Chemo-Enzymatic Modification of High-kappa Kraft Pulps with Laccase What is currentlygy being done to modify fibers to affect pulp quality?

Tree choice Chipping

O

CNH2 CH2CH n Pulping Papermaking chemicals Next Generation? • Enzymes from wood-degrading fungi can be used for altering pulp properties Cellulase •Enzymes modify fibers rendering them more flexible and aid drainage on paper machines Hemicellulase •Enzymes modify fibers to aid in bleaching Ligninases •Manggpanese peroxidase, Li gnin peroxidase, and LACCASE •Employed primarily for delignification studies •Laccase different than MnP and LiP Laccase

• Japanese lacquer tree (Rhus vernicifera) • Basidiomycete fungi and plants • Oxidoreductase enzyme • Biodelignification studies with kraft pulps • Polymerizes monoligno ls in so lu tion to form polymers with lignin-like properties Laccase •Oxidation of monomeric phenols has been shown CH2OH to result in coupling to CH CH lignin macromolecule (Lund 2001)

OCH3 OH Laccase H2O

O2 Laccase (ox)

CH OH 2 CH2OH CH2OH CH2OH CH2OH CH CH .CH CH CH CH CH CH CH CH. COUPLING . OCH . 3 OCH H OCH3 3 OCH3 OCH3 .O O O O O Rationale

• There has been limited work in pulp and paper research exploiting laccase’s polymerizing ability to modify pulps. • The high surface lignin content of high- kappa kraft pulps* makes them prime candidates for reaction with laccase.

*Lai ne, 1994 Hypothesis

O + 2H+ RED 2 Oxidizes a wide OH array ofhf phenoli c structures H2O ox • O no rxn X

Fiber Grafting? Possibilities…

• Polymerization of phenols with themselves (self-condensation)

• Polypymerization of phenols with fiber

• Pol ymer iza tion of fiber lign in Objectives

• Devise and evaluate the feasibility of a system utilizing laccase to co-polymerize (graft) water soluble compounds with high kappa kraft pulp • Determine if lignin was the main target for modification of the laccase-facilitated grafting system • Determine conditions where the laccase-facilitated grafting system was the most effective for modifying fibers • Evaluate the effects of the laccase-facilitated grafting treatment on paper strength properties and surface chemical properties Overview

Laccase + Phenolic Molecular weight Surface Chemistry Phase 1

Phase 4 Phase 3 Surface Pulp fibers Lignin Phase 2 Phase 5 SSfurface MMtilaterials Paper Chemical Structure Physical Conditions PtiProperties Phase 1: Initial Experiment

4-Hydroxybenzoic acid

•Kappa Number •Carboxylic Sygyringic acid Acid Groups •X-ray Photoelectron Spectroscopy

Vanillic acid Laccase Experimental

Kapak bag Water Pulp . Kappa 90 45oC . 20% csc Laccase . pH 4.5 1. Phenolic Phenolic 2. Laccase

Water Bath Pulp before and after treatment

Control and Brownstock Laccase 4-HdHydroxy benzo ic acid TB:20.2 TB:18.3 TB:19.2

L4Laccase+4-HdHydroxy benzo ic acid TB:15.3 Phase 1: Kappa Number • Kappa increased 110 in presence of Control 105 laccase and Laccase phenolic acids 100 • 4-hydroxybenzoic 95 acid (4-HBA) treatment with 90 laccase resulted in largest kappa 85 increase 80 LSD: C V S 4 o an yri - n H 3.75 tr illi ngic BA o l c Phase 1: Bulk Carboxylic Acid Groups

• Acid group results 0240.24 correlate with 0.22 Control kappa number Laccase results 0.2 0.18 • Laccase + 4- hydroxy benzoic 0.16 acid approx. 0.14 doubled the bulk 0.12 acid groups 0.1 0.08 C V S 4- LSD: o a yr n nilli H t in BA 0.02 meq/g ro g l c ic Phase 1: XPS Results

