's multiple range test for the effect of initial pH of media on cellulose biodegradation under optimum conditions as measured by the residual glucose content. Duncan grouping0 Mean glucose content, % of original oven-dry weight of pulp Initial pH A 50.2 6.5 AB 49.5 4.5 AB 49.2 5.5 B 48.6 3.5 "Means with the same letter are not significantly different at 0.05 level of probability. Appendix A. 193 Table A.52: Analysis of variance for initial pH of media on hemicellulose biodegradation under optimum conditions as measured by the residual xylose content. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 3 11.92 3.97 19.07***° Error 8 1.67 0.21 Corrected total 11 13.58 "indicates significance at 0.001 level of probability. Table A.53: Duncan's multiple range test for the effect of initial pH of media on hemi-cellulose biodegradation under optimum conditions as measured by the residual xylose content. Duncan grouping" Mean xylose content, % of original oven-dry weight of pulp Initial pH A 18.4 6.5 A 18.1 5.5 B 16.6 3.5 B 16.0 4.5 "Means with the same letter are not significantly different at 0.05 level of probability. Appendix A. 194 Table A.54: Analysis of variance for initial pH of media on selective biodelignification under optimum conditions of fungal-treated aspen pulp. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 3 1.93 0.64 49.19***° Error 8 0.11 0.01 Corrected total 11 2.04 "indicates significance at 0.001 level of probability. Table A.55: Duncan's multiple range test for the effect of initial pH of media on selective biodelignification index under optimum conditions of fungal-treated pulp. Duncan grouping0 Mean SBP Initial P H A 4.4 4.5 B 4.0 3.5 C 3.7 5.5 D 3.3 6.5 "Means with the same letter are not significantly different at 0.05 level of probability. h Selective biodelignification index Appendix A. 195 Table A.56: Analysis of variance for exogenously added nitrogen on lignin biodegradation under optimum conditions as measured by the residual lignin content. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 3 50.97 16.99 45.61* * * a Error 8 2.98 0.37 Corrected total 11 53.95 "indicates significance at 0.001 level of probability. Table A.57: Duncan's multiple range test for the effect of exogenously added nitrogen on lignin biodegradation under optimum conditions as measured by the residual hgnin content. Duncan grouping" Mean lignin content, % of original oven-dry weight of pulp Exogenously added nitrogen, m M A 19.7 24 B 18.3 2.4 C 15.1 1.2 C 14.9 0 "Means with .the same letter are not significantly different at 0.05 level of probability. Appendix A. 196 Table A.58: Analysis of variance for exogenously added nitrogen on cellulose biodegra-dation under optimum conditions as measured by the residual glucose content. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 3 0.10 0.03 0.06 Error 8 4.16 0.52 Corrected total 11 4.26 Table A.59: Analysis of variance for. exogenously added nitrogen on hemicellulose biodegradation under optimum conditions as measured by the residual xylose content. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 3 4.71 1.54 4.53*a Error 8 2.72 0.34 Corrected total 11 7.34 "indicates significance at 0.05 level of probability Appendix A. 197 Table A.60: Duncan's multiple range test for the effect of exogenously added nitrogen on hemicellulose biodegradation under optimum conditions as measured by the residual xylose content. Duncan grouping" Mean xylose content, % of original oven-dry weight of pulp Exogenously added nitrogen, mM A 17.2 24 B 16.4 2.4 B 15.8 1.2 B 15.7 0 "Means with the same letter are not significantly different at 0.05 level of probability. Table A.61: Analysis of variance for exogenously added nitrogen on selective biodeligni-fication index under optimum conditions of fungal-treated pulp. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 3 2.26 0.75 41.07***° Error 8 0.15 0.02 Corrected total 11 2.40 "indicates significance at 0.001 level of probability. Appendix A. 198 Table A.62: Duncan's multiple range test for the effect of exogenously added nitrogen on selective biodelignification index under optimum conditions of fungal-treated pulp. Duncan grouping0 Mean SBIb Exogenously added nitrogen, mM A 4.4 0 A 4.3 1.2 B 3.6 2.4 B 3.4 24 "Means with the same letter are not significantly different at 0.05 level of probability. ''Selective biodelignification index Table A.63: Analysis of variance for temperature on lignin biodegradation under optimum conditions as measured by the residual hgnin content. