Characterisation of the surface composition of Eucalyptus fibers by - - PowerPoint PPT Presentation

Characterisation of the surface composition of Eucalyptus fibers by immunolabelling and enzymatic peeling

Tarja Tamminen Atte Mikkelson Matti Siika-aho Kristiina Kruus Jaakko Pere Fernando Gomes Jorge L. Colodette

7th International Colloquium on Eucalyptus Pulp, May 26-29, 2015. Vitória, Espirito Santo, Brazil.

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VTT Group on the map

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Contents

• Introduction
• Materials and methods
• Xylan and pulp samples
• Characterisation methods
• Results
• Composition of the xylan samples and

xylan-deposited pulps

• Surface analysis by immunolabelling
• Surface analysis by enzymatic

treatments

• Conclusions

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Introduction

• Xylans are important for the production of printing and writing paper

grades because they facilitate pulp refining and increase fiber bonding → Interest to increase pulp xylan content.

• Grasses are a potential source of xylans to be resorbed onto

eucalyptus fibers. Elephant grass (Pennisetum purpureum) (EG) is studied here as the xylan source.

• The benefits of fiber xylan are most evident if it is situated on the

fiber surface. However, analysis of the spatial distribution of hemicelluloses in fiber is challenging.

• Enzymatic peeling using gradual total hydrolysis has been

applied.

• Immunolabelling by specific antibodies is another potential

method for the chemical mapping of fibers.

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Materials and methods: Samples

• Elephant grass (Pennisetum purpureum) (EG) was studied as the

xylan source.

• EG brown pulp (kappa 20) was produced by the Soda-AQ

process.

• Xylan was extracted by Cold Caustic Extraction (CCE) using

dosages of 400 or 700 kg NaOH/odt pulp, 10% consistency, 60 min time at room temperature.

• The xylan was precipitated from the CCE liquor by ethanol

addition followed by centrifugation and thorough washing.

• The extracted xylan was added to E. globulus brown pulp, kappa

20, produced by the Soda-AQ process

• Added as 70 g/L solution during the oxygen delignification (7 %

xylan on pulp dry weight).

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Materials and methods: Characterisation of the xylans

• Xylan composition and molar mass distribution
• Carbohydrate composition by
• acid methanolysis after purification (to remove alkali

residues)

• total enzymatic hydrolysis for hexenuronic acid-containing
• ligomeric structures
• Molar mas distribution in 1 M NaOH, followed by HPLC
• analysis. Refractive index detector was used for xylan and UV

(280 nm) for the lignin component. Calibration was done using Pullulan and PSS standards for the two compound classes, respectively.

• The EG xylan and reference Eucalyptus xylan were analysed.

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Materials and methods: Characterisation of the spatial distribution of xylan in Eucalyptus pulp fibers

• Fiber surface analysis by immunolabelling
• Primary antibody was used that recognizes a short

xylooligosaccharidic stretch containing a 4-O-methyl-glucuronic acid residue (MeGlcA-Xyl2-3).

• Enzymatic hydrolysis using three enzyme systems at 1.6% cons., pH 5.
• Total enzymatic hydrolysis: 50 FPU/g dry pulp, 40°C.
• Endoglucanase I (Cel7B) (active towards both xylan and cellulose):

0.25 mg protein/g dry pulp, 45°C.

• Purified xylanase: 500 nkat/g of dry pulp, 45°C.
• Samples taken at several time points up to 48 h, boiled to inactivate the

enzymes and hydrolysed further to deliver the total monosaccharide composition, analysed by HPAEC-PAD.

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Contents

• Introduction
• Materials and methods
• Xylan and pulp samples
• Characterisation methods
• Results
• Composition of the xylan samples and xylan-

deposited pulps

• Surface analysis by immunolabelling
• Surface analysis by enzymatic treatments
• Conclusions

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Composition of the xylan samples

• EG xylan contains ca. 10 % other carbohydrate components, whereas Euca xylan only < 2 %.
• EG xylan is highly substituted by arabinose side groups (ara/xyl = 1/15), contrary to Eucalyptus

xylan (ara/xyl = 1/800).

• The presence of arabinose in the Eucalyptus pulps as such is a potential indication of

deposited EG xylan.

• In addition to carbohydrates, the xylans contain also lignin based on gaps in material balance.

2 4 6 8 10 Elephant grass xylan Eucalyptus xylan

Xylose GlcA MeGlcA GalA Glucose Galactose Mannose Rhamnose Arabinose

100% 100%

%

• HexA-contents were 0.45 and

4.75 mg/100 mg (corresponding to 25 and 270 mmol/kg) for the EG and Eucalyptus samples, respectively.

• Deposition of EG xylan

should not significantly increase the HexA content

• f the pulp.

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Molar mass distribution of the xylans

• Average Mw of the EG and Eucalyptus xylans were determined to be 20 700 and 16 600

Da, respectively.

• Lignin was detected in the same range by the UV detector, probably bound to the xylan,

but also as smaller molecules. The average Mw of the lignin contaminant in the EG and Eucalyptus xylans were determined to be 23 400 and 11 200 Da, respectively.

• Linkages between xylan and lignin artificially increase this average value.
• The molar mass distributions were performed for the original unpurified samples. After

the purification, the samples were no longer adequately soluble in alkali to enable reliable analysis, probably due to aggregation.

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Xylan-deposited pulps

• The EG xylan was redeposited onto E. globulus kappa 20 brown fibers during

the oxygen delignification stage.

