Biomass is composed of biological material biology essay


Biomass is composed of biological material of living or recently living organisms and may be considered as a renewable energy source. It includes forestry and agriculture crops and residues, animal and industrial residues, and sewage and municipal solid waste. These sources can be divided into five distinct energy sources; garbage, wood, waste, landfill gases and alcohol fuels; and have long been identified as sustainable sources of renewable energy (Yan et al., 1997; Ong et al., 2011; Goh et al., 2010). Biomass has gained great attention due to its high energy content that may be utilized on a sustainable basis and this energy source has been exploited around the globe. If this bio-energy supply is maintained in a sustainable way, it can contribute positively to the ecological well-being of the local and global environment. Biomass energy can be converted into a large energy source such as electricity (bio-power) and heat or to a small energy source such as a wood stove or small wood-fired furnace (Sumathi et al., 2008). It supplies about 9-13% of global energy use which includes both traditional uses such as cooking and heating or modern use like producing electricity and steam. The conversion of non-edible biomass waste into fuels such ethanol, methanol, bio-oil and bio-diesel has increased significantly and could help to solve the energy shortage that has occurred in many areas worldwide. This biomass has been recognized as a major renewable energy source as it contains the most organic compounds on earth. The current high energy price has become a serious threat to mankind due to the overdependence on fossil fuel as the main source of energy for most countries and communities over the last decade. As the world economy grows, demand on energy increases and this situation has caused governments and authorities to focus on developing biomass as an alternative renewable energy resource (Ong et al., 2011). As an added windfall to the above effort, researchers are churning out a wider variety of by-products by the downstream processing of biomass that has now been developed into a promising future industry. Currently, biomass can be converted to high value commodity chemicals and fuels using the proper technology (Rass-Hansen et al., 2007). Fossil fuel generates green house gas emissions (CO2, CH4, SOX, NOX) that lead to global warming and thereby climate change. Biomass as a renewable energy source can reduce the dependency on fossil fuels and provide significant advantages in terms of carbon dioxide emissions reduction and therefore less greenhouse effect (McKendry, 2002). Biomass is basically carbon, hydrogen and oxygen-based. The principle of producing energy from biomass corresponds directly to photosynthesis where carbon dioxide and water are transformed to oxygen gas and glucose through input of energy from the sun: 6CO2 + 6H2O = C6H12O6 + 6O2Biomass is a CO2 neutral energy source based on the fact that the CO2 released during burning is taken up by growing plants provided that the rate of harvest of wood is equal to the rate of its regrowth. In particular the use of cellulose waste as fuel does not add to the CO2 footprint as it is recycled during plant growth thereby helping alleviate the climate change crises (Escobar et al., 2009). Ethanol is a prime example of biomass alcohol fuel and it is produced from starch, sugar crops and agricultural residues. However, much of current bioethanol is produced from food portion which poses ethical concerns about competition between food and feed supplies. Alternatively, the second generation of feedstock such as agriculture residues is expected to be a new source for bioethanol production. The annual global production of agricultural residue is predominantly produced by paddy straw, wheat straw, corn stover, oil palm residues, bagasse, barley straw, oat straw and sorghum straw as shown in Figure 1. 1 (Kim and Dale, 2004). In order to utilize the large amount of waste, novel technologies with improved efficiencies and reduced environmental impact are needed to be established in time (Yang et al., 2006). In addition, biomass conversion contributes significantly to national economic activities such as job creation. Figure 1: Quantities of lignocelluloses biomass in Africa, Asia, Europe, North America, Central America, Oceania and South America (million tons) (Kim and Dale, 2004).

