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 Fernando Gomes Kristiina Kruus Jorge L. Colodette Jaakko Pere 7th International Colloquium on Eucalyptus Pulp, May 26-29, 2015. Vitória, Espirito Santo, Brazil.

08/06/2015 2 VTT Group on the map

08/06/2015 3 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

08/06/2015 4 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.

08/06/2015 5 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).

08/06/2015 6 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 oligomeric 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.

08/06/2015 7 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.

08/06/2015 8 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

08/06/2015 9 Composition of the xylan samples 100% % 100% 10 Xylose  HexA-contents were 0.45 and 8 GlcA 4.75 mg/100 mg (corresponding MeGlcA to 25 and 270 mmol/kg) for the 6 EG and Eucalyptus samples, GalA respectively. Glucose 4  Deposition of EG xylan Galactose should not significantly 2 Mannose increase the HexA content Rhamnose of the pulp. 0 Arabinose Elephant grass xylan Eucalyptus xylan  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.

08/06/2015 10 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.

08/06/2015 11 Xylan-deposited pulps 100% 100% 100% 30 25 Glc, % 20 Man, % 15 Xyl, % 10 Gal, % 5 Ara, % 0 Reference CCE 400 CCE 700 pulp  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.

08/06/2015 12 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

08/06/2015 13 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 of the label.  Moderate labelling of the reference pulp fibers was observed, especially on damaged fibers, fibrillated fines and around pits.  No CLMS visible material without the immunolabelling  Fibers by optical  CLSM image for xylan  Combined image microcopy

08/06/2015 14 Surface analysis by immunolabelling Pulp CCE 400

08/06/2015 15 Surface analysis by immunolabelling Pulp CCE 700

08/06/2015 16 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

08/06/2015 17 Surface analysis by enzymatic treatments: Total hydrolysis 60 Xylan proportion in hydrolysate, % Reference 50 CCE700 50 43 45 CCE400 40 36 Potência (Reference) 40 35 Potência (CCE700) 30 26 Potência (CCE400) 30 10% hydrolysis 25 21 19 level 20 15 90% hydrolysis 15 20 level 10 5 10 - Reference CCE400 CCE700 0 0 20 40 60 80 100 Hydrolysis level, %  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.

08/06/2015 18 Surface analysis by enzymatic treatments: Arabinose as a potential marker compound 8 7  Arabinose to xylose ratio 6 Ara / xyl,, % increases as a function of time in 5 ref the reference pulp, indicating 4 400 enrichment of arabinose in the 3 700 inner part of the fiber 2 -> method to analyse spatial 1 distribution of fiber components 0 1 5 15 30 60 120 1440 2880 Time, min  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

08/06/2015 19 Surface analysis by enzymatic treatments: Endoglucanase I (Cel7B) 90 Xylan proportion in hydrolysate, % 80 90 Xylan proportion in hydrolysate, % 84 78 70 80 70 65 60 62 60 60 50 46 50 2 % hydrolysis level 40 Reference 40 14 % hydrolysis level CCE400 30 30 CCE700 20 20 Polinômio (Reference) Polinômio ( CCE400 ) 10 10 Polinômio (CCE700 ) - 0 Reference CCE400 CCE700 0 5 10 15 20 Hydrolysis level, %  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.