6

.Laccase and 4- 5 hydroxybenzoic surface acid increased nn 4 the percentage 3 of acid groups on COOH o Unextracted the surface. 2 Acetone extracted

rcent (%) rcent 1 Pe SD: 0 BS Co La H La 0.08% nt c B c A +HBA r ol Phase 1: Conclusions

• Laccase treatment with phenolic acids increases both kappa number and bulk carbliboxylic acids. • Strong evidence for laccase facilitated coupling of phenolic acids to fiber surface. • Laccase most effective in coupling of 4- hydroxybenzoic acid which may be due to absence of methoxyl groups on aromatic ring Phase 2: Treatment Conditions • Used 4-hydroxybenzoic acid to determine effect of treatment conditions • Varied presence of oxygen, dosage of 4-hba, pulp consistency, treatment time consitistency Laccase 4-Hba Dosage

time 4-Hydroxybenzoic acid

Oxygen vs. No Oxygen Full 4-hba Dose vs. Periodic Addition Phase 2: Oxygen Pressure Vs. Ambient Conditions • Laccase treatments for bio- delignification most • No real difference effective when pressurized

btbetween with O2 treatments • Compare to treatments performed in O2 perfdformed in bags reactor and vs • Treatments performed with bag identical conditions

120

100

80 Oxygen 60 No Oxygen pa Number

pp 40 Ka 20

0 Con Lac Hba Lhba Phase 2: Factorial Design

Factor P-Value •Consistency and dosage Have the most significant Dosage 0.000002 effects on grafting Consistency 0.000005 reaction (CSC) • Consistency and dosage also have the Time 0.485361 strongest interaction Dosage*CSC 0.000035 • Three factors 33 factorial with kappa number as the response Dosage* Time 0. 101097 • Variables – 5, 10, & 20 % pulp consistency – 0.1, 1, & 10 mmol dosage of 4- CSC*Time 0.980152 Hba – 2, 4, & 8 hours treatment time Phase 2: Periodic Addition of 4-Hba • Entire dose of 4-Hba added to the pulp at the beginning of the reaction vs. periodic addition of 4-Hba throughout the duration of the reaction • To decrease 4-Hba self-condtidensation and increase coupling to the pulp • Additions in two hours: – 1x – Entire dosage of 4-HBA – 2x- 1 addition per hour – 4x- 1 addition each 30 minutes – 8x- 1 addition each 15 minutes • Treatments followed by alkaline 120 110 exttitraction to remove adbddsorbed 100 4-Hba 90 80 ppa Number

aa 70 K 60 50 Pre-extraction ConLacHba1x2x4x8x Post-extraction Phase 2: Conclusions

• It is not necessary to perform the laccase/phenol/pulp reaction in a reactor

pressurized with O2 • A high consistency and high dosage of phenol (4-Hba) is necessary for obtaining higher amounts of phenol coupled to the pulp • Periodic addition was effective in increasing the amount of 4-hba coupled to the pulp Phase 3: Effects of Laccase-Grafting Treatment on Lignin

Fullyyp Bleached Pulp Laccase/4-Hba Treatment

Isolated Lignin “Lignin-Impregnated Fibers”

Laccase/4-hba Treatment

Lignin 4-HBA Molecular Weight Struc tura l Changes Phase 3: Application to Fully Bleached Pulps

• Applied 4-hba with laccase

• No significant 0.07

increase in acid ups 0.06 oo groups when 0.05 compounds applied 0.04 q/g) Acid Gr Acid ee 0030.03

with laccase (u 0.02 • Indicates pulp lignin is rboxylic

aa 0.01

necessary for C 0 coupling to fibers Con Lac 4-hba Lac+4- hba Phase 3: Kraft (kappa 92) Ligni n Impregnat ed Fibers

