Pretreatments are procedures applied prior to the major depolymerization of the covalent bonds of biomass polysaccharides. Pretreatments are intended to make biomass saccharification proceed toward greater completion, at a higher rate, and with lower enzyme loading. The broad pretreatment classifications are physical, chemical, and biological procedures, with many utilized approaches consisting of a combination of these classes. Here, we provide a brief overview of some of the most commonly employed pretreatment methods. The topic has also been extensively reviewed (Galbe et al. 2007; Hendriks et al. 2009; Agbor et al. 2011).

Physical approaches to pretreatment include chopping, shredding, grinding, and other macroscopic methods to increase biomass surface area and accessibility. A recent review of optimal milling sizes concluded that for herbaceous crops, such as switchgrass, commonly used particle sizes below ~3 mm produce no further saccharification benefits (Vidal et al. 2011). Those authors also noted that results have been variable and that particle sizes have not often been systematically varied (Vidal et al. 2011). Physical approaches also generally include treatments such as heating and increased pressure, which are typically used along with chemical pretreatments. Experiments have also been conducted with various forms of irradiation, such as treatment with microwaves and gamma waves (Agbor et al. 2011).

After chopping, chemical and physiochemical pretreatments are the most common. They can be subdivided based on pH into acidic, basic, and neutral methods (Galbe et al. 2007). These methods include dilute acid, lime, steam, ammonia fiber expansion, and the use of ionic liquids (Agbor et al. 2011). Dilute acid pretreatment is typically carried out with heated sulfuric acid at <4%. To improve cellulose access, dilute acid primarily solubilizes matrix polysaccharides; a downside is the creation of sulfuric acid waste. Lime [Ca(OH)2] treatment achieves dilute base conditions and can proceed effectively at <120°C. This method can remove acetyl groups and partially depolymerize lignin, but requires high amounts of water. Steam pretreatement, also known as steam explosion or autohydrolysis, has been the most commonly studied. The process entails exposing the biomass to pressurized steam for several seconds or minutes at 160 to 240°C between

0. 5 and 5 MPa (Agbor et al. 2011). A related approach is to use liquid hot water under similar conditions. Both of these are mostly targeting increased solubilization of matrix polysaccharides to expose cellulose.

Two promising neutral treatment methods are ammonia fiber expansion (AFEX) and ionic liquid pretreatments. These both have the advantage of being highly effective and requiring relatively low energy inputs. In AFEX, biomass is mixed with liquid ammonia at an approximately 1:1 ratio at 50 to 100°C and elevated (4 MPa) pressure. Upon rapid pressure release the ammonia volatilizes and the biomass, especially of grasses, can be converted to sugars at approaching 90% efficiency (Balan et al. 2009). Drawbacks of AFEX include the cost of ammonia and production of gaseous ammonia as a pollutant. Ionic liquid pretreatment is another highly effective method. Ionic liquids are salts that are liquids below 100°C. Ionic liquids, such as 1-ethyl-3-methylimidazolium acetate, dissolve the cellulose from biomass and also allow very high saccharification rates. Though recyclable, one major drawback is that ionic liquids are relatively costly.

Biological pretreatment refers to the use of lignocellulolytic organisms, such as white rot fungi, to initiate the cell wall depolymerization process. In planta expression of enzymes to create fully or partially "self-pretreating" plants also falls under this heading. These biological approaches will be discussed further below in the context of consolidation of bioprocessing.