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

An Auspicious Application An Auspicious Application of Laccase and of Laccase and Hydrogen Peroxidases Peroxidases Hydrogen 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

Recycling Continues to > 90% linerboard Increase > 80% newsprint 60 % Recycled > 60% white office ledger 40 < 30% mixed office waste 20 0 1987 1994 1998 2003 2012 •81 million tons of waste fiber in US in 1994

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’ ’s Talk s Talk Today � 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 Brightness After Treatment Brightness After Treatment Raw Original Formamidine Hydrogen Hydrosulfite Raw Original Formamidine Hydrogen Hydrosulfite Material Deinked sulfinic acid Peroxide (Dithionite) Material Deinked sulfinic acid Peroxide (Dithionite) Type (FAS) 0.5% 1% 2% Type (FAS) 0.5% 1% 2% Mixed Mixed 62.1 81.5 66.7 81.8 62.1 81.5 66.7 81.8 Office Office Waste Waste Colored 65.5 80.7 69.0 79.7 Colored 65.5 80.7 69.0 79.7 Ledger Ledger File Stock File Stock 54.3 54.3 72.3 72.3 61.5 61.5 71.0 71.0 Goldenrod 15.0 40.5 22.8 20.2 Goldenrod 15.0 40.5 22.8 20.2 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 Baszol Baszol 46L 46L Structure Basic Dye 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 2A- • 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. 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 1 0.8 0.6 Absorbance 0.4 0.2 0 200 300 400 500 600 -0.2 Wavelength

CIELAB Color Space CIELAB Color Space

Calculation of Dye Removal Index Calculation of Dye Removal Index Distance from Ideal Bleach Point = R 2 L=100 a=b=0 R 2 = a 2 + b 2 + (100-L) 2 R 2 Amount of Color Removal = - ∆ R 2 ∆ R 2 = R 2 2 – R 1 2 ∆ R 2 = (a 2 2 – a 1 2 – b 1 2 ) + (b 2 2 ) + [(100-L 2 ) 2 – (100-L 1 ) 2 ] R 1 Dye Removal Index (DRI) = -100 [ ∆ R 2 / R 1 2 ] DRI = Percent Color Removed a = b = L = 0 by the Treatment

Potential of Biobleaching Potential of Biobleaching Recycled Fiber Recycled Fiber O C N OH H 3 C MEDIATOR MEDIATOR

Oxidation of ß ß- -O O- -4 lignin 4 lignin dimer dimer I I Oxidation of 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 ° C 2 hour Reaction at 45 ° � 2 hour Reaction at 45 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 0.7 Basazol 46L 0.6 Treated Basl 46L Absorbance 0.5 Direct Yellow 11 0.4 Treated Yellow 11 0.3 0.2 0.1 0 280 330 380 430 480 530 580 Wavelength (nm)

Soybean Peroxidase System Soybean Peroxidase System ° C 2 hour Reaction at 45 ° C 2 hour Reaction at 45 � � 100mM Acetate buffer, pH 3.8 100mM Acetate buffer, pH 3.8 � � 1- -mM Veratryl alcohol mM Veratryl alcohol 1 � � 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 % Reduction in Abs Area 100 80 60 SBP 40 HRP 20 0 Direct Yellow Basazol 46L -20 11

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

Laccase- -Mediator System Mediator System Laccase For Treating Dyed Pulp For Treating Dyed Pulp ° C 2 hour Reaction at 45 ° � 2 hour Reaction at 45 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 � or or � 3% pulp consistency 3% pulp consistency �

Laccase Mediators Laccase Mediators Mediator Name Mediator Name Mediator Structure Mediator Structure HBT N HBT N 1- -Hydroxy Hydroxy 1 N benzotriazole benzotriazole OH NOH Violuric acid Violuric acid O O HN NH O ABTS ABTS HO 3 S S S SO 3 H 2,2'azinobis- -3 3- -ethyl ethyl- - 2,2'azinobis N N benzthiazoline- -5 5- - benzthiazoline N N sulfonate sulfonate H 2 C CH 3 H 3 C CH 2

Dissolved Direct Yellow 11 + LMS Dissolved Direct Yellow 11 + LMS 3 Dye, 1 mM HBT, Lac, to 2.5 Dye, HBT, Lac, 2hr 45°C, 10bar O2 e 2 c n a 1.5 b r o s 1 b A 0.5 20 ppm Direct Yellow 11 0 200 300 400 500 600 Wavelength, nm

Effectiveness of LMS Decolorization Effectiveness of LMS Decolorization Depends on Mediator Depends on Mediator Mediator Comparison DRI (Dye Removal Index) 70 60 50 40 30 20 10 0 Lac+HBT Lac+VA Lac+ABTS

ABTS Effective at Low ABTS Effective at Low Concentration Concentration Dose Dependence 70 DRI (Dye Removal Index) 60 50 40 30 20 10 0 0 0.005 0.01 0.05 0.5 5 mM ABTS

Post- -LMS Reaction Liquid Phase LMS Reaction Liquid Phase Post Effluents from Treated Pulps 1 White Pulp + 0.8 Lac+0.1mM ABTS Absorbance Dyed Pulp + 0.6 Lac+0.1mM ABTS 0.4 0.2 0 240 280 320 360 400 440 480 Wavelength (nm)

Laccase + ABTS Direct Yellow 11 + Laccase + ABTS After 2hr at 10bar O 2 , 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 