Recently, the bioconversion of renewable lignocellulosic biomass into fuels and chemicals as an alternative to petroleum is gaining importance due

to the realization of diminishing natural oil and gas resources. Lignocellulose into biofuels and chemicals is the most feasible conversion route strategy in terms of sustainability. In general, lignocellulosic biomass is composed of cellulose, hemicellulose and lignin. Approximately, 70% of dry mass of lignocellulosic materials are present as cellulose and hemicellulose. They are polysaccharides of the required fermentable sugars for almost fermentation process by microorganism into fuels and chemicals.

Currently more than 46 900 km2 of oil palm are cultivated in Malaysia, the worlds largest exporter of palm oil [1]. As one of the biggest exporters of palm oil and palm oil products, palm oil industries in Malaysia generate huge quantities of biomass in the form of oil palm empty fruit bunch (EFB), oil palm shells (OPS) and oil palm fibers (OPF). The potential of these are yet to be exploited. Out of these biomass, the EFB generated during processing of palm oil, can be considered as primary feedstock for the production of sugars which can be further used as carbon source for biological conversion through fermentation.

Efficient pretreatment method can significantly enhance the hydrolysis of biomass into fermentable sugars. Therefore, a detailed knowledge of pretreatment is required in order to investigate on the potential utilization of EFB as a substrate for sugars production. The structure of EFB needs to be examined to facilitate optimal utilization of this bioresource for biofuels and chemicals production.

Steam has the potential to degrade (thereby pretreat) the complex structure of lignocellulosic biomass. In steam pretreatment, the biomass is simultaneously treated at high

pressures and high temperatures steam of 140 C to 260 C, for few to several minutes. Steam pretreatment has been reported to be efficient in partially hydrolyzing hemicelluloses, modifying the lignin, increasing access to surface area, decreasing the crystallinity of cellulose and its degree of polymerization [5]. Steam pretreatment on EFB for sugars production is an economic option that can be implemented in the palm oil mill. During palm oil processing, steam is continuously being generated in the mill for electricity and sterilizing the fruits. The boilers produce superheated steam which is used to generate electricity through turbine generator. The low-pressure steam (140 C, 0.28 MPa) is used for heating purposes in the factory. Every year, palm oil mills produce 50 Gkg of OPS and OPF; but only 60 % are used as solid fuel for steam boilers [10]. This amount is sufficient to support the application of steam for the pretreatment of EFB in the palm oil mill. Overall, this pretreatment is attractive to be practiced in the palm oil mill as it include renewable resources (water, OPS and OPF), as all these could be considered as inexpensive resources and is readily available in the mill.

In this study, a pretreatment step is intended to open up the biomass (EFB) for enzymatic attack into sugars from the hemicellulose and cellulose breakdown components. Saturated steam pretreatment or commonly recognized as autohydrolysis is the process of converting lignocellulose into sugars by an exposure to the high temperature and pressure of steam with no addition of external catalysts. Lignin and other components not converted can be burned to provide the heat and electricity that needed to run the overall process involved (Wyman 2007) or to be use in the palm oil mill production. On the other hand, lignin could also be potentially used as a substrate for highly value added product of lignophenol production. Therefore, the pretreatment used is a cost-effective pretreatment method strategy which employed an excess steam from the mill to pretreat (autohydrolyse) the EFB as feasibility

study on EFB. Chemical composition, sugars concentration and structural changes were determined in order to assess the effect of steam pretreatment on the different types of EFB biomass.

A preliminary study on the different substrates and enzymes concentration together with varied temperature and pH is evaluated using common commercial cellulase enzyme, Cellulclast by Novozymes for enhancement of sugars yield. Three pretreatment conditions were chose purposely to study the effects of different parameters tested in saccharification. Later experiments were done to increase the temperatures and pressures of pretreatment for enhancement of saccharification. In this study, varied of pretreatment time is used in order to investigate the best saturated pretreatment severity factor on sugars enhancement with minor loses of polysaccharides components. The best steam pretreated EFB was then be evaluated using the scanning electron micrography, fiber analysis, FTIR spectroscopy, X-ray diffractogram, BET and thermal analysis. While the steam condensate was determined by GCMS, HPLC and colorimetric method in estimating the value of degradation products. A proposed energy balances based on mass balances calculation for the direct separation xylose and glucose from saturated steam pretreatment and saccharification is described in detail.

Since some researchers have claimed that biomass pretreatment as the second most expensive unit cost [11].