1. 2Biomass from Oil Palm

Oil palm, Elaeis guineesis, is cultivated in all tropical areas and originates from west and central Africa (Chew and Bathia, 2008). Palm oil is an important part of the agro-industry in Malaysia, Indonesia and Thailand where these countires export over 90% of world’s palm oil. Malaysia is the second largest producer of palm oil for the last 25 years with approximately 40-60% of total world palm oil production replacing Nigeria which was the chief producer before 1971 (Mongabay. com, 2007; Sumathi et al., 2008; Sumiani, 2006). In more than three decades, Malaysia has had the monopoly of being a global leader of this industry and provides more than 1. 4 million jobs (Malaysian Palm Oil Board, 2010; Craven, 2011; Malaysia Innovation Agency, 2011). The oil palm is a tropical palm tree which can be easily cultivated in Malaysia. Malaysia is blessed with favourable weather; climatic condition like being wet and humid is suitable is for planting oil palm trees. Oil palm is the highest yielding oil crop because of the increase in for demand oil and fat. The first commercial oil palm estate in Malaysia was set up in 1917 at Tennamaran Estate in Selangor (Malaysian Palm Oil Council, 2006; United Nations Development Program, 2006). In 1950, only about 38, 000 ha of the land were used for oil palm plantation. Production of a single hectare can produce 10 times more oil than other oilseeds. The fruits from oil palm have many uses such as for extraction of edible oil and for cooking. Oil palm biomass is one of the biggest resources of renewable energy and contributes to about RM6, 379 million of energy annually. There are various forms of wastes from the mills such as empty fruit bunch (EFB), palm pressed fiber (PPF), palm kernel cake (PKC), palm kernel shell (PKS), sludge cake (SC) and palm oil mill effluent (POME) (Sumiani, 2006). The quantity of waste depends on the raw materials and only EFB, PPF and PKS are considered as waste and appear in large quantities. The other waste is used as food for ruminants (cattle and goats) or left to rot for soil conservation, which increases the fertility of soil, controls erosion and provides a source of nutrients for oil palm trees (Sumathi et al., 2008; Palm Oil Industry, 2006; Aspar, 2005). The EFB has traditionally been used as fertilizer or soil conditioner as incinerated ash. However, the moisture content of EFB is 60% higher and this contributes to the ” white smoke” problem (Sumiani, 2006). The DOE discourages the incineration of EFB due to environment pollution. In the year 2005, Malaysia generated 9. 66, 5. 20 and 17. 08 million tons of fiber shell and EFBs, respectively. A fresh fruit bunch produces about 20-30% of EFB and the bulky nature of the EFB causes a high land-fill disposal cost which needs to be burnt into ash. This emits CO2, CO, NO2 and causes air pollution (Prasertsan and Prasertsan, 1996). Oil palm fiber especially EFB is rich in sugar and most research papers show that EFB has been used to produce glucose and xylose successfully (Lim et al., 1997). EFB consists of 42% C, 0. 8% N, 0. 06% P, and 2. 4% K and 0. 2% Mg (Krause, 1994). OPEFB, a by-product of the palm oil industry obtained after pressing the oil from the fruit bunch, is composed mainly of cellulose (41. 3–46. 5%), hemicellulose (25. 3–33. 8%) and lignin (27. 6–32. 5%) (Ariffin et al., 2008; Hamzah et al., 2011; Han et al., 2011; Piarpuzán et al., 2011).

1. 3Biofuel

The unpredictable supply of petroleum and increasing demand of this energy source as well as climate change concern have strengthened worldwide interest in alternatives to replace fossil-fuel from non-petroleum energy sources. The current dependence on fossil fuels as transportation fuel also has a deleterious effect on climate change, energy security and socio-economy. Fuel consumption has increased to almost double from 6, 620 million tons of oil equivalent (Mtoe) in 1980 to 11, 295 Mtoe. In 2008, an International Energy Agency estimated the increase to be 53% by 2030 (Malaysia Innovation Agency, 2011.). As a result, the need to find alternatives to petroleum has never been more urgent. The pursuit for a commercially viable method for the production of biofuel to replace fossil fuel as an energy source continues unabated on account of unpredictable petroleum prices and uncertain supplies, climate change and energy security concerns (Zecca and Chiari, 2010; Wuebbles and Jain, 2011; Escoba et al., 2009; Kim and Dale, 2004). Biofuel is a liquid fuel for the transport sector that is produced from renewable sources such as vegetable oil and biomass (Demirbas, 2007). Biofuel offers availability as it is derived from renewable sources, representing CO2 cycle in combustion, environmental-friendly, biodegradable and is a sustainable form of energy (Puppan, 2002). The difference between petroleum and biofuel is that biofuels are from a non-polluting source and contain higher oxygen content for better combustion and reduce hydrocarbon emission compared to petroleum (Demirbas et al., 2009)

1. 4First Generation Biofuel

The first generation of biofuel is derived from alcohol produced from sugar that is extracted from sugarcane, fruit and palm juice, or starch grain such as potato, rice and wheat through fermentation (Pacini and Silveira, 2011). Biofuel can help to improve domestic energy security (Naik et al., 2010). Its large-scale production has been well- proven and demonstrated successfully in Brazil where almost 50 billion litres were produced annually (Pacini and Silveira, 2011). In 1975, Brazil launched Brazilian Alcohol Program (PROALCOOL), biofuel from sugar cane and this program aimed to reduce petroleum oil imports due to the petroleum crisis (Goldemberg et al., 2004). However, the food-versus-fuel program has been roundly criticized due to rising food prices leading to geopolitical instability.

