Biological Pretreatment
In biological pretreatment, microorganisms are used to degrade lignin and hemicellulose leaving cellulose, allowing cellulose to undergo facile hydrolysis when exposed to saccharification enzymes. The most common type of microorganisms used in this pretreatment is fungi. In the early 1990s Hatakka et al. [234, 235] reported the selective delignification of wood and wheat straw by selected white-rot fungi such as Phanerochaete chrysosporium, Phlebia radiata, Dichmitus squalens, Rigidosporus lignosus, and Jungua separabil — ima. Lignin depolymerization by these fungi takes weeks to achieve significant results but can be very selective and efficient [234, 235]. White-rot fungi produce extracellular lignin-modifying enzymes, the best characterized of which are laccase (EC 1.10.3.2), lignin peroxidases (EC 1.11.1.7) and manganese peroxidases (EC 1.11.1.7). Lignin biodegradation studies have been carried out mostly using the white-rot fungus Phanerochaete chrysosporium, which produces multiple isoenzymes of lignin peroxidase and manganese peroxidase but does not produce laccase.
Many other white-rot fungi produce laccase in addition to lignin and manganese peroxidases and in varying combinations. Based on the enzyme production patterns, white-rot fungi can be categorized into three groups:
1. Lignin-manganese peroxidase group (e. g., P. chryso — sporium and Phlebia radiata)
2. Manganese peroxidase-laccase group (e. g., Dichomitus squalens and Rigidoporus lignosus)
3. Lignin peroxidase-laccase group (e. g., Phlebia ochraceo — fulva and Junghuhnia separabilima) [234]
When compared to other methods, biological pretreatments are normally conducted at low temperatures and atmospheric pressures without using expensive equipment, chemical reagents, and additional energy for lignin removal and biomass structure destruction. Therefore, it is a green, safe, and inexpensive method. However, the enzymatic reaction rates are slow, therefore long pretreatment times are required compared to other pretreatment methods.
Even though biological pretreatment technique is relatively new, it has been reported for the pretreatment of corn stover [236], rice straw [237-239], beech wood [240], pinus densiflora [241] and eucalyptus globulus [242].
White-rot fungi are mostly used for secreting ligninolytic enzymes in the biological pre-treatment process, and current research related to biological pretreatment is mainly focusing on the following five aspects:
1. Selection of white-rot fungi candidate strains for certain biomass materials.
2. Optimization of cultivation methods for white-rot fungi.
3. Characterization of fungal-treated materials.
4. Mutation breeding and crossbreeding of fungal myce- lia to obtain engineered strains.
5. Integration of fungal pretreatment with simultaneous saccharification and fermentation to produce biofuels, and evaluation of combining bio pretreatment with chemical or physicochemical approaches.
A number of currently available and under-development pretreatment methods are presented in this chapter. All these methods have their own advantages and disadvantages. A comparison of major pretreatment methods is presented in Table 5.10.
It is difficult to define the best pretreatment method as it depends on many factors such as type of lignocellulosic biomass and desired products. Pretreatments must improve the digestibility of lignocel- lulosic materials, and each pretreatment has its own effect on the cellulose, hemicellulose and lignin fractions. Looking at the current industrial activities for the production of ethanol, steam explosion and acid-based pretreatment methods are preferred, and in these processes lignin is left with the substrate and removed after the