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Improving the brightness and bleachability of douglas-fir mechanical pulps using white-rot fungi and… Chandra, Richard P. 1999

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Improving the Brightness and Bleachability of Douglas-fir Mechanical Pulps Using White-rot Fungi and Laccase Enzymes by Richard P. Chandra B . S c , 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 , 1995 A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in the Faculty of Graduate Studies Department of Wood Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January 1999 © R i c h a r d Chandra , 1999 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. 1 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 \ /Ol o o S S cU eM? The University of British Columbia Vancouver, Canada Date DE-6 (2/88) Abstract Douglas- f i r is a c o m m o n west coast tree species, possessing l o n g and strong fibers, however, the l o w brightness and poor bleachabi l i ty o f mechanica l pulps f r o m this tree have thus far l i m i t e d its use to l o w y i e l d K r a f t pulps and lumber. T h e heartwood o f D o u g l a s - f i r has been s h o w n to contain a h i g h amount o f p o l y p h e n o l i c compounds such as dihydroquercet in and/or p o l y m e r i z e d forms o f these compounds. These compounds have been s h o w n to undergo autooxidat ion, condensation, p o l y m e r i z a t i o n and metal chelat ion dur ing either the aging o f the tree or mechanica l p u l p i n g o f the w o o d , result ing i n the format ion o f dark co loured complexes i n the pulp . A l t h o u g h c h e m i c a l treatments to i m p r o v e the bleaching response o f Douglas- f i r mechanica l pulps have been unsuccessful , b i o l o g i c a l treatments have yet to be appl ied. Douglas- f i r mechanica l pulps were treated either w i t h w o o d degrading fungi or laccase enzymes to try to i m p r o v e the brightness o f mechanica l pulps or ig inat ing f r o m the p o l y p h e n o l i c chromophor ic compounds present i n the heartwood port ion o f this tree. Results o f fungal screening showed that the white-rot fungus Phanerochaete chrysosporium increased brightness o f Douglas- f i r refiner mechanica l pulps b y 1.5 pts I S O . Unfortunate ly P.chrysosporium treatment was unable to increase the brightness o f pure Douglas- f i r heartwood thermomechanica l pulps. A l t h o u g h , earlier w o r k had s h o w n that the product ion o f laccase was induced i n the fungus Trametes versicolor i n the presence o f dihydroquercet in , Trametes versicolor treatment o f heartwood mechanica l pulps c o m b i n e d w i t h c h e m i c a l extraction d i d not result i n any brightness increases greater than what c o u l d be obtained us ing c h e m i c a l extraction alone. Laccase treatments without enzyme mediators decreased the unbleached brightness o f Douglas- f i r heartwood mechanica l pulps by 4-5 pts I S O , however , after b leaching, the pulp i i brightness surpassed the control pulps by 2-3 pts I S O . W i t h the addi t ion o f o x y g e n , an e n z y m e mediator, or increasing the temperature, laccase treatments decreased both the unbleached and the bleached brightness values compared to the controls. H o w e v e r , the addi t ion o f o x y g e n alone was able to increase the brightness o f a l l pulps b y 1-2 pts I S O . P r e l i m i n a r y absorpt ion measurements o f laccase treated handsheets indicated that the product ion o f co loured quinone type structures i n the mechanica l pulps m a y be associated w i t h the posi t ive effects o f laccase treatments. i i i Table of Contents A B S T R A C T II T A B L E O F C O N T E N T S IV L I S T O F T A B L E S VI L I S T O F F I G U R E S VII L I S T O F A B B R E V I A T I O N S VIII A C K N O W L E D G E M E N T S IX 1. I N T R O D U C T I O N 1 1.1 P U L P I N G A N D B L E A C H I N G P R O C E S S E S 2 1.1.1 Chemical Pulping and Bleaching 2 1.1.2 Mechanical Pulping and Bleaching Processes 4 1.2 D O U G L A S - F I R 5 1.2.1 Chromophoric Compounds in Douglas-fir 6 1.2.2 Attempts at Solving the Low Brightness and Bleachability of Douglas-fir Heartwood Using Chemicals 8 1.3 B I O L O G I C A L C H I P A N D P U L P T R E A T M E N T 11 1.4 B I O M E C H A N I C A L P U L P I N G - F U N G A L T R E A T M E N T OF W O O D C H I P S 13 1.4.1 Optical Properties of Biomechanical Pulps 17 1.4.2 Fungal Treatment of Mechanical Pulps 19 1.5 B I O L O G I C A L B L E A C H I N G 20 7.5.7 Biological Bleaching With Lignolytic Fungi 21 1.5.2 Application of Oxidative Enzymes to Kraft Pulps 23 1.5.3 Laccase : 25 1.6 A P P L I C A T I O N S O F B I O L O G I C A L T R E A T M E N T S TO D O U G L A S - F I R 28 1.6.1 Microbial Degradation of Quercetin, Dihydroquercetin and Condensed Tannin Compounds 28 1.6.2 Applying fungi to Douglas-fir Wood Chips and TMP 29 1.6.3 Treatment of Douglas-fir Mechanical Pulps with Oxidative Enzymes 33 1.7 R E S E A R C H A P P R O A C H 35 2 M A T E R I A L S A N D M E T H O D S 36 3 R E S U L T S A N D DISCUSSION 45 3.1 S C R E E N I N G F O R F U N G I W I T H T H E A B I L I T Y T O I N C R E A S E B R I G H T N E S S O F D O U G L A S - F I R R M P 45 3.1.1 Summary 54 3.2 P H A N E R O C H A E T E C H R Y S O S P O R I U M T R E A T M E N T S OF D O U G L A S - F I R T H E R M O M E C H A N I C A L P U L P F R O M P U R E H E A R T W O O D 55 3.3 T R A M E T E S V E R S I C O L O R T R E A T M E N T OF D O U G L A S - F I R H E A R T W O O D T M P 58 iv 3.3.1 Unbleached Brightness of Douglas-fir Heartwood TMP treated with Trametes versicolor in combination with Methanol and Alkaline extraction 58 3.3.2 Hydrogen Peroxide Bleached Brightness of Douglas-fir Heartwood TMP treated with Trametes versicolor, with and without Laccase mediators, in combination Methanol and Alkaline extraction 59 3.3.3 Summary 63 3.4 L A C C A S E T R E A T M E N T S OF D O U G L A S - F I R H E A R T W O O D T M P . 6 3 3.4.1 Unbleached Optical Properties of Douglas-fir Heartwood TMP Treated with Laccase in Combination with the Mediators ABTS and HBT, and Chemical Extraction 64 3.4.2 Hydrogen peroxide Bleached Brightness of Douglas-fir Heartwood TMP treated with laccase with and without laccase mediators, in combination with Chemical Extraction 67 3.4.3 Summary 70 3.5 A D J U S T I N G T H E C O N D I T I O N S OF L A C C A S E T R E A T M E N T S O F D O U G L A S - F I R H E A R T W O O D T M P 7 3 3.5.1 The Effects of Adjusting Temperature and the Amount of Oxygen on the Unbleached Optical Properties of Douglas-fir Heartwood TMP 74 3.5.2 Hydrogen Peroxide Bleached Brightness of Douglas-fir Heartwood TMP Treated with Laccase Enzymes Under Two Different Temperatures and Bleached with Varied Levels of Sodium Hydroxide in the Bleaching Liquor With and Without Added Oxygen 76 3.5.3 The Effects of Adjusting the pH and using Different Laccases on the Unbleached and Bleached Brightness of Laccase Treatments of Douglas-fir Heartwood TMP 78 3.5.4 Diffuse Reflectance UV spectrometry of Laccase treated Handsheets 83 3.5.5 Summary 86 4 CONCLUSIONS 87 5 REFERENCES 89 v List of Tables T A B L E 1: E X A M P L E S O F T H E E N E R G Y S A V I N G S O B T A I N E D D U R I N G B I O M E C H A N I C A L P U L P I N G ( F U N G A L T R E A T M E N T PRIOR T O P R I M A R Y R E F I N I N G ) A N D D U R I N G F U N G A L T R E A T M E N T OF M E C H A N I C A L P U L P ( F U N G A L T R E A T M E N T PRIOR T O S E C O N D A R Y R E F I N I N G ) 1 6 T A B L E 2: E X A M P L E S OF S T R E N G T H I N C R E A S E S O B T A I N E D D U R I N G B I O M E C H A N I C A L P U L P I N G 1 6 T A B L E 3: B I O M E C H A N I C A L P U L P I N G OF V A R I O U S W O O D SPECIES W I T H T H E W H I T E - R O T F U N G U S CERIPORIOPSISSUBVERM1SPORA (C.S.)\EFFECTS O F F U N G A L T R E A T M E N T O N O P T I C A L P R O P E R T I E S O F M E C H A N I C A L P U L P S 1 8 T A B L E 4: S T R E N G T H P R O P E R T I E S OF D O U G L A S - F I R T H E R M O M E C H A N I C A L P U L P T R E A T E D W I T H S E L E C T E D F U N G I 4 8 T A B L E 5: I S O - B R I G H T N E S S , B E F O R E A N D A F T E R P E R O X I D E B L E A C H I N G , OF D O U G L A S - F I R R E F I N E R M E C H A N I C A L P U L P T R E A T E D W I T H S E L E C T E D F U N G I 4 9 T A B L E 6: S T A R L A B ( L * A * B * ) V A L U E S OF D O U G L A S - F I R R E F I N E R M E C H A N I C A L P U L P T R E A T E D W I T H D I F F E R E N T F U N G I B E F O R E A N D A F T E R P E R O X I D E B L E A C H I N G 5 1 T A B L E 7: H Y D R O G E N P E R O X I D E B L E A C H E D I S O - B R I G H T N E S S O F D O U G L A S - F I R H E A R T W O O D T H E R M O M E C H A N I C A L P U L P S T R E A T E D W I T H PHANEROCHAETE CHRYSOSPORIUMOVER 7 D A Y S 5 7 T A B L E 8: U N B L E A C H E D B R I G H T N E S S A N D S T A R L A B ( L * A * B * ) R E S U L T S OF D O U G L A S FIR H E A R T W O O D T M P T R E A T E D W I T H L A C C A S E I N C O M B I N A T I O N W I T H T H E M E D I A T O R S A B T S A N D H B T , A N D C H E M I C A L E X T R A C T I O N 6 6 TABLE 9:EFFECT O F T E M P E R A T U R E A N D O X Y G E N A T I O N O N T H E E F F E C T I V E N E S S O F L A C C A S E S P 5 0 4 I N I N C R E A S I N G T H E B R I G H T N E S S OF D O U G L A S - F I R H E A R T W O O D T M P 7 5 Table 10: T H E I N F L U E N C E O F P H O N T H E U N B L E A C H E D / B L E A C H E D B R I G H T N E S S OF D O U G L A S - F I R H E A R T W O O D T M P T R E A T E D W I T H F I V E D I F F E R E N T L A C C A S E S 8 2 vi List of Figures F I G U R E 1: a) D I H Y D R O Q U E R C E T I N A N D b) Q U E R C E T I N ( R I N G S L A B E L L E D "A", " B " A N D " C " . ..31 F I G U R E 2: a) P H O T O A N D b) T H E R M A L B R I G H T N E S S R E V E R S I O N R A T E S OF P U L P S T R E A T E D W I T H S E L E C T E D F U N G I 53 F I G U R E 3: H Y D R O G E N P E R O X I D E B L E A C H E D B R I G H T N E S S OF a) U N E X T R A C T E D A N D b) M E T H A N O L E X T R A C T E D D O U G L A S - F I R H E A R T W O O D TMP, T R E A T E D W I T H T. VERSICOLOR F O R 7 D A Y S , W I T H A N D W I T H O U T L A C C A S E M E D I A T O R S HBT A N D ABTS, A N D S U B S E Q U E N T A L K A L I N E E X T R A C T I O N 60 F I G U R E 4: H Y D R O G E N P E R O X I D E B L E A C H E D B R I G H T N E S S OF a) U N E X T R A C T E D A N D b) M E T H A N O L E X T R A C T E D D O U G L A S - F I R H E A R T W O O D TMP, T R E A T E D W I T H L A C C A S E F O R 7 D A Y S , W I T H A N D W I T H O U T L A C C A S E M E D I A T O R S HBT A N D ABTS, A N D S U B S E Q U E N T A L K A L I N E E X T R A C T I O N 68 F I G U R E 5: H Y D R O G E N P E R O X I D E B L E A C H E D B R I G H T N E S S OF L A C C A S E T R E A T E D A L K A L I N E E X T R A C T E D D O U G L A S - F I R H E A R T W O O D TMP W I T H A N D W I T H O U T T H E M E D I A T O R HBT A N D A S E C O N D A L K A L I N E E X T R A C T I O N PRIOR TO H Y D R O G E N P E R O X I D E B L E A C H I N G 72 F I G U R E 6: B L E A C H E D ISO B R I G H T N E S S V A L U E S (a&b) A N D H Y D R O G E N P E R O X I D E C O N S U M P T I O N (c&d) OF L A C C A S E T R E A T E D D O U G L A S - F I R H E A R T W O O D TMP W I T H S U P P L E M E N T A L O X Y G E N O R A I R . A L L S A M P L E S B L E A C H E D W I T H 6% H 2 0 2 W I T H V A R Y I N G N A O H L E V E L S I N T H E B L E A C H I N G L I Q U O R 79 F I G U R E 7: D I F F U S E R E F L E C T A N C E U V - V I S A N A L Y S I S O F a) U N B L E A C H E D A N D b) B L E A C H E D H A N D S H E E T S F R O M D O U G L A S - F I R TMP W I T H A N D W I T H O U T L A C C A S E T R E A T M E N T 85 vii List of Abbreviations c C h l o r i n e (Bleaching) E E x t r a c t i o n (Bleaching) D C h l o r i n e D i o x i d e (Bleaching) S G W Stone G r o u n d w o o d T M P T h e r m o m e c h a n i c a l P u l p R M P Ref iner M e c h a n i c a l P u l p C T M P C h e m i t h e r m o m e c h a n i c a l P u l p D H Q D i h y d r o q u e r c e t i n E D T A E t h y l e n e d i a m i n e tetraacetic a c i d B O D B i o l o g i c a l O x y g e n D e m a n d C O D C h e m i c a l O x y g e n D e m a n d M n P Manganese Peroxidase L i P L i g n i n Peroxidase v i i i Acknowledgements I have m a n y people to thank for h e l p i n g m e to make the c o m p l e t i o n o f this thesis possible. I w o u l d especial ly l i k e to thank m y supervisor, D r . Jack Saddler for g i v i n g m e the opportunity to pursue m y M S c at Forest Products b iotechnology, and D r . E d de Jong, for g i v i n g me the opportunity to try research as a summer student immediate ly f o l l o w i n g m y undergraduate degree. I also thank D r . R o d g e r Beatson for a l l o f his posi t ive technical assistance, for be ing a great f r iend and teacher, and for m a k i n g research fun even w h e n the results were not posi t ive . T h e support and fr iendship o f D r . C a r l Johansson is gratefully a c k n o w l e d g e d a long w i t h D r . Colette B r e u i l , and a l l o f the rest o f Forest Products B i o t e c h n o l o g y . I w o u l d also l i k e to thank the P u l p and Paper Research Institute o f Canada. W i t h o u t their faci l i t ies and assistance I never w o u l d have been able to conduct this research. I a m thankful for the supply o f laccase enzymes that I received f r o m D r . C l a u s F e l b y o f N o v o N o r d i s k and D r . L i i s a V i i k a r i o f V T T F o o d and B i o t e c h n o l o g y . T h e f inancia l support f r o m the Science C o u n c i l o f B . C . and M a c M i l l a n B l o e d e l Research was also greatly appreciated. I g ive m y greatest thanks to m y f a m i l y and friends for being w i t h m e dur ing the fun times, and h e l p i n g me through a l l the tough t imes, especial ly m y m o m and dad and m y grandmother that have supported me through anything. I w o u l d also l i k e to thank m y closest fr iend Grace w h o has helped me b e y o n d words. I w o u l d also l i k e to thank m y grandfather, but I a m sorry that he passed away this past spring without seeing me graduate. I dedicate this w o r k to h i m . i x 1. Introduction There w i l l be a cont inuing demand for affordable w o o d as a fiber source for paper products i n the pulp and paper industry i n B r i t i s h C o l u m b i a . W i t h continued pressure to m a k e use o f a l l o f the fiber sources w i t h i n B . C . , it is probable that there w i l l be a decrease i n the amount o f p u l p w o o d avai lable that possesses desirable properties such as h i g h bleachabi l i ty and l o n g fibers. T o more eff iciently make use o f the fiber supply , the use o f newer or different p u l p i n g methods c o m b i n e d w i t h u t i l i z i n g feedstocks w i t h less desirable c o l o u r and fiber properties, such as Douglas- f i r should be explored. D o u g l a s - f i r is a c o m m o n west coast tree species constituting approximately 9 % o f the standing v o l u m e and 1 0 % o f the lumber p r o d u c t i o n i n B . C . ( C O F I 1995). Douglas- f i r is c o m m o n l y used for the product ion o f lumber and l o w e r y i e l d c h e m i c a l pulps such as Kraf t pulps ( V a n der Zee 1996). A l t h o u g h it has been s h o w n that h i g h y i e l d mechanica l pulps w i t h l o n g , strong fibers can be produced f r o m us ing Douglas- f i r as a furnish, these mechanica l pulps also have very l o w brightness and bleachabi l i ty . T h e l o w brightness and bleachabi l i ty o f Douglas- f i r mechanica l pulps is due to the p o l y p h e n o l i c chromophor ic compounds res iding i n the heartwood port ion o f the tree, w h i c h become further darkened dur ing mechanica l p u l p i n g processes ( G u p t a 1976, V a n der Zee 1996). A l t h o u g h various chemicals have been used to treat Douglas- f i r mechanica l pu lp and chips to try and obtain acceptable brightness levels, these efforts have been largely unsuccessful . H o w e v e r , there have been few attempts at assessing the potential o f b i o l o g i c a l treatments to i m p r o v e both the brightness and bleachabi l i ty o f Douglas- f i r mechanica l pulps . T h i s is the m a i n theme o f this thesis, w h i c h has l o o k e d at the potential o f fungi and their redox enzymes to enhance the brightness and bleachabi l i ty o f Douglas- f i r mechanica l pulps. 1 B i o l o g i c a l treatments w i t h white-rot fungi and their oxidat ive enzymes, have been re lat ively successful i n decreasing the mechanica l energy necessary for p r o d u c i n g m e c h a n i c a l pulps , and increasing the brightness and bleachabi l i ty o f c h e m i c a l pulps ( A k h t a r 1998, Pa ice 1995). T h e p o l y p h e n o l i c character and resemblance to phenol ic l i g n i n structures exhibi ted b y the c h r o m o p h o r i c compounds present i n Douglas- f i r mechanica l pulps make them p r i m e targets for treatment w i t h the oxidat ive enzymes ( H a n c o c k 1957), such as laccase, that are produced b y m a n y o f the wood-degrading fungi . It has been s h o w n that the oxidat ive l i g n o l y t i c enzymes, such as laccase, produced by white-rot fungi , preferential ly degrade phenol ic c o m p o u n d s ( M u l h e i m 1992). Thus , one o f the p r i m a r y objectives o f the w o r k described here was to assess the abi l i ty o f white-rot fungi and their enzymes to remove or alter the p o l y p h e n o l i c chromophore derived co lour i n D o u g l a s - f i r mechanica l pulps to increase p u l p brightness and bleachabi l i ty . I n order to understand the rationale behind m y w o r k , the f o l l o w i n g sections contain essential background i n f o r m a t i o n o n mechanica l and c h e m i c a l p u l p i n g and b leaching. T h i s includes a d iscuss ion o f the inherent co lour problems o f D o u g l a s - f i r m e c h a n i c a l pulps , b i o l o g i c a l treatments w i t h white-rot fungi and laccase enzymes, and an assessment o f h o w white-rot fungi and laccase enzymes m i g h t be appl ied to Douglas- f i r mechanica l pulps . 1.1 Pulping and Bleaching Processes 1.1.1 Chemical Pulping and Bleaching It is k n o w n (Fengel 1989) that c h e m i c a l p u l p i n g p r i m a r i l y concentrates o n s o l u b i l i z i n g the l i g n i n "adhes ive" between papermaking fibers us ing chemicals , w h i l e mechanica l p u l p i n g p r i m a r i l y involves the use o f a p h y s i c a l force to break apart the w o o d into fibers ( S m o o k 1992). 2 E x a m p l e s o f c h e m i c a l p u l p i n g methods include K r a f t , sulfite and organosolv , w h i c h differ i n the chemicals used. K r a f t c h e m i c a l pulps are the most prevalent, accounting for approximately 7 0 % o f the w o r l d ' s total pu lp output and approximately 4 3 % o f the C a n a d i a n pulp output (Bajpai 1992, S m o o k 1992). K r a f t c h e m i c a l pulps possess l o n g , pure fibers and are relat ively easy to bleach ( S m o o k 1992). U n l i k e mechanica l pulps , Kraf t pulps and other c h e m i c a l pulps are produced by d i s s o l v i n g the l i g n i n between fibers. D u r i n g Kraf t p u l p i n g approximately 9 0 % o f l i g n i n is removed (Bajpai 1992, F e n g e l 1989) w i t h approximate ly 2 - 4 % residual l i g n i n r e m a i n i n g i n the Kraf t pu lp after the p u l p i n g procedure, depending o n the w o o d species used as a feedstock (Fengel 1989). The residual l i g n i n is m a i n l y responsible for the b r o w n c o l o u r o f K r a f t pulps pr ior to b leaching w h i c h consists o f conjugated c h e m i c a l structures such as quinones, c o m p l e x e d catechols, chlacones, and stilbenes (Fengel 1989). Unfortunate ly , portions o f the hemice l lu lose are also d isso lved dur ing K r a f t and other c h e m i c a l p u l p i n g procedures (Fengel 1989). Therefore, c h e m i c a l p u l p product ion results i n an approximate 4 5 % y i e l d ( S m o o k 1992, F e n g e l 1989). H o w e v e r , c h e m i c a l pulps possess excellent strength properties, are relat ively easy to bleach, and usual ly have l itt le problems w i t h brightness stabil i ty w h e n exposed to heat and l ighted condit ions. B l e a c h e d K r a f t pulps usual ly sel l for 1 0 - 2 0 % higher prices than their unbleached counterparts w i t h chlor ine, hypochlor i te , chlorine d iox ide , o x y g e n , ozone and h y d r o g e n peroxide as the p r i n c i p a l b leaching agents e m p l o y e d for b leaching (Bajpai 1992, S m o o k 1992). A t y p i c a l b leaching sequence for K r a f t pulps is a D E D E D sequence where the init ia ls stand for chlor ine d i o x i d e ( D ) , a lkal ine extraction ( E ) , a second chlor ine d i o x i d e stage ( D ) , a second a lka l ine extraction (E) f o l l o w e d by a second appl icat ion o f chlor ine d i o x i d e ( D ) ( S m o o k 1992). T h e effluents produced dur ing K r a f t p u l p i n g and bleaching are a major environmental concern, and 3 must be treated before their release, because o f their h i g h organic l o a d and tox ic chlorinated organics (Sykes 1994). 