1. 5Second Generation Biofuel

Malaysia produced a large amount of lignocellulosic waste mainly palm oil waste which makes up the bulk of cheap and abundant non-food material available. The lignocellulosic waste comprises mainly of cellulose, hemicelluloses and lignin, and the conversion of these material to second-generation biofuel offers the opportunity to replace fossil fuels without competing with food-fuel (Goh et al., 2010). However, second generation of biofuel is still non-commercial because of the lack of technology to make this process cost and energy-efficient. The study and research on second generation biofuel show that in order to make the cost of biofuels more comparable with standard petrol and diesel, a more cost- effective route needs to be developed. In addition, a renewable low-carbon energy to be used for road transport clearly needed. Second generation biofuel in the form of cellulosic ethanol could produce 75% less CO2 than normal gasoline, whereas corn, cassava or sugarcane ethanol reduces CO2 levels by just 60% (Patumsawad, 2011).

1. 6National Biomass Policy

The implementation of a renewable energy policy is of strategic importance for many countries as a means to diversify and wean away from the use of fossil fuels such petroleum. Greenhouse gases emission and geo-politics are contributing factors in the effort to enhance energy security (Mohammed et al., 2011). To enhance energy security, the US, European Union countries, ASEAN countries, Brazil and most of the countries in the world have shifted their dependency on fossil fuel to biofuel as an alternative for energy generation. First generation biofuel’s impact on land use, biodiversity, carbon balance and rising food prices have cast a negative perception on the biofuel industry. Second generation biofuel that depends on lignocelluloses feedstock is touted as a saviour to the continuing hopes of the biofuel industry (Naik et al., 2010). The drive for research and development of second generation biofuel is dictated by lower cost and greater abundance of material resources for biofuel production. As the technology for second generation biofuel is yet to mature, research on biofuel production from lignocelluloses biomass continues to show positive growth throughout the world. In Malaysia, petroleum is highly subsidized where subsidies in 2006 totalled US$4. 3 billion and in 2007 totalled US$4. 7 billion. The price for petroleum set by the government is far lower compared to the international price. The integration of biofuel initiative was meant to lower the amount spent on petroleum subsidies (Hashim and Ho, 2011). Countries with abundant lignocelluloses biomass resources such as Malaysia, have taken the lead in developing national biomass strategies. The Malaysian government has been experimenting with biofuel production since 1982 (Craven, 2011). Oil palm cultivation in Malaysia provides an abundant and renewable supply of waste lignocellulosic biomass, a key bioresource that has been identified for exploitation under the National Biomass Strategy’s wealth creation scheme (Malaysia Innovation Agency, 2011; Sorda et al., 2010)The demand for biofuel in the European market was estimated to jump from 3 million tonnes in 2005 to 10 million tonnes by 2010 (Craven, 2011). Biomass Power Generation & Cogeneration Project (Biogenic) was jointly funded by the Government of Malaysia, United Nations Development Program, Global Environment Facility and the Malaysian private sectors to reduce the GHG emission from fossil fuel combustion processes and reduce waste residues from palm oil (PTM, 2004). Currently, the project on new wealth creation from the Malaysian’s palm oil industry predicts that biofuel production from lignocelluloses biomass will be commercially viable between 2013 and 2015. Despite uncertainties and setbacks on the development of the technology to produce the lignocellulosic biofuel, the Malaysian government continues to be optimistic towards the implementation of the second generation biofuel for commercialization. Bio-based chemicals derived from lignocelluloses biomass are also poised for commercialization between 2015 and 2020 with the objectives to increase total biomass production from 80 million tonnes in 2015 to 100 million tonnes with the proportion of products being 18% bio based chemicals, 12% bio fuel, 32% pelletization, 10% wood product and the other 28% bioenergy. This programme will add RM30 billion more to Malaysia’s Gross National Income (GNI) in the year 2020 and create 66000 of job opportunities (Malaysia Innovation Agency, 2011).

1. 7Research Objective

This research is in tandem with current efforts in several parts of the world which focus on discovering new chemical methods for hydrolysing cellulose to sugars in order to ensure a sustainable supply of transportation fuel and avert climate change. The objective of this research is to optimise the parameters of operating conditions for perchloric acid hydrolysis of cellulosic biomass derived fro m oil palm in order to maximize the yield of glucose. The glucose produced will then be subjected to fermentation for ethanol production.

1. 8Research Scope

To investigate the effect of perchloric acid to hydrolyse OPEFB biomass by varying the following parameters: Acid concentrationReaction timeii)To compare the potency of different acids for hydrolysis of OPEFB. iii)To compare yield of ethanol after detoxification. iv)To optimise the combination process of acid hydrolysis parameters in order to obtain highest yield of glucose.

1. 9Organisation of the Study

This thesis is organised into five chapters. Chapter one provides background of the study and discusses the generation of bioethanol and strategy of governments to overcome in uncertainties of petroleum supply. Chapter two reviews the literature on previous study which deals with treatment, hydrolysis and fermentation of lignocellulosic biomass. It involves details in content on lignocellulosic biomass and as well as researchers involved in this area. Chapter three describes the materials and methodology involved in conversion of OPEFB to sugars and ethanol. The analytical method includes comparisons between acids, parameters to hydrolyze OPEFB, detoxification and fermentation process. Chapter four presents results and discussion. The results on hydrolysis between comparative acids and condition parameters of acid are reported. The effectiveness of hydrolysis is compared and the highest yield of glucose is identified. The optimum values for the variables are obtained respectively. Chapter five concludes the study. It presents the conclusion of the objective and recommendation for further improvement of this work in the future.



