Statistical significance of the results

Figure 1-1: Major application fields of nanomaterials. From Tsuzuki, 2009 ... 1-3 Figure 1-2: Classification of nanomaterials in accordance with ISO TS 27687:2008 and ISO TS 80004-4:2011. From NanosafePACK (Dobón-Lopez, 2013) ... 1-4 Figure 1-3: Classification of nanomaterials according to their chemical composition.

From USA-Canada Regulatory Cooperation Council, 2013... 1-5 Figure 1-4: Schematic representation of cellulose chains and their intra- and intermolecular bonding structure. From Lin and Dufresne, 2014. ... 1-8 Figure 1-5: Ultrastructure of lignocellulosic fibers. From Postek et al., 2011 ... 1-9 Figure 1-6: Transmission electron microscope images of A)NFC, B)NCC. From Klemm et al., 2011. ... 1-14 Figure 1-7: AFM image of nanofibrillar cellulose (NFC), from Ferrer et al., 2012) (A) and NFC hydrogel, from Inventia AB (Sweden) (B). ... 1-15 Figure 1-8: SEM micrograph of a NCC film showing chiral nematic organization, from Majoinen et al., 2012, (A). NCC film showing birefringence from Kelly et al., 2013 (B).

... 1-18 Figure 1-9: Plant xylan showing action sites of the different xylanase activities. From Beg et al. 2001. ... 1-23 Figure 1-10: Schematic representation of the action of the three main cellulolytic activities. From Wikipedia commons. ... 1-24 Figure 1-11: General overview of the present thesis ... 1-26 Figure 2-1: Chemical principle of the Spiro method for quantification of reducing sugars in a solution. ... 2-3 Figure 2-2: Cylindrical blade-stirred reactor. ... 2-5 Figure 2-3: AHIBA Easydye apparatus with its 250 mL vessels. ... 2-6

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Figure 2-4: Thermostatic bath with magnetic stirring (left), samples during hydrolysis (right). ... 2-7 Figure 2-5: General overview of NCC preparation protocol. ... 2-8 Figure 2-6: Scheme of the sequential testing plan, from Pepió and Polo, 2000 ... 2-10 Figure 2-7: Experimental setup for acetylation reaction in a thermostatic bath with reflux refrigerated by water. ... 2-14 Figure 2-8: Mütek particle charge detector. ... 2-16 Figure 2-9: Tared 25 mL glass beakers with NCC suspension samples. ... 2-22 Figure 2-10: Experimental setup for potentiometric titration (left) and chemical principle of the titration from Abitbol et al., 2013 (right). ... 2-24 Figure 3-1: ISO brightness (A) and Kappa number (KN) and hexenuronic acid content (HexA) (B) of samples. ... 3-5 Figure 3-2: Viscosity (bars, left axis) and wet zero-span tensile index (points, right axis) (A) and correlation between both variables (B). ... 3-7 Figure 3-3: SEM micrographs of cellulose fibers. Images correspond to: Eucalyptus ECF (A), TCF (B); flax ECF (C), TCF (D); sisal ECF (E), TCF (F) and cotton linters (G). ... 3-10 Figure 4-1: Optical microscope images of enzymatically treated fibers (left) and control fibers (right). ... 4-9 Figure 4-2: Size (A) and sulfur content (B) of NCC samples expressed as a function of yield for 62% (diamonds) and 64% (triangles) H²SO⁴. ... 4-11 Figure 4-3: Acid hydrolysis mechanism (A) and sulfate esterification on NCC surface (B). From Lu and Hsieh, (2010). ... 4-12 Figure 4-4: Yield of NCC preparation process from C1, KC1 and initial fibers at the different studied conditions. ... 4-15 Figure 4-5: NCC average size vs yield. Circles indicate data with 62% and 64% wt.

