An Auspicious Application An Auspicious Application of Laccase and - - PowerPoint PPT Presentation

An Auspicious Application An Auspicious Application

• f Laccase and
• f Laccase and

Hydrogen Hydrogen Peroxidases Peroxidases for Biobleaching of for Biobleaching of Recalcitrant Paper Dyes Recalcitrant Paper Dyes

Kristina Knutson and Arthur Ragauskas, Kristina Knutson and Arthur Ragauskas, Institute of Paper Science and Institute of Paper Science and Technology, Atlanta, GA Technology, 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

20 40 60 1987 1994 1998 2003 2012 % Recycled

Research Objectives Research Objectives

• Test effectiveness of virgin pulp

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

+

Enzyme

Today Today’ ’s Talk s Talk

• Dyes Direct Yellow 11 & Basazol 46L

Dyes Direct Yellow 11 & Basazol 46L

• Measurement of Color

Measurement of Color

• Laccase &

Laccase & peroxidases peroxidases

• Peroxidases

Peroxidases experimental results experimental results

• Laccase experimental results

Laccase experimental results

• Summary

Summary

Recycling Colored Papers Recycling Colored Papers

20.2 20.2 22.8 22.8 40.5 40.5 15.0 15.0 Goldenrod Goldenrod 71.0 71.0 61.5 61.5 72.3 72.3 54.3 54.3 File Stock File Stock 79.7 79.7 69.0 69.0 80.7 80.7 65.5 65.5 Colored Colored Ledger Ledger 81.8 81.8 66.7 66.7 81.5 81.5 62.1 62.1 Mixed Mixed Office Office Waste Waste Hydrosulfite Hydrosulfite (Dithionite) (Dithionite) 2% 2% Hydrogen Hydrogen Peroxide Peroxide 1% 1% Formamidine Formamidine sulfinic acid sulfinic acid (FAS) 0.5% (FAS) 0.5% Original Original Deinked Deinked Raw Raw Material Material Type Type

Brightness After Treatment Brightness After Treatment

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

Structure Direct Yellow 11 Structure Direct Yellow 11

Structure most problematical dye commonly used for office paper

Structure Basic Dye Structure Basic Dye Baszol Baszol 46L 46L

United States Patent Number 4,256,458 March 17, 1981 Methine Dyes for Paper and Amonically-Modified Fibers Patent 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 Dye Example Dye

Structure Direct Yellow 11 Structure Direct Yellow 11

Example Spectra Example Spectra

ph8.5 No PEG+B40 Con

• 0.2

0.2 0.4 0.6 0.8 1 200 300 400 500 600

Wavelength Absorbance

CIELAB Color Space CIELAB Color Space

Calculation of Dye Removal Index Calculation of Dye Removal Index

Distance from Ideal Bleach Point = R2 R2 = a2 + b2 + (100-L) 2 Amount of Color Removal = -∆R2 ∆R2 = R2

2 – R1 2

∆R2 = (a2

2 – a1 2) + (b2 2 – 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 Fiber Recycled Fiber

