Scanning Electron Microscopy

A scanning electron microscope (SEM) is an electron microscope that images a sample by scanning it with a beam of electrons in a raster scan pattern. The electrons bombard the atoms of the sample and the signal produced reflects information about the sample’s surface topography, composition and other prop­erties such as electrical conductivity.

For SEM analysis of grass biomass samples which were subjected to various mild acid pre-treatments as described earlier (a study done by the authors A. O’Donovan and V. K. Gupta), the treated grass biomass residues were oven dried at 60 °C and adhered onto a stainless steel specimen holder called a specimen stub with the aid of an adhesive carbon tab. As grass biomass is non-conductive it is coated with an ultrathin coating of electrically conducting material, deposited on the sample either by low-vacuum sputter coating or by high-vacuum evaporation. Non-conductive specimens tend to charge when scanned by the electron beam, and especially in secondary electron imaging mode, this causes scanning faults and other image artefacts. In the below images, the grass biomass was gold coated using a gold EM Scope SC500 Au coater but materials such as gold/palladium alloy, platinum, osmium, iridium, tungsten, chromium and graphite can also be used. The biomass must be electrically conductive, at least at the surface, and electrically grounded to prevent the accumulation of electrostatic charge at the surface. For additional information on SEM analysis refer to a review available on the Internet at http://serc. carleton. edu/research_education/geochemsheets/techniques/SEM. html. This online review also refers to the literature that further explores SEM.

SEM analysis is a useful tool to examine the effects of pre-treatments and enzymatic hydrolysis on the structure of the plant cell wall and has been used by several researchers for this purpose (Gomez et al. 2008; Jieben et al. 2011).

The gold coated biomass samples were analysed using a Hitachi S-570 SEM and suitably magnified images were recorded. Dilute acid pretreatment may affect biomass structure by solubilising or altering hemicelluloses, altering lignin struc­ture and increasing the available surface area and pore volume of the substrate. The effects of various mild acid pretreatments are shown in images A to N, Fig. 4.10.

Images A and B show untreated grass. It is clear that the cells are well struc­tured the fibres that make up the structure of the grass are connected very tightly. After treatment with just water (hot liquid pretreatment), the cells are generally still well structured and the fibres are still tightly connected (images C & D). After acid hydrolysis, the SEM images show the grass biomass has been affected by all acid pretreatments. In images E, F, G, H, K and L, the cells seem less structured and organised and the fibres seem less tightly connected. However, treatment with nitric acid seems to have had a very destructive effect on the grass biomass. This would correlate with the results of Fig. 4.9 which shows greatest sugar release was achieved by treatment with nitric acid. Image I shows how the grass fibres have completely come apart and in image J areas of the structure have become weak­ened, with pores starting to appear. Treatment with sulphuric acid also seems to

Fig. 4.10 Continued

Fig. 4.10 The effects of various mild acid pretreatments are shown in images (A-N). The grass biomass was pretreated with a range of acids of different concentrations (0.5 and 2 %) at 10 % solids loading. The treatment conditions were 121 °C for 30 min. The biomass residue was separated from the pretreatment hydrolysate, oven dried and gold plated before SEM analysis

have been a very effective pretreatment as it is clear some cell tissues have been destroyed and very definite pores have appeared in the grass structure (images M and N). The destruction of the grass structure shown in these images may be attributed to the preferential degradation of the labile components such as hemi — celluloses and acid soluble lignin.

Pre-treating grass biomass with dilute acid is a favorable process as it helps remove the hemicelluloses fraction and disrupt the grass structure which allows greater accessibility for the cellulase enzymes. It may also help lead to less hemicelluloses and lignin content in the cellulose preparation for the acid hydrolysed perennial rye grass.

4.3 Conclusion

The effect of pretreatments is very dependent on the biomass composition. This chapter focused on reviewing in detail the composition of the grass plant cell wall. Mild acid pre-treatments were reviewed and several mild acid pretreatment con­ditions were tested. The resulting acid hydrolysate sugar yields were noted and the pretreated biomass residues were subjected to SEM analysis to take a closer look at the effects of acid pretreatments, specifically on the plant cell wall. These pre­treatment processes should make the lignocellulosic biomass more susceptible to enzymatic attack, where crystallinity of cellulose, its accessible surface area and protection by lignin and hemicellulose are the main factors in order to obtain an efficient hydrolysis.

Acknowledgments I would like to acknowledge Pierce Lalor and the Centre for Microscopy and Imaging at the National University of Ireland Galway for the use of and assistance with a Scanning Electron Microscope

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Part III

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