34 kDa
3.1 Introduction
Chapter 3
Selection of cellulose and hemicellulose rich substrates and efficient pretreatment process for bioethanol production
cellulose that is high molecular weight polymer consisting of glucose chains rigidly held together as bundles of fibres (Sun and Cheng, 2002). Hemicelluloses (20-40%, w w-1) found in agricultural residues are shorter polymers of various sugars that bind cellulose bundles together (Sun and Cheng, 2002). Lignin (10- 30%, w w-1) present in plant cell wall consists of a three-dimensional polymer of propyl-phenol that is bound to hemicellulose to provide rigidity (Ralph et al., 2001). The structural carbohydrate analysis of various agricultural residues (leafy biomass) showed that the percent fraction of cellulose is maximum followed by hemicellulose and lignin (Sun and Cheng, 2002). Easy and ample availability of leaves from trees of jamun (Syzygium cumini), asoka (Saraca indica), bamboo (Bambusa dendrocalamus), poplar (Populus nigra) and eucalyptus (Eucalyptus marginata) and weeds like wild grass (Achnatherum hymenoides) and water hyacinth (Eichhornia crassipes) in northern India envisaged interest in exploiting these as substrates for making and retrieval of many valuable products such as bioethanol (Das et al., 2012).
Albeit lignocellulose being the most abundant renewable resource available, its rigid structure and crystalline nature prevents it from its efficient utilization for hydrolysis (Sun and Cheng, 2002). An effective pretreatment strategy is necessary for the liberation of the cellulose and hemicellulose from the lignin seal so as to render it accessible for a subsequent hydrolysis. To date, a fair number of readily available pre- treatment techniques are reported in literature (Barrett et al., 2009). Physical pretreatment, often called size reduction breaks down the substrate physically.
Chemical pretreatment disrupts chemical bonds aiding in enhanced enzymatic attack to the carbohydrate polymers (Barrett et al., 2009). Steam explosion is a prompt
discharged to a vessel operated at lower pressure (Sharma et al., 2007). Alkali pretreatment refers to the application of alkaline solutions such as sodium hydroxide and calcium hydroxide to remove lignin and a part of the hemicellulose and efficiently increase the accessibility of enzyme to the cellulose (Okeke and Obi, 1995). Wet oxidation separates the cellulosic fraction from lignin and hemicellulose (Palonen et al., 2003). Phosphoric acid (H3PO4) – acetone pretreatment of lignocellulosic biomass can effectively remove hemicellulose, producing C5 sugar monomers in the liquid (Li et al., 2009). Pretreatment by ammonia fibre expansion (AFEX) method has the advantage over others that it does not produce inhibitors for the downstream processes at high temperature (> 90°C) and pH (< 12.0), which, minimizes the formation of degraded products from sugar resulting in higher yields (Holtzapple et al., 1991). Organosolv pretreatment using an organic or aqueous organic solvent decomposes the network of lignin and possibly a part of the hemicellulose (Geng et al., 2012). The pH controlled hot water and dual step dual temperature (DSDT) mild acid hydrolysis pretreatment under high pressure can penetrate into the biomass, hydrate cellulose, and remove hemicellulose and part of lignin (Mosier et al., 2005; Bosch et al., 2010). Microwave assisted alkali (MAA) pretreatment loosens cellulose more effectively than hemicellulose and lignin (Zhu et al., 2006).
Electron microscopy and Fourier transform infrared (FT-IR) spectroscopy have been used for the analysis of morphological and structural modifications in the biomass after the pretreatment (Nada et al., 1998; Kelly et al., 2004; Rodrigues et al., 2007; Rezende et al., 2011). The hydrolytic activity of cellulases with the maximum release of utilizable sugars is a crucial factor in bioethanol production. The prime
hindrance in the usage of commercial fungal enzymes is, due to its high cost. Also, there is absence of prominent β-glucosidase activity in most of the readily available enzymatic pools, directed towards an efficient saccharification process (Schulein, 1988). The thermophilic Clostridium thermocellum cellulosome displays 50-fold higher specific activity than the corresponding Trichoderma reesei system against crystalline cellulose (Demain et al., 2005). Glycoside hydrolases are a group of enzymes which includes cellulases and hemicellulases. According to CAZy database, glycoside hydrolase family 5 (GH5) exhibits activities of cellulase (EC 3.2.1.4);
licheninase (EC 3.2.1.73), glucan endo-β-(1→6)-glucosidase (EC 3.2.1.75) and cellulose β-(1→4)-cellobiosidase (EC 3.2.1.91), whereas glycoside hydrolase family 43 displays activities of β-(1→3)-xylosidase (EC 3.2.1.-), β-xylosidase (EC 3.2.1.37), α-L-arabinofuranosidase (EC 3.2.1.55), xylanase (EC 3.2.1.8) and arabinanase (EC 3.2.1.99). Saccharomyces cerevisiae possesses the intrinsic ability of utilizing various substrates for ethanol production apart from high ethanol tolerance and endurance to metabolic inhibitions (Casey and Ingledew, 1986). Candida shehatae possesses key enzymes, xylitol dehydrogenase and xylose reductase to metabolize pentose sugars for ethanol production by pentose phosphate pathway (Kadam and Schimdt, 1997). In simultaneous saccharification and fermentation (SSF) process, the enzymatic hydrolysis of complex polysaccharides and the fermentation of monomeric sugars are performed in a single step whereas in separate hydrolysis and fermentation (SHF) the hydrolysis and fermentation are performed in separate reactors (Hamelinck et al., 2005; Sangkharaket al., 2011).
In the present study, cellulose and hemicellulose rich substrate was selected
structural carbohydrate composition. The efficiency of different pretreatments was evaluated in terms of complex carbohydrate breakdown of lignocellulosic wild grass and water hyacinth. The efficiency of mixed microwave assisted alkali (MAA) with organosolv pretreatment in degradation of wild grass and water hyacinth was confirmed by field emission scanning electron microscopy (FESEM) and FT-IR analyses. The efficacy of recombinant C. thermocellum hydrolytic GH5 cellulase along with S. cerevisiae in terms of bioethanol production was determined by two modes of fermentation, SHF and SSF. Also, the efficiency of C. thermocellum hydrolytic GH43 hemicellulase (α-L-arabinofuranosidase) along with C. shehatae was evaluated by SHF and SSF modes of fermentation.
3.2 Materials and Methods