Chapter 6. Simultaneous Saccharification and Fermentation in shake flask at higher concentration of pretreated wild grass and
1. Quest for alternative energy
1.3 Pretreatment
1.3.1 Physical pretreatment 1.3.1.1 Mechanical size reduction
The first step for ethanol production from agricultural solid wastes is comminution of the selected substrates by means of milling, grinding or chipping.
This improves the surface to volume ratio making the biomass more accessible to chemical or biological treatment and also reduces cellulose crystallinity and improves the efficiency of downstream processing (Cheng and Sun, 2002). Wet milling, dry milling, vibratory ball milling and compression milling are usually done for this purpose. The initial and final particle sizes, moisture content and the nature of waste (hardwood, softwood, fibrous, etc.) determines the power requirements for mechanical comminution of agricultural materials (Talebnia et al., 2010). Albeit size reduction provide better results, very fine particle size may impose negative effects on the subsequent processing such as pretreatment and enzymatic hydrolysis (Bjerre et al., 1996).
1.3.1.2 Pyrolysis
Pyrolysis has also been used for pretreatment of lignocellulosic materials.
When the materials are treated at temperatures greater than 300˚C, cellulose rapidly decomposes to gaseous products and residual char (Kilzer and Broido, 1965;
Shafizadeh and Brad-bury, 1979). The decomposition is much slower with less volatile product formation at lower temperatures. Mild acid hydrolysis (1 N sulphuric acid, 97˚C, 2.5 h) of the residues from pyrolysis pretreatment had resulted in 80–85%
conversion of cellulose to reducing sugars with more than 50% glucose (Fan et al.,
1987). The process can be enhanced in the presence of oxygen (Shafizadeh and Bradbury, 1979). When sodium carbonate is added as a catalyst, the decomposition of pure cellulose occurs at a lower temperature (Shafizadeh and Lai, 1975).
1.3.2 Physicochemical pretreatment 1.3.2.1 Steam explosion
Steam explosion is a promising and simple method of pretreatment which makes biomass more accessible to cellulase attack (Sharma et al., 2007). This method of pretreatment does not employ any catalyst and fractionates the biomass to yield levulinic acid, xylitol and alcohols (Balat et al., 2008). In this method, 60% moisture is maintained in 1 g of biomass (600 µL water for 1 g biomass) which is then heated using high pressure steam (20-50 bar, 160-290ºC) for a few minutes; the reaction is then stopped by sudden decompression to atmospheric pressure (Sharma et al., 2007).
When steam is allowed to expand within the lignocellulosic matrix it separates the individual fibres (Balat et al., 2008). The high recovery of xylose (45-65%) makes the steam-explosion pretreatment economically attractive.
1.3.2.2 Ammonia fibre expansion
Ammonia fibre expansion (AFEX) is an alkaline thermal pretreatment which exposes the lignocellulosic materials to high temperature and pressure followed by rapid pressure release (Mosier et al., 2005). It involves liquid ammonia and steam explosion (Balat et al., 2008). This system exposes the polymers (hemicellulose and cellulose) to enzymatic attack without liberating any sugars. This pretreatment has the drawbacks of being less efficient for biomass containing higher lignin contents (e.g.
softwood, newspaper) as well as of causing solubilisation of only a very small fraction of solid material particularly hemicellulose.
1.3.3 Chemical pretreatment 1.3.3.1 Acid hydrolysis
Concentrated acids such as sulphuric acid (H2SO4) and hydrochloric acid (HCl) have been used to treat lignocellulosic materials. Although they are powerful agents for cellulose hydrolysis, concentrated acids are toxic, corrosive and hazardous and require reactors that are resistant to corrosion. In addition, the concentrated acids must be removed after hydrolysis to make the process economically feasible (Sivers and Zacchi, 1995). Dilute acid hydrolysis has been successfully developed for pretreatment of lignocellulosic materials. The dilute sulphuric acid pretreatment can achieve high reaction rates and significantly improve cellulose hydrolysis (Esteghlalian et al., 1997). At moderate temperature, direct saccharification suffers from low yields because of sugar decomposition. High temperature in dilute acid treatment is favorable for cellulose hydrolysis (McMillan, 1994). Recently developed dilute acid hydrolysis processes use less severe conditions and achieve high xylan to xylose conversion yields. There are primarily two types of dilute acid pretreatment processes: high temperature (T greater than 160˚C), continuous-flow process for low solids loading (5-10% [weight of substrate/weight of reaction mixture]) (Converse et al., 1989), and low temperature (T less than 160˚C), batch process for high solids loading (10–40%) (Cahela et al., 1983; Esteghlalian et al., 1997). Although dilute acid pretreatment can significantly improve the cellulose hydrolysis, its cost is usually
AFEX. A neutralization of pH is necessary for the downstream enzymatic hydrolysis or fermentation processes.