1.1.2 Mechanical Pulping and Bleaching Processes M e c h a n i c a l p u l p i n g methods include Stone G r o u n d w o o d ( S G W ) , T h e r m o m e c h a n i c a l p u l p i n g ( T M P ) , Ref iner M e c h a n i c a l P u l p i n g ( R M P ) and C h e m i t h e r m o m e c h a n i c a l p u l p i n g ( C T M P ) ( S m o o k 1992). M e c h a n i c a l pulps constitute approximately 4 0 % o f Canada 's p u l p output as compared to o n l y 15% i n the U n i t e d States ( S m o o k 1992). T h e r m o m e c h a n i c a l p u l p i n g uses h i g h pressure steam to pre-soften w o o d chips pr ior to f iber izat ion i n a refiner, compared to Kraf t c h e m i c a l p u l p i n g w h i c h dissolves the l i g n i n w i t h i n the w o o d chips us ing N a O H and Na2SC*4; therefore thermomechanical p u l p i n g results i n a 9 0 - 9 5 % y i e l d ( S m o o k 1992)*compared to 4 5 - 5 0 % y i e l d for K r a f t p u l p i n g . Thus , thermomechanical p u l p i n g appears to be a better method for more eff ic iently u t i l i z i n g the pulp w o o d supply. T h e r m o m e c h a n i c a l p u l p i n g c o m b i n e d w i t h the use o f alternative fiber sources c o u l d be one w a y o f part ia l ly a l lev iat ing a future fiber supply p r o b l e m . A s w i t h a l l mechanica l p u l p i n g processes, the result ing p u l p is produced i n a h i g h y i e l d relative to the feedstock, however most pulps have a l o w brightness. Therefore the majority o f the post mechanica l pu lp b leaching w o r k has focused o n increasing the brightness o f the pulp without decreasing the pulp y i e l d . T h e b leaching o f mechanica l pulps is often referred to as " b r i g h t e n i n g " since the l i g n i n i n mechanica l pulps is not degraded dur ing the b leaching process ( S m o o k 1992). Instead, d u r i n g the b leaching o f mechanica l pulps , l i g n i n is decolour ized reduct ive ly b y chemicals such as s o d i u m hydrosulf i te (Na2S204) and ox idat ive ly by chemicals such as h y d r o g e n peroxide (H2O2) ( S m o o k 1992). A l t h o u g h the h i g h y i e l d o f pu lp result ing f r o m mechanica l p u l p i n g m a k e this 4 process attractive compared to c h e m i c a l p u l p i n g , mechanica l p u l p i n g also has several disadvantages. Those inc lude; di f f icult b leaching to h i g h brightness levels (higher than 8 5 % I S O ) , high-energy requirements for f iberizat ion; shorter and weaker fibers, and unstable brightness (colour reversion) under l ight or heat ( S m o o k 1992). M a n y o f these problems stem f r o m the h i g h l i g n i n ( 2 0 - 3 0 % ) and extractives content i n m e c h a n i c a l pulps as compared to c h e m i c a l pulps. H o w e v e r , the h i g h chromophor ic extractives content i n the heartwood o f Douglas- f i r also contribute to the dark colour, l o w brightness and l o w bleachabi l i ty o f mechanica l pulps f r o m this tree ( B u b l i t z 1974, V a n der Zee 1996). 1.2 Douglas-fir A s mentioned earlier, the co lour p r o b l e m lies m a i n l y i n the heartwood p o r t i o n o f Douglas- f i r , since sapwood T M P can be bleached to a brightness level 14 points I S O higher than that o f the heartwood ( V a n der Zee 1996). Therefore, o n l y Douglas- f i r sapwood T M P has suitable brightness properties for the product ion o f newsprint ( V a n der Zee 1996). Further p r o o f o f the detrimental effects o f heartwood to mechanica l pulps is demonstrated by the decrease i n brightness o f result ing mechanica l pulps as more heartwood chips are added to the p u l p w o o d supply ( G u p t a 1976). B l e a c h i n g w i t h H 2 0 2 at charges less than 2 % has negative (i.e. l o w e r i n g brightness) effects o n heartwood ( G u p t a 1976). T h i s m a y be due to the pulp darkening at the a lkal ine condit ions e m p l o y e d dur ing b leaching to activate H2O2 since the h i g h amount chromophores i n D o u g l a s - f i r heartwood can consume the H2O2 to negate the b leaching reaction. Unfortunately D o u g l a s - f i r trees possess a narrow sapwood section (4-22 annual r ings at breast height), due to early deve loping heartwood (6-10 yr.) ( V a n der Zee 1996). Thus , the result ing industrial w o o d chip pi les generally consist o f 3 5 - 4 0 % heartwood chips ( V a n der Zee 1996). I n 5 order for Douglas- f i r to be used effectively as a mechanica l p u l p feedstock, the l o w brightness and bleachabi l i ty must be addressed b y f i n d i n g w a y s o f altering or r e m o v i n g the p o l y p h e n o l i c der ived co lour i n heartwood pulps. 1.2.1 Chromophoric Compounds in Douglas-fir It has been s h o w n that the heartwood o f D o u g l a s - f i r contains approximately 2 . 4 - 8 . 8 % o f its weight as extractives ( V a n der Zee 1996). It has also been s h o w n that d ihydroquercet in ( D H Q ) and quercetin constitute 8 0 - 9 0 % o f the phenol p o r t i o n o f the extractives (Del lus 1997, V a n der Zee 1996) w i t h the d ihydroquercet in content o f Douglas- f i r approximately 20 t imes that o f quercetin ( B u b l i t z 1974). F l a v o n o i d s , condensed tannins and phlobaphenes are the names g i v e n to these p o l y p h e n o l i c extractives (Del lus 1997, V a n der Zee 1996). F l a v o n o i d s and condensed tannins belong to the group o f part ia l ly water soluble extractives k n o w n as the proanthocyanidins, w h i l e extractives soluble i n organic solvents are ca l led phlobaphenes ( D e l l u s 1997, Hergert 1960). T h e insoluble phenol ic acids and l i g n i n - l i k e compounds represent the f i n a l class o f extractives (Fengel 1989). The f lavonoids and condensed tannins are k n o w n to be p r i m a r i l y responsible for the l o w brightness and bleachabi l i ty o f D o u g l a s - f i r T M P pulps ( B e t z 1974, Hergert 1960, V a n der Zee 1996). T h e concentration o f the f lavonoids increase f r o m the p i t h outwards, reaching a m a x i m u m value at the heartwood/sapwood boundary and then decreasing i n the sapwood ( H a n c o c k 1957). It has been suggested that the p a r e n c h y m a cel ls at the heartwood/sapwood boundary undergo a p e r i o d o f intensif ied act ivi ty just pr ior to their death, w h i c h results i n the d i f fus ion o f their c e l l contents into the surrounding w o o d , thus " t r a n s f o r m i n g " the sapwood into heartwood at the boundary ( H a n c o c k 1957). O n c e i n the heartwood, po lyphenols such as D H Q undergo autooxidative reactions that result i n their 6 p o l y m e r i z a t i o n to co loured compounds ca l led tannins and phlobaphenes (Hergert 1960). These p o l y m e r i c p o l y p h e n o l s are thought to be the c h i e f source o f co lour i n Douglas- f i r heartwood (Hergert 1960). A s a result, any attempts to produce mechanica l pulps f r o m older D o u g l a s fir trees pose a s ignif icant b leaching p r o b l e m , due to the increased heartwood content o f the furnish ( G u p t a 1976). It has been s h o w n that the addi t ion o f quercetin to h e m l o c k mechanica l pulps resulted i n the same poor b leaching response as Douglas- f i r , however, addi t ion o f d ihydroquercet in had litt le effect ( B u b l i t z 1974). The ineffectiveness o f d ihydroquercet in to decrease h e m l o c k p u l p brightness is s ignif icant since it indicates that D H Q undergoes changes d u r i n g ag ing o f the tree and p u l p i n g o f the w o o d , result ing i n the formation o f co loured components i n the pulp . E x p e r i m e n t a l evidence that D H Q m a y play a role i n contr ibut ing to the l o w brightness and bleachabi l i ty o f Douglas- f i r mechanica l pulps , is indicated b y the addi t ion o f d ihydroquercet in to cel lulose filter paper w h i c h results i n co lour changes under heated condit ions that m i m i c those o f thermomechanical p u l p i n g (Troughton 1973). H e a t i n g o f unextracted Douglas- f i r w o o d m e a l and subsequent differential reflectance spectrophotometry (450 and 550 n m ) also s h o w e d increased co lour intensity compared to Douglas- f i r w o o d meal samples that were pre-extracted before heating (Troughton 1973). V a r i o u s c h e m i c a l treatments have been appl ied, w i t h l i m i t e d success, to remove or alter p o l y p h e n o l i c chromophores such as d ihydroquercet in i n D o u g l a s - f i r chips and mechanica l pulps i n an attempt to increase the brightness o f the chips and pulps f r o m Douglas- f i r heartwood. 7 1.2.2 Attempts at Solving the Low Brightness and Bleachability of Douglas-fir Heartwood TMP Using Chemicals T h e h i g h temperature condit ions o f thermomechanical p u l p i n g have been s h o w n to promote the o x i d a t i o n and condensation o f the p o l y p h e n o l i c chromophores i n D o u g l a s - f i r , result ing i n the format ion o f darker co loured pulps (Betz 1974, B u b l i t z 1974, V a n der Z e e 1996). A possible so lut ion to this p r o b l e m m a y be to add a reducing agent to the p u l p i n g mixture ( w o o d chips) to prevent the o x i d a t i o n o f the polyphenols and increase p u l p brightness. E x p e r i m e n t s us ing ascorbic a c i d as a reducing agent have s h o w n little or no effect o n heartwood samples ( 0 . 4 % brightness improvement) whereas the brightness o f sapwood samples was i m p r o v e d b y 2 .7% I S O brightness ( V a n der Zee 1996). These results suggest that a large p o r t i o n o f the o x i d a t i o n and p o l y m e r i z a t i o n o f compounds such as D H Q and quercetin takes place w i t h i n the tree pr ior to thermomechanical p u l p i n g . It is probable that, as the tree ages, the f lavonoids i n the heartwood undergo c h e m i c a l changes such as autooxidat ion and condensation result ing i n coloured, unextractable, and nonreactive complexes . C o l o u r e d complexes can be generated f r o m the o x i d a t i o n o f or tho-dihydroxy groups present o n extractives such as quercet in and dihydroquercet in (Betz 1974, V a n der Zee 1996). T h e h y d r o x y groups are i n i t i a l l y o x i d i z e d to f o r m benzoquinones, w h i c h can be further p o l y m e r i z e d to f o r m dark coloured and inso luble complexes ( V a n der Zee 1996). These complexes themselves, and modi f icat ions o f these complexes dur ing thermomechanica l p u l p i n g , result i n the l o w brightness o f D o u g l a s - f i r heartwood T M P f r o m D o u g l a s - f i r (De l lus 1997). 8 A n o t h e r source o f brightness loss m a y occur dur ing p u l p i n g w h e n metal ions, especial ly i r o n , react w i t h ortho-hydroxy phenol ic extractives such as d ihydroquercet in , to f o r m darkly coloured, h i g h l y stable metal-complexes ( V a n der Zee 1996). T h e metal ions are present i n w o o d chips, and/or m a y originate f r o m the p u l p i n g process waters, or f r o m the p u l p i n g equipment (Fengel 1989). E t h y l e n e d i a m i n e tetraacetic a c i d ( E D T A ) has been added to r a w w o o d chips pr ior to thermomechanical p u l p i n g for chelat ion o f metal ions to prevent the formation o f co loured complexes ( V a n der Zee 1996). Unfortunate ly , E D T A addit ion d i d not result i n brightness improvements ( V a n der Zee 1996). T h e addi t ion o f E D T A and i r o n to w o o d was s h o w n to be he lpful i n i m p r o v i n g brightness compared to samples supplemented w i t h o n l y i r o n ( V a n der Zee 1996). F r o m these results, it is evident that large portions o f the co loured complexes are formed i n the w o o d pr ior to the thermomechanica l p u l p i n g process. A s ment ioned earlier, various c h e m i c a l treatments have been appl ied, w i t h l i m i t e d success, to remove the p o l y p h e n o l i c co loured complexes i n Douglas- f i r mechanica l pulps . Extract ion o f Douglas- f i r heartwood pulps us ing either e thyl acetate, acetone, hot water and c o l d water has s h o w n v a r y i n g results. The inconsistency o f extraction results can be attributed to the different pulp samples used as w e l l as the different solvents e m p l o y e d since the extractive content o f Douglas- f i r and the c h e m i c a l characteristics o f each solvent varies. E x t r a c t i o n o f heartwood g r o u n d w o o d w i t h ethyl acetate resulted i n a decrease i n both the bleached and unbleached brightness values ( B u b l i t z 1974). It was thought that this effect might be due to the extraction o f co loured compounds f r o m the interior o f the w o o d and redeposit ion o n the fiber surface ( B u b l i t z 1974). Extract ion w i t h acetone resulted i n approximately 2 and 1 point unbleached and bleached I S O brightness gains respectively ( B u b l i t z 1974, G u p t a 1976). A l t h o u g h the f lavonoids were s h o w n to be soluble i n basic m e d i a , u p o n exposure to basic 9 conditions, these compounds underwent air oxidation and self-polymeriztion, or oxidative grafting onto wood components, thus inhibiting extraction (Betz 1974). Although extraction with hot water had a detrimental effect, since hot water mimics the conditions employed in the thermomechanical pulping process (Bublitz 1974), extraction with cold water resulted in brightness increases of 2 pts ISO after bleaching (Bublitz 1974). Although pulping the wood chips under cold water conditions to decrease the colour problems would be favourable, hot water is typically employed, as one way of minimizing energy consumption during refining to fiberize wood chips (Bublitz 1974). Further extraction experiments have utilized other organic solvents such as cyclohexane and methanol alone and in combination with water (Bublitz 1974, Dellus 1997, Gupta 1976). These results showed that the most effective methanol treatments employed a lower proportion of water at lower temperatures. Cooking with methanol: water (1:1) at 150°C resulted in a brightness decrease from 37.4 to 17.2 % ISO brightness (Van der Zee 1996). The optimum results were obtained when using a solvent combination of methanol: water (7:3) at room temperature for 48 h which resulted in a 2.5 pt ISO brightness increase prior to bleaching and 12 pt ISO increase after bleaching with 5% peroxide (Van der Zee 1996). However, bleaching at room temperature for 48 h is not a method that could be applied at an industrial scale because it is both uneconomical and time consuming. A method must be found to increase the brightness of Douglas-fir mechanical pulps that is both rapid and applicable at an industrial scale. Overall, it has been shown that extraction of mechanical pulps with solvents has had limited success, primarily because of the strong affinity of the chromophoric polyphenolic compounds for pulp components (Betz 1974). A s mentioned earlier, the primary goal of my work was to assess the potential of biological methods for removing or altering the colour causing compounds such as the flavonoids, in Douglas-fir, so that Douglas-fir can be bleached to higher brightness levels. 10 1.3 Biological Chip and Pulp Treatment Several biological treatments have been shown to improve dewatering, decrease pitch problems, improve fiber characteristics, and improve brightness of pulp (Barker 1997). Biomechanical pulping and biological bleaching are particularly relevant for the application to Douglas-fir T M P to improve pulp brightness and bleachability. Growing fungi on wood chips to decrease the fiberization energy while improving fiber characteristics has been the focus of biomechanical pulping (Akhtar 1993), while increasing the brightness of chemical pulps using fungi or enzymes, to replace chemical bleaching, has been the focus of biological bleaching (Paice 1995). The two methods differ since they involve the treatment of different starting materials (wood chips for mechanical pulp vs. chemical pulp), however both involve the use of fungi and their enzymes, or enzymes alone, to achieve their goals. In order to use these methods as tools to improve the brightness of Douglas-fir mechanical pulps, the mechanisms of each method should be fully understood. Since both biomechanical pulping and biological bleaching primarily involve the use of white-rot fungi and their enzymes, it would be useful to describe these organisms in greater detail. White-rot fungi are used both in the processes of biomechanical pulping and biological bleaching. White-rot fungi have the ability to almost completely degrade the lignin located between both the papermaking fibers and within the cell wall (Blanchette 1995). The effect is often called a "bleached white" appearance. The ability to degrade lignin has made white-rot fungi a prime choice for application to wood chips, to mechanical pulps and for delignification of chemical pulps (to be discussed later). It is thought that the white-rot fungi primarily degrade lignin in order to access the hemicellulose and cellulose, by producing lignolytic enzymes that 11 oxidatively degrade lignin. White-rot fungi can be separated into two main groups of degradation types called simultaneous and sequential. Simultaneous white-rot fungi degrade all cell wall components uniformly during all decay stages. Examples of this type of decay fungi include Phanerochaete chrysosporium, Trametes versicolor and, Dichomitus squalens (Blanchette 1995). Sequential white- rot fungi attack cell wall components selectively. Examples of this type of decay fungi include Ceriporiopsis subvermispora, Phellinus pini and Heterobasidion annosum (Blanchette 1995, Daniel 1994). The degradation begins with hemicellulose and lignin followed by cellulose. A general feature shared by all white-rot fungi is the ability to ultimately consume all cell-wall components (approximately 95-97% under optimal conditions). During the initial degradation of wood, the mycelia of the white-rot fungi are concentrated on the cell wall surfaces, enhancing their enzymes' ability to penetrate from the lumen outward into the plant cell. General methods that screen for white-rot fungi usually distinguish white-rot fungi from other fungi on the basis of their ability to completely degrade lignin, and the white colour produced during degradation of wood. One example of a biochemical test capable of screening for lignolytic fungi involves the decolourization of a dye Poly R-478 (antrapyridine chromophore) (Gold 1988). Fungi are grown in media containing Poly R-478 dye and the decolourization of the dye is measured spectrophotometrically by comparing the ratio of absorbance at a wavelength of 520 nm to 350 nm (Gold 1988). It has been shown that the rate of decrease in the absorbance ratio has a positive correlation with oxidative lignolytic enzyme activity (Gold 1988). Another unique screening method by Nishida et al. (1988) used the formation of coloured zones on wood powder agar plated supplemented with guaiacol as an indicator of lignolytic activity. Apparently, the test monitored the ability of lignolytic enzymes produced by the fungi to react with guaiacol, a compound representing a lignin model compound. Although various groups continue to try to elucidate the actual enzymatic mechanisms involved in lignin modification, much of the research carried out in this 12 area has i n v o l v e d the assessment o f the direct c o m m e r c i a l feasibi l i ty o f this approach and the actual benef ic ia l changes that can be derived f r o m b i o l o g i c a l treatments such as b i o m e c h a n i c a l p u l p i n g and b i o l o g i c a l b leaching. 1.4 Biomechanical Pulping - Fungal Treatment of Wood Chips T h e benefits that have been c l a i m e d o n the various p u l p i n g process parameters incurred by " B i o m e c h a n i c a l " p u l p i n g inc lude a decrease i n f iber izat ion energy, improvement i n paper strength properties, decrease i n c h e m i c a l usage, lesser effluent problems, and p i t c h r e m o v a l (Akhtar 1994, F ischer 1994). " B i o p u l p i n g " w h i c h has p r i m a r i l y a imed at p i t c h r e m o v a l has generally used fungi such as the ascomycete Ophiostoma, w h i c h are different f r o m the group o f fungi that have been used to achieve benefits such as energy savings, increased paper strength, effluent improvement and enhancement o f f iber properties (Fischer 1994). T h e latter class o f fungi , white-rot fungi , w h i c h consist o f most ly basidiomycetes , are used p r i m a r i l y i n " b i o m e c h a n i c a l " p u l p i n g and are o f greater interest to this study, since these fungi s h o w p r o m i s i n g capabil i t ies that m a y help alleviate the problems associated w i t h D o u g l a s - f i r mechanica l pulps. White-rot fungi have been appl ied at various stages i n the p u l p i n g process, i n c l u d i n g addi t ion to w o o d chips pr ior to p r i m a r y ref ining (before f iberization) (Leatham 1990, A k h t a r 1994), addi t ion pr ior to secondary ref ining o f m e c h a n i c a l pulps (Bar- lev 1992), and addit ion pr ior to b leaching stage o f K r a f t pulps ( b i o l o g i c a l bleaching) (Paice 1995). S ince a large port ion o f the appl icat ion o f fungal treatments dur ing and after mechanica l p u l p i n g has been appl ied to w o o d chips pr ior to i n i t i a l f iberizat ion, the past w o r k that has assessed effects o f b iomechanica l p u l p i n g o n various pulp properties is discussed i n some detai l . 