2. 1Lignocellulose

Lignocelluloses material in plant fiber mainly consists of cellulose and hemicellulose of sugar polymer; and usually accounts for 65-70 percent of the plant dry weight. These polymer sugars mainly consist of D-glucose, D-mannose, D-galactose, D-xylose, L-arabinose, D-glucuronic acid, L-rhamnose and D-fucose. Table 2. 1 shows the sugar content of different plant holocelluloses (Park and Kim, 2012). Table 2. 1: Sugar content of different plant holocelluloses (Park and Kim, 2012)

EucalyptusLarix LeptolepisPinus RigidaRice StrawBarley StrawCellulose41. 843. 443. 139. 135. 9Hemicellulose18. 724. 423. 723. 629. 1Lignin30. 128. 929. 012. 115. 4Others9. 43. 34. 225. 219. 6Lignocellulose can be liberated by hydrolysis for fermented by microorganisms to form different chemicals. Cellulose, hemicellulose, and lignin in plant biomass can be converted to energy resources such as alcohol, methane, furfural and organic acids (Park and Kim 2012).

2. 2Cellulose

Cellulose is a straight chain polymer of D-glucose unit with linkage through several hundred to over ten thousand of β (1→4)-glycosidic bonds (Balat el at., 2008, Rowell et al., 2005). It has hydrophilic properties, insoluble in water and most organic solvents, with the contact angle of 20–30 chiral and is biodegradable. These glucose units can be broken down chemically by high concentrated acids or low concentrated acid at high temperature. Compared with starch, starch consists of D-glucose unit link with α (1→4)-glycosidic bonds, and no coiling or branching occurs. In cellulose, the bond was linked by hydrogen from hydroxyl groups on the glucose with oxygen atoms on the same or on a neighbour chains firmly together side-by-side as shown in Figure 2. 1. This bond makes cellulose results in a high tensile strength, crystalline, strong and difficult to depolymerize (Ga´mez, 2006; Patumsawad, 2011). Cellulose chain length depends on total glucose units that were linked to form polymer such as wood pulp which consist of 300 to 1700 units, of cotton and other plant fibers and a have chain length from 800 to 10, 000 units. Figure 2. 1: The chemical structure of cellulose.

2. 3Hemicellulose

Hemicelluloses are a heteropolymer with matrix polysaccharides in almost all plant cell walls. Hemicellulose has a random structure of branched sugars polymer such as xylose and arabinose. These amorphous polysaccharides make hemicelluloses hydrolyzed easily by dilute acid, base and hemicellulose enzymes. Compared to cellulose it contains only glucose unit, whereas hemicelluloses contains many different pentose sugar monomer which includes xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan as shown in Figure 2. 2. In hemicellulose, xylose is a major D-pentose sugar monomer followed by L-sugars of arabinose. In addition, hemicelluloses also contains small amount of mannuronic acid and galacturonic acid (Gírio et al., 2010). Figure 2. 2: Chemical structure of hemicellulose.

2. 4Lignin

Lignin is an amorphous highly complex aromatic structure polymer connected mainly by three-dimensional cross-linked C-C and C-O-C of p-hydroxyphenylpropanaid units. It is hydrophobic and can be classified by its variable structural element. There are three basic building blocks of precursor alcohol of guaiacyl, syringyl, and p –hydroxyphenyl moieties in plant lignin. The guaiacyl units is consist of 4-hydroxy-3-methoxycinnamyl (coniferyl) alcohol; syringly unit is 3, 5-dimethoxy-4-hydroxycinnamyl (sinapyl) alcohol, and p-hydroxyphenyl unit of p-hydroxycinnamyl (coumaryl) as shown in Figure 2. 3 (Lewis and Yamamoto, 1990, Rencoret et al., 2011; Boerjan et al., 2003; Ralph et al., 2004). Lignins are soluble in base solution which it makes it easy for to be removed from lignocellulose plant biomass. Figure 2. 3: Chemical structure of lignin.