H²SO⁴. All samples were obtained from initial, C1 and KC1 fibers. ... 4-16 Figure 4-6: Sulfur content of NCC, as % of elemental sulfur (bars, left axis). Surface charge of NCC as cationic demand of suspensions (dots, right axis). ... 4-17

Figure 4-7: SEM images of dried NCC films. A: C1_NCC 62 % wt.; B: KC1_NCC 62 % wt.; C: C1_NCC 64 % wt.; D: KC1_NCC 64 % wt. All samples on images were filtered through Whatman 41 filter. ... 4-21 Figure 5-1: Fiber length (mm) distribution of initial, cellulase treated (C) and control (KC) fibers indicated as % of total. ... 5-6 Figure 5-2: Yield data predicted by models with 62% wt. sulfuric acid (A), and with 65% wt. sulfuric acid (B). Grey and black charts represent data in presence and absence of cellulase, respectively. ... 5-8 Figure 5-3: Particle size data predicted by models with 62% wt. sulfuric acid (A), and with 65% wt. sulfuric acid (B). Grey and black charts represent data in presence and absence of cellulase, respectively. ... 5-10 Figure 5-4: Surface charge data predicted by models with 62% wt. sulfuric acid (A), and with 65% wt. sulfuric acid (B). Grey and black charts represent data in presence and absence of cellulase, respectively. ... 5-11 Figure 5-5: Sulfur content (as % S) of samples at studied conditions in presence and absence of cellulase. ... 5-13 Figure 5-6: Zeta Potential (A) and Polydispersity Index (PDI) (B) of samples at studied conditions in presence and absence of cellulase ... 5-14 Figure 5-7: TEM images of NCC. Images correspond to: cellulase, 25 min, 47 ºC 62%

acid (optimal point, A); control, 25 min, 47 ºC, 62% acid (B); cellulase, 50 min, 60ºC, 62% acid (C); cellulase, 25 min, 47 ºC, 65% acid (D); cellulase, 50 min, 47 ºC, 65% acid (E). Scale bar: 100 nm... 5-17 Figure 5-8: FTIR spectra of (A) cellulase pretreated fibers, (B) NCC (no cellulase, 62%

wt. acid, 25 min), (C) NCC (cellulase, 65% wt. acid, 50 min). ... 5-18 Figure 6-1: Models relating enzymatic treatment yield (A) and fiber length (B) to enzyme dose and enzymatic treatment time. ... 6-7 Figure 6-2: Fiber length distribution of samples after enzymatic treatments. ... 6-8 Figure 6-3: Models relating total released glucose (A) and fiber viscosity (B) to enzyme dose and enzymatic treatment time. ... 6-9

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Figure 6-4: Total crystallinity index of fibers (TCI) of fibers during enzymatic treatments. ... 6-10 Figure 6-5: Model relating NCC hydrolysis yield to enzyme dose and enzymatic treatment time. ... 6-11 Figure 6-6: Model relating total yield to enzyme dose and enzymatic treatment time………...………6-12 Figure 6-7: Model relating NCC surface charge to enzyme dose and enzymatic treatment time. ... 6-13 Figure 6-8: Model relating NCC total crystallinity index (TCI) to enzyme dose and enzymatic treatment time. ... 6-14 Figure 6-9: Cellulase quantitative effects. Total released sugars (as glucose equivalents) and fibers viscosity expressed in front of NCC yield (A), NCC yield increase expressed versus chain scission number (CSN), both calculated from initial fibers (B) and NCC surface charge expressed versus NCC yield (C). ... 6-15 Figure 6-10: Enzymatic hydrolysis rate, as mg glucose released per minute as a consequence of enzymatic treatments expressed in front NCC hydrolysis yield. ... 6-16 Figure 6-11: Proportion of each oligosaccharide released during enzymatic hydrolysis.

... 6-17 Figure 7-1: Work scheme of the present chapter ... 7-5 Figure 7-2: A- Xylan content (%); B- ISO Brightness (%) and C – HexA content (µmol/g odp). Mean values are represented, error bars indicate confidence intervals. ... 7-10 Figure 7-3: KN values along xylanolytic treatments. Specific values of KN due to HexA and to lignin are also indicated. ... 7-11 Figure 7-4: Accumulated concentration (i.e. adding the amount released each hour) of reducing sugars on effluents of treatments with X (A: X treatment, B: Xs treatment).

