An Auspicious Application An Auspicious Application of Laccase and of Laccase and

Hydrogen Hydrogen PeroxidasesPeroxidasesfor Biobleaching of for Biobleaching of

Recalcitrant Paper DyesRecalcitrant Paper Dyes

Kristina Knutson and Arthur Ragauskas, Kristina Knutson and Arthur Ragauskas, Institute of Paper Science and Institute of Paper Science and

Technology, Atlanta, GATechnology, Atlanta, GA

•81 million tons of waste fiber in US in 1994

>90% linerboard

>80% newsprint

>60% white office ledger

<30% mixed office waste

Recycling Continues to Increase

0

20

40

60

1987 1994 1998 2003 2012

% R

ecyc

led

Research ObjectivesResearch Objectives

Test effectiveness of virgin pulp Test effectiveness of virgin pulp biobleaching technologies on color biobleaching technologies on color removal from dyed pulpsremoval from dyed pulps

+ Enzyme

TodayToday’’s Talks Talk

Dyes Direct Yellow 11 & Basazol 46LDyes Direct Yellow 11 & Basazol 46LMeasurement of ColorMeasurement of ColorLaccase & Laccase & peroxidasesperoxidasesPeroxidasesPeroxidases experimental resultsexperimental resultsLaccase experimental resultsLaccase experimental resultsSummarySummary

Recycling Colored PapersRecycling Colored Papers

20.220.222.822.840.540.515.015.0GoldenrodGoldenrod71.071.061.561.572.372.354.354.3File StockFile Stock

79.779.769.069.080.780.765.565.5Colored Colored LedgerLedger

81.881.866.766.781.581.562.162.1Mixed Mixed Office Office WasteWaste

Hydrosulfite Hydrosulfite (Dithionite) (Dithionite)

2%2%

Hydrogen Hydrogen Peroxide Peroxide

1%1%

FormamidineFormamidinesulfinic acid sulfinic acid (FAS) 0.5% (FAS) 0.5%

Original Original DeinkedDeinked

Raw Raw Material Material TypeType

Brightness After TreatmentBrightness After Treatment

Reference: Ferguson, L.D. 1996 TAPPI Deinking Short Course “Bleaching Wastepaper”

Structure Direct Yellow 11Structure Direct Yellow 11

Structure most problematical dye commonly used for office paper

Structure Basic Dye Structure Basic Dye BaszolBaszol 46L46LUnited States Patent Number 4,256,458 March 17, 1981

Methine Dyes for Paper and Amonically-Modified FibersPatent applies to methine dyes of the formula

•A is an organic or inorganic ion•R is hydrogen, methyl or ethyl•Q groups are the same or different substituted or unsubstituted aryl or hetaryl radicals•X represents a bridge member selected from the group consisting of aliphatic,

aromatic and heteroaromatic radicals.

2A-

Structure of most common yellow dye used in directory paper

Example DyeExample Dye

Structure Direct Yellow 11Structure Direct Yellow 11

Example SpectraExample Spectra

ph8.5 No PEG+B40 Con

-0.2

0

0.2

0.4

0.6

0.8

1

200 300 400 500 600

Wavelength

Abs

orba

nce

CIELAB Color SpaceCIELAB Color Space

Calculation of Dye Removal IndexCalculation of Dye Removal IndexDistance from Ideal Bleach Point = R2

R2 = a2 + b2 + (100-L) 2

Amount of Color Removal = -∆R2

∆R2 = R22 – R1

2

∆R2 = (a22 – a1

2) + (b22 – b1

2) + [(100-L2) 2 – (100-L1) 2]

Dye Removal Index (DRI) = -100 [∆R2 / R1

2] DRI = Percent Color Removed

by the Treatment

L=100 a=b=0

R2

R1

a = b = L = 0

Potential of Biobleaching Potential of Biobleaching Recycled FiberRecycled Fiber

N OHC

O

H3C

MEDIATORMEDIATOR

Oxidation of Oxidation of ßß--OO--4 lignin 4 lignin dimerdimer I I by laccase/HBT systemby laccase/HBT system

Peroxidase Catalytic CyclePeroxidase Catalytic Cycle

Clear applications for color destruction that have not yet been examined

Experimental ResultsExperimental Results

Horseradish Peroxidase SystemHorseradish Peroxidase System

2 hour Reaction at 452 hour Reaction at 45°°C C 100mM Acetate buffer, 100mM Acetate buffer, pH 3.8pH 3.811--mM Veratryl alcohol mM Veratryl alcohol 10ppm Dye10ppm Dye5 Units Horseradish 5 Units Horseradish peroxidase /ml peroxidase /ml 0.1 mM Hydrogen 0.1 mM Hydrogen peroxideperoxide