• The enrichment of the xylan-deposited pulps is clearly seen. More enrichment

was detected at higher charge.

5 10 15 20 25 30 Reference pulp CCE 400 CCE 700 Glc, % Man, % Xyl, % Gal, % Ara, %

100% 100% 100%

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Contents

• Introduction
• Materials and methods
• Xylan and pulp samples
• Characterisation methods
• Results
• Composition of the xylan samples and xylan-

deposited pulps

• Surface analysis by immunolabelling
• Surface analysis by enzymatic treatments
• Conclusions

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Surface analysis by immunolabelling Reference pulp

• Fibers were labelled with

anti-MeGlcA (Xyl 2-3) and Alexa Fluor 633. CLSM with red laser (638 nm) was used for the detection

• f the label.
• Moderate labelling of the

reference pulp fibers was

• bserved, especially on

damaged fibers, fibrillated fines and around pits.

• Fibers by optical

microcopy

• CLSM image for xylan
• Combined image
• No CLMS visible

material without the immunolabelling

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Surface analysis by immunolabelling Pulp CCE 400

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Surface analysis by immunolabelling Pulp CCE 700

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Contents

• Introduction
• Materials and methods
• Xylan and pulp samples
• Characterisation methods
• Results
• Composition of the xylan samples and xylan-

deposited pulps

• Surface analysis by immunolabelling
• Surface analysis by enzymatic treatments
• Conclusions

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Surface analysis by enzymatic treatments: Total hydrolysis

• Total hydrolysis releases very fast material from fiber surface.
• Arbitrarily, the composition of the hydrolysate at 10 % hydrolysis level can be interpreted to

represent the surface material.

• The increased amount of surface xylan as a function on xylan deposition amount is detected.

Hydrolysis level, % Xylan proportion in hydrolysate, %

26 36 43 15 19 21

• 5

10 15 20 25 30 35 40 45 50 Reference CCE400 CCE700 10% hydrolysis level 90% hydrolysis level 10 20 30 40 50 60 20 40 60 80 100 Reference CCE700 CCE400 Potência (Reference) Potência (CCE700) Potência (CCE400)

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Surface analysis by enzymatic treatments: Arabinose as a potential marker compound

• Isolated EG xylan contains arabinose, contrary to Euca xylan
• > potential marker for deposited grass xylan.
• No clear enrichement of ara in the total enzymatic hydrolysis, indicating that there is arabinose

in the Euca pulp, even if not in the extracted xylan

• > Arabinose cannot be used as a marker for the grass xylan

1 2 3 4 5 6 7 8 1 5 15 30 60 120 1440 2880

ref 400 700

• Arabinose to xylose ratio

increases as a function of time in the reference pulp, indicating enrichment of arabinose in the inner part of the fiber

• > method to analyse spatial

distribution of fiber components

Time, min Ara / xyl,, %

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Surface analysis by enzymatic treatments: Endoglucanase I (Cel7B)

• Pure endoglucanase I (Cel7B) is known to have high activity on both xylan and cellulose.
• The hydrolysates consisted mainly of xylose, but the degree of hydrolysis remained low due to the lack of

assisting enzymes.

• Even if the order of xylan enrichment in the three samples was as expected, the results cannot be

used for the evaluation of the surface enrichment in comparison to the bulk composition.

Xylan proportion in hydrolysate, % Hydrolysis level, % 65 78 84 46 60 62

• 10

20 30 40 50 60 70 80 90 Reference CCE400 CCE700 2 % hydrolysis level 14 % hydrolysis level Xylan proportion in hydrolysate, % 10 20 30 40 50 60 70 80 90 5 10 15 20 Reference CCE400 CCE700 Polinômio (Reference) Polinômio ( CCE400 ) Polinômio (CCE700 )

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Surface analysis by enzymatic treatments: Xylanase

• Pure xylanase is selective for xylan
• > Hydrolysate composition does not provide much information
• The final level of released xylose as such can be used as an indication of

fiber surface xylan.

Xylan proportion in hydrolysate, % Hydrolysis level, % 10 20 30 40 50 60 70 80 90 100 2 4 6 8 10 Reference CCE 400 CCE 700 Xylose hydrolysed at 24h, mg/100 mg

3.2 6.0 7.4 2 4 6 8

Reference CCE 400 CCE 700

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Conclusions

• Xylan can be extracted from EG by the CCE method and deposited on

Eucalyptus pulp.

• The isolated xylan contains mainly xylan with high degree of

substitution by arabinose but low HexA content. It is contaminated by lignin.

• Immunolabelling revealed xylan at sites of various fiber defects and,

especially in the xylan-deposited samples, as separate patches.

• Total enzymatic hydrolysis as a function of time is a practical tool to follow

the surface xylan content.

• The method also gave an indication of the xylan substitution pattern as

a function of fiber wall thickness.

• Pure xylanase hydrolysis provided information of the total amount of xylan

that is accessible to the enzyme as an indication of fiber surface xylan.

• Surface xylan content of pulp fibers is relevant for the paper technical

properties of the pulp. Therefore it is important to monitor the spacal distribution of xylan in addition to its total content.

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The research leading to these results has received funding from the European Community's Seventh Framework Programme FP7/2007-2013 under grant agreement no KBBE-2009-3-244362 LignoDeco (EU/Brazil co-operation project). Acknowledgements