13 T h e benefits o f b i o m e c h a n i c a l p u l p i n g have been described above, and examples o f energy savings w h e n white-rot fungal treatment is appl ied pr ior to p r i m a r y re f in ing are w e l l documented (Table 1). It has been s h o w n that softening o f w o o d chips results i n a reduct ion i n the mechanica l energy required for f iber izat ion and less f iber shearing, result ing i n a higher proport ion o f longer fibers and increased paper strength (Setc l i f f 1990). T h e partial degradation o f l i g n i n also increases interfiber b o n d i n g since l i g n i n coats fibers i n the m i d d l e l a m e l l a (Setc l i f f 1990). E x a m p l e s o f strength increases are also w e l l documented (Table 2). A n increase i n interfiber b o n d i n g due to the degradation o f l i g n i n coat ing m e c h a n i c a l pu lp fibers results i n increased fiber b o n d i n g thus increasing burst and tensile strength, w h i l e the higher proport ion o f longer fibers result ing f r o m the decreased f iber shearing dur ing b i o m e c h a n i c a l p u l p i n g results i n higher tear strength values. A t the fiber l eve l , m i c r o s c o p y studies o f b i o m e c h a n i c a l pulps indicated dramatic differences w h e n compared to conventional mechanica l pulps . I n a study b y Sachs et al (1990), it was s h o w n that aspen b i o m e c h a n i c a l p u l p had fibers o f higher u n i f o r m i t y , increased f ibr i l la t ion , less f iber stiffness, and less w o o d c e l l w a l l debris than convent ional aspen mechanica l pulps such as R M P , T M P , C T M P and S G W . Effluents o f b i o m e c h a n i c a l pulps have also been s h o w n to have less tox ic i ty , l o w e r B O D and l o w e r C O D than conventional m e c h a n i c a l p u l p i n g effluents (Sykes 1993). In most o f the recent publ ished w o r k , the sequential ly degrading white-rot fungus Ceriporiopsis subvermispora, appears to be the organism o f choice for biomechanica l p u l p i n g since C. subvermispora treatments results i n strength improvements c o m b i n e d w i t h energy savings (Akhtar 1993; 1994; 1995; 1997, Setc l i f f 1990). T o f u l l y understand h o w the white-rot fungi have been used to achieve the benef ic ia l effects o f b i o m e c h a n i c a l p u l p i n g it is necessary to discuss the methodology e m p l o y e d dur ing the process. There have been several detailed publ icat ions descr ibing the procedures o f b i o m e c h a n i c a l p u l p i n g at both the laboratory and m i l l scale (Akhtar 1993; 1994; 1995; 1997, 14 Setc l i f f 1990). O v e r a l l , the process involves an i n i t i a l inoculat ion onto s m a l l w o o d c h i p samples, f o l l o w e d b y a larger scale inoculat ion to a substantial quantity o f autoclaved (steril ized) chips (Akhtar 1993; 1994, L e a t h a m 1990; Sachs 1989). Recent ly , changes to the tradit ional process described i n these papers have been used to save steri l izat ion energy and t ime. P r i o r to inoculat ion, the chips are steam treated for steri l izat ion rather than us ing autoclaving, w h i l e c o r n steep l i q u o r (a by-product f r o m the corn w e t - m i l l i n g process) is n o w added as a nutrient source to decrease the amount o f fungal i n o c u l u m necessary for sufficient fungal c o l o n i z a t i o n ( A k t h a r 1997). T h e disadvantages o f this process inc lude l o n g incubat ion t imes, h i g h requirement o f energy required for steri l izat ion o f w o o d chips and decreases i n the unbleached pulp brightness (Akhtar 1993; 1994, L e a t h a m 1990, Sachs 1989). B i o m e c h a n i c a l p u l p i n g has been successful ly used w i t h respect to decreasing ref ining energy and i m p r o v i n g strength and f iber characteristics, however the effects o f white-rot fungal treatment o n the opt ica l properties o f mechanica l pulps is o f direct relevance to m y w o r k . 15 T A B L E 1: E X A M P L E S OF T H E ENERGY SAVINGS OBTAINED DURING BIOMECHANICAL PULPING (FUNGAL T R E A T M E N T PRIOR TO PRIMARY REFINING) A N D DURING FUNGAL T R E A T M E N T OF MECHANICAL PULP (FUNGAL T R E A T M E N T PRIOR TO SECONDARY REFINING) W o o d Species F u n g u s R e f i n i n g Stage E n e r g y savings(%) Reference A s p e n Phanerochaete P r i m a r y 38 L e a t h a m 1990 chrysosporium L o b l o l l y p ine Ceriporiopsis P r i m a r y 42 A k h t a r 1993 subvermispora Spruce/Red IZU-154 Secondary 35 K a s h i n o 1993 pine Jute Ceriporiopsis Secondary 38 Sabbharwal 1995 subvermispora T A B L E 2: E X A M P L E S OF STRENGTH INCREASES OBTAINED DURING BIOMECHANICAL PULPING W o o d Species F u n g u s Burst (%increase) Tear (%increase) Tensi le (%increase) Reference A s p e n Ceriporiopsis 10 98 15 Setc l i f f 1990 subvermispora L o b l o l l y Ceriporiopsis 15 60 N / g a A k h t a r 1994 P i n e subvermispora Spruce IZU-154 0 0 35 K a s h i n o 1993 Jute Ceriporiopsis 100 20 25 Sabbharwal 1995 subvermispora D a t a unavai lable 16 1.4.1 Optical Properties of Biomechanical Pulps T h e opt ica l properties o f b i o m e c h a n i c a l pulps have not yet become a major focus o f research, since the m a i n goal o f b i o m e c h a n i c a l p u l p i n g is to reduce the energy necessary for p r i m a r y and secondary ref ining (Akhtar 1994; 1997). R e d u c t i o n o f re f ining energy, improvements i n paper strength properties, and m o d i f i c a t i o n o f p u l p effluent have been accompl ished by b i o m e c h a n i c a l p u l p i n g , however these improvements have frequently come at the expense o f reducing the opt ica l properties o f the pulps ( A k h t a r 1993; 1994, Sykes 1993). I n a l l cases, b i o m e c h a n i c a l p u l p i n g has decreased the unbleached brightness o f result ing pulps . H o w e v e r , there are conf l i c t ing reports that suggest that pulps treated w i t h fungi pr ior to p r i m a r y or secondary ref ining can or cannot be easi ly bleached to the brightness levels o f m e c h a n i c a l pulps without fungal treatment (Akhtar 1998, B a r - l e v 1982). W h e n one discusses opt ica l properties, there are three important measurements to take into consideration. Brightness is the ref lect ivity o f a sheet o f paper under an incident l ight at 457 n m ( P i l o n 1982). L i g h t scattering coefficient relates to the scattered l ight f r o m a sheet exposed to an incident l ight beam and it is inversely proport ional to the amount o f internal b o n d i n g occurr ing w i t h i n the paper sheet ( P i l o n 1982). O p a c i t y is the property o f a sheet to obstruct the passage o f l ight and to prevent one f r o m seeing through it ( P i l o n 1982). M a n y workers (Table 3) have assessed the effects o f b i o m e c h a n i c a l p u l p i n g o n the opt ica l properties o f the result ing m e c h a n i c a l pulps . M o s t b iomechanica l p u l p i n g treatments result i n a decrease i n unbleached brightness, however the results o f fungal treatment o f mechanica l pulps , w h i c h are direct ly pertinent to m y w o r k , are not as consistent w i t h respect to changes i n brightness. 17 T A B L E 3: BIOMECHANICAL PULPING OF VARIOUS WOOD SPECIES WITH THE WHITE-ROT FUNGUS CERIPORIOPSIS SUBVERMISPORA: EFFECTS OF FUNGAL TREATMENT ON OPTICAL PROPERTIES OF MECHANICAL PULPS Chip Source Control C. subvermispora Control C.subvermispora C .subvermispora Control Reference Brightness Pulp Brightness Opacity Opacity L.S.C. a (m2/kg) L.S.C." (%ISO) (%ISO) (m2/kg) Loblolly Pine 45.8 31.9 (with agitation) 93.8 91.0 30.8 46.3 Akhtar 1993 Loblolly Pine 45.8 35.7 (static) 93.8 89.7 32.0 46.3 Akhtar 1993 Aspen 62.4 49.2 96.3 94.5 41.1 65.3 Akhtar 1994 White Birch 50 46.5 N/g b N/g 67.4 64.3 Setcliff 1990 Black Spruce 54.2 37.2 N/g N/g 41.6 55.8 Setcliff 1990 Norway 54.7 41.9 N/g N/g 43.6 50 Setcliff Spruce 1990 Eucalyptus 46.9 45.5 N/g . N/g 59.4 76.5 Setcliff 1990 a L . S . C . = L i g h t Scattering Coef f ic ient b N/g= D a t a unavai lable 18 1.4.2 Fungal Treatment of Mechanical Pulps F u n g a l treatment o f mechanica l pulps is usual ly performed w i t h the objective o f reducing the re f in ing energy necessary for subsequent ref ining for the development o f p u l p fibers (secondary ref ining) . In some cases, fungal treatment o f mechanica l pulps showed s i m i l a r results to those obtained after treatment o f w o o d chips. Treatment o f spruce-pine-fir p u l p ( 6 0 % -1 0 % - 3 0 % ) w i t h P.chrysosporium, T. versicolor, P. ostreatus, and P. pini resulted i n decreased brightness and l ight scattering coefficients ( P i l o n 1982). T h e largest effect was seen w i t h P.chrysosporium, w h i c h decreased brightness f r o m 52.5 % I S O to 32.2 % I S O and l ight scattering coefficient f r o m 69.2 m 2 / k g to 56.0 m 2 / k g , w h i l e l eav ing the opacity unchanged ( P i l o n 1982). T h e treatment o f n o n - w o o d y jute fibers w i t h the fungus C.subvermispora also resulted i n a brightness decrease f r o m 44.7 to 33.2 % I S O . Therefore the darkening effects^of fungal treatment o n mechanica l pu lp fibers was not l i m i t e d to o n l y w o o d y fibers ( H a r m o h i n d e r 1995). A study o f fungal de l igni f icat ion o f mechanica l pulps u t i l i z i n g P.chrysosporium to treat L o b l o l l y p ine C T M P (chemithermomechanical pulp) resulted i n a decrease i n brightness f r o m 52.8 % I S O to 17.9 % I S O after 2 weeks incubat ion ( P e l l i n e n 1989). A f t e r peroxide b leaching the brightness increased to o n l y 41.6 % I S O , compared to the untreated control that had a bleached brightness o f 62.4 % I S O ( P e l l i n e n 1989). A l k a l i n e extraction o f the pulps to remove s m a l l l i g n i n fragments that m a y have been liberated by the fungal treatment, resulted i n a further decrease i n brightness and loss o f pu lp y i e l d ( P e l l i n e n 1989). I n another study, red alder (Alnus rubra Bong) T M P was treated w i t h P. chrysosporium to reduce energy requirements for secondary ref ining ( B a r - l e v 1982). Treatments were performed w i t h and without added nutrients. T h e fungal treatments d i d not result i n any brightness changes compared to the contro l T M P w h e n treatments were f o l l o w e d by an a lkal ine extraction (Bar- lev 1982). U n l i k e the cases 19 described prev ious ly , this fungal treatment decreased secondary ref ining energy w h i l e preserving the brightness o f the p u l p (Bar- lev 1982). C o m p a r i n g the w o r k o f P e l l i n e n et a l . (1989), B a r - l e v et a l . (1982) and P i l o n et a l . (1982) illustrates that the use o f different methodology , different pulps , fungal species and/or w o o d species can either reduce brightness losses, increase brightness, or further decrease brightness. A n y in format ion that can elucidate the effects o f white-rot fungal treatment o n mechanica l pulps is s ignif icant to this study, since white-rot fungal treatment o f m e c h a n i c a l pulps f r o m Douglas- f i r comprises a large part o f m y w o r k . A l t h o u g h the effects o f fungal treatments o n mechanica l pu lp brightness discussed here were inconsistent, there have been enough definite successes observed after the appl icat ion o f white-rot fungi and their enzymes w i t h regard to increasing brightness and bleachabi l i ty o f c h e m i c a l pulps , to warrant the examinat ion o f their act ion o n Douglas- f i r mechanica l pulps . 1.5 Biological bleaching B i o l o g i c a l b leaching can be def ined as the growth o f fungi or direct appl icat ion o f enzymes for degradation o f residual l i g n i n and other chromophores, result ing i n the b leaching o f c h e m i c a l l y produced pulps. B i o l o g i c a l applications that have been assessed to date have revealed three m a i n types o f applications, w i t h fungi and their enzymes that have s h o w n to have a b leach boost ing effect. These methods inc lude, appl icat ion o f hemicel lulases, growth o f l i g n o l y t i c white-rot fungi , and the direct appl icat ion o f d e l i g n i f y i n g enzymes (Paice 1995). H e m i c e l l u l a s e s such as xylanase are currently appl ied c o m m e r c i a l l y dur ing m i l l - s c a l e b leaching, and they have been s h o w n to increase the brightness and decrease the c h e m i c a l consumpt ion during b leaching o f Kraf t pulps (Jean 1994,Scott 1993). X y l a n a s e is thought to operate b y h y d r o l y z i n g 20 redeposited hemice l lu lose , a l l o w i n g larger fragments o f l i g n i n to diffuse f r o m the f iber w a l l (Kante l inen 1993, Paice 1992). H o w e v e r , xylanase is l i m i t e d i n its abi l i ty to increase brightness, since it does not speci f ica l ly attack l i g n i n , therefore brightness gains are, i n most cases, i n the range o f 3-5 pts I S O (Dunlop-Jones 1994). A s the p o l y p h e n o l i c compounds responsible for the l o w brightness and bleachabi l i ty o f Douglas- f i r T M P have a s imi lar c h e m i c a l structure to phenol ic l i g n i n , m y research has focused o n the appl icat ion o f white-rot fungi and their l i g n o l y t i c enzymes, w h i c h have the capabi l i ty to degrade phenol ic and non-phenol ic l i g n i n and c h r o m o p h o r i c structures. 1.5.1 Biological Bleaching With Lignolytic Fungi M o s t o f the past w o r k o n b i o l o g i c a l b leaching w i t h l i g n o l y t i c fungi has i n v o l v e d the growth o f white-rot fungi o n hardwood K r a f t pulps pr ior to c h e m i c a l b leaching, w i t h the goal o f degrading the residual l i g n i n result ing i n increased pulp brightness and savings o f b leaching chemicals . Softwoods are m u c h less responsive to b i o l o g i c a l b leaching w i t h fungi than hardwoods, because o f their increased l i g n i n content and their abundance o f g u a i a c y l l i g n i n sub-units ( R e i d 1990). S o f t w o o d l i g n i n is w e l l bonded and more susceptible to the b l o c k i n g reactions that restrict softwood de l igni f icat ion (Fengel 1989). A f t e r K r a f t p u l p i n g , the amount o f residual l i g n i n i n softwoods is 2-3 times higher that o f hardwoods ( R e i d 1990). T o date, the most frequently e m p l o y e d , and one o f the most effective fungal species for b i o l o g i c a l b leaching o f Kraf t pulps has proven to be Trametes versicolor. T h e b leaching effect o f white-rot fungi such as T. versicolor is carried out by extracel lular oxidat ive enzymes and is mediated b y fungal metabolites whose product ion is induced dur ing growth o n a l i g n o c e l l u l o s i c substrate (Paice 1995). 21 It has been s h o w n that the l i g n o l y t i c enzymes secreted b y Trametes versicolor d u r i n g b i o l o g i c a l b leaching are laccase and manganese peroxidase ( M n P ) ( R e i d 1990). Laccase and M n P are oxidat ive i n nature and the addit ion o f these enzymes should increase the speci f ic i ty and speed o f the l i g n o l y t i c reaction, w h i l e the use o f defined e n z y m e mixtures w h i c h do not contain extraneous enzymes w i l l prevent unnecessari ly degradation o f other p u l p components such as cel lulose. I n some cases, the degradation o f carbohydrates dur ing fungal treatment has been negated by the addi t ion o f glucose to the pulp to repress the product ion o f h y d r o l y t i c cellulases and hemicel lulases (Bajpai 1992, R e i d 1990). A previous screening experiment compar ing 9 different strains o f white-rot fungi for their abi l i t ies to del igni fy h a r d w o o d K r a f t pulps and i m p r o v e brightness, showed that Trametes (Coriolus) versicolor increased bleached pulp brightness values and removed the most l i g n i n , compared to a l l other species tested, and the untreated control pu lp (Paice 1989). In this w o r k , T.versicolor b leached the pulp f r o m 33.5 to 48.0 % I S O (Paice 1989). H o w e v e r , T.versicolor treatment o f softwood (spruce) pulps d i d not result i n any br ightening effect. The ineffectiveness o f T.versicolor o n softwood pulps m a y be attributed to the fact that this fungus o n l y attacks hardwoods i n nature ( A d d l e m a n 1995, Paice 1989). E n z y m e assays o n the supernatant o f the h a r d w o o d pulp treatments revealed o n l y the act ion o f laccase (Paice 1989). A l t h o u g h enzyme assays d i d not show the presence o f any cellulase act iv i ty , the pulps showed a marked v iscos i ty loss, w h i c h indicated an attack o n the cel lulose component (Paice 1989). These results may be dece iv ing since cel lulase enzymes m a y remain attached to the pulps dur ing the e n z y m e assays result ing i n an i n a b i l i t y to measure their true activity i n the supernatants ( K a t a g i r i 1995, Paice 1989). Laccases m a y also o x i d i z e cel lulose i n the pulps result ing i n a decrease i n the interaction o f cel lulose w i t h cupriethlyene diamine dur ing the measurement o f v iscos i ty thus decreasing v iscos i ty measurements wi thout degrading cel lulose (Fengel 1989). H o w e v e r , contrary to the effects o f cel lulose attack, the tear, 22 burst, breaking length, and stretch values o f pulps a l l increased, w h i c h w o u l d seem to indicate the absence o f cel lulose degrading enzymes (Paice 1989,Reid 1990). I n this study, the addi t ion o f the culture supernatant to freshly steri l ized h a r d w o o d K r a f t pulps had no effect (Paice 1989). Therefore, it can be assumed that there was either a rapid turnover o f the enzymes, the enzymes were cel l-associated, or there were other requirements for b leaching w i t h the supernatant. T h e past w o r k i n this area has generally focused o n the specif ic degradation o f l i g n i n i n c h e m i c a l pulps. H o w e v e r , as mentioned earlier, D o u g l a s - f i r mechanica l pulps possess large amounts o f p o l y p h e n o l i c chromophores and l i g n i n compared to c h e m i c a l pulps. O n e o f the goals o f m y project is to use a s i m i l a r strategy to see i f these fungi or enzymes can be used to speci f ica l ly alter or part ia l ly degrade the p o l y p h e n o l i c chromophores present i n D o u g l a s - f i r mechanica l pulp . 1.5.2 Application of Oxidative Enzymes to Kraft Pulps A p p l i c a t i o n o f enzymes to decrease the amount o f residual l i g n i n and i m p r o v e brightness o f K r a f t pulps has become an attractive opt ion compared to the growth o f the actual organisms that produce these enzymes, since direct appl icat ion o f enzymes has i m p r o v e d the speci f ic i ty and speed o f b i o l o g i c a l b leaching (Paice 1995). T h e two m a i n enzymes o f interest w h e n discuss ing b i o l o g i c a l b leaching w i t h oxidat ive enzymes are laccase and manganese peroxidase ( M n P ) , (Bourbonnais 1997, Paice 1995). Laccases w i l l be the focus o f most o f the discuss ion here since these enzymes have been s h o w n to be the most effective dur ing b i o l o g i c a l b leaching o f K r a f t pulps w h e n c o m b i n e d w i t h a mediator ( A n d e r 1998). Laccase has also been s h o w n to speci f ica l ly act u p o n phenol ic compounds, w h i c h predominate i n the compounds responsible for the dark colour o f Douglas- f i r ( V i i k a r i 1998). A l t h o u g h l i g n i n peroxidase ( L i P ) is also produced 23 by l i g n o l y t i c fungi and has s h o w n to be effective i n l i g n i n degradation, this e n z y m e has not, as yet, been s h o w n to p lay a dominant role i n b i o l o g i c a l b leaching o f K r a f t pulps w i t h l i g n o l y t i c fungi (Paice 1995). Studies u t i l i z i n g the white-rot fungus T.versicolor have s h o w n that laccase, M n P and cel lobiose:quinone oxidoreductase ( C B Q ) are produced dur ing b i o l o g i c a l b leaching but not L i P ( A r c h i b a l d 1992, F u k u s h i m a 1995, Paice 1995). The absence o f L i P d u r i n g b leaching w i t h T.versicolor is s t i l l under debate ( A r c h i b a l d 1992, I i m o r i 1996, K a t a g i r i 1995). Other studies e m p l o y i n g T.versicolor, w h i c h measure the enzyme act ivi ty o f enzymes adsorbed onto the pulps , suggest that L i P is present dur ing b i o l o g i c a l b leaching ( I i m o r i 1996, K a t a g i r i 1995). It has been s h o w n that C B Q oxid izes ce l lobiose and reduces the radicals formed dur ing l i g n i n o x i d a t i o n by peroxidases ( A n d e r 1990, K a t a g i r i 1995, Paice 1993, R o y 1993; 1994). T h e p r o d u c t i o n o f methanol can be used to moni tor the degree o f de l igni f icat ion dur ing b i o l o g i c a l b leaching, s ince demethylat ion o f m e t h o x y l groups occurs before the b leaching reaction (Bourbonnais 1992). It has also been s h o w n that the product ion o f methanol increases past the leve l o f pulps treated w i t h M n P w h e n laccase is suppl ied w i t h the mediator A B T S (2,2 ethylbenzthiazoline-6-sulfonate) (Paice 1995). A n o t h e r reason w h y laccase w i l l be the c h i e f oxidat ive enzyme e m p l o y e d i n m y w o r k is because it does not require added manganese, a chelator or a careful ly moni tored source o f hydrogen peroxide as is the case w i t h M n P . A s w i l l be discussed later, laccase has also been impl icated i n the specif ic degradation o f the p o l y p h e n o l i c compounds responsible for the dark co lour o f D o u g l a s - f i r mechanica l pu lp ( P i c k a r d 1969, 1970). Because o f the s ignif icant role laccase enzymes p lay i n b i o l o g i c a l l y b leaching K r a f t pulps w i t h fungi and enzymes, and their potential for appl icat ion to Douglas- f i r mechanica l pulps to i m p r o v e brightness, laccase enzymes deserve further discussion. 