1.3.3.2 Alkaline hydrolysis
Some bases can also be used for pretreatment of lignocellulosic materials and the effect of alkaline pretreatment depends on the lignin content of the materials (Fan et al., 1987). The alkaline hydrolysis is based on the mechanism of saponification of intermolecular ester bonds crosslinking xylan hemicelluloses and other components, as in lignin and other hemicellulose. The porosity of the lignocellulosic materials increases with the removal of the crosslinks (Tarkow and Feist, 1969). Dilute sodium hydroxide (NaOH) treatment of lignocellulosic materials cause swelling, leading to an increase in internal surface area, a decrease in the degree of polymerization, a decrease in crystallinity, separation of structural linkages between lignin and carbohydrates and disruption of the lignin structure (Fan et al., 1987). The digestibility of sodium hydroxide treated hardwood increased from 14% to 55% with the decrease in lignin content from 24-55% to 20% (Millet et al., 1976). However, no effect of dilute NaOH pretreatment was observed for softwoods with lignin content greater than 26% (Millet et al., 1976). Dilute NaOH pretreatment was also effective for the hydrolysis of straws that have relatively low lignin content of 10-18% (Bjerre et al., 1996).
1.3.3.3 Wet oxidation
Wet oxidation is well suited for loosening the surface structures of the lignocellulosic biomass aiding in hemicellulose degradation (Pedarson et al., 2002).
In wet oxidation, the feedstock material is mixed with water (1 L of water per 6 g of biomass) and heated in a high pressure reactor either in the presence of oxygen or nitrogen at temperatures above 120˚C (Martín et al., 2007). This technique facilitates the transfer of partially hydrolysed hemicellulose molecules from solid phase to the liquid phase. Hemicellulose hydrolysis causes the release of sugar oligomers into the broth (Ahring et al., 1999).
1.3.3.4 Organosolv pretreatment
Organic solvent or organosolv pulping processes are alternative methods for the delignification of lignocellulosic materials. This technique uses organic solvent/water mixtures and thus eliminates the need to burn the liquor and allows the isolation of the lignin (by distillation of the organic solvent). Examples of such pretreatment include using 90% formic acid and pressurized carbon dioxide in combination (50% alcohol/water mixture and 50% carbon dioxide) (Cardona et al., 2010). Other various organic solvents which can be used for delignification are methanol, ethanol, acetic acid, performic acid, per-acetic acid and acetone (Zhao et al., 2009).
1.3.4 Biological pretreatment
Biological pretreatment process was performed to prevent the loss of cellulose, by a cellulase-less mutant of Sporotrichum pulverulentum developed for the degradation of lignin in wood chips (Ander and Eriksson, 1977). Hatakka (1983) studied the pretreatment of wheat straw by 19 white-rot fungi and found that 35% of
Similar conversion was obtained in the pretreatment by Phanerochaete sordida 37 and Pycnoporus cinnabarinus 115 in four weeks (Hatakka, 1983). However, further studies illustrated the use of brown-, white-, and soft-rot fungi in the degradation of lignin and hemicellulose in waste materials (Fan et al., 1987). Brown rots mainly attack cellulose, while white and soft rots attack both cellulose and lignin. White-rot fungi are most effective Basidiomycetes for biological pretreatment of lignocellulosic materials (Fan et al., 1987). The white-rot fungus P. chrysosporium produces lignin- degrading enzymes, lignin peroxidases and manganese-dependent peroxidases for the degradation of wood cell walls (Waldner et al., 1988). These enzymes were produced during secondary metabolism in response to carbon or nitrogen limitation (Boominathan and Reddy, 1992). Akin et al., (1995) also reported the improvement in biodegradation of Bermuda grass stems by 29-32% using white-rot fungi Ceriporiopsis subvermispora and 63-77% using Cyathus stercoreus after 6 weeks.
The advantages of biological pretreatment include low energy requirement and mild environmental conditions. However, the rate of hydrolysis in most biological pretreatment processes is very low.