Blend er Filter paper Acetone extract

Lignin extraction Lign in+fil ter paper fib ers .9:1 dioxane:1N HCL 20% csc in dioxane .2 hours w/acid ppprecipitation

Roto-evaporate React With Lignin impregnated fibers l+4laccase+4- Hba Phase 3 Lignin Impregnated Fibers :31P NMR 0.5

Non-Condensed structures at C5 • Carboxyllic acid groups increased 0.4 indicating coupling of 4-hydroxybenzoic (mmole/g lignin) acid to lignin gnin ii 1 0.3

0.8 ol/g l

mm 0.2

0.6 m

0.4 0.1 0.2 BS Contr La 4-HBALa cca c+ 4 ol se -H 0 B B C L 4 L A S ontrol ac -HBA ac+4-HBA cas e Both non-condensed and condensed phenolic groups decreased in the presence of laccase indicating the predominant reaction was the reaction of laccase with phenols Phase 3 Ligni n Impregnat ed Fibers: Molecular Weight

15500

15000 (Mw) tt 14500

ular W 14000 cc 13500 Mole 13000 n n n a a a b Co Lac Syr V H c+V c+Hba LSD: ac+Syr a a L L L 1545 g/mol Phase 3: Conclusions • NMR data suggests increase in acid groups that indicate the attachment of 4- hydroxybenzoic acid to lignin. • NMR data also show s that laccase decreases both the non-condensed and C5 condense d pheno ls diduring grafting. • Sensitivity of molecular weight analysis may be insufficient to illustrate changes imparted by grafting of 4-hba to lignin Phase 4: Pulp Surface Materials

High-kappa kraft pulp

Laccase –Grafting Treatment

Isolate Surface Material

Molecular Weight Carboxylic Acid Groups Phase 4: Isolation of Pulp Surface Material

Suspend at 10% Consistency

35 g pulp Sample

Filter 5x through Whatman no.41 Disintegg,rate for 200,000 filter paper ret ai ni ng >20 um Revolutions material and freeze dry

Yield of 40-50 mg Per sample * Heijnesson et al 1995 Phase 4: Pulp Surface Material Molecular Weight

• Pulps treated with • Molecular weight increased with laccase and 4-hba laccase+4-hba treatment fllfollowe d by iso ltilation o f • Indicates laccase-facilitated surface material coupling of hba to the fiber 3500 surface • Dissolved and ) acetylated with ww 3000 DMSO/DMF/Pyridine 2500 ight (M ight

mixture ee 2000 • Molecular weight 1500 measured by gel 1000 cular W

permeation ee chromatography 500 Mol 0 Control Lac 4-hba 4- LSD: hba+lac 752 g/mol COH Phase 4 CH2 Pulp Surface Material: SO3Na Acid Group Titration OH 4-hydroxy phenylacetic acid CH2O • Pulps pre-treated with OH Guaiacol Sulfonate laccase with ttiyrosine, 0.8 4-hydroxyphenylacetic 0.7

tent tent 060.6 acid, and guaiacol nn sulfonate 0.5

eq/g 0.4 • Acid groups on surface Co oup rr MM 0.3 material increased with 0.2

laccase-grafting Acid G 0.1 treatments 0 H OH r s y Pa Gs Con Lac T Tyr +Pa +G HO C C O c+ ac ac a L L NH2 L Tyrosine Tyr=tyrosine, Pa=Phenylacetic acid Gs= Guaiacol Sulfonate Phase 4: Conclusions • Laccase-facilitated grafting treatments result in increases in the molecular weight and carboxylic acid groups on material isolated from the pulp fiber surface Phase 5: Paper Physical Properties

Tensile Tear Burst

High-kappa pulp Laccase grafting treatment

Wet-tensile Zero-span Z-direction Tensile Phase 5 Compounds Studied Compounds applied to high-kappa kraft pulps: O C OH