24 1.5.3 Laccase Laccases (benzenediol: o x y g e n oxidoreductase) are copper containing enzymes that o x i d i z e phenol ic structures w h i l e reducing o x y g e n to water (Paice 1989). A d d i n g a mediator w i t h laccases enhances K r a f t pu lp treatments. T h i s is p r i m a r i l y because the mediator a l l o w s for the o x i d i z a t i o n o f both phenol ic and non-phenol ic l i g n i n structures (Bourbonnais 1990), as w e l l as the o x i d a t i o n o f l i g n i n w i t h i n the fibers. T h i s is required as the laccase enzyme is very large (50-80,000 k D a ) and cannot penetrate the f iber i tse l f ( X u 1997). M e d i a t o r s such as the d y e A B T S (2,2 ethylbenzthiazoline-6-sulfonate), w h e n c o m b i n e d w i t h laccase, have been s h o w n to be readi ly o x i d i z e d f o r m i n g stable cat ion radicals that decrease k a p p a number and increase methanol product ion, indicat ing increased ox idat ion o f l i g n i n (Bourbonnais 1995). These compounds are ca l led mediators rather than catalysts since u n l i k e catalysts, the mediators are usual ly depleted dur ing the reaction w i t h laccases. T h e addi t ion o f laccase without a mediator such as A B T S has been s h o w n to actual ly p o l y m e r i z e rather than d e p o l y m e r i z e l i g n i n (Bourbonnais 1995, Paice 1995). T h e format ion and p o l y m e r i z a t i o n o f cat ion radica l phenol ic l i g n i n sub-units is the most l i k e l y explanation for the increase i n molecular weight o f l i g n i n w h e n laccase is added without a mediator (Bourbonnais 1995). A l t h o u g h the m e c h a n i s m whereby A B T S or other laccase mediators prevent l i g n i n p o l y m e r i z a t i o n is not yet f u l l y understood, it has been speculated that the phenoxy radicals produced b y l i g n i n o x i d a t i o n by l a c c a s e - A B T S f o r m complexes w i t h the mediator ( A B T S ) rather than c o m b i n i n g to f o r m p o l y m e r s (Bourbonnais 1995). Research to elucidate the m e c h a n i s m o f laccase-mediator b iobleaching is o n g o i n g , however, there are other concerns to be addressed before this technology can be appl ied at an industr ial scale. 25 Unfortunate ly , laccases are not yet avai lable i n suff ic iently large quantities for c o m m e r c i a l use, w h i l e the A B T S mediator is s t i l l best described as an expensive analyt ica l c h e m i c a l rather than a " c o m m o d i t y " c h e m i c a l (Paice 1995). A l s o , dur ing treatment w i t h laccase, A B T S attaches to the pulp and decreases the brightness p r i o r to b leaching (Paice 1995). These problems must be solved before laccase can be appl ied at an industr ia l scale. H o w e v e r , it has been s h o w n that o n l y 1 U laccase/g o f pu lp c o m b i n e d w i t h the mediator A B T S is required to g a i n m a x i m u m del igni f icat ion o f softwood K r a f t p u l p (Paice 1995). It has also been s h o w n that 6 0 % o f laccase enzyme act ivi ty can be recovered after b i o l o g i c a l b leaching treatment (Bourbonnais 1996). Laccase functions at temperatures as h i g h as 60°C and at a broad 3.5-6.5 p H range (Paice 1995). A recent study has s h o w n that appl icat ion o f laccase and A B T S to softwood K r a f t p u l p f o l l o w e d b y an a lkal ine extraction stage, results i n the r e m o v a l o f co loured l i g n i n - A B T S complexes attached to the pulp (Bourbonnais 1996). Other options for i m p r o v i n g the performance o f laccase that can result i n de l igni f icat ion without brightness decrease, inc lude us ing other mediators such as 1 -hydroxybenzotr iazole ( H B T ) , or c o n t r o l l i n g the redox potential dur ing the laccase o x i d a t i o n ( C a l l 1991). N e w lab-trials u t i l i z i n g laccase for de l igni f i cat ion o f Kraf t pulps have p r i m a r i l y u t i l i z e d H B T as the mediator, since H B T is colourless and has been s h o w n to be more effective than A B T S w h e n c o m b i n e d w i t h laccase for de l ign i f i ca t ion o f K r a f t pulps ( C a l l 1994). D e l i g n i f i c a t i o n values o f up to 5 5 % have been achieved u t i l i z i n g laccase and H B T ( C a l l 1994). Unfortunately laccase act iv i ty is d i m i n i s h e d after H B T is o x i d i z e d to B T (benzotriazole), therefore the laccase dosage must be increased w h e n u t i l i z i n g H B T as a mediator for pu lp de l igni f i cat ion (Bourbonnais 1997). Recent ly , a naturally occurr ing mediator i n the fungus Pycnoporus cinnabarinus has been isolated (Eggert 1996). T h e isolated mediator, 3 - H A A (3-hydroxyanthrani l ic acid) , w h e n c o m b i n e d w i t h laccase has been s h o w n to be effective i n c leav ing a, P-O-4 d i m e r o f g u a i a c y l 26 sub-units. H o w e v e r , this mediator has yet to be s h o w n to be effective i n pulp de l igni f i cat ion w i t h laccase (Eggert 1996; 1998). Screening o f mediators through o x i d a t i o n o f a p - O - 4 d imer o f l i g n i n subunits revealed that H B T was the most effective laccase mediator tested ( L i 1997). A n o t h e r extensive study revealed that A B T S and H B T possessed different reaction mechanisms and were the most effective mediators i n de l igni f i cat ion (Bourbonnais 1997). T h e same study also showed that laccases f r o m different fungi had v a r y i n g l i g n o l y t i c capabil it ies w h e n c o m b i n e d w i t h H B T (Bourbonnais 1997). T h i s can be attributed to v a r y i n g degrees o f tolerance to the deactivating effect o f H B T seen w i t h different laccases (Bourbonnais 1997). M o r e o v e r , laccases possessing higher redox potentials have been s h o w n to be more effective i n o x i d a t i o n o f m o d e l compounds and i n de l igni f i cat ion (Bourbonnais 1997, Eggert 1998, X u 1997). T h e difference i n redox potential between different laccases has been s h o w n to be a result o f a difference i n a single a m i n o a c i d residue 10 amino acids f r o m the active ( T l ) site o f the e n z y m e (Eggert 1998). A m o n g laccases f r o m various organisms, this single a m i n o a c i d residue has been s h o w n to be either phenylalanine, leucine, or methionine (Eggert 1998). T h e redox potential o f the e n z y m e has been s h o w n to be highest w h e n a phenylalanine residue occupies this pos i t ion , f o l l o w e d by leucine and has been s h o w n to be lowest w i t h methionine (Eggert 1998). A l t h o u g h b i o l o g i c a l b leaching u t i l i z i n g laccase enzymes is currently rece iv ing a lot o f attention, as indicated by the predominance o f this topic at the recent " B i o t e c h n o l o g y i n the P u l p and Paper Industry" conference i n V a n c o u v e r , there is s t i l l considerable r o o m for improvement . Factors such as treatment condit ions, enzyme sources and n e w mediators are currently be ing investigated ( L i 1997). N e w mediators must be both inexpensive, and effective i n p u l p de l igni f icat ion, i f laccase b i o l o g i c a l b leaching is to eventually be appl ied at an industr ial scale. H o w e v e r , there has been enough progression i n this area to warrant an assessment o f white-rot 27 fungi and their enzymes for i m p r o v i n g the brightness and bleachabi l i ty o f D o u g l a s - f i r mechanica l pulps . 1.6 Applications of Biological Treatments to Douglas-fir There has been no w o r k reported thus far o n attempts to apply microorganisms and their enzymes to D o u g l a s - f i r mechanica l pulps to i m p r o v e optical properties. The p o l y p h e n o l i c character o f the co loured compounds responsible for the l o w brightness o f D o u g l a s - f i r heartwood makes them very s i m i l a r to the phenol ic components o f l i g n i n . Therefore, as mentioned earlier, there is the potential that the enzymes and m i c r o o r g a n i s m s w h i c h degrade/alter l i g n i n can also degrade/alter the flavonoids, tannins and phlobaphenes responsible for the l o w brightness o f Douglas- f i r mechanica l pulps. 1.6.1 Microbial Degradation of Quercetin, Dihydroquercetin and Condensed Tannin Compounds Quercet in is t y p i c a l l y found w i t h i n the tissues o f the Douglas- f i r tree attached to the carbohydrate rutinose [ 6 - 0 - ( 6 - d e o x y - a - L - m a n n o p y r a n o s y l - ) - D - g l u c o s e ] and this material is co l lect ive ly ca l led rut in. Quercet in degradation has been s h o w n to occur i n the fungus Asperigillus flovus v i a the dioxygenase enzyme quercetinase. T h e system produces external enzymes that first h y d r o l y t i c a l l y cleave the ester l inkage between the carbohydrate and quercet in and then oxidizes the C r i n g o f quercetin (figure 1) to produce carbon m o n o x i d e and r i n g o x i d a t i o n products ( C h i l d 1963, H a y 1961, K r i s h n a m u r t y 1970, O k a 1971). Cul ture filtrates o f 28 Trametes versicolor have been s h o w n to degrade condensed tannins and transform catechin, w h i c h is a s i m i l a r c o m p o u n d to dihydroquercet in ( A r c h a m b a u l t 1996, L o r u s s o 1996). L a c c a s e enzymes, isolated f r o m T. versicolor, have also been s h o w n to o x i d a t i v e l y transform dihydroquercet in and quercetin to coloured compounds v i a o x i d a t i o n o f the c a r b o n y l group o n the C r i n g o f quercetin and dihydorquercet in (figure 1) without the product ion o f carbon m o n o x i d e u n l i k e the degradation scheme o f A. flavus ( P i c k a r d 1969). T h e product ion o f laccase by T. versicolor was also s h o w n to be induced by the addi t ion o f d ihydroquercet in to the g r o w t h m e d i u m , but induced to a lesser degree by added quercetin ( P i c k a r d 1970). A l t h o u g h the products formed b y the induced laccase o x i d a t i o n o f quercetin and dihydroquercet in have not yet been revealed, it has been hypothesized that the co loured products formed by o x i d a t i o n were incorporated into c e l l material ( P i c k a r d 1970). T h e fungus Phanerochaete chrysosporium has been s h o w n to have the abi l i ty to degrade tannins i n the leaves o f oak (Quercus incana). It was found that there was a 6 6 % decrease i n condensed tannin content f r o m fermentation w i t h P. chrysosporium for 10 days, a l though there was also a substantial decrease i n cel lulose and hemicel lu lose content w h i c h is undesirable for pulp applicat ions ( M a k k a r 1994). B o t h T.versicolor and P.chrysosporium were appl ied i n this study w i t h the hope that these fungi c o u l d effect the direct degradation o f the specif ic compounds responsible for the l o w brightness o f Douglas- f i r heartwood T M P . 1.6.2 Applying fungi to Douglas-fir Wood Chips and TMP I n the w o r k reported i n this thesis, I have assessed the potential o f white-rot fungi to alter or remove the coloured precursors such as D H Q , and more important ly the p o l y m e r i c p o l y p h e n o l i c co loured material responsible for the l o w brightness o f Douglas- f i r heartwood mechanica l pulps. A s mentioned before, there have been no reports o n the pretreatment o f 29 Douglas- f i r w o o d chips or mechanica l pulps w i t h fungi to reduce the energy necessary for p r i m a r y and secondary ref ining. A l t h o u g h , as reported earlier, appl icat ion o f fungi to other softwood species has y i e l d e d successful results w i t h respect to reduct ion o f re f in ing energy and improvement o f paper strength properties, these improvements were usual ly accompanied b y signif icant losses i n p u l p brightness. The appl icat ion o f white-rot and brown-rot fungi to softwood mechanica l pulps to reduce the energy required for secondary ref ining has also resulted i n darker co loured pulps ( P e l l i n e n 1989, P i l o n 1982), except for a single report, where the or ig ina l p u l p brightness was recovered w h e n alkal ine extraction was carried out subsequent to fungal treatment w i t h P.chrysosporium (Bar- lev 1982). A s prev ious ly mentioned, the l i g n i n degrading enzymes o f white-rot fungi are oxidat ive i n nature and degrade l i g n i n by r e m o v i n g an electron f r o m the aromatic structures and/or the al iphatic side chains creating cat ion radicals that can break apart the l i g n i n p o l y m e r into o l igomers and further into monomers . Results suggest that although the white-rot fungi and their oxidat ive enzymes degrade l i g n i n and chromophores to increase brightness dur ing treatment o f c h e m i c a l pulps (Paice 1995), the partial oxidat ive degradation o f l i g n i n and chromophores by these fungi and/or enzymes dur ing treatment o f mechanica l pulps is actual ly i n v o l v e d i n a loss o f brightness pr ior to b leaching ( P e l l i n e n 1989). 30 OH O FIGURE 1: a) DIHYDROQUERCETIN A N D b) QUERCETIN (RINGS L A B E L L E D " A " , " B " A N D " C " ) 31 It has been suggested that the format ion o f co lour dur ing b i o m e c h a n i c a l p u l p i n g was due to the format ion o f m e l a n i n (Pe l l inen 1989) w h i c h is a pigment made up o f phenol ic residues l i n k e d to carbohydrates and protein ( P e l l i n e n 1989). T h i s pigment is usual ly associated w i t h fungal m y c e l i a ( P e l l i n e n 1989). M o r e recently, p o l y p h e n o l oxidases have been suggested as causal agents for the darkening o f pulps after treatment o f chips or pulps themselves (Setc l i f f 1990). P o l y p h e n o l oxidases (such as laccase) are produced i n some fungi (such as white-rot fungi) and are i n v o l v e d i n biosynthesis , m o d i f i c a t i o n , p o l y m e r i z a t i o n , and i n certain cases biodegradation o f l i g n i n (Akhtar 1994, E r i k s s o n 1982, Fuj i ta 1993, Sachs 1990, S e t c l i f f 1990). These enzymes m a y o x i d i z e l i g n i n , and phenol ic extractives such as g a l l i c and tannic acids, f o r m i n g quinone structures, turning the pulps reddish-brown, result ing i n lower brightness values ( E r i k s s o n 1982, P i l o n 1982, Setc l i f f 1990). T h i s is a plausible explanation since mechanica l pulps and w o o d chips contain a m u c h higher proport ion o f l i g n i n and extractives compared to c h e m i c a l pulps (Fengel 1989). T h i s is evident by the fact that dur ing the early stages o f degradation b y white-rot fungi , the w o o d exhibits a b r o w n colour, indicat ive o f o x i d i z e d l i g n i n (Blanchette 1995). A s mentioned prev ious ly , white-rot fungal treatment o f softwoods results i n the format ion o f a darker c o l o u r than treatment o f hardwoods, because softwoods contain a higher l i g n i n content (Blanchette 1995, S m o o k 1992 Setc l i f f 1990). Other w o r k has s h o w n that P.chrysosporium treatment o f Kraf t pulps prepared f r o m Douglas- f i r resulted i n decreased unbleached brightness values, however the post-bleach brightness was not measured ( D a w s o n -A n d o h 1991). M o s t l i k e l y , the decreased unbleached brightness o f the Douglas- f i r K r a f t pulps was due to the format ion o f quinone structures w h i c h have been reported to be easi ly bleached to 32 regular brightness levels b y hydrogen peroxide b leaching (Paice 1995). A l t h o u g h the b i o m e c h a n i c a l p u l p i n g treatments described here have been s h o w n to decrease the unbleached brightness values o f mechanica l pulps , the basis for m y w o r k was to use the white-rot fungi and their oxidat ive enzymes to speci f ica l ly affect the p o l y p h e n o l i c chromophores i n Douglas- f i r mechanica l pulps to increase the brightness and bleachabi l i ty . 1.6.3 Treatment of Douglas-fir Mechanical Pulps with Oxidative Enzymes Other avenues o f b i o l o g i c a l treatment i n v o l v e the direct appl icat ion o f ox idat ive enzymes to decolourize or degrade the p o l y m e r i c po lyphenols responsible for the l o w brightness o f Douglas- f i r mechanica l pulps. O x i d a t i v e enzymes produced b y l i g n i n degrading fungi , such as laccases, have been described earlier as the c h i e f enzymes responsible for b i o l o g i c a l b leaching o f c h e m i c a l pulps (Paice 1995). These enzymes are presumed to be successful because o f their abi l i ty to degrade the residual l i g n i n present i n c h e m i c a l pulps pr ior to c h e m i c a l b leaching (Fuj i ta 1991; 1993, M u r a t a 1992, Paice 1995). Presently, there are a l i m i t e d number o f publ icat ions o n the appl icat ion o f oxidat ive enzymes to mechanica l pulps. H o w e v e r , crude enzymes isolated f r o m fungi g r o w i n g o n bagasse i n b u l k storage have been appl ied to bagasse mechanica l pulps result ing i n an increase i n peroxide bleached brightness by 2-3 pts I S O (Prasad 1996). A l t h o u g h this represents a treatment w i t h a mixture o f crude enzymes, rather than treatment w i t h a specif ic enzyme, it demonstrates the potential for enzymat ic improvement o f brightness o f m e c h a n i c a l pulp . A s previous ly described, the appl icat ion o f enzymes such as laccase and M n P to softwood Kraf t pulps without subsequent c h e m i c a l b leaching has been successful i n increasing brightness up to 75.5 % I S O (Bourbonnais 1996, K o n d o 1994). T h e increased l i g n i n and 33 phenol ic extractives content o f Douglas- f i r mechanica l pulps compared to Kraf t pulps presents dif f icult ies to the b i o l o g i c a l b leaching o f Douglas- f i r mechanica l pulps. Laccases without mediators have been appl ied to T M P fibers o f beech (Fagus sylvatica) to enhance auto adhesion o f hot-pressed fiberboards ( F e l b y 1997). It has been hypothesized that laccases react at the fiber surface p r o d u c i n g phenoxy radicals f r o m l i g n i n , w h i c h contact surfaces o f other fibers result ing i n the format ion o f f iber-fiber bonds. P h e n o x y radicals produced b y laccase o x i d a t i o n have also been described as laccase mediators c y c l i n g between their radical and phenol forms thereby enhancing the o x i d a t i o n o f beech fibers b y laccase ( F e l b y 1997). It should be noted that d u r i n g laccase treatment o f beech fibers, the treated samples suffer s ignif icant brightness decreases ( F e l b y 1997). T h e b leaching response o f laccase treated beech fibers was not revealed, since decreases i n brightness are not important for the product ion o f f iberboards ( F e l b y 1997). D i r e c t l y relevant to m y w o r k w i t h respect to the p o l y p h e n o l i c chromophores i n D o u g l a s - f i r mechanica l pulps , laccases both w i t h and without mediator compounds have been s h o w n to preferential ly o x i d i z e phenol ic compounds ( M u l h e i m 1992, V i i k a r i 1998). A l s o , as p r e v i o u s l y mentioned, the product ion o f laccase enzymes was induced i n the white-rot fungus Trametes versicolor dur ing growth i n m e d i a containing d ihydroquercet in ( P i c k a r d 1970). O n e o f the objectives o f this thesis was to assess the treatment o f D o u g l a s - f i r mechanica l pulps w i t h oxidat ive enzymes for its potential to increase pulp brightness and bleachabi l i ty . I hoped to determine i f oxidat ive enzymes such as laccase, can speci f ica l ly degrade the p o l y p h e n o l i c extractives responsible for the l o w brightness o f Douglas- f i r mechanica l pulps. 34 1.7 Research Approach A range o f w o o d degrading fungi were appl ied to Douglas- f i r m e c h a n i c a l pulps to screen for a fungus or fungi capable o f increasing the brightness and bleachabi l i ty o f D o u g l a s - f i r mechanica l pu lp by speci f ica l ly reacting w i t h the p o l y p h e n o l i c chromophores i n the pulp . It was hoped that the fungus w o u l d increase the pulp brightness without sacr i f ic ing the strength and brightness stabil ity o f the result ing paper sheets. The best fungus or fungi f r o m the fungal screening experiment were then appl ied to thermomechanical pulps consist ing o f pure D o u g l a s -fir heartwood, since this pu lp represents a worst case scenario w i t h respect to the h i g h chromophore content o f Douglas- f i r mechanica l pulps. Phanerochaete chrysosporium, Trametes versicolor and laccase enzymes were also appl ied to pure heartwood Douglas- f i r T M P , i n c o m b i n a t i o n w i t h c h e m i c a l extraction, since P.chrysosporium, T.versicolor, and laccase enzymes have been i m p l i c a t e d i n the degradation o f D H Q and condensed tannins ( M a k k a r 1994, P i c k a r d 1970), w h i l e methanol extraction has been appl ied successful ly to increase the brightness o f Douglas- f i r mechanica l pulps ( K y r i a c o u 1998). In an attempt to o p t i m i z e laccase treatments, the condit ions o f treatments were adjusted w i t h respect to factors such as temperature, the avai labi l i ty o f o x y g e n and e n z y m e mediators, p H , and different laccase enzymes. 35 2 MATERIALS AND METHODS 2.1 Fungal Screening 2.1.1 Microorganisms T h e f o l l o w i n g fungi were tested for co lour r e m o v a l , strength effects and brightness stabil i ty o f Douglas- f i r mechanica l pulps: Bjerkandera sp. B O S 5 5 C I M W 1.91, Ceriporiopsis subvermispora (Pilat) G i l b e r t s o n & R i v . (syn. Poria subvermispora) C B S 347.63, Dichomitus squalens (Karst .) R e i d (syn. Polyporus anceps) C B S 432.34, Hypholoma fasciculare (Hudsson:Fr . ) K u m m e r (syn. Naematoloma fasciculare) C I M W 10.92, Lentinula edodes (Berk.) Pegler C B S 833.87 ( D S M 2989) , Phanerochaete chrysosporium B u r d s . B K M F-1767 A T T C 24725, Trametes versicolor ( L . : F r . ) P i la t (syn. Coriolus versicolor) P a p r i c a n 52 ( A T T C 20869) , Gloeophyllum trabeum (Pers. :Fr.) M u r r i l l (syn. Lenzitus trabed) 4 7 D (Forintek) , Postia placenta (Fr.) M . L a r s & L o m b . (syn. Poria placenta) 120F (Forintek) , Lepista nuda ( B u l l i a r d : F r . ) C o o k e (syn. Clitocybe nuda) C B S 512.85, Ophiostoma piliferum strain Cartapip 97 f r o m Clar iant Corporat ion , Charlotte , N C , Penicillium simplicissimum (Oudem.) T h o r n C B S 170.90. 