2. 5Process Conversion of Lignocellulosic Biomass

Biomass consists of complex carbohydrates polymers cellulose, hemicellulose and lignin. In lignocelluloses biomass, lignin has a reputation of hindering OPEFB saccharafication into monomeric sugars. The main function of pre-treatment is to break the ether bonds that cross-link lignin and hemicellulose, decrease crystalline of cellulose and increase biomass surface area (Mosier et al., 2005). Enzymes cannot efficiently convert the lignocelluloses biomass due to complex structure of lignocellulose in plant. A variety of pre-treatment methods are applied to lignocelluloses feedstocks in order to enhance the sugar yield for ethanol production. The production of bioethanol from cellulosic biomass generally follows well-established practices: grinding and milling of crop residues to powder, chemical pre-treatment, chemical and/or enzymatic hydrolysis followed by fermentation (Chandel et al., 2007). The treatments include thermal treatment and chemical treatment; such as lime treatment, acid treatment, ammonia fiber explosion (AFEX) and aqueous ammonia recycle (ARP). Each pre-treatment method may have different effect on glucose and xylose yield upon enzymatic and chemical hydrolysis depending on lignocellulosic structure in plant biomass and formation of inhibitory compounds. Pre-treatment or delignification releases lignin from polysaccharides which involves the cleavage of non-phenolic β-O-linkage and phenolic α-O-4 linkage.

2. 5. 1Physical Treatment

The biomass is required to undergo necessary size reduction, in order to make it adequate for further downstream processing. The particle size of the biomass will influence the penetration of acid into solid biomass particle (Najafpour et la., 2007). Practically, physical pre-treatment was conducted by mechanical such as chipping, grinding and explosion. A combination of chipping, grinding and milling in biomass reduces cellulose crystalline for further process. Usually, size of the biomass materials is 10–30 mm after chipping and 0. 2–2 mm after milling or grinding. Compared to ordinary ball milling, vibratory ball milling is more effective for improving digestibility by breaking down the cellulose crystalline of biomass such as spruce and aspen chips (Abasaeed et al., 1991). However, it depends on size and waste biomass characteristics.

2. 5. 2Hydrothermal

Hydrothermal is the process of using liquid hot water (auto hydrolysis) and steam explosion treatment. The process of treatment involves compressing hot air into material at a temperature of between 150-230˚C (Garrote et al., 1999). The reaction takes place when hydronium ions generate in-situ by water auto ionization. Steam explosion is the most commonly used method for pre-treatment to breakdown the structural component by heat in the form of steam and force shear. Initially, the chipped material wetting takes place for several seconds to a few minutes at a temperature of 160–260˚C with pressure 0. 69–4. 83 MPa. After several seconds to few minutes, pressure is released to the atmospheric pressure and biomass structure explodes. The desegregation of inter and intra molecular linkage causes hemicellulose degradation and lignin transformation to cellulose hydrolysis. The efficiency of this process depends on several factors such as the rate of reaction, temperature, size of biomass and moisture content. The process can be achieved by either high temperature of 270˚C for 1 min or lower temperature of 190˚C for 10 min (Gírio et al., 2010; Dale and Moelhman, 2010; Jin and Chen, 2006).

2. 5. 3Alkaline treatment

The alkaline treatment can be done by using alkaline earth metals based agents and ammonia. Most lime treatment use is sodium, calcium and potassium. Alkaline treatment readily removes lignin and increases efficiency of enzyme saccharafication compared to acid treatment (Sun and Cheng, 2002). However, without proper delignification, there will be loss of some of the sugars and the production of bio ethanol is lower (Yadav et al., 2011). Calcium hydroxide and sodium hydroxide are the most common lime used for pre-treatment because they are relatively low-cost and safer reagent compared to other alkalis (Kaar and Holtzapple , 2010; Saha and Cotta, 2008 ). Lime wills saponification the uronic ester linkages in 4-O-methyl-D-glucoronic acids cross along xylan hemicellulose and other polymeric materials (Misson et al., 2009). For example, the alkali treatment with 10 M sodium hydroxide at room temperature efficiently removes lignin from the dilute sulphuric acid-treated EPFB fiber and decreases lignin concentration. Sodium hydroxide treatment also efficiently removes lignin from EPFB fiber where 85. 2 g cellulose and 1. 8 g hemicellulose per 100 g is obtained in treated EPFB fiber (Kim and Kim, 2012). After lime pre-treatment, solid separation step and pH reduction is necessary to reduce the cost of the process (Park and Kim, 2012). Among the chemical pre-treatment methods, alkaline treatment has been proven to effectively remove lignin for conversion by biological lignocelluloses to sugar. It was promising improvement of anaerobic digestion of newspaper, corn stalk, hardwoods, softwood, and paper tubes (Fox et al., 2003; Qingming, 2005; Teghammar et al., 2009; Mirahmadi et al, 2010). Pre-treatment can be successfully used for high concentration or low concentration of NaOH (Mirahmadi et al., 2010). Typically, low concentration of NaOH, ranging between 0. 5-4%, needs a high temperature and pressure to disintegrate lignin and hemicellulose for destruction of lignocellulose (Taherzadeh and Karimi, 2008). However, in this process, no NaOH is reusable and several different inhibitors are formed during the process. In contrast, pre-treatment at high-concentration of NaOH pre-treatment, ranging between 6–20% is the only required process at ambient pressure and relatively low temperature. This process is efficient for the reduction of cellulose crysatillinity (Mirahmadi et al., 2010). In addition, high concentration NaOH pre-treatment is economical due to the possibility of reusing the NaOH solution for environmental impact in the process.