X3, X2 and X1 stand for xilotriose, xilobiose and xylose, respectively. ... 7-13 Figure 7-5: Reduction in hemicelluloses content (A), HexA content (B) and KN (C) compared to initial pulp, and increase in brightness (D) produced by combined treatments, indicated as a contribution of each enzymatic step. ... 7-14

Figure 7-6: Wet zero-span index values expressed in front of viscosity for (a) xylanase treatments (X, Xs) and (b) Carbohydrase mixture (Cx) + xylanase (X) treatments. ... 7-16 Figure 7-7: Concentration of sugars on effluents (expressed in relation to pulp mass) of combined Cx and X treatments. Previous treatment, when available, is indicated between parenthesis and only data of effluents of the second treatment is shown. X3, X2 and X1 stand for xilotriose, xilobiose and xylose, respectively; C3, C2 and C1 stand for cellotriose, cellobiose and glucose, respectively. ... 7-17 Figure 7-8: Xylans (A) and HexA content (B) reduction from initial pulp after different enzymatic steps and alkaline extractions with NaOH. Error bars indicate confidence intervals. ... 7-19 Figure 7-9: SEM images of treated fibers. Pictures represent following samples: A) X2, B) KX2, C) Cx, D) KCx, E) Cx24, F) KCx24, G) X2 and H) X2+E ... 7-20 Figure 7-10: CSN (A) and xylans content reduction (as %) (B), represented in front of reactivity increase. Variations were calculated in comparison to the previous stage, not initial pulp. ... 7-22 Figure 8-1: Workflow scheme of the different treatments performed. A: NCC from HCC fibers (chapter 7) using optimal conditions (chapters 5 and 6). B: NCC from sisal fibers. C: NCC from dissolving-grade fibers. ... 8-5 Figure 8-2: Fiber length distribution of initial fibers and after Fa (20 U/g odp, 2 h) and Fc (10 U/g odp 24 h) treatments from cotton linters (A) and sisal (B). ... 8-7 Figure 8-3: Yield of NCC samples obtained from initial (In), enzymatically treated (Fa, Fc) and alkali extracted (E) cotton linters (A) (data reproduced from chapter 6) and sisal fibers (B). All samples were prepared with 62% wt. H²SO⁴ at 47ºC during 25 min. .... 8-9 Figure 8-4: Average particle size (A) and sulfur content (B) of NCC samples obtained from initial (In), enzymatically treated (Fa, Fc) and alkali extracted (E) sisal fibers. All samples were prepared with 62% wt. H²SO⁴ at 47ºC during 25 min. ... 8-10 Figure 8-5: NCC from sisal fibers using three different sulfuric acid concentrations and different enzymatic treatments with Celluclast enzyme (N). Samples were prepared with 45 min of acid hydrolysis at 45 ºC using each indicated acid concentration. .... 8-11

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Figure 8-6: NCC extraction yield from dissolving fibers (D) using H²SO⁴ 63% wt at 45ºC during 45 min. Initial, and Celluclast (N) treated samples are indicated. ... 8-13 Figure 9-1: Structure of the general discussion. ... 9-2 Figure 9-2: NCC yield predicted by models at 25 minutes and 47 ºC in presence and absence of cellulase pretreatment and with 62 % wt. (optimal point) and 65 % wt.

H²SO⁴. Data reproduced from chapter 5. ... 9-8 Figure 9-3: Total yield values predicted by models for NCC obtained from initial, control and enzymatically pretreated fibers under different conditions. All samples were hydrolyzed during 25 minutes at 47 ºC with 62 % wt. H²SO⁴. Data reproduced from chapter 6. ... 9-10 Figure 9-4: Crystallinity increases produced by sulfuric acid during NCC isolation (fibers to NCC) and by enzymatic treatments on fibers and on NCC in studies from chapters 5 and 6. Increases produced by enzyme are calculated from the value showed by each control sample. ... 9-13 Figure 9-5: Hemicelluloses content of TCF sisal fibers after xylanase treatments (X, Xs), cellulase treatments (Cx2h, Cx24h) and combined treatments (Cx2h + X5h). Data reproduced from chapter 7. ... 9-15 Figure 9-6: Hemicelluloses content of initial or Cx treated TCF sisal fibers after cold alkaline extraction with 4 % (E4%) or 9 % (E9%) w/v NaOH. Data reproduced from chapter 7. ... 9-17