CH2

HBA PA 4-Hydroxybenzoic acid Vanillic acid OH Sygyringic acid 4-hydroxy phenylacetic acid O COOH SO3Na H H2N C OH N HO C C O OH OH NH2 CH O HOO N CH2CH3 2 OH GA Tyrosine OH OH CH2CH3 Guaiacol Sulfonate Celestine Blue Gallic Acid O Phase 5 C OH Paper Physical Properties: CH2 Apparent Density HBA PA OH 0.48 0.4 0.46 0.44 0.35 04200.4.42 0.3

0.38 3 g/cm3 0.36 g/cm 0.25 0.34 0.2 0.32 0.3 0.15 n c a a c a a b b n a Co L H La P +H Co Lac+P Lac Kappa 91, Refined 2000 rev. PFI Kappa 91, Refined 1000 rev. PFI COOH Phase 5 Paper Physical Properties: OH OH OH Apparent Density GA

0.35 0.5

0.3 ty ity ii 0.4 0.25 0.2 0.3

rent Dens 0.15 Dens rent g/cm3) g/cm3) 0.2 (( (( aa aa 0.1

App 0.1 App 0.05 0 0 Con Lac Gal LGA Con Lac Gal LGA

Kappa 91, No Refining Kappa 91, Refined 2000 rev. PFI Phase 5 O C OH

Paper Physi cal Properti es: CH2 Burst HBA PA 3.1 2.5 OH 2.9 2.7 2 g // 252.5 1.5 mm 2.3

2.1 kN/g. 1 kPa.m2 1.9 0.5 1.7 1.5 0 a a ac ac Con L Hba Hb Con L a + P ac+P ac L L Kappa 91, Refined 2000 rev. PFI Kappa 91, Refined 1000 rev. PFI COOH Phase 5 Paper Physical Properties: OH OH OH Burst GA

1.5 2.8 1.4 2.6 2/g) 2/g) 1.3 2.4 mm mm 1.2 2.2 1.1 2 1 1.8 ex (KPa. 0.9 ex (KPa. dd 0.8 dd 161.6 0.7 1.4

Burst In 0.6 1.2 Burst In Burst 0.5 1 Con Lac GA LGA Con Lac GA LGA

Kappa 91, No Refining Kappa 91, Refined 2000 rev. PFI Phase 5 Paper Physical Properties: ZDT HBA 280 260 240 220 200

kPa 180 160 140 120 100 a n c a b Co La Hb H Lac+ O Phase 5 C OH Paper Physical Properties: COOH CH2 Tear Resistance

OH OH PA OH OH 3.5 2.5 GA 3 2 2.5 gg gg 2 1.5 n.m2/ n.m2/ 1.5 1 mm mm 1 0.5 0.5 0 0 a ac c A P on a G Con L +Pa C L GA L ac L Kappa 91, Refined 1000 rev. PFI Kappa 91, No Refining Phase 5 Paper Physical Properties: COOH Zero Span OH OH HBA OH GA 120 120 100 100 )

gg 80 80 60 60 N.m/g S (N.m/ SS ZZ Z 40 40 20 20 0 0 n a a o b b C Lac H H Lac GA GA + Con L c La Kappa 91, Refined 2000 rev. PFI Kappa 91, No Refining O C OH Phase 5 CH2 Paper Physical Properties: Tensile Strength

OH PA HBA

36 45 34 40 32 30 35 28 .m/g N.m/g NN 26 30 24 25 22 20 20 c Con Con Lac Pa +Pa La +Hba ac Hbaac L L Kappa 91, Refined 2000 rev. PFI Kappa 91, Refined 1000 rev. PFI Phase 5 COOH Paper Physical Properties:

OH OH Tensile Strength OH GA

25 45 24 23 40 22 (N.m/g) (N.m/g) 21 35 20 19 30

18 Index ile ile Index ss ss 17 25 Ten Ten 16 15 20 Con Lac GA LGA Con Lac GA LGA Kappa 91, No Refining Kappa 91, Refined 2000 rev. PFI Phase 5 COOH Paper Physical Properties:

OH OH Wet-Tensile Strength OH GA

1.8 2.5 1.6 1.4 2 1.2 (N.m/g) (N.m/g) 1 151.5

0.8 1 0.6 ile Index ile ile Index ss ss 040.4 050.5 Ten Ten 0.2 0 0 Con Lac GA LGA Con Lac GA LGA Kappa 91, No Refining Kappa 91, Refined 2000 rev. PFI Phase 5: Conclusions

• Addition of phenolic acids to the pulp resulted in the best performing papers during paper strength testing • Laccase treatment with gallic acid provided the largest increases in wet and dry tensile strength of all the compounds tested • The increases in strength with laccase/gallic acid treat ment were not accompani ed by changes in sheet density Phase 6: Further Investigation

COOH

Laccase + OH OH Radical Scavenger OH

Contact Angle Vertical Wicking Phase 6 Radical Scav enger • Observe if gallic acid coupling to kraft pulp is due to radical coupling reactions • Radical scavenger OH OH • Added in 10:1 molar ratio O to gallic acid due to H2C C O laccase • Has been shown to H scavenge radicals of gallic acid in grape juice (Tulyathan 1989) HO OH • Reduces quinones formed by laccase Ascorbic Acid oxidation back to phenols (Bocks 1967) Phase 6 Radical Scavenger • Lac+Gal ineffective in the presence of ascorbic acid (asc) • Wet-tensile strength of original pulp preserved with ascorbic acid g) 250.0 0 2 // 1.8 200.0 1.6

ol/g) 1.4 x (N.m ee mm 150.0 0 121.2 1 100.0 0.8 OH (u OH

0.6 Ind ile ss OO 50.0 0 040.4 C 0.2 0.0 0 et Ten et

A al WW on lac G GA sc sc al c L a la ascg Bulk COOH

lac+asc+g Wet Tensile Strength

Unrefined Pulp Phase 6 Contact Angle

49 • Compatibility of )) sheet surface with 48

water egrees 47 dd • Measured with 46 camera shooting 45 Angle ( Angle tt 1 frame/0 .067 sec 44 • Slight decrease in 43 Contac contact angle 42 observed with gallic Con Lac GA LGA acid treatment • Higher dosages of gallic acid could not be measured since θ there was no drop holdout Phase 6 Vertical Wicking • Important for absorbent Paper Strip characteristics of non-woven structures • Kineti c rela tions hip can be used to relate stttlructural and surface properties Wetting Front

Water Reservoir Phase 6 Vertical Wicking • Lucas-Washburn Wicking Equation: h=distance traveled r=capillary ra dii h= rσ cos θ 1/2 √t =k√t t=time 2 2µ2 µ σ=surface tension =tortuosity factor µµy= viscosity

• Square-root time should be linear to vertical distance traveled (Hodgson and Berg 1988) • Increasing slope of distance vs. square root time curve should indicate enhanced liquid absorbing capability of material

• Increased compatibility with H2O may indicate propensity for hydrogen bonding during paper sheet formation Phase 6 Vertical Wicking Laccase+Gallic Acid 9 •Steepest slope 8 •Lowest contact angle 7 Control 6 Laccase cm) (( 5 Gallic 4 R2 = 0.9775 Lac+gallic

stance DLac+gal ii 3 D 2 Linear (Control) 1 0 0 5 10 15 20 S(/)Square root time (sec1/2)