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 2 43.22 21.61 77 ig*** 0 Error 6 1.68 0.28 Corrected total 8 44.90 "indicates significance at 0.001 level of probability. Appendix A. 199 Table A.64: Duncan's multiple range test for the effect of temperature on lignin biodegra-dation under optimum conditions as measured by the residual hgnin content. Duncan grouping" Mean lignin content, % of original oven-dry weight of pulp Temperature 0 c A 20.2 32 B 19.1 22 C 15.1 28 "Means with the same letter are not significantly different at 0.05 level of probability. Table A.65: Analysis of variance for temperature on cellulose biodegradation under op-timum conditions as measured by the residual glucose content. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 2 0.55 0.27 1.08 Error 6 1.52 0.25 Corrected total 8 2.07 Appendix A. 200 Table A.66: Analysis of variance for temperature on hemicellulose biodegradation under optimum conditions as measured by the residual xylose content. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 2 11.31 5.65 25.83**° Error 6 1.31 0.22 Corrected total 8 12.62 "indicates significance at 0.01 level of probability. Table A.67: Duncan's multiple range test for the effect of hemicellulose on hemicellulose biodegradation under optimum conditions as measured by the residual xylose content. Duncan grouping" Mean xylose content, % of original oven-dry weight of pulp Temperature 0 C A 18.1 32 A 17.8 22 B 15.6 28 "Means with the same letter are not significantly different at 0.05 level of probability. Appendix A. 201 Table A.68: Analysis of variance for temperature on selective biodelignification index under optimum conditions of fungal-treated pulp. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 2 1.50 0.75 74.16***" Error 6 0.06 0.01 Corrected total 8 1.56 "indicates significance at 0.001 level of probability. Table A.69: Duncan's multiple range test for the effect of temperature on selective biodelignification index under optimum conditions of fungal-treated pulp. Duncan grouping" Mean Temperature SBIb °c A 4.3 28 B 3.5 32 B 3.4 22 "Means with the same letter are not significantly different at 0.05 level of probability! ''Selective biodelignification index Appendix A. 202 Table A.70: Analysis of variance for the effect of fungal treatment(treatments 21, 24 and control) on brightness of pulp handsheets. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 2 110.05 55.03 4746:74***° Error 27 0.31 0.01 Corrected total 29 110.36 "•indicates significance at 0.001 level of probability. Table A.71: Duncan's multiple range test for the effect of fungal treatment (treatments 21, 24 and control) on brightness of pulp handsheets. Duncan grouping0 Mean Fungal brightness, % treatment A 54.1 24 B 53.5 21 C 49.8 control "Means with the same letter are not significantly different at 0.05 level of probability. Appendix A. 203 Table A.72: Analysis of variance for the effect of fungal treatment(treatments 21, 24 and control) on (dry)zero-span breaking length of pulp handsheets. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 2 8.61 4.30 180.97***° Error 27 0.64 0.02 Corrected total 29 9.24 "indicates significance at 0.001 level of probability. Table A.73: Duncan's multiple range test for the effect of fungal treatment(treatments 21, 24 and control) on (dry)zero-span breaking length of pulp handsheets. Duncan grouping" Mean (dry)zero-span breaking Fungal length, km treatment A 7.1 21 B 6.8 24 C 5.8 control "Means with the same letter are not significantly different at 0.05 level of probability. Appendix A. 204 Table A.74: Analysis of variance for the effect of fungal treatment(treatments 21, 24 and control) on (wet)zero-span breaking length of pulp handsheets. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 2 14.85 7.43 311.31***° Error 27 0.64 0.02 Corrected total 29 15.49 "indicates significance at 0.001 level of probability. Table A.75: Duncan's multiple range test for the effect of fungal treatment(treatments 21, 24 and control) on (wet)zero-span breaking length of pulp handsheets. Duncan grouping" Mean (wet)zero-span breaking Fungal length, km treatment A 5.7 control A 5.6 21 B 4.2 24 "Means with the same letter are not significantly different at 0.05 level of probability. Appendix A. 205 Table A.76: Analysis of variance for the effect of fungal treatment(treatments 21, 24 and control) on tensile index of pulp handsheets. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 2 11.86 5.93 344.57***° Error 27 0.47 0.02 Corrected total 29 12.33 "indicates significance at 0.001 level of probability. Table A.77: Duncan's multiple range test for the effect of fungal treatment(treatments 21, 24 and control) on tensile index of pulp handsheets. Duncan grouping" Mean tensile Fungal index, Nm/g treatment A 2.2 - 21 B 1.7 control C 0.7 24 . "Means with the same letter are not significantly different at 0.05 level of probability. Appendix A. 206 Table A.78: Analysis of variance for the effect of fungal treatment(treatments 21, 24 and control) on burst index of pulp handsheets. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 2 1.26 0.63 72 Q4***° Error 27 0.24 0.01 Corrected total 29 1.50 "indicates significance at 0.001 level.of probability. Table A.79: Duncan's multiple range test for the effect of fungal treatment(treatments 21, 24 and control) on burst index of pulp handsheets. Duncan grouping" Mean burst Fungal index, kPa. m 2/g treatment A 0.7 21 B 0.6 control C 0.2 24 "Means with the same letter are not significantly different at 0.05 level of probability. Appendix A. 207 Table A.80: Analysis of variance for the effect of fungal treatment(treatments 21, 24 and control) on tear index of pulp handsheets. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 2 4.26 2.13 221.54***° Error 27 0.26 0.01 Corrected total 29 4.53 "indicates significance at 0.001 level of probability. Table A.81: Duncan's multiple range test for the effect of fungal treatment(treatments 21, 24 and control) on tear index of pulp handsheets. Duncan grouping" Mean tear Fungal index, mN. m 2/g treatment A 1.0 21 A 1.0 control B 0.2 24 "Means with the same letter are not significantly different at 0.05 level of probability. Appendix A. 208 Table A.82: Analysis of variance for the effect of fungal treatment(treatments 21, 24 and control) on density of pulp handsheets. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 2 14658.70 7329.35 462.65***a Error 27 427.73 15.84 Corrected total 29 15086.43 "indicates significance at 0.001 level of probability. Table A.83: Duncan's multiple range test for the effect of fungal treatment(treatments 21, 24 and control) on density of pulp handsheets. Duncan grouping11 Mean Fungal density, kg/m3 treatment A 293.2 24 B 257.3 control C 241.0 21 "Means with the same letter are not significantly different at 0.05 level of probability. Appendix A. 209 Table A.84: Analysis of variance for the effect of fungal treatment (treatments 21, 24 and control) on opacity(Tappi) of pulp handsheets. Source of variation Degrees of freedom Sum of squares Mean square F value Treatment 2 33.01 16.51 255.53***° Error 27 1.74 0.06 Corrected total 29 34.75 "indicates significance at 0.001 level of probability. Table A.85: Duncan's multiple range test for the effect of fungal treatment(treatments 21, 24 and control) on opacity of pulp handsheets. Duncan grouping" Mean Fungal opacity(Tappi), % treatment A 93.2 control B 92.9 21 C 90.8 24 "Means with the same letter are not significantly different at 0.05 level of probability. Appendix A. i CO CD 210 o \ 2 - it E G> \ — ^ . o — u. • UJ r j UJ 0 a. «/i ix u i UJ UJ \ '.n CL UJ z -•I U« »- •' c f - _ l 0 c IT-S' in tr. •o hi 0 T rf Y UJ I f ' I I 0. u cn o UJ Ul o z •I E UJ U'I O m •I ct i UJ (/I •I O 1/1 ao CD u if o —J > X UJ 1/1 c o I-a. ^ vwian lunnyvjl* calif p. n 03 906362 00 Figure A.31: Chromatogram from the separation of standard solution of six common wood sugars and the internal standard, ribose. Appendix A. 211 Figure A.32: Chromatogram from the separation of hydrolysates of aspen refiner me-chanical pulp. 

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