2. 5. 4Ammonia Treatment

The aqueous ammonia treatment reaction mechanism is similar to lime treatment where the biomass swelling and cleave the ether and ester bonds in lignin. Aqueous ammonia can solubilize lignin to 65-85% without degradation of cellulose fraction and loss of glucan. Aqueous ammonia is known to remove lignin and to enhance saccharification and acid hydrolysis of cellulose to sugar for production of ethanol (Jung et al., 2011a; Li and Kim, 2011). The research on optimization pre-treatment condition of aqueous ammonia for EFB on temperature, time, solid to liquid ratio and ammonia concentration by enzymatic digestibility test were studied. Aqueous ammonia with concentration of 21% at 60˚C for 12 h can recover about 78. 3% of glucan and remove 41. 1% of lignin, respectively (Jung et al., 2011b). However, this reagent has hazardous, malodorous and corrosive properties for equipment. In contrast to ammonia fiber explosion (AFEX) process, the concept of AFEX is similar to steam explosion where process need pressure to break lignocellulose structure. Typically, AFEX process was conducted at 90˚C in residence time for 30 min. It was used for the pre-treatment with low lignin content in lignocelluloses materials such as wheat straw, wheat chaff, rice straw (Vlasenko et al., 1997), kenaf newspaper (Holtzapple et al., 1992), switchgrass (Reshamwala et al., 1995), aspen chips and bagasse (Holtzapple et al., 1991). In addition, small particle size of biomass is not required in AFEX pre-treatment compared to other pre-treatment processes (Holtzapple et al., 1990). After pre-treatment, aqueous ammonia can be recycled to reduce the cost and protect the environment. In an ammonia recovery process, a temperature up to 200˚C was used to heat residual ammonia to vaporize and then it was withdrawn from the system by a pressure controller.

2. 5. 5Acid treatment

The common acid treatments have been successfully developed for pre-treatment of lignocelluloses materials by using sulphuric acid in the range of 0. 5-1. 0 %wt at 50˚C. The dilute sulphuric acid is used efficiently to improve cellulose hydrolysis and achieve high reaction rates (Esteghlalian et al., 1997). Temperature and acid concentration play an important role for pre-treatment process. Without proper pre-treatment, direct saccharification can suffer from low yields because of sugar decomposition. 4% (v/v) sulphuric acid treatment extracted almost 50% of the biomass as a soluble fraction. The dilute acid treatment was more effective in extracting the cellulose and hemicellulose fractions than lignin in EPFB fibers. After dilute acid pre-treatment, 12. 1% yield of hemicellulose content remained in the residual biomass and the removal of lignin is achieved by about 87. 9%. Besides that, acid pre-treatment of hydrolyzing hemicelluloses to form xylan and less formation of inhibitor such as furfural is carried out. Less than 1 g/L furfural and 2. 6 g/L acetic acid is regenerated in acid extract solution and other compounds are not detected in the solution depending on process temperature. Formation of high xylan in lignocellulosic materials as a third of the total carbohydrate to xylose is necessary to achieve favourable overall process. In order to remove some toxic compounds from the surface of dilute acid pre-treated EFB fiber, is soaked in water for 1 hour as this weakens and softens the fiber structure for hydrolysis process (Kim and Kim, 2012).

2. 5. 6Hydrolysis

Hydrolysis is a decomposition process in which a chemical bond of compound is broken down by a reaction with water. The hydrolysis of cellulose can be defined as a cellulolysic process while the hydrolysis of cellulose or starch into glucose (sugar) may be defined as a saccharification process. The hydrolysis of cellulose to glucose only occurs at economically viable yields when a catalyst is used. Due to the robust lignocellulosic architecture, the biomass cannot be hydrolyzed easily except under strong conditions (Yunus et al., 2010). Concentrated and dilute acids under appropriate conditions are able to penetrate the lignin-polysaccharide much better and break down cellulose and hemicellulose polymer into monomeric sugars without a pre-treatment process. Lignin has the reputation of hindering OPEFB saccharafication into monomeric sugars (Meunier-Goddik and Penner, 1999). The acid hydrolysis is shown to be more effective in the hydrolysis of crystalline structure of cellulose compared to enzymatic hydrolysis. The reason is because enzyme hydrolysis requires high porosity, specific surface area and a higher degree of hydration due to the stearic hindrance caused by lignin-polysaccharides linkage that limits access in particular fibrocystic enzymes to specific carbohydrates moieties leading to lower yields. In addition, to improving the enzymatic hydrolytic efficiency, the lignin-hemicellulose network has to be loosened for the better amenability of cellulase to residual carbohydrate fraction for sugar recovery. Pre-treatment steps are required to remove lignin to enhance the enzymatic susceptibility of cellulose. Various mineral acids such as dilute and concentrated (Rahman et al., 2007; Chin et., al 2010), hydrochloric acid (Herera et., al 2004), phosphoric acid (Lenihan et al., 2010) and nitric acid (Hamilton et al., 2004; Rodriguez–Chong et al., 2004), have been used for hydrolysis of polysaccharides, performing at varying degree of efficiency. A previous study using phosphoric acid and sulphuric acid for hydrolysis of lignocellulosic and the neutralization of acids points to the advantage of introducing essential elements such as sulphur and phosphorus that is beneficial for microbial growth during the fermentation process (Zhang et al., 2012). However, acid hydrolysis can cause glucose to degrade rapidly and produce less desirable compound such as furfural from dehydration of pentoses and hydroxymethylfurfural from dehydration of hexoses during hydrolysis.