2.1.2 Fungal Growth conditions T h e strains were maintained o n glucose/malt extract (20 g/L malt extract, 35 g/L glucose and 18 g/L agar) slants at 4 ° C and transferred to petri dishes containing the same m e d i u m before inoculat ion. T h e plates were inoculated w i t h 4 - m m agar plugs at the centre o f the plates. A f t e r inoculat ion the plates were incubated i n a stationary incubator at 28°C. T h e m e d i a used for pre-growth o f fungus contained 20 g/L glucose, 5.0g/L peptone, 3.0 g/L, yeast extract, 1.0 g/L 36 KH2PO4, 0.5g/L M g S 0 4 at p H 5.5 and was inoculated w i t h 2 agar plugs. T h e w h o l e preculture (approximately 0.3 g dry weight) was h o m o g e n i z e d for 3 seconds i n a W a r i n g blender ( N e w Hart ford , Connect icut) o n the l o w setting. A l l strains were g r o w n for 7 days except for P. placenta and L. edodes, which were incubated, for 14 days and L. nuda, w h i c h was incubated for 21 days. 2.1.3 Douglas-fir Refiner Mechanical Pulp Medium Douglas- f i r (Pseudotsuga menziesii (Franco) M i r b . ) Ref iner M e c h a n i c a l P u l p ( R M P ) was prepared at M a c M i l l a n B l o e d e l Research (Vancouver , B . C . ) . T h e pulp was prepared b y steam pre-treatment o f w o o d chips and subsequent ref ining (Sprout W a l d r o n ) under atmospheric pressure. T w e n t y - f i v e grams ( O D ) o f pu lp ( p H 4.5 and 1 0 % consistency) was autoclaved for 20 m i n . at 121 ° C i n p o l y p r o p y l e n e containers (10 c m diameter, 500 m l ) and then inoculated w i t h the homogenized p r e g r o w n fungal culture. T h e control was autoclaved but d i d not receive fungal inoculat ion. T h e Douglas- f i r R M P was incubated for up to 7 days i n a stationary incubator at 28°C. E v e r y second day the p u l p mixture was stirred w i t h a glass rod. C o n t r o l s were Douglas- f i r pulps without fungal inoculat ion. M y c e l i u m i n the containers p r o v i d e d v i s u a l evidence o f growth. A complete label led p o l y p r o p y l e n e (10 c m diameter, 500 m l ) container was harvested at 0, 2 and 7 days. T h e samples were extracted w i t h 0.1 M N a O H for 30 m i n . at r o o m temperature immediate ly after harvesting. T h e fibers were col lected b y filtration and subsequently the p u l p received a 1% water wash. Af ter each filtration and w a s h stage the water was passed through the fi lter twice to col lect the fines. 37 2.1.4 Bleaching Sequence E a c h o f the pulp samples were chelated before b leaching w i t h peroxide. T h e condit ions for chelat ion were: start p H o f 5.5, 3 % consistency, 1% E D T A at 50°C for 30 m i n . Af ter chelat ion the f i ltered p u l p received a 1% water w a s h and the fines were passed through twice . T h e peroxide b leaching was carried out at a 1 0 % consistency, 4 % H2O2, 0 . 0 5 % M g S 0 4 . 7 H 2 0 , 2 % N a O H and 5 % N a S i 0 3 for 3 hours at 80 ± 2 ° C . The exact peroxide concentration and the peroxide consumpt ion were determined by t itration w i t h N a 2 S 2 0 3 . 2.1.5 Optical and Physical Testing T h e unbleached pulps were used to make p h y s i c a l handsheets, w h i l e both unbleached and bleached pulp samples were used to make opt ica l handsheets. P h y s i c a l handsheets were prepared according to T A P P I Test M e t h o d T 2 0 5 - o m 8 8 . F o r the determination o f the burst index ( M u l l e n Tester, B . F . Perk ins and Sons, H o l y o k e , M A , U S A ) , tear index (Series 4 0 0 Moni tor/Tear , Test ing M a c h i n e s Inc. , A m i t y v i l l e , N Y , U S A ) and zero-span breaking length (TroubleShooter, P u l m a c Instruments Int., M o n t p e l l i e r , V T , U S A ) , tests were conducted according to T A P P I Test M e t h o d s T231 c m - 8 5 , T 5 3 8 om-88 and T 2 2 0 o m - 8 8 , respectively. ISO-brightness (the reflectance o f the paper sheet at 457 nm) and co lour co-ordinates ( L * indicat ing l ightness, a* indicat ing redness, and b * indicat ing ye l lowness) were determined w i t h a Technibri te T B - 1 C (Technidyne C o r p . , N e w A l b a n y , I N , U S A ) us ing 4.0 g handsheets made according to T A P P I Test M e t h o d T 2 7 2 o m - 9 2 . A l l ISO-brightness and L * , a*, b * values are the average o f two handsheets and each handsheet was read 10 t imes (5 t imes at each site). 38 2.1.6 Thermal and Photo Reversion T h e thermal reversion test was performed according to T A P P I T 4 5 3 Test M e t h o d pm-85 us ing a 105°C c irculat ing o v e n i n a 23°C and 5 0 % relative h u m i d i t y r o o m . A t ime course w i t h 25 hour intervals was run for a total o f 125 hours to determine the rate o f aging. T h e bleached handsheets were tested for photo reversion i n a photochemica l reactor (The Southern N e w E n g l a n d U l t r a v i o l e t C o m p a n y , H a m d e n , Connect icut) c o m p r i s e d o f eight 300-400 n m lamps. T h e apparatus was located i n a 23 ° C and 5 0 % relative h u m i d i t y r o o m and the samples were exposed to a 4-hour t ime course. Handsheets were r e m o v e d at 1 hour intervals and condi t ioned i n the same r o o m for several hours before reading the brightness. 2.2 Treatment of Douglas-fir Heartwood TMP with Phanerochaete chrysosporium 2.2.1 Douglas-fir Heartwood Thermomechanical Pulp N o n - c o m p r e s s i o n heartwood chips ( N C H ) f r o m 129 year o l d Douglas- f i r tree were obtained f r o m the 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 demonstration forest i n H a n e y , B . C . . C h i p s were separated and ref ined to produce non-compress ion heartwood thermomechanical pu lp ( T M P ) at the P u l p and Paper Research Institute o f C a n a d a located i n V a n c o u v e r B . C . . 2.2.2 Treatment of Douglas-fir Heartwood TMP with Phanerochaete Chrysosporium Treatments were performed the same as described for Sect ion 2.1 replac ing Douglas- f i r refiner mechanica l pu lp (heartwood/sapwood m i x ) w i t h Douglas- f i r heartwood T M P except o n l y brightness testing was performed o n Douglas- f i r heartwood T M P treated w i t h P.chrysosporium. 39 2.3 Treatment of Douglas-fir Heartwood TMP with Trametes versicolor 2.3.1 Methanol Extraction of Douglas-fir Heartwood TMP N o n - c o m p r e s s i o n heartwood thermomechanical P u l p (8g O D ) was extracted w i t h methanol i n a soxhlet extractor according to K y r i a c o u et a l (1998) for 24 hours or u n t i l there was no more co lour r e m o v a l . T h e pulp was subsequently g i v e n a 1% water w a s h and p H was adjusted to 5.0 w i t h N a O H . P u l p was then added to specif ied treatments w i t h T. versicolor. 2.3.2 Trametes versicolor Growth Tversicolor was prepared o n agar plates (1 .5% agar, 0 . 5 % glucose, 0 . 3 5 % malt extract) and incubated 7 days at 30°C. T w o agar plugs from these plates were used to inoculate glucose peptone m e d i a ( 1 % peptone, 2 % malt extract, 1% glucose, 0 . 3 % yeast extract) for further incubat ion at 30°C for 7 days. T w e n t y m i l l i l i t e r s o f culture was h o m o g e n i z e d i n a W a r i n g B l e n d e r for 4 seconds and used to inoculate each culture. 2.3.3 Trametes versicolor Treatment Conditions N o n - c o m p r e s s i o n heartwood T M P (methanol extracted heartwood T M P used w h e n specif ied) was suspended at 2 % consistency and p H adjusted to 5.0 w i t h N a O H . M e d i a t o r s were added i n the order o f 1% based o n the pulp dry weight, a long w i t h fungal i n o c u l u m . Samples were incubated w i t h shaking (200 rpm) for 7 days at 28°C. A p p r o p r i a t e samples were a lkal ine extracted (see be low) . A l l pulps were then bleached w i t h h y d r o g e n peroxide (see be low) . 40 2.3.4 Alkaline Extraction of Douglas-fir Heartwood TMP after Fungal Treatment Speci f ied pulp samples treated w i t h T.versicolor (8g O D ) were a lkal ine extracted us ing 2 % N a O H at 60°C for 20 m i n . w i t h st irring. Immediate ly after a lkal ine extraction, the p u l p was f i l tered, re-suspended at 1% consistency and p H adjusted to p H 5.0 w i t h H C 1 . 2.3.5 Hydrogen Peroxide Bleaching of Trametes versicolorTreated Pulps A l l pu lp samples were washed w i t h water at 1% consistency and p H adjusted to 5.5 w i t h N a O H and H C 1 pr ior to chelat ion. C h e l a t i o n was performed at 2 % consistency w i t h 0 . 3 % E D T A for 30 m i n . at 50°C. A f t e r a 1% water wash, pulps were bleached w i t h 8 % h y d r o g e n peroxide, 1.5 % N a O H ( 8 % N a S i 0 3 and 0 . 0 5 % M g S 0 4 . 7 H 2 0 ) for 3 hours at 80°C. S o d i u m Si l icate ( 8 % N a S i O s ) and m a g n e s i u m sulfate (0.05 % M g S 0 4 ) were added as stabilizers. T h e exact peroxide concentration and the peroxide consumpt ion were determined b y t itration w i t h N a 2 S 2 0 3 . 2.3.6 Handsheet Analysis ISO-brightness and co lour co-ordinates ( L * , a*, b * ) were determined w i t h a Technibr i te T B - 1 C (Technidyne C o r p . , N e w A l b a n y , I N , U S A ) us ing 3.0 g handsheets made according to T A P P I Test M e t h o d T 2 7 2 o m - 9 2 . A l l ISO-brightness and L * , a*, b * values were the average o f two handsheets and each handsheet was read 10 t imes (5 t imes at each site). 2.4 Laccase Treatments of Douglas-fir Heartwood TMP 2.4.1 Laccase Assays Laccase was assayed spectrophotometrical ly according to B o u r b o n n a i s and Paice (1995) at 4 2 0 n m i n 0.1 M s o d i u m acetate buffer ( p H 5.0) and 0.5 m M A B T S (2,2 az ino-bis-6-thiazol ine-3-sul fonic acid). T h e o x i d a t i o n o f A B T S was moni tored by determining the increase i n A420 41 (e420= 3.6 x 10 M " c m " ) and 1 U = 1 u m o l o f A B T S o x i d i z e d per m i n . Laccase was added to p u l p samples at 5 U/g pulp a long w i t h 1% A B T S or H B T mediators. 2.4.2 Laccase Treatments Samples (8g) o f methanol extracted and unextracted Douglas- f i r non-compress ion heartwood T M P were suspended i n water at 5 % consistency and the p H was adjusted to 5.0 w i t h N a O H . M e d i a t o r s were added i n the order o f 1% based o n the pulp dry weight , together w i t h enzymes to the appropriate samples (see be low) . Samples were incubated w i t h shaking (200 rpm) for 2 h at 25°C. Samples were b o i l e d for 25 m i n to deactivate the enzyme. Appropr ia te samples were a lkal ine extracted (see be low) . A l l samples were bleached w i t h h y d r o g e n peroxide (see be low) . A l l treatments were performed i n the presence o f air unless otherwise specif ied. 2.4.3 Laccase Treatments in the Presence of Added Oxygen F o r specif ied treatments, o x y g e n was bubbled into the reaction vessel at atmospheric pressure (i.e. o x y g e n saturated condit ions) . A l l other treatments were performed without o x y g e n added to the reaction vessel. 2.4.4 Adjusting Temperature and Oxygen Conditions Speci f ied laccase treatments were performed using 50°C. A l l other treatments were performed at 25°C. 42 2.4.5 Using Different Laccases At pH 5.0 and pH 7.0 Laccase S P 5 0 4 was used i n previous treatments except w h e n otherwise specif ied. Laccases S P 5 0 4 , N S 1 0 0 2 , and N S 1 0 0 3 were a l l obtained f r o m C l a u s F e l b y o f N o v o N o r d i s k . Laccase S P 5 0 4 was produced f r o m Trametes villosa w h i l e N S 1 0 0 2 & N S 1 0 0 3 were der ived f r o m Aspergillus. Laccases were also obtained f r o m D r . L i i s a V i i k a r i o f V T T B i o t e c h n o l o g y and F o o d Research, E s p o o , F i n l a n d . The V T T laccases (crude and pure) were der ived f r o m Trametes hirsuutum. Treatments were performed at p H 5.0 and 7.0. Samples were subsequently bleached as described b e l o w w i t h 6 % H2O2 and 3 % N a O H . 2.4.6 Adjusting Bleaching Conditions A l l samples were bleached according to section 2.3.6, except for the w o r k adjusting the bleaching condit ions, us ing different laccase enzymes at different p H , v a r y i n g o x y g e n and temperature. D u r i n g these experiments, samples were bleached w i t h 6 % h y d r o g e n peroxide w h i l e v a r y i n g s o d i u m h y d r o x i d e f r o m 2 to 3 to 4 to 5 % i n the b leaching l iquor. T h e exact peroxide concentration and the peroxide consumpt ion were determined b y titration w i t h N a 2 S 2 0 3 . 2.4.7 Diffuse Reflectance Analysis of Handsheets O p t i c a l handsheets for reflectance testing were prepared according to T a p p i 272 om-92 and analyzed us ing P e r k i n E l m e r 1600 spectrometer w i t h diffuse reflectance accessory. H a l o n was used to calibrate the spectrometer. V a l u e s were calculated for figures 7a) and 7b) according to the K u b l e l k a - M u n k E q u a t i o n assuming l ight scattering remained constant (Schmidt 1993): 43 k/s= O R ) 2 2R k= absorption s= scattering R= reading from spectrophotometer 3 Results and Discussion 3.1 Screening for Fungi with the Ability to Increase Brightness of Douglas-fir RMP T h e m a i n goal o f this i n i t i a l fungal screening experiment was to f i n d a w o o d degrading fungus or fungi capable o f increasing the brightness o f Douglas- f i r refiner mechanica l pu lp . W e also wanted to determine the effects o f treatments w i t h different w o o d degrading fungi o n the strength properties and the brightness stabil ity o f the Douglas- f i r refiner mechanica l pu lp . It has been prev ious ly s h o w n that the b i o m e c h a n i c a l p u l p i n g o f w o o d chips increases the strength properties o f the result ing mechanica l pulps ( A k h t a r 1993; 1994, E r i k s s o n 1982, L e a t h a m 1990, W e g n e r 1991). T h e tear index, burst index and zero-span breaking length have a l l been s h o w n to increase after fungal pre-treatment o f w o o d chips w i t h C. subvermispora or P. chrysosporium (Akhtar 1993; 1994, Setc l i f f 1990). T h e increases i n the strength properties have a lways been achieved w h e n the w o o d chips were inoculated w i t h fungi pr ior to the p r i m a r y ref ining stage. F u n g a l pre-treatment has also been impl icated i n the softening o f w o o d chips and loosening o f w o o d fibers, thus faci l i tat ing f iber izat ion and a l l o w i n g for preservation o f the longer fiber fraction ( L e a t h a m 1990). T h e degree o f f i b r i l l a t i o n o f w o o d fibers has been measured us ing the dye S imon's stain. P u l p absorbs S i m o n ' s stain result ing i n a blue co lour i f the fibers are not f ibr i l lated. A s the p u l p fibers become increasingly f ibr i l lated, they become y e l l o w and/or orange co loured ( A k h t a r 1993; 1995, Blanchette 1992). The degree o f f i b r i l l a t i o n over t ime can be monitored by this method, as w e l l as the rapid evaluation o f different fungal strains for process o p t i m i z a t i o n (Akhtar 1993). In the w o r k reported here, Douglas- f i r R M P was treated w i t h various wood-degrading fungi and subsequently measured to determine the changes to the burst strength, tear strength and zero-span breaking length. 45 It was apparent that there were no signif icant improvements i n paper strength properties result ing f r o m any o f the fungal treatments (Table 4). M a n y white-rot and brown-rot fungal treatments suffered severe decreases i n a l l o f the paper strength properties after 7 days o f incubat ion (Table 4). A s expected, treatment w i t h the brown-rot fungus, G.trabeum, resulted i n the most substantial reduct ion i n paper strength properties, since brown-rot fungi extensively degrade carbohydrates (Blanchette 1992). M o s t o f the white-rot fungal treatments also resulted i n decreased strength properties w i t h the exception o f C.subvermispora and P.chrysosporium. A s mentioned i n the introduct ion, C.subvermispora is k n o w n to selectively degrade l i g n i n and has been s h o w n to be an ideal fungal strain for b i o m e c h a n i c a l p u l p i n g o f w o o d chips ( A k h t a r 1993; 1994). P.chrysosporium was also further assessed as this fungus left pu lp strength properties intact w h i l e increasing p u l p brightness. T h e separate bleached and unbleached brightness values o f the controls and the pulps to w h i c h the fungi were i n i t i a l l y added (i.e. at t ime zero), were the same. S ince there was a potential for loss o f brightness dur ing autoclaving to steril ize the p u l p due to thermal reversion, a l l samples were autoclaved for the same amount o f t ime so that brightness losses were approximately equal for a l l samples. T h e exception was P. simplicissimum as this i n o c u l u m contained green spores, result ing i n decreased brightness at zero t ime and at a l l o f the measured t ime intervals (Table 5). T h e bleached and unbleached brightness values o f handsheets remained the same after treatment w i t h L. edodes and O. piliferum, and increased after treatment w i t h P. chrysosporium (Table 5). A l l other fungal treatments resulted i n decreases i n both bleached and unbleached brightness values. A n observed decrease i n unbleached brightness a lways resulted i n a decrease i n the corresponding bleached brightness values (Table 5). P.chrysosporium increased both the unbleached and the bleached brightness (Table 5). A s ment ioned i n the 46 introduct ion, b iomechanica l p u l p i n g o f w o o d chips t y p i c a l l y results i n a decrease i n unbleached brightness. H o w e v e r , it is c l a i m e d that b i o m e c h a n i c a l pulps are usual ly bleachable to the leve l o f bleached control pulps (Akhtar 1998). I n the w o r k described here, the results obtained f r o m the fungal treatment o f D o u g l a s - f i r R M P showed that the brightness values o f pulps treated for 7 days w i t h the white-rot fungi C.subvermispora, H.fasciculare, T.versicolor and D.squalens c o u l d not be recovered to the leve l o f the control w h e n us ing the same h y d r o g e n peroxide charge (Table 5). Treatment w i t h L. edodes also resulted i n a substantial decrease i n the unbleached brightness values; however, the brightness c o u l d be recovered to a comparable leve l to the other fungal treated pulps after h y d r o g e n peroxide bleaching. A l t h o u g h the brightness measurements were o f the greatest interest to the objectives o f this study, further details o n the changes to the co lour characteristics o f the paper sheet that occurred dur ing fungal treatment c o u l d be obtained by observing the values o f the co lour co-ordinates i n the S T A R L A B system. T h i s method gives an indicat ion o f the lightness/darkness ( L * ) o f the handsheet, w h i l e +a indicates redness, -a indicates greeness, +b indicates ye l lowness and -b indicates blueness. M o s t o f the unbleached and bleached handsheets f r o m the other fungal treatments showed the same co lour characteristics as T. versicolor except for a few exceptions (Table 6). 47 T A B L E 4: STRENGTH PROPERTIES OF DOUGLAS-FIR THERMOMECHANICAL PULP TREATED WITH DIFFERENT FUNGI. ^ Incubation time (days) T i m e (Days) Zero-span (km) Burst index (kPa*m 2 /g) Tear index ( m N * g / m 2 ) Bjerkandera sp. B O S 5 5 0 8.12 ± 0 . 2 3 1.02 ±0.08 4.00 ± 0.22 2 7.65 ± 0.30 0.92 ± 0.05 4.39 ± 0 . 0 4 7 7.30 ± 0 . 5 0 0.71 ± 0.02 3.25 ± 0.05 C. subvermispora 0 8.69 ± 0 . 1 1 1.17 ±0.05 4.35 ± 0.00 2 8.90 ± 0 . 5 2 1 . 1 6 ± 0.05 4.32 ± 0 . 5 6 7 8.29 ± 0 . 1 5 1.02 ±0.05 3.88 ± 0.40 D. squalens 0 8.61 ± 0 . 3 5 1.03 ±0.05 4.34 ± 0 . 2 2 2 7.90 ± 0 . 1 5 0.93 ± 0.02 4.31 ± 0 . 1 8 7 6.71 ± 0 . 5 1 0.65 ± 0.04 2.25 ± 0.04 P. chrysosporium 0 8.27 ± 0 . 5 5 1 . 1 4 ± 0 . 0 7 3.79 ± 0 . 2 1 2 7.67 ± 0 . 3 7 0.95 ± 0.05 3.59 ± 0 . 0 4 7 8.40 ± 0 . 2 4 1.04 ±0.05 3.76 ± 0 . 1 7 T. versicolor 0 8.06 ± 0 . 1 5 0.90 ± 0.05 4.03 ± 0.05 2 7.93 ± 0.37 0.94 ± 0.05 3.78 ± 0 . 1 3 7 7.03 ± 0.41 0.75 ± 0.05 3.37 ± 0 . 1 7 G. trabeum 0 8.37 ± 0 . 2 5 1.02 ±0.09 3.92 ± 0 . 2 7 2 7.62 ± 0.25 0.90 ± 0.04 4.39 ± 0 . 6 5 7 5.60 ± 0 . 2 7 0.74 ± 0.02 2.69 ±0.00 L. nuda 0 8.18 ± 0 . 7 9 1.03 ± 0 . 0 4 4.25 ± 0.00 2 8.13 ± 0 . 5 7 1.04 ± 0 . 0 7 3.99 ± 0 . 1 3 7 8.22 ± 0.26 0.97 ± 0.07 4.26 ± 0 . 1 7 C o n t r o l 0 0 8.45 ± 0.25 1.06 ± 0 . 1 6 4.25 ± 0 . 1 6 2 8.34 ±0.28 1.04 ± 0 . 0 4 4.20 ±0.08 7 8.00 ± 0 . 3 4 1.03 ± 0 . 0 4 4.62 ± 0 . 2 1 G r o w t h Condit ions are described i n the Materials and Methods Section The strains H. fasciculare, L. edodes, P. placenta, O. piliferum Cartapip 97 and P. simplicissimum a l l showed no changes i n the strength properties. T h e control values are the average o f four independent experiments 48 T A B L E 5: ISO-BRIGHTNESS, BEFORE AND AFTER PEROXIDE BLEACHING, OF DOUGLAS-FIR REFINER MECHANICAL PULP TREATED WITH DIFFERENT FUNGI A Unbleached P u l p ( % I S O ) b Bleached P u l p ( % I S O ) b Incubation time (days) 0 2 7 0 2 7 Bjerkandera sp. B 0 S 5 5 36.6 37.4 37.3 64.4 64.4 64.2 C. subvermispora 37.1 36.6 32.6 64.2 63.2 56.6 D. squalens 37.6 36.8 32.7 66.1 64.1 60.9 H. fasciculare 36.7 35.2 29.9 63.7 63.0 54.4 L. edodes 37.7 37.0 34.9 63.9 63.4 62.4 P. chrysosporium 36.7 37.4 37.5 62.4 63.6 64.1 T. versicolor 35.1 34.0 28.5 64.3 61.5 54.4 G. trabeum 36.2 36.7 31.9 64.2 64.7 53.8 P. placenta 38.5 37.2 37.6 63.1 63.8 63.2 L. nuda 35.6 35.1 32.7 63.0 62.4 59.6 O. piliferum Cartapip 97 37.5 36.6 36.3 64.9 64.8 64.4 P. simplicissimum 34.4 34.6 34.7 50.6 51.8 51.4 C o n t r o l 0 36.9 36.4 36.2 64.1 64.5 63.7 ± 0 . 