Test Duration = 5 min Lac+gallic wicked 20% further SlSample weihtight =0015.15g +/- 0010.01g and absorbed 30% more H O 3 3 2 Sample density= .27g/cm +/- .01 g/cm than control sample Average of 2 tests Phase 6: Conclusions • Ascorbic acid inhibits the laccase/gallic treatment strongly suggesting a free- radical mechanism • Laccase/ galli c acid trea tmen t resu lts in an increase in hydrophilicity of fibers Thesis Conclusions • It is ppppossible to use laccase to couple phenolic compounds to high-kappa kraft pulps • Coupling imparts significant changes to the pulp fiber surface • Lignin is the main site of coupling for laccase generated phenoxy radicals • Coupling of gallic acid to the fiber results in tremendous increases in wet/dry tensile strength coupled with an increase in hydrophilicity of fibers. • Strength improvements may be a combination of improvement of surface characteristics and cross- linking of phenoxy radicals between fibers in the sheet Publications and Presentations • Chandra, R.P . and Ragauskas, A.J ., Enzyme and Microbial Technology June 2002, Vol. 30 (7), 855-861, “Evaluating the Ability of Laccase to Couple Phenols to High-Yield Kraft Pulps With Laccase.” • Chandra, R.P., Felby, C.L. and Ragauskas, A.J., submitted to: Journal of Wood Chemistry and Technology “Improving Laccase-facilitated Coupling of Phenolic Acids to High-Kappa Kraft Pulps” • Chandra, R.P. and Ragauskas, A.J., 11th International Symposium in Wood and Pulppging Chemistry , June 11, 200, Nice, France, “Sculppgting the Molecular Weight of Lignin via Laccase.” • Chandra, R.P. and Ragauskas, A.J., Tappi Pulping Conference, November 4-7, 2001, Seattle, Washington, U.S.A. Paper accepted for presentation, “ Laccase: Renegade of Fiber Modification” • Chandra, R.P. and Ragauskas A.J., 8th International Conference on Biotechnology in the Pulp and Paper Industry, Invited Paper and Book Chapter, pp.165-172, June 4, 2001, Helsinki, Finland “Elucidating the Effec ts o f Laccase Trea tmen ts on the Physi cal Proper ties o f High -Kappa Kraft Pulps.” • Chandra, R.P. and Ragauskas, A.J., American Chemical Society National Meeting 2002,,p Invited Speaker and Published Book Chap ter for Anselme Payen Award Symposium, April 2002, Orlando, Florida, “Bio- grafting Celestine Blue to High-Kappa Kraft Pulps with Laccase” • Chandra, R.P., Lehtonen, L.K., and Ragauskas, A.J., Accepted in Biotechnology Progress. “Modification of High-Yield Kraft Pulps with Laccase to Improve Strength Properties: Laccase Treatment in the Presence Gallic Acid” Publications and Presentations

• Chandra, R.P. and Ragauskas, A.J., submitted to Journal of Agriiiculture and Food Chemistry. “Modifiificat ion o f H igh-Yifield Kraft Pulps with Laccase: Enhancing the Effects of Laccase Treatment with Xylanase” • Chandra, R.P. and Ragauskas, A.J., American Chemical Society National Meeting 2002, Invited Speaker for Anselme Payen Award Symposium, April 2002, Orlando, Florida, “Fiber Modification with Laccase: You Say You Want a Revolution?” • Chandra, R.P ., Chakar, F.S ., Allison, L., Kim, D.H ., Elder, T., and Ragauskas, A.J., 10th International Conference on Biotechnology in the Pulp and Paper Industry, June 4, 2001, Helsinki, Finland, “Delving into the Fundamental LMS Delignification of High-kappa Kraft PPulpsulps.” • Chandra, R.P. and Ragauskas, A.J., American Chemical Society Pacifichem Conference 2000, December 2000, Honolulu, Hawaii, “ Parsinggygg Laccase’s Effects on Modifying Lignin.” • Chandra, R.P. , Dyer, T.J. and Ragauskas, A.J., Publication to be submitted, “The Aptitude of Laccase to Attach Compounds to Bleached Chemical Pulps “ Future Tip of the iceberg • Many new avenues – New grafting agents and substrates – Various application methods – Chemo-enzymatic opens door for novel fiber modification possibilities • Energy effic ien t • Environmentally friendly • Untapped potential