2. 5. 7Dilute Acid Hydrolysis

Dilute acid hydrolysis of biomass has successfully degraded lignocelluloses materials into sugars. Dilute acid hydrolysis is a simple process and no acid recovery is needed. The process needs an optimum condition like high pressure and temperature to break down the cellulose to glucose and to prevent lower yields of sugar from being produced. Compared with concentrated acid, dilute acid is relatively low acid consumption and less corrosion of equipment (Gírio et al., 2010). Usually, dilute acid hydrolysis is conducted at two stages due to the different structure of cellulose and hemicellulose. In the first stage the process needs a milder condition to hydrolyze hemicelluloses followed by the second stage where the process needs a much harsher condition to hydrolyze cellulose. The advantage of dilute acid is to avoid fermentation in inhibitor production such as furan compound, weak carboxylic acids and phenolic compounds.

2. 5. 8Concentrated Acid Hydrolysis

Unlike dilute acid hydrolysis, concentrated acid hydrolysis of lignocelluloses usually yields a near-theoretical sugar value but with fewer degradation products. The most widely used and tested mineral acid for the hydrolysis process is hydrochloric acid, sulphuric acid and also phosphoric acid. The concentrated acid hydrolysis uses relatively mild temperatures, but is conducted at a very high concentration of sulphuric acid and at a minimum pressure (Gírio et al., 2010). This process provides complete and rapid conversion of cellulose to glucose and hemicellulose to xylose with low degree of degradation (Lenihan et al., 2010). High sugar recovery efficiency is the primary advantage of the concentrated acid hydrolysis process. Up to 90 % of cellulose and hemicelluloses are degraded to their sub units after the treatment process. The low temperatures and pressure of the concentrated acid hydrolysis leads to minimised sugar degradation (Chandel et al., 2007). Sugars derived from this hydrolysis process are easily fermented by microorganism.

2. 5. 9Sulphuric Acid

The most commonly used method to hydrolyze the lignocelluloses materials is the sulphuric acid hydrolysis method. Higher concentration of sulphuric acid is usually used in the first stage and is succeeded by a dilution with water in the second stage to hydrolyze and dissolve the substrate. 60 % to 75 % concentrated sulphuric acid is used mostly for the first stage of sulphuric acid hydrolysis while 10 % and 30 % of concentrated sulphuric acid at temperature of 80oC is used in the second stage of the hydrolysis to treat the substrates. Estimation of glucose yield depends on the acid concentration and also the reaction period. Based on the study of sulphuric acid hydrolysis of wood chip, about 80% of sulphuric acid is used to hydrolyze the small grinded mixed wooden chip before it is diluted with distilled water to obtain 26% (w/w) acid. After hydrolization process, filteration is needed to separate insoluble solid from sugar substrate. The pH of filtrate is highly acidic and needs to be neutralizing to around pH 6 to 8 for further fermentation process (Chin et al., 2010). Based on Rahman et al., (2006), the hydrolysis of OPEFB biomass is produced by using 2% diluted sulfuric acid at 120˚C and 31. 1 g/L of xylose from hemicellulose and 4. 0 g/L of glucose. Estimation of sugars yield depends on the acid concentration and also the reaction period. Figure 2. 4, it shows the trend of acid concentration over period of time. As acid concentration increases, xylose concentration release tends to peak much faster but decreases easily if prolonged over a period of time. Figure 2. 4: Effects of sulfuric acid concentration and reaction over time on yield of xylose of OPEFB fiber at 120oC (Rahman et al., 2006). In contrast, concentration of glucose increases over time and hydrolysis due to the structure of cellulose which is more crystalline and complex compared to hemicellulose. It needs a longer period of time to dilute acid to degrade cellulose to glucose as shown in Figure 2. 5Figure 2. 5: Effects of sulfuric acid concentration and reaction over time on yield of glucose of OPEFB fiber at 120oC (Rahman et al., 2006).

