To further decrease the cost of the pretreatment part of any biomass conversion process it is essential that sugar yields are increased (sugar losses are minimized), solids concentrations are as high as possible, and the capital cost of the pretreatment reactors and associated equipment are kept as low as possible. It is also desirable that the pretreatment process increases the ease with which the cellulose in the pretreated biomass can be saccharified so that it can be accomplished with lower cellulase loadings or in a shorter digestion time.

Development and improvement of existing pretreatment processes is the subject of in­tensive research at several institutions in the USA and overseas. One approach that is re­ceiving more attention is the investigation of the effects of pretreatment at a more funda­mental level; increasingly research is looking at the effects of biomass pretreatment at the cellular, ultrastructural and even molecular level of the plant cell wall. The plant cell wall is highly complex at all length scales and especially chemically heterogeneous at the molec­ular level. For example, in nature dozens of glycosyl hydrolases are involved in plant cell wall deconstruction. These glycosyl hydrolases act on cell wall polysaccharides specifically and synergistically; however, the biochemistry of these unique catalytic events, caused by
individual enzymes acting on biomass as well as the consequences of thermal chemical pretreatment on these reactions, remains poorly understood. There is an increasing focus on characterization of biomass’s molecular structure and ultrastructure in order to gain sufficient understanding of the relationships between pretreatment chemistry and enzyme digestibility. There is also a desire to determine the effects that cellulases and other enzymes have on biomass ultrastructure.

Researchers at Michigan State University have been studying the changes that occur at the cellular level in corn stover upon AFEX pretreatment (78). After staining with a lignin — specific dye, safranin, they observed a change in the distribution of lignin in the cells by confocal laser microscopy. Scanning electron micrographs also appeared to indicate a change in distribution of lignin-like compounds and they postulated that the ammonia partially cleaved the lignin resulting in a decrease in its glass transition temperature so that the lignin could be relatively easily mobilized at temperatures close to 100oC. They also saw a 10-30% decrease in the oxygen:carbon ratio by X-ray photoelectron spectroscopy (XPS) at the surface of the cells indicating an increase in carbon-rich species such as lignin. A redistribution of lignin to the surface of plant cell walls caused by pretreatment could have a significant effect on the digestibility of the cellulose.

Similar effects have been observed in dilute acid pretreatment of corn stover. Dilute acid pretreatments are performed at much higher temperatures than AFEX pretreatments (140- 200oC versus about 90oC). The level of lignin redistribution is therefore much higher. In dilute acid pretreatment, droplets are seen to form that appear to be at least lignin derived. The droplets appear in the liquid hydrolyzate, adhere to the surface of the biomass and also collect in spaces between cells (Figure 14.1). Visually, these microscopic changes appear very significant; however, it is not known at this time how significant they are in altering the digestibility or accessibility of the cellulose in the pretreated materials.

The plant cell wall microfibril, the primary target substrate of bioconversion, is believed to consist of a cellulose-elementary-fibril core surrounded by a hemicellulose sheath forming a macromolecular composite (79). In acidic pretreatments hydrolysis and solubilization of the

1.5% H2SO4 ; 140°C

hemicellulose is thought to be the main mechanism by which accessibility of the cellulose to enzymes is increased. Changes in xylan distribution are being studied by probing pretreated materials with xylan-specific monoclonal antibodies. The distribution of xylan in cells and within cell walls can then imaged by binding the antibody with a secondary fluorescent dye (Alexa-488) for confocal laser scanning microscopy (CLSM) or a gold nanoparticle for transmission electron microscopy (TEM). In dilute acid pretreatment of corn stover rind differences in the pattern of fluorescence have been observed that are attributable to changes in the distribution of xylan in the cell wall (Figure 14.2). The cause of this is still being investigated and the effect of differences in xylan distribution on cellulose digestibility is still being interpreted.

It is anticipated that studies such as those described above will lead to significant ad­vances in our understanding of how pretreatment can overcome the natural recalcitrance of biomass and increase the digestibility of biomass in cost-effective pretreatments. An im­proved understanding should then allow for design of improved pretreatments that decrease the cost of converting biomass feedstocks into fermentable monomeric sugars.

Acknowledgment

This work was supported by the US DOE Office of the Biomass Program.