Cellulose, a principle component of all plant materials, is considered one of the most abundant renewable resources in the world. Cellulose is made of linked glucose molecules connected by 1, 4 glycosidic bonds. Hemicellulose is a complex, heterogeneous mixture of sugars and sugar derivatives that form a highly branched network. The monomers that comprise hemicelluloses are hexoses (glucose, galactose and mannose) and pentoses (arabinose and xylose). Hemicellulose can be classified into three groups, namely xylans, mannans and galactans based on the polymer backbone that is very often homopolymeric with 1, 4 linkage. Cellulosic materials also contain lignin, a three dimensional phenylpropane polymer with phenylpropane units held together by ether and carbon-carbon bonds (Sun, Y 2002).

The structures of the lignocellulosic biomass, especially cellulose crystallinity, the sheathing of hemicelluloses, and the lignin barrier, make it more recalcitrant enzymatic hydrolysis compared to corn starch. Mechanical or chemical pretreatment is used to break down the hemicelluloses and lignin structures in order to improve the substrate digestibility (Sun, Y 2002).

Cellulose is a polymer of glucose, a 6-carbon sugar. Hemicellulose is consists of a mixture of 5-carbon and 6-carbon sugar such as xylose, mannose, glucose, arabinose, galactose and uronic acids. Lignin is a phenolic polymer that makes up the bulk of the remaining dry mass and cannot be utilized by ethanol fermenting microorganisms (Greer 2005).

The varied raw materials used in manufacture of ethanol via fermentation are classified into three main types of raw materials. There are sugars (from sugarcane, sugar beets, molasses and fruits), starches (from corn, cassava, potatoes and root crops) and lignocellulosic biomass (from wood, agricultural residues and paper mills). Starches and cellulose must first be hydrolyzed to fermentable sugars by the action of enzymes or mineral acids (Lin & Tanaka 2006).

2.2

LIGNOCELLULOSIC BIOMASS

Lignocellulosic biomass is principally composed of the compounds cellulose, hemicelluloses and lignin.

The African oil palm, Elaeis guineensis is used as commercial agriculture in the production of palm oil in Malaysia (Ibrahim 2006). At present, Malaysia is the largest producer and exporter of oil palm produced about 51% of all oil palm obtained in the world (Rodrguez et al 2007). However, palm residues are largely wasted. Palm oil production generates 8.8 106 t y-1 of empty fruit bunches, 5.4 106 t y-1 of fibre and 2.3 106 t y-1 of shell (Husain et al 2002). Oil palm empty fruit bunch (OPEFB) is the major component of all solid wastes at the mill. According to Kittikun (2002), in one ton of fresh fruit bunches (FFB) composed of 230-250 kg of empty fruit bunch. Recently, it was reported that, 15 million tons

of OPEFB waste is generated annually in Malaysia by oil palm mills. In the past, OPEFB often be used as fuel to generate steam at the mills which creates environmental pollution problems in nearby localities (Rahman et al 2007). However, burning is now prohibited by regulations to prevent air pollution. Therefore nowadays, OPEFB can be used as an alternative raw material to produce bioethanol due to it contains of cellulose and hemicellulose that can be degraded into fermentable sugars through enzymatic hydrolysis. The OPEFB has a fibrous structure and the fibers stick together to form vascular bundles. The chemical composition of OPEFB is shown in Table 1 (% dry weight, w/w). The conversion of lignocellulosic biomass to ethanol is more challenging than regular food biomass due to the complex structure of the plant cell wall (Rebecca et al 2007). In order to obtain high fermentable sugars, the structure of OPEFB has to be altered or removed using suitable pretreatment method. The pretreatment method is used to facilitate the enzymatic hydrolysis of lignocellulosic materials (hgren et al 2007). It has been noted that heating lignocellulosic materials to high temperature changes its physical and chemical properties. Heat treatment using moist heat or steam on lignocellulosic materials has been applied by many researchers as a pretreatment in lignocellulosic bioethanol production (hgren et al 2007, Linde et al 2007, Viola et al 2007, Negro et al 2003, Kaar et al 1998).

In the palm oil mill, sterilization process on fresh fruit bunch (FFB) has been used to inactivate the natural enzymes, loosen the fruits off the bunch and soften the mesocarp, resulting in easier extraction of oil. Typical steam temperature used in a horizontal direct contact steam sterilizer at the mill is 140oC for 50 minutes (Prasertsan & Prasertsan 1996). In this study, the effect of heat treatment using high pressure steam generated from the boiler in the mill on OPEFB as a pretreatment will be investigated.