2. 5. 10Hydrochloric Acid Hydrolysis

Depending on biomass characterisation, hydrochloric acid (HCl) hydrolysis can be done at low concentration or even at high concentration of HCl. It was reported that low concentration of HCl reduced the sugar concentration which only accounts for 20 g/L and results in 10% HCl in EFB. While high concentration of HCl can generate the highest sugar concentration which can account for about 20 g/L. In spite of this, high acid concentration can decrease the sugar concentration when it reacts at elevated temperatures. When the reaction takes place for a much longer time when it is associated with high temperature it is known that deformation of sugar to unwanted products such as furfural and hydroxyl methyl furfural (HMF) can take place which subsequently reduces the sugar concentration. Hydroxyl methyl furfural and furfural originate from xylose and glucose decomposition and is usually submerged or produced when hydrolysis is conducted with the presence of acid catalyst and at high temperatures (Najafpour et al., 2007)Moderate reaction with ambient pressure and temperature is preferred when concentrated hydrochloric acid hydrolysis is conducted. EFB lignocellulose fiber conversion of 36, 60, 65 and 80 % is achieved when 5 % solid is used with 15, 20, 25 and 30 % concentrated HCl is used for over a reaction time of 40 minutes. However, when 10% solid was used for a reaction time of over 60 min, the sugar concentration from the acid hydrolysis is known to account for a value of 22. 5 g/l. (Najafpour et al., 2007)Based on Herrera et al., 2003, the effect of various concentration of HCl acid at atmospheric pressure on the hydrolysis of sorghum straw was studied in order to look on the production of glucose, xylose, furfural and also acetic acid. From the studies, when the hydrolysis is performed with 6% HCl at 100°C, xylose concentration peaks at 180 min of reaction time with a value of 19. 7 g/L and then decreases over the time period such as shown on Figure 2. 6. While glucose, furfural and acetic acid are released concentration is marked up to 5. 3, 1. 7 and 3. 6 g/L at 180 min, respectively. Figure 2. 6: Yield of glucose, xylose, furfural and acetic acid at 6 % HCl against time of reaction at 100˚C (Herrera et al., 2003).

2. 5. 11Perchloric Acid

Perchloric acid (HClO4) usage has the potential for hydrolysis process of lignocelluloses material and this is preferable since the perchloric acid has a function as a hydrolysing agent and also as an oxidising agent. The oxidizing function of HClO4 can help in delignification and can reduce the reaction time and energy if compared to the other acid pre-treatment that are used. In addition, neutralisation of the access HClO4 with KOH can lead to precipitation of the insoluble KClO4, and this has an advantage as it can be recycled to HClO4. Theoretically, perchloric acid hydrolysis can be conducted with high concentrations and followed by diluted perchloric acid. This process is preferred to be carried out in two stages because higher sugar degradation can be avoided from the hydrolyzed materials when conducted in two stages rather than single stage. On top of that, when two-stages of hydrolysis is conducted, formation of fermentation inhibitors can be lessened and reduce the heating time (Ismail et al., 2012).

2. 5. 12Fermentation

Fermentation is a process where the breakdown of carbohydrate such as sugar takes place with organic compounds; bacteria, yeast or other microorganism; into acid or alcohol through oxidation reaction. In this scope of research, ethanol is the main objective of research as it is essential for bio fuel energy as a renewable energy for future power generation. Fermentation process is dependent on time and also conditions of the fermentation to give a high yield of fermentation product (Chin et al., 2011). The pH and temperature has a significant influence on fermentation due to its effect on yeast growth, fermentation rate and by-product formation (Pramanik., 2003). From previous research conducted on the effect of temperature on the fermentation process, it is known that the temperature has proven to yield a complex mixture of products. The most efficient ethanol fermentation conditions are determined by high yield of ethanol in the shortest fermentation time. Saccharomyces cerevisiae is a microorganism that is known to give significant effect on the fermentation process. The microorganisms has attracted considerable attention in recent years for the simultaneous saccharification and fermentation (SSF) process of bio-ethanol from agricultural wastes since it is known to have a higher tolerance to both ethanol and also inhibitors that is present in hydrolysates of the lignocellulosic materials. Based on Millati et al., (2011), the medium has been pH adjusted and sterilized to ensure that the fermentation process yields a high efficiency. Other known fungus used for fermentation process is Mucor indicus. From the research, it was reported that both S. cerevisiae and M. indicus are proven to ferment the hydrolyzates from the corresponding stages despite of the presence of HMF and furfural. The yields of ethanol from the fermentation process are 0. 46 g ethanol/glucose by S. cerevisiae and 0. 45 g ethanol-1 xylose by M. indicus (Millati et al., 2011).