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Sago hampas is an example of an agricultural waste that has been widely utilised as animal feed, compost for mushroom culture, for particleboard manufacture and for hydrolysis to confectioners’ syrup ( (Singhal et al., 2008). Apart from that, the hampas which contains starch and lignocellulose, has also shown its capability to be converted into sugar through enzymatic and acid hydrolysis (Awg-Adeni et al., 2010). These sugars can be further fermented and converted into value added products such as bioethanol.
The problems associated with natural lignocellulose that are directly hydrolysed by enzymes are the inefficient enzymatic hydrolysis which leads to low sugar yield. This is due to the nature of the lignocellulosic structure in the sago hampas. This low yield directly affects the cost of production where it is not cost effective as high amount of substrate is used but low amount of sugar is produced. Therefore, the raw material requires some form of pretreatment to open up the structure of lignocellulosics as well as starch to ensure efficient enzymatic and acidic hydrolysis is accomplished.
The beneficial effects of pretreatment of lignocellulosic materials have been recognized for a long time. The goal of the pretreatment process is to remove lignin and hemicellulose, reduce the crystallinity of cellulose, and increase the porosity of the lignocellulosic materials (Kumar et al., 2009). Pretreatment must meet the following requirements: (1) improve the formation of sugars or the ability to subsequently form sugars by hydrolysis, (2) avoid the degradation or loss of carbohydrate, (3) avoid the formation of by-products that are inhibitory to the subsequent hydrolysis and fermentation processes, and (4) be cost-effective (Kumar et al., 2009).
Steam pretreatment (SP) is the most extensively studied and commonly applied physicochemical method of biomass pretreatment where the major chemical and physical changes to lignocellulosic biomass are often attributed to the removal of hemicellulose. This improves the accessibility of the enzymes to the cellulose fibrils.
The order of the SP (before or after enzymatic hydrolysis on starch) in this experiment will be compared in order to determine whether it plays a significant role in producing a higher sugar yield. The experiment will be divided into two, where the first experiment will be done by conducting enzymatic hydrolysis of the sago hampas first before it undergoes simultaneous SP and acid hydrolysis, while the second experiment will be done with the sago hampas undergoing simultaneous SP and acid hydrolysis followed by enzymatic hydrolysis. The parameter involved in both the experiment that would be manipulated to determine the optimum condition is the substrate load which is varied at 7%, 10% and 15%.
The objectives for this experiment includes:
- To determine the optimum parameters for SP of sago hampas
- To analyse the sugar production upon completing SP
- To observe the change of the microstructure of sago hampas before and after the SP
2.0 Literature Review
2.1 Sago Hampas
Sago ‘hampas’ is an inexpensive, starchy lignocellulosic, copious fibrous residue left behind after most of the starch has been washed out of the rasped pith of the Metroxylon sagu (sago palm) (Singhal et al., 2008). On a dry basis, sago hampas contains 58% starch, 23% cellulose, 9.2% hemicellulose, and 4% lignin (Awg-Adeni et al., 2013).
Starch is a water insoluble granule that compose the major reserve of polysaccharide in higher plants (Dumitriu, 2005). Starch comprises of two essential polysaccharides, amylopectin and amylose. Both polysaccharides are formed based on chains of 1→4 linked α-D-glucose where amylopectin is extremely branched consisting on average one branch point which is 1→4→6 linked for every 20-25 straight chain remnants, while amylose is significantly linear (Dumitriu, 2005).
Cellulose is found in an organized fibrous structure where it is the main component of plant cell wall which confers the structural support for the cell (Agbor et al., 2011). β-(1,4)-glycosidic bonds link the D-glucose subunits to each other and thus making up the linear polymer while the long-chain cellulose polymers are joined together by van der Waals and hydrogen bonds. This causes the cellulose to be packed into microfibrils which is covered by hemicelluloses and lignin (Kumar et al., 2009).
Hemicellulose is a branched, heterogeneous polymer of hexoses (galactose, glucose, mannose), pentoses (arabinose, xylose), and acetylated sugars (Agbor et al., 2011). The branches consist of short lateral chains which is easily hydrolysed and they have a lower molecular weight when compared to cellulose (Agbor et al., 2011). The backbone of hemicellulose is either a heteropolymer or a homopolymer where the short branches are connected by β-(1, 4)-glycosidic bonds and sometimes β-(1, 3)-glycosidic bonds (Kumar et al., 2009).
