Changes in cellulose crystallinity can be quantitatively interpreted using the lateral order index readings from ATR-FTIR spectra and X-ray diffraction. In addition to this, carbohydrate-binding modules have specific recognition capabilities for cellulose and other polysaccharides in plant cell walls, but have yet to be used in a comparative and quantitative manner to interpret the changes in cell wall crystallinity after the industrial mercerization treatments. Intact Gossypium hirsumtum lines FM966 and Cooker were treated with increasing NaOH concentrations and then labelled with CBMs for cellulose of varying crystallinities. These his-tagged proteins bind to cellulose, and can then be further tagged with a fluorochrome probe to allow detection under UV excitation.
The micrographs shown in figure 5.1 shows the in situ fluorescence imaging of CBMs directed to the intact surfaces of FM966 and Cooker lines of cotton fibres after NaOH mercerization treatments. Four specific CBMs were used to visualise the crystallinity changes seen with the increasing NaOH concentrations (0.0-8.0 mol dm-3) for both fibre lines, shown in Figure 5.2. With increasing alkali treatments, the binding intensities of CBM2a and CBM3a, which bind to crystalline cellulose, were shown to decrease. This can be correlated with a decrease in the cellulose crystallinity of the cotton fibres. Additionally, the significant decrease in binding intensity of CBM2a and CBM3a in both cooker and FM966 lines were seen occurring between 1.0 mol dm-3 and 2.0 mol dm-3 NaOH. In contrast, CBM17 and CBM4-1, which are directed to amorphous cellulose on intact fibres showed an opposite effect. With increasing NaOH
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mercerization treatments, the binding intensity of CBM17 and CBM4-1 also increases, correlating with a decrease in the fibre cellulose crystallinity.
Figure 5.2: In situ fluorescence imaging of four cellulose-directed CBMs (CBM2a, CBM3a, CBM4-1 and CBM17) to FM966 and Cooker cotton fibres after pre-treatments with a series of NaOH concentrations (0.0−8.0 mol dm-3).
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The observations shown in figure 5.2 are qualitative, so using Image J; quantitative data sets were generated from the CBM micrographs for comparison with the ATR-FTIR data. Figure 5.3 shows the quantitative changes in CBM binding intensities after increasing NaOH concentrations for both fibre lines; cooker and FM966. In Figure 5.3a and Figure 5.3b, the changes in the lateral order index of cotton fibres using ATR-FTIR were shown after increasing NaOH concentrations. The most significant changes in the LOI occurred between 2.0 mol dm-3 and 4.0 mol dm-3 NaOH treatments, which correlate with the cellulose I transformation to cellulose II. Post 4.0 mol dm-3 NaOH, no further changes in the cotton cellulose were seen, most likely because cellulose II is the most stable and energetically favourable class of cellulose. For the crystalline cellulose-binding CBM3a and CBM2a (Figure 5.3a & Figure 5.3b) the observed changes were seen up to 2.0 mol dm-3 NaOH less than was seen with ATR-FTIR. With CBMs, a steeper decrease in crystallinity was also seen. In Figure 5.3c and Figure 5.3d, the HBI changes seen with increasing NaOH concentrations were compared with the intensity readings for CBM17 and CBM4-1. The significant changes in cellulose HBI were observed between 3.0 mol dm-3 and 4.0 mol dm-3 NaOH. This is correlated with the increase in hydrogen bonds, yet a decrease in crystallinity is seen through the conversion of cellulose I to cellulose II. In comparison, amorphous cellulose directed CBM17 and CBM4-1 (Figure 5.3c & Figure 5.3d) had very similar binding intensities for both FM966 and cooker fibre lines with the increasing NaOH treatments. The binding intensities steadily increased along with the NaOH concentration, especially between 2.0 mol dm-3 and 6.0 mol dm-3 NaOH. In comparison to the ATR-FTIR readings, this data shows that using
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CBMs visualises a less sudden transition in the generation of amorphous cellulose II.
Figure 5.3: Quantification of fluorescence micrographs and ATR-FTIR analyses in response to NaOH treatments. (a) CBM2a and CBM3a directed to crystalline regions of Cooker cotton fibres with increasing NaOH concentration in comparison with LOI (ATR-FTIR). (b) CBM2a and CBM3a directed to crystalline regions of FM966 cotton fibres with increasing NaOH concentration in comparison with LOI (ATR-FTIR). (c) CBM4-1 and CBM17 directed to amorphous regions of Cooker cotton fibres with increasing NaOH concentration in comparison with HBI (ATR-FTIR). (d) CBM4-1 and CBM17 directed to amorphous regions of Cooker cotton fibres with increasing NaOH concentration in comparison with HBI (ATR-FTIR). Error bars show plus and minus of standard deviation of fluorescence quantification. Produced in collaboration with Alenka Kljun (Kljun et al., 2011).
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As previously mentioned, X-ray diffraction is a non-destructive analytical technique that can reveal information about the crystallinity, chemical composition and physical properties of cellulose rich materials, such as cotton fibres. The process is based on measuring the scattering of X-ray beams after they hit a fibre sample, taking into account the scatter angle, wave polarization and energy (Segal et al., 1959).
The binding of CBM3a and CBM2a directed to crystalline cellulose, was compared with the CrI values calculated from the X-ray diffraction readings, shown in Figure 5.4. The CrI changes observed with increasing NaOH mercerisation treatments roughly followed the results from the ATR-FTIR crystallinity analysis.
Figure 5.4: Quantification of cellulose crystallinity using carbohydrate-binding module labelling to cotton fibres and X-Ray diffraction analyses in response to NaOH treatments: (a) CBM2a and CBM3a directed to crystalline regions of Cooker cotton fibres with increasing NaOH concentration in comparison with X- Ray diffraction. (b) CBM2a and CBM3a directed to crystalline regions of FM966 cotton fibres with increasing NaOH concentration in comparison with X-Ray diffraction. Produced in collaboration with Alenka Kljun (Kljun et al., 2011)
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5.3 Industrial pre-treatments on cotton fibre
During processing, cotton fibres undergo scouring and mercerisation treatments to homogenise the structural properties and reactivity to dyeing processes. In Figure 5.5, mature scoured and mercerized FM966 fibres were stained with the β-glycan binding stain, Calcofluor white (Hughes and Mccully, 1975). As previously mentioned, the mature fibre primary cell wall is divided into two layers which differ in their composition on each surface. The outer surface is mostly made up of waxes and pectins, specifically HG, which masks the majority of epitopes of the underlying cellulose and hemicelluloses (Vaughn & Turley 1999). This can be seen in Figure 5.5 (UNTR) in which the fibre is untreated and as a result dyes very poorly with Calcofluor white. The scouring process (Figure 5.5 SCR) removes most of the waxes and pectins of the primary cell wall which allows a greater amount of access to the cellulose by the dye, hence a more intense binding was seen. Finally, the mercerized mature fibres (Figure 5.5 MERC) have a lower overall crystallinity and more cellulose II than the untreated fibre, as shown by the previous ATR-FTIR, CBM and X-ray diffraction results in this chapter.
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Figure 5.5: Industrial pre-treatments and the effect on Calcofluor fluorescence cellulose staining. Scale 50 µm.
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5.4 Comparative analysis of crystallinity changes in cellulose II