7 ± 0 . 4 ± 0 . 5 ± 1.1 ± 1.2 ± 1.1 G r o w t h conditions are described i n Materia ls and Methods C o n t r o l values ± standard deviation are the average o f four independent experiments E a c h ISO-brightness value is the average o f two handsheets and each handsheet was read 10 t imes (5 t imes o n each side) 49 A unique result was obtained w i t h the handsheets treated w i t h L. edodes since this fungus o n l y inf luenced the lightness value ( L * ) (Table 6) . ' P.chrysosporium was the o n l y fungus whose treatment resulted i n a reduct ion i n both ye l lowness and redness (Table 6). In contrast, treatment w i t h G. trabeum resulted i n a substantial increase i n both ye l lowness and redness w h i c h were retained after b leaching (Table 6). T h i s m a y be the result o f modi f icat ions o f l i g n i n and/or p o l y p h e n o l i c extractives by G. trabeum, since it has been reported that brown-rot fungi cause modi f icat ions to the l i g n i n macromolecule such as demethlyat ion (Blanchette 1995). M o d i f i c a t i o n s o f the p o l y p h e n o l i c extractives and l i g n i n by the fungi appl ied i n this screening study m a y also have an effect o n the brightness stabil ity o f paper sheets made f r o m D o u g l a s - f i r mechanica l pu lp . T h e resistance o f Douglas- f i r mechanica l pu lp handsheets to brightness reversion b y U V - l i g h t and heat exposure was also o f interest to this study. C h r o m o p h o r e s , such as the condensed tannins and phlobaphenes associated w i t h the l o w brightness and bleachabi l i ty o f Douglas- f i r mechanica l p u l p , may also contribute to the l o w brightness stabil ity o f the p u l p (Sykes 1993, T a c h i b a n a 1992). Chromophores associated w i t h l i g n i n and extractives m a y undergo br ightening dur ing h y d r o g e n peroxide b leaching, however, u p o n exposure to l ighted and heated condit ions, these compounds can revert to their co loured f o r m , result ing i n the y e l l o w i n g o f mechanica l pulps (Sykes 1993). 50 T A B L E 6: STARLAB (L*A*B*) VALUES OF DOUGLAS-FIR REFINER MECHANICAL PULP TREATED WITH DIFFERENT FUNGI BEFORE AND AFTER PEROXIDE BLEACHING A Unbleached P u l p b Bleached P u l p Fungus Incubation t ime (days) L * a* b * L * a* B * Lentinula 0 77.9 2.46 17.7 92.0 -3.03 14.7 Edodes 2 77.5 2.64 18.0 91.8 -2.95 14.8 7 71.8 2.73 16.5 91.6 -2.86 15.4 Phanerochaete 0 73.3 2.53 16.4 91.1 -2.25 14.4 Chrysosporium 2 73.4 2.39 16.1 91.5 -2.42 13.9 7 73.3 2.16 15.8 91.7 -2.47 13.8 Trametes 0 76.8 2.79 19.2 92.1 -2.75 14.4 Versicolor 2 76.5 3.12 20.1 91.2 -2.54 15.4 7 73.9 3.91 23.9 89.2 -2.11 19.2 Gloeophyllum 0 77.5 2.69 18.9 91.9 -2.72 14.2 Trabeum 2 77.9 2.65 19.1 92.0 -2.82 13.9 7 76.0 3.52 22.3 88.1 -1.07 17.5 C o n t r o l 0 0 78.3 2.35 19.0 92.1 -2.80 14.1 ± 0 . 1 4 ± 0 . 1 7 ± 0 . 1 6 ± 0 . 3 5 ±0.08 ± 0 . 4 6 2 78.1 2.32 19.4 92.1 -2.94 14.0 ± 0 . 0 9 ± 0 . 1 1 ± 0 . 0 7 ±0.08 ± 0 . 1 2 ± 0 . 5 1 7 77.8 2.39 19.4 92.1 -2.91 14.0 ± 0 . 2 5 ± 0 . 1 5 ± 0 . 4 0 ± 0 . 2 7 ± 0 . 1 3 ± 0 . 3 7 G r o w t h conditions are described i n Mater ia ls and M e t h o d s Starlab C I E L * a * b * system, i n w h i c h L * is the measure o f lightness, +a indicates redness, -a indicates greenness, +b indicates yel lowness and -b indicates blueness. C o n t r o l values ± standard deviat ion are the average o f four independent experiments 51 In m y w o r k , it was hoped that treatments w i t h fungi , w h i c h m o d i f y the chromophores i n Douglas- f i r mechanica l pulps to increase brightness, c o u l d also retard the photo and thermal brightness revers ion o f the pulp . A l t h o u g h , handsheets f r o m a l l fungal treatments were subjected to U V - l i g h t and thermal testing, o n l y those that showed m a r k e d effects o n the brightness stabil i ty o f Douglas- f i r R M P are described here (figures 2 a & 2 b ) . A f t e r exposure to U V l ight for one hour, the o n l y fungus that retarded the brightness reversion o f bleached D o u g l a s - f i r R M P was L.edodes (figure 2a). Pretreatment w i t h several species, i n c l u d i n g P. chrysosporium, resulted i n i n i t i a l reversion rates comparable to the control , however a l l o f these pre-treatments resulted i n l o w e r brightness values after prolonged U V exposure (figure 2a). N o n e o f the fungal strains decreased the rate o f thermal reversion (figure 2b) and i n fact, G. trabeum and P. chrysosporium treatments resulted i n an increased rate o f c o l o u r format ion dur ing the thermal reversion testing. O v e r a l l , none o f the fungal treatments were successful i n retarding the photo and thermal brightness reversion o f the mechanica l pulps. It is probable that the inabi l i ty o f fungal treatments to retard brightness reversion is associated w i t h the decrease i n brightness obtained d u r i n g fungal treatment. T h i s probably results i n the alteration o f the l i g n i n and p o l y p h e n o l i c extractive derived chromophores i n the pulp . S i m i l a r to m y results, Sykes (1993) conc luded that mechanica l pulps treated w i t h C. subvermispora were more susceptible to photo- and thermal-aging than controls (Sykes 1993). 52 a) 65 to (/) (D C D) CO 6 CO 60h 55h 50 h 45 40 - • — Control - A — Lentinus edodes • •• Phenerochaete chrysospoium • Trametes versicolor 1 2 3 Time (hr) b) 65 60 h ^ 55 co CO CD c 50 .c CQ 45 ^ 40 35 • i • i • i 1 i 1 i - • — Control - A - - Phanerochaete chrysosporium-• Gleoephyllum trabeum -20 0 20 40 60 80 100 120 140 160 Time (hr) FIGURE 2 a) PHOTO A N D b) T H E R M A L REVERSION R A T E S OF S E L E C T E D F U N G A L T R E A T M E N T S OF D O U G L A S - F I R REFINER M E C H A N I C A L PULP (TREATMENTS SHOWN ARE THE RESULT OF 7 DAYS OF F U N G A L TREATMENT) 53 H o w e v e r , Tachibana et a l (1992) have s h o w n that fungal treatments o f unbleached T M P w i t h P. chrysosporium and T. versicolor f o l l o w e d by alkal ine h y d r o g e n peroxide b leaching resulted i n an 8 0 % decrease i n heat-induced (thermal) brightness reversion (Tachibana 1992). T h e y also showed that P. ostreatus and T. versicolor treatments c o u l d repress l ight- induced co lour-reversion by 5 0 % and a treatment w i t h a crude enzyme solut ion o f Stereum hirsutum c o u l d repress the reversion by more than 9 0 % . The experimental des ign o f T a c h i b a n a et a l . (1992) was comparable to the one described here except Tachibana et a l . fa i led to specify the tree species f r o m w h i c h they acquired their mechanica l pulps , and they d i d not per form a post-treatment alkal ine extraction step (Tachibana 1992). The difference i n results m a y be due to the h i g h concentration o f c h r o m o p h o r i c extractives present i n Douglas- f i r m e c h a n i c a l pulps , w h i c h m a y counteract the benef ic ia l effects o f the fungal treatment described i n the w o r k by T a c h i b a n a et a l (1992). 3.1.1 Summary O v e r a l l , the results indicated that treatment o f D o u g l a s - f i r R M P w i t h the fungi selected here, either reduced brightness and strength properties, o r had no effect w i t h the except ion o f P.chrysosporium. Treatment o f the pulp w i t h P.chrysosporium resulted i n a sl ight brightness increase w i t h no signif icant changes i n the strength properties. A s mentioned i n the introduct ion, it has been reported that treatments o f mechanica l p u l p fibers o f red alder w i t h P.chrysosporium pr ior to secondary re f in ing resulted i n increased paper strength properties w i t h no effect o n brightness (Bar- lev 1982). H o w e v e r , contradictory results were reported w h e n l o b l o l l y pine C T M P was treated w i t h P.chrysosporium, as the pulp strength increased but suffered severe decreases i n pulp brightness and bleachabi l i ty ( P i l o n 1982). I n related w o r k , w h e n D o u g l a s - f i r Kraf t pu lp was treated w i t h P. chrysosporium, the paper strength properties were i m p r o v e d by 54 5 0 - 1 0 0 % ( D a w s o n - A n d o h 1991). Unfortunate ly , the effects o f subsequent b leaching were not measured ( D a w s o n - A n d o h 1991). Nevertheless, dur ing this fungal screening experiment, the results obtained d u r i n g treatment o f Douglas- f i r R M P w i t h Phanerochaete chrysosporium were the most encouraging, since treatments increased brightness by 1.5 pts I S O w h i l e preserving the strength o f the paper sheet. F r o m the w o r k reported here, it was apparent that fungal treatments pr ior to fiberization (pr imary ref ining) m a y be more effective at increasing the paper strength properties since most fungal treatments decreased paper strength. C. subvermispora p r o v e d to be an except ion as it decreased brightness w h i l e the paper strength properties were left unchanged. T h i s strain is k n o w n to cause selective degradation o f the l i g n i n whether the substrate is w o o d chips or mechanica l pu lp (Blanchette 1995). I n m y w o r k , fungal treatments to increase p u l p brightness and bleachabi l i ty were o f the highest pr ior i ty , therefore further w o r k focused o n u t i l i z i n g Phanerochaete chrysosporium to increase the brightness o f D o u g l a s - f i r heartwood thermomechanical pu lp . 3.2 Phanerochaete chrysosporium treatments of Douglas-fir Thermomechanical pulp from Pure Heartwood A s described i n the previous section, fungal screening showed that P. chrysosporium was the most effective fungus for increasing the brightness o f D o u g l a s - f i r refiner m e c h a n i c a l pu lp . T h e Douglas- f i r heartwood R M P used i n the previous section was a mixture o f sapwood and heartwood (approx. 7 0 % sapwood). A s described i n the introduct ion, the heartwood p o r t i o n o f Douglas- f i r trees is responsible for the l o w brightness o f D o u g l a s - f i r mechanica l pulps . Therefore, it was o f interest to this study to treat Douglas- f i r heartwood thermomechanica l p u l p 55 ( T M P ) w i t h P.chrysosporium to see i f the benef ic ia l effects o f this fungus c o u l d be extended to a pure heartwood mechanica l pu lp . Douglas- f i r heartwood T M P (Table 7) has s igni f icant ly l o w e r bleached brightness values than d i d the Douglas- f i r R M P (Table 5) used i n the i n i t i a l screening experiment (See C o n t r o l V a l u e s o n Tables 5 and.7). T h i s illustrates the detrimental effect that the presence o f a substantial amount o f Douglas- f i r heartwood w o u l d have o n the brightness o f a m e c h a n i c a l pu lp . Douglas- f i r heartwood T M P represents a worst-case scenario w i t h respect to the l o w brightness and bleachabi l i ty o f Douglas- f i r mechanica l pulps , therefore this p u l p was chosen for a l l further experiments. Douglas- f i r heartwood T M P was treated, focus ing p r i m a r i l y o n brightness results, us ing the same protocol as the fungal screening, replac ing the R M P w i t h heartwood T M P . It was apparent that the fungal treatment reduced the brightness o f D o u g l a s - f i r heartwood T M P by up to 2 pts I S O after 7 days treatment (Table 7). Unfortunate ly , u n l i k e treatment o f Douglas- f i r R M P , P.chrysosporium was ineffective i n increasing brightness o f D o u g l a s - f i r heartwood T M P . A l t h o u g h , as ment ioned i n the introduct ion, P.chrysosporium has been s h o w n to be able to degrade condensed tannin compounds i n oak ( M a k k a r 1994), it is possible that P.chrysosporium m a y not have the abi l i ty to degrade the condensed co loured compounds present i n Douglas- f i r heartwood T M P . 56 T A B L E 7: H Y D R O G E N PEROXIDE B L E A C H E D I S O BRIGHTNESS OF DOUGLAS-FIR H E A R T W O O D THERMOMECHANICAL PULPS TREATED WITH PHANEROCHAETE CHRYSOSPORIUM OVER 7 DAYS A Treatment D a y 0 D a y 2 D a y 7 C o n t r o l 59.7 ±1.1 59.6 ±0.8 59.5 ±1.0 P. chrysosporium 58.2 ±1.0 58.0 ±1.3 57.6 ±1.2 a Treatment condit ions are described i n M a t e r i a l s and M e t h o d s Each handsheet scanned 10 times Results are the average of 3 treatments 57 P r e v i o u s l y , it was s h o w n that laccase product ion c o u l d be induced b y the addi t ion o f D H Q to the m e d i a w h e n g r o w i n g the white-rot fungus Trametes versicolor ( P i c k a r d 1969; P i c k a r d 1970). T.versicolor has also been s h o w n to degrade some condensed tannin compounds ( P i c k a r d 1970). Therefore, T.versicolor and laccase enzymes were chosen for further experiments i n an attempt to increase the specif ic i ty o f enzymat ic act ion o n the p o l y p h e n o l i c chromophores i n D o u g l a s - f i r heartwood T M P . 3.3 Trametes versicolor Treatment of Douglas-fir Heartwood TMP 3.3.1 Unbleached Brightness of Douglas-fir Heartwood TMP treated with Trametes versicolor in combination with Methanol and Alkaline extraction It is k n o w n that Trametes versicolor produces laccases and manganese peroxidases w h e n it is g r o w n o n K r a f t pulps and that these l i g n o l y t i c enzymes react p r i m a r i l y w i t h the phenol ic components o f l i g n i n to achieve the observed brightness g a i n ( A r c h i b a l d 1992, Paice 1993,1995). H o w e v e r , o f direct relevance to Douglas- f i r pulps , T.versicolor has also been s h o w n to m o d i f y d ihydroquercet in ( P i c k a r d 1969; P i c k a r d 1970). T h u s , T.versicolor was i n i t i a l l y a p p l i e d to D o u g l a s - f i r heartwood T M P i n an attempt to determine whether this fungus c o u l d speci f ica l ly alter, or part ial ly degrade either the l i g n i n or D H Q derived p o l y p h e n o l i c chromophores that are responsible for the l o w brightness o f D o u g l a s - f i r heartwood mechanica l pulps . Unfortunate ly , any brightness g a i n that might have been achieved o n the unbleached p u l p c o u l d not be measured, since the abundant growth o f T.versicolor i n the shaking cultures interfered w i t h both the handsheet formation and the brightness measurements. 58 3.3.2 Hydrogen Peroxide Bleached Brightness of Douglas-fir Heartwood TMP treated with Trametes versicolor, with and without Laccase mediators, in combination Methanol and Alkaline extraction Since it has been s h o w n that pr ior methanol extraction o f D o u g l a s - f i r T M P c o u l d remove some o f the l o w molecular weight chromophor ic material present i n the pulp ( K y r i a c o u 1998), w e next compared the effectiveness o f T. versicolor treatments i n c o m b i n a t i o n w i t h pr ior methanol and subsequent a lkal ine extraction (figures 3a and 3b). A s was found i n earlier w o r k w i t h Douglas- f i r , methanol extraction o f heartwood T M P pr ior to h y d r o g e n peroxide b leaching was able to increase the f inal pu lp brightness f r o m 59.6 to 63 % I S O (figure 3 a & 3 b ) ( K y r i a c o u 1998). T h i s result differs f r o m G u p t a et a l . (1976) where extraction o f D o u g l a s - f i r heartwood p u l p w i t h mixtures o f acetone:water and alcohol:benzene d i d not result i n any increases i n bleached brightness values. H o w e v e r , this discrepancy m a y be caused b y the difference i n solvents used for extraction, the age difference o f the trees (65 year-o ld heartwood compared to the 129 year o l d heartwood used i n these experiments), or changes i n the chemistry o f the w o o d that occur dur ing re f in ing . G u p t a et a l . (1976) used D o u g l a s - f i r R M P whereas Douglas- f i r heartwood T M P was used i n m y study. A l k a l i n e extraction was also performed f o l l o w i n g fungal treatments, since it had been reported prev ious ly that a lkal ine extraction enhances brightness and bleachabi l i ty b y r e m o v i n g the l o w molecular weight compounds released dur ing fungal treatments o f K r a f t pulps (Bourbonnais 1996, K o n d o 1994). 59 a) 64-62 H - 60-^ 58-co (U 56-C £ 54-| O) £ 5 2 H o 5 0 • c/5 — 48-46-Tv= Trametes versicolor ireatmeni A = ABTS mediator H = HBT mediator ae = alkaline extraction -9E-Tv-A Tv-A-ae Tv-H Tv-H-ae Tv-ae Tv ae Heartwood PULP TREATMENT b) 64 62 ^ > 60 CO | 56 -| I 54 -| g> . O 5 0 ^ 48 46 me = methanol extraction Tv= Trametes versicolor treatment A = ABTS mediator H = HBT mediator ae = alkaline extraction -3Er -5-me-Tv-A me-Tv-A-ae me-tv me-Tv-H me-Tv-ae me-ae me-Tv-H-ae PULP TREATMENT F I G U R E 3: H Y D R O G E N PEROXIDE BLEACHED BRIGHTNESS OF a) UNEXTRACTED A N D b) M E T H A N O L E X T R A C T E D DOUGLAS-FIR HEARTWOOD T M P , TREATED WITH T. VERSICOLOR FOR 7 DAYS, WITH A N D WITHOUT LACCASE MEDIATORS H B T AND A B T S , A N D SUBSEQUENT A L K A L I N E EXTRACTION (TREATMENTS ARE T H E RESULT OF 2 TRIALS PERFORMED IN T H E ORDER OF SYMBOLS SHOWN UNDER E A C H BAR) 60 T h e increases i n brightness achieved by alkal ine extraction were comparable to those obtained by methanol extraction (figures 3 a & 3 b ) , w h i l e a sequential methanol/alkal i extraction resulted i n a lower f ina l bleached brightness than w h e n us ing methanol alone. A s there was no cumulat ive or addit ive effect w h e n us ing two different extractions, this i m p l i e d that methanol and a lkal ine extraction r e m o v e d the same substances. It was apparent that T.versicolor (Tv) treatment o f heartwood p u l p caused the bleached brightness to decrease f r o m 59.6 to 58.1 % I S O (figure 3a) w h i l e fungal treatment o f pre-methanol extracted pulps (me-Tv) resulted i n a m u c h larger decrease, f r o m 63 to 59.7 % I S O (figure 3b). It is k n o w n that T.versicolor produces laccase, l i g n i n peroxidase, and manganese peroxidase enzymes, however, it has been s h o w n that o n l y laccase and manganese peroxidase are produced d u r i n g b i o l o g i c a l b leaching o f Kraf t pulps ( A d d l e m a n 1995, A r c h i b a l d 1992, Paice 1993). It has been suggested that dur ing treatment, these oxidat ive l i g n o l y t i c enzymes o x i d i z e the l i g n i n and phenol ic extractives present i n mechanica l pulps to coloured quinone and condensed quinone products, result ing i n decreased f inal brightness ( P e l l i n e n 1989, P i l o n 1982). A l t h o u g h a lkal ine extraction o f the sample after T.versicolor treatment (Tv-ae) resulted i n a further 1 pt drop i n f inal brightness, a lkal ine extraction after fungal treatment o f the methanol extracted p u l p (me-Tv-ae) (figure 3a) was able to restore the f ina l pu lp brightness to 63 % I S O (figure 3b). It appears that, once the extractives have been r e m o v e d , o x i d a t i o n o f the pulp by T.versicolor resulted i n the format ion o f a dark, water insoluble , co lour that c o u l d not be readi ly bleached. H o w e v e r , m u c h o f the co lour is soluble i n a l k a l i , as a lkal ine extraction restored m u c h o f the p u l p bleachabi l i ty . A p p a r e n t l y , the presence o f extractives dur ing T.versicolor treatment resulted i n the format ion o f co lour more strongly bound to the pulp . It is probable that this is the result o f p o l y m e r i z a t i o n o f the extractives by laccase, analogous to that s h o w n to occur w h e n 61 laccase is appl ied to l i g n i n without an enzyme mediator such as A B T S or H B T ( B o u r b o n n a i s 1995). Previous w o r k had s h o w n that the addi t ion o f a mediator greatly faci l i tated the abi l i ty o f T.versicolor der ived laccases to degrade residual l i g n i n i n K r a f t pulps . H o w e v e r , w h e n mediators were added to the Douglas- f i r heartwood T M P treatments (figures 3a & 3b), the addit ion o f A B T S ( T v - A ) decreased the final brightness f r o m 59.6 to 48.1 % I S O and f r o m 63.1 to 52.6 % I S O for the methanol extracted T M P ( m e - T v - A ) . A l t h o u g h a lkal ine extraction o f the pulps pr ior to peroxide b leaching increased the brightness value to 53.4 % I S O for the T M P ( T v -A - a e ) and to 55.6 % I S O for the methanol extracted pulp ( m e - T v - A - a e ) , the final brightness values o f these samples were s t i l l lower than those o f the untreated controls ( T M P 59 % , methanol extracted T M P , 63 % ) . P r e v i o u s l y it has been s h o w n that A B T S i n the presence o f laccase results i n the format ion o f a purple co lour that is b o u n d to l i g n i n (Bourbonnais 1996). Since mechanica l pulps have a h i g h l i g n i n content that is not r e m o v e d dur ing peroxide b leaching, this b o u n d coloured c o m p l e x is probably a signif icant contributor to the l o w e r brightness that was observed. T h i s contrasts w i t h the l a c c a s e / A B T S treatment o f K r a f t pulps where the l i g n i n a long w i t h the colour was r e m o v e d d u r i n g subsequent a l k a l i extraction (Bourbonnais 1996). T h e addi t ion o f H B T to laccase treatments o f the pulp also decreased the brightness f r o m 59.6 to 55.6 % I S O for the T M P ( T v - H ) and f r o m 63.1 to 62.0 % I S O for the methanol extracted T M P ( m e - T v - H ) (figure 3b). It is probable that an early deplet ion o f H B T dur ing the seven day treatment leaves the oxidat ive enzymes produced by T.versicolor wi thout a mediator and, as a result, the laccase and other oxidat ive enzymes may o x i d i z e and/or p o l y m e r i z e l i g n i n and extractives, result ing i n a brightness decrease. Subsequent a lkal ine extraction o f the T.versicolor 62 and H B T treated pulps recovered the brightness leve l to 57 % I S O for the T M P (Tv-H-ae) and to 64.5 % I S O for the methanol extracted T M P (me-Tv-H-ae) . W i t h the pre-methanol extracted pulp , the a lkal ine extraction probably l iberated some o f the co loured components b o u n d to the p u l p , a l l o w i n g the pulps to be bleached to b e y o n d the 6 3 % I S O leve l o f the control . I n the case o f the p u l p without pre-extraction w i t h methanol , p o l y m e r i z e d extractives produced d u r i n g fungal treatment were most l i k e l y incomplete ly removed. 