Peroxidase Decolorizes Basic DyePeroxidase Decolorizes Basic Dye

HRP Treatment

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

280 330 380 430 480 530 580

Wavelength (nm)

Abs

orba

nce

Basazol 46L

Treated Basl 46L

Direct Yellow 11

Treated Yellow11

Soybean Peroxidase SystemSoybean Peroxidase System

2 hour Reaction at 452 hour Reaction at 45°°C C 100mM Acetate buffer, pH 3.8100mM Acetate buffer, pH 3.811--mM Veratryl alcohol mM Veratryl alcohol 0.1M Calcium chloride0.1M Calcium chloride10ppm Dye10ppm Dye1 Units Soybean peroxidase 1 Units Soybean peroxidase /ml /ml 0.1 mM Hydrogen peroxide0.1 mM Hydrogen peroxide

Comparison of SBP and HRPComparison of SBP and HRP

-20

0

20

40

60

80

100

Direct Yellow11

Basazol 46L

% R

educ

tion

in A

bs A

rea

SBP HRP

SBP Effective Over Wide Ph RangeSBP Effective Over Wide Ph Range

89.489.489.789.789.089.08.58.5

90.690.687.487.489.789.74.54.5

86.986.985.385.385.185.13.83.8

67.367.335K35K

60.260.210K10K

69.269.2NonNon

ee2.42.4Basazol Basazol

46L46L

% % DecDec

..PEGPEG% Dec.% Dec.PEGPEG

% % DecDec

..PEGPEGpHpHSBP TreatSBP Treat

LaccaseLaccase--Mediator SystemMediator SystemFor Treating Dyed PulpFor Treating Dyed Pulp

2 hour Reaction at 452 hour Reaction at 45°°C C 10bar Oxygen pressure10bar Oxygen pressure100mM Acetate buffer, 100mM Acetate buffer, pH 4.5pH 4.5Mediator 0.005Mediator 0.005--5mM 5mM 50 50 µµkatkat/mL Laccase/mL Laccase10ppm Direct Yellow 11 10ppm Direct Yellow 11 oror3% pulp consistency3% pulp consistency

Laccase MediatorsLaccase Mediators

ABTSABTS2,2'azinobis2,2'azinobis--33--ethylethyl--benzthiazolinebenzthiazoline--55--sulfonatesulfonate

Violuric acidVioluric acid

HBTHBT11--Hydroxy Hydroxy benzotriazolebenzotriazole

Mediator StructureMediator StructureMediator NameMediator Name

S

N

S

N

H2C CH3 CH2H3C

N N

SO3HHO3S

HN NH

O

OO

NOH

N

N

N

OH

Dissolved Direct Yellow 11 + LMSDissolved Direct Yellow 11 + LMS

0

0.5

1

1.5

2

2.5

3

200 300 400 500 600

Wavelength, nm

20 ppm Direct Yellow 11

Abs

orba

nce

Dye, 1 mM HBT, Lac, to

Dye, HBT, Lac, 2hr 45°C, 10bar O2

Effectiveness of LMS Decolorization Effectiveness of LMS Decolorization Depends on MediatorDepends on Mediator

Mediator Comparison

0

10

20

30

40

50

60

70

Lac+HBT Lac+VA Lac+ABTS

DR

I (D

ye R

emov

al In

dex)

ABTS Effective at Low ABTS Effective at Low ConcentrationConcentration

Dose Dependence

010203040506070

0 0.005 0.01 0.05 0.5 5

mM ABTS

DR

I (D

ye R

emov

al In

dex)

PostPost--LMS Reaction Liquid PhaseLMS Reaction Liquid Phase

Effluents from Treated Pulps

0

0.2

0.4

0.6

0.8

1

240 280 320 360 400 440 480

Wavelength (nm)

Abs

orba

nce

White Pulp +Lac+0.1mM ABTSDyed Pulp +Lac+0.1mM ABTS

Laccase + ABTS

Direct Yellow 11 + Laccase + ABTSAfter 2hr at 10bar O2, 45 ºC

Purified Direct Yellow 11

ConclusionConclusion

Laccase/ABTS system can decolorize stilbene Laccase/ABTS system can decolorize stilbene dye Direct Yellow 11dye Direct Yellow 11PeroxidasesPeroxidases are ineffective for Direct Yellow 11are ineffective for Direct Yellow 11PeroxidasesPeroxidases do decolorize basic dye Basazol 46Ldo decolorize basic dye Basazol 46LLaccase decolorizes Basazol 46LLaccase decolorizes Basazol 46L