N OH C O H3C

MEDIATOR MEDIATOR

Oxidation of Oxidation of ß ß-

• O

O-

• 4 lignin

4 lignin dimer dimer I I by laccase/ HBT system by laccase/ HBT system

Peroxidase Catalytic Cycle Peroxidase Catalytic Cycle

Clear applications for color destruction that have not yet been examined

Experimental Results Experimental Results

Horseradish Peroxidase System Horseradish Peroxidase System

• 2 hour Reaction at 45

2 hour Reaction at 45°

°C

C

• 100mM Acetate buffer,

100mM Acetate buffer, pH 3.8 pH 3.8

• 1

1-

• mM Veratryl alcohol

mM Veratryl alcohol

• 10ppm Dye

10ppm Dye

• 5 Units Horseradish

5 Units Horseradish peroxidase /ml peroxidase /ml

• 0.1 mM Hydrogen

0.1 mM Hydrogen peroxide peroxide

Peroxidase Decolorizes Basic Dye Peroxidase Decolorizes Basic Dye

HRP Treatment

0.1 0.2 0.3 0.4 0.5 0.6 0.7 280 330 380 430 480 530 580

Wavelength (nm) Absorbance

Basazol 46L Treated Basl 46L Direct Yellow 11 Treated Yellow 11

Soybean Peroxidase System Soybean Peroxidase System

• 2 hour Reaction at 45

2 hour Reaction at 45°

°C

C

• 100mM Acetate buffer, pH 3.8

100mM Acetate buffer, pH 3.8

• 1

1-

• mM Veratryl alcohol

mM Veratryl alcohol

• 0.1M Calcium chloride

0.1M Calcium chloride

• 10ppm Dye

10ppm Dye

• 1 Units Soybean peroxidase

1 Units Soybean peroxidase /ml /ml

• 0.1 mM Hydrogen peroxide

0.1 mM Hydrogen peroxide

Comparison of SBP and HRP Comparison of SBP and HRP

• 20

20 40 60 80 100

Direct Yellow 11 Basazol 46L

% Reduction in Abs Area

SBP HRP

SBP Effective Over Wide Ph Range SBP Effective Over Wide Ph Range

89.4 89.4 89.7 89.7 89.0 89.0 8.5 8.5 90.6 90.6 87.4 87.4 89.7 89.7 4.5 4.5 86.9 86.9 85.3 85.3 85.1 85.1 3.8 3.8 67.3 67.3 35K 35K 60.2 60.2 10K 10K 69.2 69.2 Non Non e e 2.4 2.4 Basazol Basazol 46L 46L % % Dec Dec . . PEG PEG % Dec. % Dec. PEG PEG % % Dec Dec . . PEG PEG pH pH SBP Treat SBP Treat

Laccase Laccase-

• Mediator System

Mediator System For Treating Dyed Pulp For Treating Dyed Pulp

• 2 hour Reaction at 45

2 hour Reaction at 45°

°C

C

• 10bar Oxygen pressure

10bar Oxygen pressure

• 100mM Acetate buffer,

100mM Acetate buffer, pH 4.5 pH 4.5

• Mediator 0.005

Mediator 0.005-

• 5mM

5mM

• 50

50 µ µkat kat/mL Laccase /mL Laccase

• 10ppm Direct Yellow 11

10ppm Direct Yellow 11

• r
• r
• 3% pulp consistency

3% pulp consistency

Laccase Mediators Laccase Mediators

ABTS ABTS 2,2'azinobis 2,2'azinobis-

• 3

3-

• ethyl

ethyl-

• benzthiazoline

benzthiazoline-

• 5

5-

• sulfonate

sulfonate Violuric acid Violuric acid HBT HBT 1 1-

• Hydroxy

Hydroxy benzotriazole benzotriazole Mediator Structure Mediator Structure Mediator Name Mediator Name

S N S N H2C CH3 CH2 H3C N N SO3H HO3S

HN NH O O O NOH

N N N OH

Dissolved Direct Yellow 11 + LMS Dissolved Direct Yellow 11 + LMS

0.5 1 1.5 2 2.5 3 200 300 400 500 600

Wavelength, nm 20 ppm Direct Yellow 11 A b s

• r

b a n c e Dye, 1 mM HBT, Lac, to Dye, HBT, Lac, 2hr 45°C, 10bar O2

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

Mediator Comparison

10 20 30 40 50 60 70

Lac+HBT Lac+VA Lac+ABTS DRI (Dye Removal Index)

ABTS Effective at Low ABTS Effective at Low Concentration Concentration

Dose Dependence

10 20 30 40 50 60 70 0.005 0.01 0.05 0.5 5 mM ABTS DRI (Dye Removal Index)

Post Post-

• LMS Reaction Liquid Phase

LMS Reaction Liquid Phase

Effluents from Treated Pulps

0.2 0.4 0.6 0.8 1 240 280 320 360 400 440 480

Wavelength (nm) Absorbance

White Pulp + Lac+0.1mM ABTS Dyed Pulp + Lac+0.1mM ABTS

Laccase + ABTS Direct Yellow 11 + Laccase + ABTS After 2hr at 10bar O2, 45 ºC Purified Direct Yellow 11

Conclusion Conclusion

• Laccase/ABTS system can decolorize stilbene

Laccase/ABTS system can decolorize stilbene dye Direct Yellow 11 dye Direct Yellow 11

• Peroxidases

Peroxidases are ineffective for Direct Yellow 11 are ineffective for Direct Yellow 11

• Peroxidases

Peroxidases do decolorize basic dye Basazol 46L do decolorize basic dye Basazol 46L

• Laccase decolorizes Basazol 46L

Laccase decolorizes Basazol 46L