In this study, steaming and mechanical treatment might be combined to effectively disrupt the cellulosic structure and make it more susceptible to enzymatic hydrolysis. The conversion of hemicelluloses and cellulose through enzymatic hydrolysis result in the production of pentoses and hexoses sugars. Enzymatic hydrolysis of cellulose to glucose consists of the cellulase adsorption onto the surface of the cellulose, the biodegradation of cellulose to fermentable sugars and the desorption of the cellulose. Enzymatic degradation of cellulose to glucose is generally accomplished by at least three major classes of enzymes: endo-glucanases, exo-glucanases and -glucosidase. These enzymes are usually called together cellulose or cellulolytic enzymes (Wyman 1996). Whereas since hemicellulose contains different sugar units, the hemicellulytic enzymes are more complex and involve at least endo-1,4--D-xylanases, exo-1,4--D-xylosidases, endo-1,4--D-mannanases, -

mannosidases, acetyl xylan esterases, -glucuronidase, -L-arabinofuranosidases and galactosidases (Taherzadeh & Karimi 2007).

Current industrial fermentation organisms are design to only convert glucose to ethanol and other products. In order to utilize lignocellulosic biomass which contains 5- and 6- carbon sugars, the microorganism that can ferment efficiently 5- and 6- carbon sugars must be used and developed. The co-fermentation using single or consotia of yeast is oriented to complete assimilation of all the sugars previously released during the hydrolysis of lignocellulosic biomass. Hence, in this study, the utilizing of local yeast isolates that capable of assimilating both hexoses and pentoses in an optimal way could allow high conversion and ethanol yield.

2.4

BIOMASS PRETREATMENT

The purpose of preatment of lignocellulosic materials is to remove lignin and hemicelluloses, reduce cellulose crystallinity and increase the porosity of the materials. Pretreatment must meet the following requirements: (1) improve the formation of sugars or the ability to subsequently form sugars by enzymatic hydrolysis; (2) avoid the degradation or loss of carbohydrate; (3) avoid the formation of byproducts inhibitory to the subsequent hydrolysis and fermentation processes; be cost-effective. For the pretreatment of lignocellulosics, several physical, physical-chemical, chemical and biological processes have been developed (Snchez & Cardona 2007). The main pretreatment methods reported in literature are shown in Table 2. Methods Procedures Examples of pretreated materials / Remarks References

Table 2: Pretreatment methods of lignocellulosic biomass for fuel ethanol production 2.5 ENZYME HYDROLYSIS

Cellulose can be hydrolysed by the action of cellulases into glucose units. The classical cellulase system includes endoglucanase, exoglucanase and cellobiase (-glucosidase). Endoglucanase attacks more or less randomly at sites within (1-4)--D-glucan chains in amorphous regions of cellulose or at the surface of microfibrils. Exoglucanase releases cellobiose from non-reducing ends of -D-glucan chains. Cellobiase hydrolyzes cellobiose and water-soluble cellodextrins to glucose (Lee, J 1997).

2.6

SACCHARIFICATION

OR

FERMENTATION

OF

BIOMASS

HYDROLYZATES

Once the cellulose and hemicelulose have been broken down to simple sugars, fermentation can then take place (Murphy & McCarthy 2005). The classical configuration employed for fermenting biomass hydrolyzates involves a sequential process where the hydrolysis of cellulose and fermentation are carried out in different units. This configuration is known as separate hydrolysis and fermentation (SHF). In the alternative variant, the simultaneous saccharification and fermentation (SSF), the hydrolysis and fermentation are performed in a single unit (Snchez & Cardona 2007).

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Rahman, S.H.A., Choudhury, J.P. & Ahmad, A.L. 2007. Production of xylose from oil palm empty fruit bunch fiber using sulfuric acid. Biochemical Engineering Journal 30:97103. Rani, K.S, Swamy, M.V. & Seenayya, G. 1998. Production of ethanol from various pure and natural cellulosic biomass by Clostridium thermocellum strains SS21 and SS22. Process Biochemistry 33(4):435-440. Srinivas, D., Rao, K.J., Thdore, K. & Panda, T. 1995. Direct conversion of cellulosic material to ethanol by the intergenetic fusant Trichoderma reesei QM 9414/Saccharomyces cerevisiae NCIM 3288. Enzyme and Micobial Technology 17:418-423. Wyman, C.E. 2007. What is (and is not) vital to advancing cellulosic ethanol. Trends in Biotechnology 25(4):153-157. Yu, Z. & Zhang, H. 2003. Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysate with Saccharomyces cerevisiae. Bioresource Technology 90:95-100.