3.3.3 Summary O v e r a l l , T. versicolor treatments o f pulps resulted i n a decrease i n brightness. T h i s was probably due to a non-specif ic o x i d a t i o n o f the large amount o f l ignin/phenol ic extractives present i n D o u g l a s - f i r T M P result ing i n the format ion o f co loured products that were not readi ly bleached b y h y d r o g e n peroxide. T h e next step was to use a laccase e n z y m e preparation to treat pure D o u g l a s - f i r heartwood T M P i n an attempt to obtain a more specif ic attack o n the p o l y p h e n o l i c extractives responsible for the l o w brightness o f the pulp . 3.4 Laccase Treatments of Douglas-fir Heartwood TMP I n earlier w o r k it was s h o w n that laccase product ion by T. versicolor c o u l d be induced b y the addit ion o f d ihydroquercet in to the growth m e d i a ( P i c k a r d 1969). A s described i n the introduct ion, autooxidat ion o f the c h r o m o p h o r i c compounds i n D o u g l a s - f i r heartwood d u r i n g aging and p u l p i n g results i n the formation o f co loured complexes , and thus decreased brightness i n the result ing pulp , however, dur ing this w o r k w e hoped that the specif ic o x i d a t i o n o f the phenol ic chromophores by laccase m a y alter or part ia l ly degrade these compounds to increase pulp brightness and bleachabi l i ty . It has also been recognized that laccase is a pract ica l choice for appl icat ion to pulps since, u n l i k e manganese peroxidase, it does not require co-factors such 63 as manganese, a chelat ion agent, and peroxide. Therefore, to try and alleviate some o f the problems encountered w h e n us ing the fungus itself, the effects o f laccase treatment o n the brightness o f extracted and unextracted heartwood thermomechanica l pulps were assessed. Laccase S P 5 0 4 obtained f r o m N o v o N o r d i s k was used for treatment since this enzyme was s h o w n to react effectively w i t h T M P fibers to enhance auto-adhesion o f fibers i n the p r o d u c t i o n o f fiber-boards ( F e l b y 1997). A s mentioned earlier, i n previous w o r k it has been s h o w n that methanol extraction c o u l d effectively remove some o f the c h r o m o p h o r i c material associated w i t h Douglas- f i r and western red cedar ( K y r i a c o u 1998). I n addit ion, it has been s h o w n that a lka l ine extraction facil itated b i o l o g i c a l b leaching o f c h e m i c a l pulps w i t h T versicolor or laccase b y r e m o v i n g some o f the l o w molecular weight compounds released dur ing fungal and e n z y m e treatment. T h e brightness o f methanol extracted unbleached Douglas- f i r heartwood T M P was first assessed, f o l l o w e d by the effects o f laccase treatments i n the presence o f mediators A B T S and H B T i n c o m b i n a t i o n w i t h methanol extraction and/or a lkal ine extraction. T h e brightness results o f both the unbleached and bleached samples are discussed f o l l o w i n g this scheme. 3.4.1 Unbleached Optical Properties of Douglas-fir Heartwood TMP Treated with Laccase in Combination with the Mediators ABTS and HBT, and Chemical Extraction M e t h a n o l extraction o f heartwood T M P was able to raise the brightness f r o m 40.1 to 43.1 % I S O (Table 8), however the laccase treatment drastical ly reduced brightness to 34.1 % I S O . W h e n the pulp was methanol extracted pr ior to laccase treatment, the brightness was s t i l l l o w e r than the untreated control by approximately h a l f o f a brightness point (Table 8). It is probable that this observed decrease i n brightness dur ing laccase treatments is due to the o x i d a t i o n o f l i g n i n result ing i n the formation o f co loured quinone structures ( P e l l i n e n 1989, 64 P i l o n 1982). T h e addi t ion o f mediators d i d l itt le to alleviate this p r o b l e m as the largest decrease i n brightness, f r o m 40.1 to 32.2 % I S O for the unextracted pulp and f r o m 43.1 to 36.3 % I S O for the methanol extracted p u l p , occurred w h e n the p u l p was treated w i t h laccase c o m b i n e d w i t h the mediator A B T S . T h e h i g h a* value o f l a c c a s e / A B T S treated pulps is indicat ive o f the red component o f the dist inct ive purple co lour that was obtained, a result that had prev ious ly been observed d u r i n g the l a c c a s e / A B T S treatment o f K r a f t pulps (Bourbonnais 1996). T h i s earlier w o r k suggested that the formation o f the purple co lour was due to the o x i d i z e d A B T S , w h i c h was covalent ly b o u n d to the l i g n i n w i t h i n the pulp . H o w e v e r , w i t h K r a f t p u l p , the purple c o l o u r was readi ly r e m o v e d w i t h subsequent a lkal ine extraction and bleaching steps ( B o u r b o n n a i s 1996). A s discussed earlier, mechanica l pulps possess a s igni f icant ly higher l i g n i n content that is not substantially reduced dur ing b leaching stages, therefore it is probable that the purple l i g n i n - A B T S complexes w o u l d be m u c h harder to remove. A l t h o u g h replacement o f A B T S w i t h H B T darkened the p u l p to a lesser degree, the a* value o f 3.79 (Table 8) result ing f r o m l a c c a s e / H B T treatment indicated that a more intense red co lour was formed. It was apparent that laccase treatment o f Douglas- f i r heartwood mechanica l pu lp w i t h or without mediators d i d not increase the unbleached brightness. 65 T A B L E 8: U N B L E A C H E D B R I G H T N E S S A N D S T A R L A B ( L * A * B * ) R E S U L T S O F D O U G L A S - F I R H E A R T W O O D T M P T R E A T E D W I T H L A C C A S E S P 5 0 4 I N C O M B I N A T I O N W I T H T H E M E D I A T O R S A B T S A N D H B T , A N D C H E M I C A L E X T R A C T I O N A P u l p Sample I S O Br ightness (%) L * a* b * H e a r t w o o d T M P 4 0 . U 0 . 4 8 1 . 4 1 2 . 5 3 1 9 . 4 5 m e 4 3 . 1 ± 0 . 9 8 2 . 1 4 2 . 0 4 1 9 . 1 4 L 3 4 . 1 ± 0 . 7 7 6 . 7 2 2 . 7 8 2 0 . 9 5 m e - L 3 9 . 5 ± 0 . 5 7 9 . 5 8 3 . 0 9 1 8 . 8 9 L - H 3 6 . 2 ± 0 . 1 7 7 . 3 5 3 . 7 9 1 9 . 1 5 m e - L - H 3 8 . 7 ± 0 . 3 7 9 . 1 4 3 . 1 6 1 9 . 0 2 L - A 3 2 . 2 ± 0 . 4 7 4 . 5 4 . 6 8 1 6 . 9 1 m e - L - A 3 6 . 3 ± 0 . 3 7 8 . 0 2 3 . 3 0 1 9 . 6 7 (me = methanol extracted, L=laccase treated, H= HBT mediator, A= ABTS mediator) (Results are the average of duplicates with treatments performed in the order shown) Treament condit ions are discussed i n M a t e r i a l s and M e t h o d s section 6 6 3.4.2 Hydrogen Peroxide Bleached Brightness of Douglas-fir Heartwood TMP Treated with Laccase With and Without Laccase Mediators, in Combination with Chemical Extraction A l t h o u g h the laccase treatment o f p u l p resulted i n an increase i n peroxide bleached brightness f r o m 58 to 61 % I S O ( L ) (figure 4a) no change i n brightness was obtained through laccase treatment o f the pre-methanol extracted pulp ( m e - L = 63 % I S O ) (figure 4b). It should be noted that the unbleached brightness o f the laccase treated p u l p was 34.1 compared to 40.1 for the untreated control (Table 8). Thus , a l though the laccase treatment decreased the p u l p brightness pr ior to b leaching, it apparently l iberated or m o d i f i e d the c h r o m o p h o r i c c o m p o u n d s present i n the D o u g l a s - f i r T M P , a l l o w i n g it to be bleached to a higher brightness l e v e l than the control . T h e observation that laccase treatment o f methanol extracted pulp ( m e - L ) resulted i n approximately equal brightness levels to that o f the methanol extracted control (me), indicated that the methanol ic extractives i n the Douglas- f i r heartwood T M P were the m a i n site o f the reaction for laccase i n the unextracted pulps . E a r l i e r it was s h o w n that laccases wi thout mediators act speci f ica l ly u p o n the phenol ic components o f l i g n i n ( M u l h e i m 1992). Laccase o x i d a t i o n o f the extractives and/or l i g n i n undoubtedly rendered t h e m m o r e water and a l k a l i soluble, enhancing their r e m o v a l pr ior to and dur ing b leaching. I n addi t ion, the laccases m a y be m o d i f y i n g the p o l y p h e n o l i c chromophor ic extractives or phenol ic l i g n i n i n the p u l p ( M u l h e i m 1992), thus increasing their b leachabi l i ty w i t h h y d r o g e n peroxide. 67 a) CO CO CD c -*—' -C O) CO O 64-62 H 60-58-56-54-| 52-50-48-46-L = laccase treatment H = HBT mediator A = ABTS mediator ae = alkaline extraction -3Er L-A L-A-ae L-H L-ae L-H-ae ae Heartwood PULP TREATMENT b) 0 s 70-69-68-67-66-65-Cfl R4-CD 63-*E 62-D> 61 -O 5 9" C/3 58-— 57-56-55-•3-me = methanol extraction L = laccase A = ABTS mediator H = HBS mediator me-L-A me-L-H-ae me-L-H-ae me-L-ae me-L me-L-H PULP TREATMENT FIGURE 4: HYDROGEN PEROXIDE BLEACHED BRIGHTNESS OF a) UNEXTRACTED AND b) METHANOL EXTRACTED DOUGLAS-FIR HEARTWOOD T M P , TREATED WITH LACCASE SP504 FOR 7 DAYS, WITH AND WITHOUT LACCASE MEDIATORS H B T AND A B T S , AND SUBSEQUENT ALKALINE EXTRACTION (TREATMENTS ARE THE RESULT OF 2 TRIALS PERFORMED IN THE ORDER OF SYMBOLS SHOWN UNDER EACH BAR) 68 A l k a l i n e extraction o f the laccase treated pulps increased the brightness f r o m 59.4 to 62.4 % I S O for pulps without methanol extraction. H o w e v e r , a lkal ine extraction d i d not have a s ignif icant effect o n the laccase treated methanol extracted pulps (figure 4b) indicat ing that methanol extraction affected the same pulp components as a lkal ine extraction. Laccase treatment supplemented w i t h A B T S resulted i n a s ignif icant decrease i n brightness for both the unextracted pulp ( L - A ) and the p u l p pre-extracted w i t h methanol ( m e - L -A ) (figures 4 a & 4b). A s discussed prev ious ly , this c o u l d be caused b y the b i n d i n g o f o x i d i z e d A B T S to the pulp . A l k a l i n e extraction part ia l ly recovered the brightness o f the unextracted p u l p ( L - A - a e ) but had no effect o n the methanol extracted pulps ( m e - L - A - a e ) . These results are consistent w i t h what was observed w i t h the unbleached pulps (Table 8), again s h o w i n g that the presence o f l i g n i n and methanol ic extractives has a profound effect o n the possible b i n d i n g o f the A B T S co lour c o m p l e x to the p u l p dur ing laccase treatment. It was apparent that laccase treatment supplemented w i t h H B T resulted i n increased brightness for the methanol extracted p u l p ( m e - L - H ) and decreased brightness for the p u l p without pre-extraction w i t h methanol ( L - H ) , w h e n compared to untreated controls and methanol extracted heartwood T M P respectively (figures 4 a & 4b). It is probable that the brightness increase was due to the l a c c a s e - H B T c o m b i n a t i o n reacting speci f ica l ly w i t h the b o u n d unextractible chomophores that m a y have grafted onto pulp l i g n i n and/or carbohydrates and are thus not r e m o v e d dur ing the methanol extraction process, whereas, for the unextracted pulps , l a c c a s e - H B T reacted w i t h extractives to create co lour and/or b i n d to the pulp . A l t h o u g h , subsequent a lkal ine extraction increased the brightness o f the unextracted p u l p ( L - H - a e ) , it decreased the brightness o f the pulps pre-extracted w i t h methanol ( m e - L - H - a e ) . It appears that the co lour formed b y the reaction o f the l a c c a s e - H B T w i t h the extractives is o n l y part ia l ly a l k a l i 69 soluble. S i m i l a r to the treatments appl ied to pulps pre-extracted w i t h methanol , laccase treatments o f a lkal ine extracted pulps were performed i n an attempt to determine the effect o f laccase o n the b o u n d non-extractable c h r o m o p h o r i c compounds. S o m e o f the treatments were g i v e n a subsequent a lkal ine extraction after enzyme treatment to remove any a l k a l i soluble co lour that the e n z y m e m a y have l iberated f r o m the p u l p , as performed w h e n b i o l o g i c a l l y b leaching K r a f t pulps (Bourbonnais 1996). H o w e v e r , none o f the laccase treatments resulted i n bleached brightness values b e y o n d that o f the a lkal ine extracted control (figure 5). S i m i l a r to the results obtained w i t h the pulps pre-extracted w i t h methanol , laccase treatments both w i t h and without mediators had a m i n i m a l effect. O v e r a l l , laccase treatments o f pre-alkal ine extracted pulps were ineffective i n increasing the bleached brightness values. It has been s h o w n that extraction o f T M P fibers w i t h water decreases the effectiveness o f laccase treatments for the enhancement o f auto-adhesion dur ing the product ion o f fiber-boards (Hassingboe 1998). Therefore, the i n a b i l i t y o f laccase treatments to increase the brightness o f extracted pulps m a y be due to the absence o f p o l y p h e n o l i c extractable chromophores, w h i c h are the most l i k e l y target for reaction dur ing treatment o f Douglas- f i r mechanica l pulps w i t h laccase. 3.4.3 Summary U n l i k e treatments w i t h T. versicolor, laccase treatment o f methanol extracted pulps w h e n c o m b i n e d w i t h H B T was able to increase the h y d r o g e n peroxide bleached brightness o f Douglas- f i r heartwood T M P . H o w e v e r , the most interesting result was obtained w h e n u t i l i z i n g 70 the laccase e n z y m e without c h e m i c a l extraction or mediators as this treatment increased bleached brightness by 3 pts I S O . T h i s result contrasts w i t h the previous f indings obtained w i t h K r a f t pu lp where the presence o f a mediator such as H B T or A B T S was essential for enhanced bleaching w i t h laccase (Bourbonnais 1996, Paice 1995, Sealy 1997). A s mentioned i n the introduct ion, the increased l i g n i n (23-25%) and the presence o f c h r o m o p h o r i c extractives i n Douglas- f i r heartwood mechanica l p u l p , as compared to K r a f t pulps represent a m u c h more dif f icult task for increasing the brightness and bleachabi l i ty us ing laccase enzymes. T h e differences between the act ion o f laccase o n T M P and Kraf t probably reflect total ly different mechanisms. I n K r a f t b leaching, the laccase/mediator system enhances de l igni f icat ion whereas, f r o m the prev ious ly described w o r k w i t h Douglas- f i r heartwood T M P , it appears that laccase aids i n the r e m o v a l or alteration o f c h r o m o p h o r i c extractives such as D H Q and/or phenol ic l i g n i n i n the pulp . 71 64 H 62 H CO CO CD C 60 •*-> sz -Q 58 o CO 56 H L = laccase treatment ae = alkaline extraction H = HBT mediator Heartwood TMP ae ae-L ae-L-ae ae-L-H ae-L-H-ae Pulp Treatment F I G U R E 5: H Y D R O G E N P E R O X I D E B L E A C H E D B R I G H T N E S S OF L A C C A S E SP504 T R E A T E D A L K A L I N E E X T R A C T E D D O U G L A S - F I R H E A R T W O O D T M P W I T H A N D W I T H O U T T H E M E D I A T O R H B T A N D A S E C O N D A L K A L I N E E X T R A C T I O N PRIOR T O H Y D R O G E N P E R O X I D E B L E A C H I N G ( T R E A T M E N T S A R E T H E R E S U L T S O F 2 T R I A L S P E R F O R M E D IN T H E O R D E R OF S Y M B O L S S H O W N O N F I G U R E ) 72 T h e abi l i ty o f laccase alone to react w i t h and degrade the p o l y p h e n o l i c extractives is consistent w i t h the prev ious ly reported abi l i ty o f laccase to react w i t h phenol ic l i g n i n ( M u l h e i m 1992), depolymer ize large l ignosulphonate complexes ( L e o n o w i c z 1985) and s l ight ly d e p o l y m e r i z e l i g n i n ( V i i k a r i 1998). I n the case o f D o u g l a s - f i r heartwood T M P the p o l y p h e n o l i c extractives and l i g n i n are located throughout the pulp fibers, thus faci l i tat ing the access o f laccase enzymes to phenol ic extractives/lignin without the need for a mediator c o m p o u n d . 3.5 Adjusting the Conditions of laccase treatments of Douglas-fir Heartwood TMP I n an effort to both i m p r o v e the b leaching o f D o u g l a s - f i r heartwood T M P w i t h laccase and to obtain a better understanding o f the mechanisms, laccase i n the absence o f a mediator, was added to the p u l p , w h i l e v a r y i n g temperature, o x y g e n a v a i l a b i l i t y , b leaching condit ions, p H o f treatments, source o f laccase enzymes. T h e effects o f the treatments o n p u l p brightness, pr ior and subsequent to peroxide b leaching and peroxide c o n s u m p t i o n were monitored. Treatments were performed at 50°C and 25°C. A l t h o u g h 50°C is k n o w n to be o p t i m a l for laccase act iv i ty , our previous w o r k had s h o w n that incubat ion at 25°C p r o v i d e d a c o m p r o m i s e between laccase act ivi ty and possible thermal darkening o f the T M P (Chandra 1998). A s o x y g e n is essential for the catalytic act ion o f laccase (Bourbonnais , 1990), an increase i n the supply o f o x y g e n was expected to i m p r o v e the abi l i ty o f laccase to degrade the organics. Samples were supplemented w i t h addit ional o x y g e n i n the reaction vessel w h i l e those samples wi thout addit ional o x y g e n were l i m i t e d to the o x y g e n i n the air o f the reaction vessels. 73 3.5.1 The Effects of Adjusting Temperature and the Amount of Oxygen on the Unbleached Optical Properties of Douglas-fir Heartwood TMP W e next wanted to determine i f the addi t ion o f o x y g e n or the temperature o f incubat ion had a s ignif icant influence o n the effectiveness o f the N O V O S P 5 0 4 laccase (used i n a l l previous experiments) i n increasing the brightness o f the heartwood T M P . T h e unbleached brightness o f the heartwood T M P decreased w i t h laccase treatment at both 25 and 50°C, by up to 5 pts I S O (Table 9). T h i s was probably due to the o x i d a t i o n o f l i g n i n and extractives (such as D H Q ) to co loured quinone groups o n the surface or w i t h i n T M P fibers, as the format ion o f stable l o w molecular weight phenoxy radicals f r o m c o l l o i d a l l i g n i n i n so lut ion had been p r e v i o u s l y reported during the treatment o f beech w o o d fibers and T M P w i t h laccase enzymes ( F e l b y , 1997). It has been proposed that phenoxy radicals m a y act as mediators between the laccase enzyme and the l ignin/extractives i n the f iber- l ignin matr ix (Fe lby 1997, Hass ingboe 1998), since laccase enzymes are too large (55-80 k D a ) to penetrate the fiber w a l l ( X u 1997). W i t h Douglas- f i r T M P , it appears that there is either sufficient accessible reactive material at the fiber surface for laccase enzymes to give a posit ive effect i n the absence o f an added mediator, or p o l y p h e n o l i c extractives and/or l i g n i n m a y be acting as mediators themselves between the laccase and the fiber-lignin-extractives throughout the fiber. 74 T A B L E 9: E F F E C T O F T E M P E R A T U R E A N D O X Y G E N A T I O N O N T H E E F F E C T I V E N E S S OF L A C C A S E SP504 I N I N C R E A S I N G T H E B R I G H T N E S S O F D . F I R H E A R T W O O D T M P A ISO Brightness L* a* b* P u l p Treatment 25°C 50°C 25°C 50°C 25°C 50°C 25°C 50°C C o n t r o l 40.1 39.8 80.45 79.34 2.98 3.58 18.55 19.29 L a c c a s e 34.8 32.3 76.91 75.47 4.81 5.43 20.40 21.40 C o n t r o l + 0 2 41.1 4 0 80.68 79.91 3.45 3.77 18.92 19.39 Laccase + O2 36.4 33.2 78.25 75.86 4.67 5.11 20.59 20.63 Treatments condit ions described i n M a t e r i a l s and M e t h o d s 75 T h e unbleached brightness values o f the control heartwood pulps (no enzyme treatment) at 25°C were s l ight ly higher than were the values obtained at 50°C (Table 9). T h e a* and b * values o f the unbleached pulps increased w i t h laccase treatment and at higher temperatures (50°C), indicat ing the format ion o f red and y e l l o w colours respectively (Table 9). It is probable that the increases i n a* and b * were due to the enzymat ic and thermal p o l y m e r i z a t i o n and/or o x i d a t i o n o f l i g n i n or c h r o m o p h o r i c compounds such as d ihydroquercet in ( D H Q ) to quinone structures, w h i c h resulted i n increased co lour i n the p u l p ( P e l l i n e n 1989). A s the p o l y m e r i z a t i o n o f d ihydroquercet in has been i m p l i c a t e d i n the format ion o f Douglas- f i r brownsta in , it is probable that D H Q also contributes to the co lour o f the D o u g l a s - f i r heartwood T M P ( L a v e r , 1996, V a n der Zee , 1996). T h e addi t ion o f o x y g e n increased the unbleached brightness o f a l l the pulps compared to their counterparts without added o x y g e n b y approximately 1 pt I S O i n each case (Table 9). B o t h laccase treatment and higher temperatures increased a* and b * i n the absence o f o x y g e n (Table 9). H o w e v e r , w i t h added o x y g e n , increases i n a* and b * were to a lesser extent. Changes to the unbleached brightness i m p o s e d b y laccase treatments w i t h and without added o x y g e n had an effect o n both the bleached brightness and h y d r o g e n peroxide consumed dur ing b leaching w h i c h w i l l be discussed i n the f o l l o w i n g sections. 