Lignin is a multiplex, large molecular construction comprising of cross-linked polymers of phenolic monomers which is available in primary cell wall, conferring structural support, resistance against microbial attack, and impermeability (Kumar et al., 2009). It is insoluble in water and due to its close relationship with cellulose microfibrils, lignin has been recognized as a main inhibitor to microbial and enzymatic hydrolysis of lignocellulosic biomass (Agbor et al., 2011).
2.2 Pretreatment of Lignocellulosic Biomass
In theory, the ideal pretreatment activity generates a disrupted, hydrated substrate that is smoothly hydrolysed but prevents the generation of fermentation inhibitor and sugar deterioration products (Agbor et al., 2011). Pretreatment alter the construction of cellulosic biomass to cause cellulose in the plant fibres to be exposed and more accessible. Pretreatment process can be roughly divided into different categories; physical, physicochemical, chemical, biological, electrical, or a combination of these (Kumar et al., 2009).
2.2.1 Steaming pretreatment
Steam pretreatment (SP) or steam explosion is the most extensively studied and commonly applied physicochemical method of biomass pretreatment (Agbor et al., 2011). The major chemical and physical changes to lignocellulosic biomass by SP are often attributed to the removal of hemicellulose. This improves the accessibility of the enzymes to the cellulose fibrils (Mosier et al., 2005). In this method, biomass is treated with high-pressure saturated steam, typically initiated at a temperature of 160-260 °C (corresponding pressure, 0.69-4.83 MPa) for several seconds to a few minutes, and then the pressure is suddenly reduced, which makes the materials undergo an explosive decompression (Kumar et al., 2009). The biomass/steam mixture is held for a period of time to promote hemicellulose hydrolysis, and the process is terminated by an explosive decompression. The process causes hemicellulose degradation and lignin transformation due to high temperature, thus increasing the potential of cellulose hydrolysis (Kumar et al., 2009).
2.3 Sugar Production from Hydrolysis
Acids or enzymes can be used to break down the cellulose into its constituent sugars. Enzyme hydrolysis is widely used to break down cellulose and starch into its constituent sugars while acid hydrolysis hydrolyses hemicellulose to xylose and other sugars (Yang et al., 2011).
2.3.1 Enzymatic Hydrolysis
Amylases and glucoamylases are common enzymes used for enzymatic hydrolysis of starch while cellulase and β-glucosidase are usually used for enzymatic hydrolysis of cellulose (Ramos, 2003). Successful enzymatic hydrolysis of cellulosic remnants has been achieved using extremely specific enzymes, however saccharification rate of raw materials that are untreated are generally less than 10% (Chen, 2014). Therefore, efficient enzymatic hydrolysis requires some form of pretreatment to open up the structure of lignocellulosics as well as starch to enhance its efficiency and rate of hydrolysis (Ramos, 2003).
2.3.2 Acid Hydrolysis
Acid accelerates autohydrolysis of lignocellulose raw materials (Chen, 2014). It can be used either as the actual method of hydrolysing to fermentable sugars, or as a pretreatment of lignocellulose for enzymatic hydrolysis (Taherzadeh & Karimi, 2008). Enzymatic hydrolysis of lignocellulosic materials can be improved efficiently with the treatment of acid at high temperature (Taherzadeh & Karimi, 2008). Dilute acid hydrolysis has been investigated using a wide range of catalysts such as hydrogen fluoride, sulphuric acid, nitric acid, and hydrochloric acid (Ramos, 2003). Dilute sulphuric acid treatment of lignocellulosic raw materials hydrolyses hemicellulose to xylose and other sugars and thus improves the digestion of cellulose in residual solids (Chen, 2014). The acid pretreatment can operate either under a low temperature and high acid concentration (concentrated-acid pretreatment) or under a high temperature and low acid concentration (dilute-acid pretreatment). The lower operating temperature in concentrated-acid pretreatment (e.g. 40 °C) is a clear advantage compared to dilute-acid processes. However, high acid concentration (e.g. 30-70%) in the concentrated-acid process makes it extremely corrosive and dangerous (Taherzadeh & Karimi, 2008). At an elevated temperature (e.g. 140-190 °C) and low concentration of acid (e.g. 0.1- 1% sulphuric acid), the dilute-acid treatment can accomplish high reaction rates and improve hydrolysis of cellulose significantly (Taherzadeh & Karimi, 2008). Almost 100% removal of hemicellulose is possible through dilute-acid pretreatment. The pretreatment can disrupt lignin and increases the cellulose’s susceptibility to enzymatic hydrolysis but it is not effective in dissolving lignin (Taherzadeh & Karimi, 2008).