3.5.2 Hydrogen Peroxide Bleached Brightness of Douglas-fir Heartwood TMP Treated with Laccase Enzymes under Two Different Temperatures and Bleached with Varied Levels of Sodium Hydroxide in the Bleaching Liquor With and Without Added Oxygen T h e h y d r o g e n peroxide bleached brightness levels o f the pulps were e x a m i n e d next, w h i l e v a r y i n g the proport ion o f s o d i u m h y d r o x i d e i n the b leaching l iquor , to determine i f there are any differences i n the bleaching o f laccase treated pulps as compared to controls . A l k a l i n e condit ions were e m p l o y e d dur ing h y d r o g e n peroxide b leaching to shift the h y d r o g e n 76 peroxide/perhydroxyl e q u i l i b r i u m to the p e r h y d r o x y l anion ( H O O ") side, since the p e r h y d r o x y l anion was the active b leaching species (Fengel 1989, S m o o k 1992). T h e s o d i u m h y d r o x i d e concentration was var ied to obtain the highest brightness. W h e n no supplemental o x y g e n was added (figure 6a), a 3 % s o d i u m h y d r o x i d e concentration resulted i n the highest brightness for both enzyme and non-enzyme treated pulps at both 25 and 50 °C. T h e bleached brightness values discussed i n this w o r k were those obtained w h e n bleaching w i t h 6 % h y d r o g e n peroxide and 3 % s o d i u m hydroxide . T h e bleached brightness o f the pulp treated w i t h laccase at 25°C was 56. 7 % I S O whereas the corresponding control o n l y reached a brightness value o f 54. 9 % I S O (figure 6a). A s indicated earlier, a l though the unbleached brightness o f the p u l p treated w i t h laccase at 25°C was 3 4 . 8 % I S O , w h e n compared to 4 0 . 1 % I S O for the corresponding unbleached control (Table 9), a s ignif icant brightness enhancement was achieved despite an i n i t i a l darkening o f the p u l p , as the brightness g a i n after laccase treatment was 26.9 pts I S O , compared to o n l y 18.1 pts I S O for the control . Concurrent w i t h this h i g h brightness g a i n a decrease i n peroxide consumpt ion f r o m 7 4 % to 4 5 % was also observed (figure 6c). A l t h o u g h a s i m i l a r trend was also obtained for treatments at 50°C; the i n i t i a l pu lp darkening was greater at 50°C than at 25°C and the f inal bleached brightness was s l ight ly less than observed w i t h the corresponding control (figure 6a). It appears that the c h r o m o p h o r i c materials formed o n reaction o f the p u l p w i t h laccase at l o w e r temperature (25°C), are more readi ly bleached b y a lkal ine h y d r o g e n peroxide. It is probable that the darkening o f pulps , w h i c h becomes more pronounced at higher temperature is caused, by the o x i d a t i o n and p o l y m e r i z a t i o n o f c h r o m o p h o r i c extractives such as D H Q . Attempts to boost the ox idat ion act ivi ty o f the enzyme b y adding o x y g e n to the reaction vessel , resulted i n an approximate l p t I S O h y d r o g e n peroxide bleached brightness increase w i t h a l l samples at both 50°C and 25°C (figure 6b). These results were consistent w i t h 77 those obtained w i t h the unbleached pulps where the brightness o f a l l samples increased b y approximately 1 pt I S O after treatment w i t h o x y g e n (Table 9). H o w e v e r , w h e n compared to the controls, laccase i n the presence o f o x y g e n d i d not i m p r o v e the final brightness o f the pulps . I n fact, decreases i n the order o f 1 pt I S O were obtained, a l though laccase treatment w i t h added o x y g e n d i d provide a higher reduct ion i n peroxide c o n s u m p t i o n than d i d those treatments performed without added o x y g e n , especial ly at 25°C (figures 6 c & d ) . A l t h o u g h treatments w i t h laccase alone and o x y g e n alone i m p r o v e d h y d r o g e n peroxide bleached brightness, their actions were not additive. 3.5.3 The Effects of Adjusting the pH and using Different Laccases on the Unbleached and Bleached Brightness of Laccase Treatments of Douglas-fir Heartwood TMP A s this i n i t i a l w o r k indicated that the highest brightness values were obtained after laccase treatment at 25°C, i n the absence o f supplemental o x y g e n , w e next assessed the effectiveness o f different laccases for their potential to enhance the brightness o f D o u g l a s - f i r T M P . T h e enzymes f r o m N o v o were e m p l o y e d because o f the successful results o f F e l b y at a l (1997) w h o treated beech mechanica l p u l p fibers. T h i s treatment o f mechanica l pulps was s i m i l a r to the treatment o f Douglas- f i r heartwood T M P described i n the w o r k reported here. 78 a) _ 60-^ 58 H w co 56 H L U 1 ? 54 H x o 52 J g 5<H o co 48 H 46 J Bleached Brightness (%ISO) A i r Supplemental O x y g e n b) 1 3 % NaOH 60-] 8^ 58-co 56-C O L U z 54-I o 52-BRI 50-O 48-C O 46-1 3 % NaOH Hydrogen Peroxide Consumption (%) A i r Supplemental O x y g e n c) _ 100 # 95 z 90 g 85 £ 80 | 75 (Q 70 65 60 55 50 8 1 3 %NaOH d) _ 100-# 95-z 90-85-80. § 75. co 70. 65 • 60 • 55. 50 • i -Q. 8 X 1— 3 % NaOH FIGURE 6: B L E A C H E D I S O B R I G H T N E S S V A L U E S (a&b) A N D H Y D R O G E N P E R O X I D E C O N S U M P T I O N (c&d) OF D O U G L A S - F I R H E A R T W O O D T M P T R E A T E D W I T H L A C C A S E N O V O S P 5 0 4 W I T H A I R O R S U P P L E M E N T A L O X Y G E N . A L L S A M P L E S B L E A C H E D W I T H V A R Y I N G N A O H L E V E L S IN T H E B L E A C H I N G L I Q U O R . (-A-) C O N T R O L 2 5 ° C ; ( - • - ) C O N T R O L 5 0 ° C ; ( - • - ) L A C C A S E 2 5 ° C ; ( - • - ) L A C C A S E 5 0 ° C 79 Laccases obtained f r o m V T T were also used because o f their effectiveness i n act ing u p o n phenols w h e n used to bleach K r a f t pulps ( P o p p i u s - l e v l i n et a l , 1997). Therefore, w e anticipated that the Apergillus der ived laccases obtained f r o m V T T w o u l d react more speci f ica l ly w i t h the p o l y p h e n o l i c compounds responsible for the l o w brightness o f D o u g l a s - f i r heartwood T M P . A l t h o u g h the o p t i m u m p H for a l l the laccases was p H 5, since potential c h r o m o p h o r i c extractives such as d imethoxyphenols have a p K a o f 7.0-8.7, w e expected that treatments at higher p H (7.0) to increase the ox idat ion o f compounds such as d imethoxyphenols that might be more readi ly o x i d i z e d by laccases i n their i o n i z e d f o r m ( X u 1997), thereby targeting the o x i d i z i n g act ion o f laccase toward some o f the p o l y p h e n o l i c compounds responsible for the l o w brightness and bleachabi l i ty i n Douglas- f i r heartwood T M P . A s was observed earlier, after treatment w i t h S P 5 0 4 (table 9), a l l o f the unbleached brightness values decreased after laccase treatment at p H 5 (table 10). T h e Apsergillus der ived N S 1 0 0 2 laccase gave the highest bleached brightness values (58.4 % I S O ) w h i c h were s l ight ly higher than those achieved w i t h the other enzymes. It is possible that the source o f laccase m i g h t have a sl ight effect o n its performance dur ing treatment o f D o u g l a s - f i r heartwood T M P , reflecting s m a l l differences i n the protein structure o f the laccase enzymes w i t h respect to the size and the active site o f the enzyme (Bourbonnais 1997, X u , 1997). Differences at the active sites m a y occur, for example where a reducing substrate such as D H Q becomes o x i d i z e d by laccase (T2/T3 cooper site) or at the T l copper site o f the laccase e n z y m e affecting the o x i d a t i o n potential o f the enzyme. P r e v i o u s l y it was s h o w n , that variations i n a specif ic single a m i n o a c i d residue o f a laccase can change the o x i d a t i o n potential o f the entire enzyme (Eggert, 1998). 80 A l t h o u g h , laccase enzymes have been s h o w n to p r i m a r i l y o x i d i z e phenol ic compounds i n the absence o f a mediator, and to also possess act ivi ty at p H 7.0 ( M u l h e i m , 1992 ), exposure to a higher p H ( p H 7.0) had a slight negative effect o n the unbleached brightness o f a l l samples. T h i s c o u l d be due to either darkening reactions i n the pulp at the higher p H leve l , the higher amounts o f enzyme added to the treatments performed at p H 7.0, or a change i n the enzymat ic reaction at p H 7.0 compared to p H 5.0. W h e n enzyme treatments were performed at p H 5.0, N o v o N S 1 0 0 2 f r o m Asperigillus obtained the highest bleached brightness 58.4 % I S O (Table 10). Treatment w i t h N o v o S P 5 0 4 reached a bleached brightness level very close to that obtained after treatment w i t h N O V O N S 1 0 0 2 . These results indicate that the source o f laccase might have a slight effect o n its performance dur ing treatment o f Douglas- f i r heartwood T M P , w h i c h m a y reflect the differences i n protein structure between the laccase enzymes w i t h respect to the size and the active site o f the enzyme (Bourbonnais 1 9 9 7 , X u 1997). L e o n o w i c z et a l (1984) also reported that laccase reactions w i t h l i g n i n m o d e l compounds at var ied p H levels resulted i n the format ion o f different products. These workers reported m u c h more f o r m a t i o n o f p o l y m e r i z e d quinone compounds f r o m reaction o f v a n i l l i c and s y r i n g i c a c i d w i t h laccase at higher p H levels (>6) compared to the same reaction at l o w e r p H levels (3-5) ( L e o n o w i c z 1984). T h i s agrees w i t h the results obtained i n this w o r k since the reactions at p H 7.0 decreased p u l p brightness (Table 10) more than d i d treatments at p H 5.0. T h i s m a y be the due to the format ion o f more p o l y m e r i z e d co loured compounds at p H 7.0. A l l enzyme treatments performed at p H 5.0 reached a s l ight ly higher h y d r o g e n peroxide bleached brightness than those performed at p H 7.0 except for N O V O S P 5 0 4 (Table 10). Samples treated w i t h N O V O S P 5 0 4 laccase reached approximately equal h y d r o g e n peroxide bleached brightness at p H 5.0 and 7.0 (Table 10). 81 Table 10: T H E I N F L U E N C E O F P H O N T H E U N B L E A C H E D / B L E A C H E D B R I G H T N E S S OF D O U G L A S - F I R H E A R T W O O D T M P T R E A T E D W I T H F I V E D I F F E R E N T L A C C A S E S A Laccase p H 5 p H 7 U n b l e a c h B l e a c h % % H 2 0 2 U n b l e a c h B l e a c h % % H 2 0 2 % I S Q I S O C o n s u m e d % I S Q ISQ. Consumed C o n t r o l 4 0 . U 0 . 1 6 57.6 ± 1.0 75.6 ± 4 39.7 ± 0.4 56.5 ± 0.6 74.9 ± 9 (no enzyme) N o v o S P 5 0 4 35 ±0.25 58.2 ±0.8 64.4 ± 8 34.3 ± 1.2 58.6 ±0.8 66.4 ± 4 N o v o 35.5 ±0.45 58.4 ± 0.3 67.4 ± 5 35.3 ± 0.3 57.7 ± 0.9 66.7 ± 7 N S 1 0 0 2 N o v o 35.8 ± 0 . 1 6 58.0 ± 0.2 66.6 ± 3 35.6 ± 0 . 6 57.3 ± 1.0 65.1 ± 8 N S 1 0 0 3 V T T C r u d e 34.6 ± 0.2 57.4 ± 0.4 65.2 ± 6 32 ± 0.8 57.6 ± 1.2 67 ± 4 T.hirsuutum V T T Pure 35.5 ±0.45 57.6 ± 0.7 68.2 ± 6 34.2 ± 0 . 6 57.3 ± 0.8 65.6 ± 9 T.hirsuutum Treatments C o n d i t i o n s described i n Mater ia ls and M e t h o d s 82 Samples treated w i t h N O V O S P 5 0 4 also reached the highest bleached brightness o f a l l the enzymes at p H 7.0 (Table 10). R e s i d u a l peroxide levels were s imi lar for a l l enzyme treatments w i t h every treatment result ing i n an approximate 10 % reduct ion i n h y d r o g e n peroxide consumpt ion compared to control pulps. It was surpris ing that N O V O N S 1 0 0 3 laccase d i d not outperform the other enzymes d u r i n g treatments at p H 7.0, since this enzyme was spec i f ica l ly engineered to have substantial enzyme act ivi ty at higher p H levels. A l t h o u g h there were very s m a l l differences i n the results o f the treatments at p H 5 and 7, treatments at p H 7.0 proved to be less effective because o f the higher amounts o f enzymes needed to p r o v i d e adequate act iv i ty at p H 7.0. 3.5.4 Diffuse Reflectance UV Spectrometry of Laccase Treated Handsheets I n an attempt to determine the mode o f act ion o f laccase, diffuse reflectance ultraviolet-v i s i b l e spectrometry analysis o f handsheets was used to per form a p r e l i m i n a r y invest igat ion o f the effects o f laccase treatments o n the surface chemistry o f the paper sheets. B l e a c h e d and unbleached handsheets obtained f r o m laccase treatments at 25°C were analyzed, since this treatment showed post-bleached brightness enhancement o f D o u g l a s - f i r heartwood T M P (figure 7a). T h e laccase treated (25°C) unbleached handsheets showed an increased absorption (k) i n the 400-700 n m reg ion compared to the untreated control , i l lustrat ing the increase i n co lour after laccase treatment (Schmidt J . A . 1993) (figure 7a). T h i s was also noted i n the decrease i n unbleached brightness ( % I S O ) as absorption is inversely proport ional to I S O brightness measurements. B l e a c h i n g w i t h hydrogen peroxide reduces absorption i n the co lour absorbing regions w h i c h coincides w i t h the observed brightness increase d u r i n g b leaching (figure 7b). It was apparent that bleached pulps f r o m laccase treatments have a l o w e r absorption i n the v i s i b l e absorbing region than bleached control pulps (figure 7b). T h i s direct ly corresponds to the 83 increase i n bleached p u l p brightness obtained after laccase treatments. A s a result, the D o u g l a s -f ir heartwood T M P treated w i t h laccase became darker and showed strong absorption before b leaching. H o w e v e r after b leaching, the brightness o f laccase treated pulps surpassed the controls and showed less absorption w h e n compared to the untreated controls. It is l i k e l y that the increased bleachabi l i ty o f the laccase treated pulps was due to the higher amount o f quinone structures i n the pulp before b leaching. W h e n l o o k i n g at the difference between the absorpt ion curves it can be seen that there was a stronger absorption i n the 4 3 0 n m reg ion w i t h the laccase treated unbleached pulps compared to the control w h i c h corresponds to the p r o d u c t i o n o f co loured quinone structures (Inset graph o n figure 7a). H o w e v e r , the control showed absorption i n the 4 3 0 n m region, relative to the laccase treated p u l p , after b leaching. (Inset graph o n f igure 7b). It is probable that laccase pre-treatment at l o w temperatures m a y a i d h y d r o g e n peroxide b leaching by part ia l ly o x i d i z i n g p o l y p h e n o l i c chromophores to quinones, thereby enhancing further o x i d a t i o n by h y d r o g e n peroxide (i.e. increasing b leachabi l i ty) and consequently reducing the c o n s u m p t i o n o f h y d r o g e n peroxide. 84 300 400 500 600 Wavelength 700 800 400 500 600 Wavelength (nm) 800 FIGURE 7: DIFFUSE REFLECTANCE U V - V I S ANALYSIS OF a) U N B L E A C H E D A N D b) B L E A C H E D HANDSHEETS FROM DOUGLAS-FIR T M P WITH A N D WITHOUT L A C C A S E T R E A T M E N T (INSET GRAPHS ARE T H E CONTROL PLOT MINUS T H E L A C C A S E TREATED PLOTS) 85 3.5.5 Summary It was apparent that treatment o f Douglas- f i r heartwood T M P w i t h laccase enzymes at lower temperatures resulted i n a decrease i n the unbleached brightness values, increased bleached brightness values and a reduct ion i n h y d r o g e n peroxide c o n s u m p t i o n d u r i n g b leaching. T h e laccase enzymes seem to be acting o n the pulp b y o x i d i z i n g the c h r o m o p h o r i c compounds o n the fiber surface, or alternatively by us ing a mediator m e c h a n i s m where l i g n i n or extractives i n the reaction l i q u i d act as the mediator for laccase enzymes. T h e increased bleachabi l i ty o f the pulps m a y be due to the format ion o f quinone structures at the p u l p surface or w i t h i n the p u l p w h i c h are more amenable to h y d r o g e n peroxide bleaching. T h e o p t i m u m alkal ine levels i n the h y d r o g e n peroxide b leaching l iquors d i d not differ between the control and laccase treated pulps . T h e addi t ion o f o x y g e n to the laccase treatments further reduced the peroxide c o n s u m p t i o n during h y d r o g e n peroxide bleaching. H o w e v e r , w i t h the addi t ion o f o x y g e n , laccase treatments s l ight ly decreased the p u l p brightness. Laccase and o x y g e n treatment d i d not have an addit ive effect i n increasing p u l p brightness, indicat ing that they probably affected the same chromophor ic material w i t h i n the pulp . A l t h o u g h slight differences i n the eff icacy o f the var ious laccases indicated possible differences i n the protein structure o f the active site o f the enzymes, these differences were not significant. Treatments at higher p H showed litt le improvement w h i l e increased amounts o f e n z y m e were needed to obtain an act iv i ty equivalent to what c o u l d be obtained at the e n z y m e s ' o p t i m u m o f p H 4.5-5.5. 86 4 Conclusions T h e results o f fungal screening indicated the potential for the use o f fungi to i m p r o v e the brightness o f Douglas- f i r mechanica l pulps , since the white-rot fungus Phanerochaete chrysosporium increased brightness o f Douglas- f i r refiner mechanica l pulps by 1.5 pts I S O . Unfortunately subsequent P.chrysosporium treatment was unable to increase the brightness o f Douglas- f i r heartwood T M P . Therefore the colour component o f Douglas- f i r heartwood mechanica l pulps p r o v e d more di f f icult to remove or alter than that o f the sapwood or a mixture o f sapwood and heartwood. E a r l i e r w o r k had s h o w n an associat ion between the p r o d u c t i o n o f laccase and the abi l i ty to degrade the chromophor ic compounds such as dihydroquercet in , w h i c h contribute to the l o w brightness o f Douglas- f i r mechanica l pulps i n the fungus Trametes versicolor. Surpr is ing ly , treatment o f heartwood mechanica l pulps w i t h T versicolor c o m b i n e d w i t h c h e m i c a l extraction d i d not result i n any brightness increases greater than what c o u l d be obtained us ing c h e m i c a l extraction alone. O v e r a l l fungal treatments p r o v e d to be ineffective i n increasing the brightness o f Douglas- f i r heartwood mechanica l pulps. T h i s was probably due to the lack o f speci f ic i ty o f fungal attack since T. versicolor produces several different enzymes that are a l l capable o f altering the characteristics o f Douglas- f i r mechanica l pulps. A l t h o u g h laccase treatments without an enzyme mediator decreased the unbleached brightness o f Douglas- f i r heartwood mechanica l pulps , after b leaching the pulps brightness surpassed the control pulps by 2-3 pts I S O . Laccase treated pulps also consumed 10-15% less hydrogen peroxide dur ing b leaching. T h i s was most l i k e l y due to the partial o x i d a t i o n o f phenol ic components i n the pulps to coloured quinone structures thereby increasing the p u l p 87 bleachabi l i ty result ing i n brightness increases c o n s u m i n g less peroxide. W h e n supplemented w i t h an enzyme mediator, laccase treatment decreased both the unbleached and the bleached brightness values. T h i s c o u l d be due to the b i n d i n g o f mediator compounds to the h i g h levels o f l i g n i n present i n the pulps. A s s h o w n o n figure 6, both increasing the temperature and/or adding o x y g e n to laccase treatments resulted i n a decrease i n brightness w h e n compared to the controls. It is probable that increasing the temperature decreased the brightness o f the pulps because o f the format ion o f n e w chromophores at higher temperatures. H o w e v e r , o x y g e n was able to increase the brightness o f a l l pulps b y 1-2 pts I S O . N e i t h e r changing the reaction p H nor us ing different types o f laccase had a large effect o n the brightness gains and decrease i n h y d r o g e n peroxide c o n s u m p t i o n obtained dur ing laccase treatments al though treatments were most successful at p H 5.0. O v e r a l l , methanol and a lkal ine extraction were the most benef ic ia l w i t h respect to increasing the brightness o f the heartwood mechanica l pulps. In this w o r k , laccase treatment without mediators proved to be the best b i o l o g i c a l method for increasing the brightness and bleachabi l i ty o f Douglas- f i r heartwood T M P . Further w o r k should focus o n further o p t i m i z i n g laccase treatments, testing the effects o f laccase treatments o n other paper properties such as strength and brightness reversion as w e l l as t r y i n g to elucidate the m e c h a n i s m whereby o x y g e n alone increases the brightness o f mechanica l pulps. 88 5 References A d d l e m a n , K . , D u m o n c e a u x , Paice, M . G . , B o u r b o n n a i s , R. , and A r c h i b a l d , F . S . , 1995. P r o d u c t i o n and Character izat ion o f Trametes versicolor mutants unable to b leach H a r d w o o d Kraf t P u l p . 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