Renewable energy is now capturing a good share of the worldwide headlines because of concerns about declining supplies of fossil fuels, escalating population and industrialization triggering ever-increasing demand of fuels.
Worldwide ethanol demand is increasing continuously. Conventional crops such as corn and sugarcane are unable to meet the global demand of bioethanol production due to their primary value of food and feed. Therefore, lignocellulosic substances such as agricultural wastes are attractive feedstocks for bioethanol production and they are too cost effective, renewable and abundant.
Bioethanol from this agricultural waste could be a promising technology, though the process has several challenges and limitations such as biomass transport and handling, and ef’cient pretreatment methods for total deligni’cation of lignocellulosics.
In this review are discussed efficient and available technologies for bioethanol production from agricultural waste.
Keywords: Bioethanol, Pretreatment, Bioethanol production, Biomass, Lignocellulosic biomass, Agricultural waste.
Fossil energy sources such as oil, coal, natural gas, etc are important factors for the world’s present economy development, they make everything work from fuel to electricity and other goods. (Uihlein A. et al., 2009).
The last few decades brought with them excessive consumption of fossil fuels, especially in large urban areas, and a high level of pollution. (Ballesteros I et al., 2006).
The global production of oil is decreasing significantly from year to year and that’s due to the expansion of human population and increasing of industrial property, which is also generating a high energy consumption. (Campbell CH, Laherrere JH, 1998). Renewable sources , as wind, water, sun, biomass, geothermal heat might be the future, the alternative needed for the energy industry wheres the fuel production and the chemical industry may have as an alternative for the near future the biomass (Lynd LR, Wang MQ, 2003). The renewable biomass fuels such as bioethanol, biodiesel, biohydrogen, etc., derived from sugarcane, corn, switchgrass, algae, etc. can become the perfect substitutes for all petroleum-based fuels.
Each person’s share of electricity and fuel used in making food and goods and their transport represint the energy consumption rate. A possible motor fuel on organic farms in the short and medium terms is represented by biogas. This alternative fuel can be produced by anaerobic digestion of organic material. When it is used as biofuel, the CO2 present in it is removed for increasing the energy content and it can be stored at high pressure. Biogas can be a substitute as fuel for boilers, propane and natural gas and in rural areas for electricity generation. Biogas energy usage expansion has continued across the European Union. According to Eur Observ’ER, about 13. 4 million tonnes oil equivalent (Mtoe) of biogas primary energy were produced during 2013, which is 1.2 Mtoe more than in 2012 representing a 10.2% growth. However, the biogas sector’s momentum was more sluggish than in 2012 (16.9% between 2011 and 2012, giving an additional 1.8 Mtoe) and it is expected to lose some of its impetus in 2014 in a number of countries whose sector expansion controlling policy changes will limit the future use of energy crops. 52.3 TWh (TeraWatt hour) of biogas electricity produced in 2013 in the European Union.
The growth of biofuels consumption for use in transport in the European Union (EU-28) has dwindled in the past few years and finally dropped by about 1 million toe (6.8%) between 2012 and 2013 according to EurObserv’ER, to a consumption level of 13.6 million toe. Nevertheless, sustainable biofuel consumption, certified and thus eligible for inclusion in European targets increased slightly by 1.1% to 11.8 Mtoe (EurObserv’ER Database 2013).
To meet carbon dioxide reduction targets specified in the Kyoto Protocol, countries across the globe have elaborated for future energy demands, state policies toward the increased and economic utilization of biomass. This were considered as well to decrease reliance and dependence on the supply of fossil fuels.
Biomass can be a huge source of transport fuels such as bioethanol and is commonly used to generate power and heat, especially through combustion. In present ethanol is the most widely used liquid biofuel for motor vehicles (Demirbas A., 2005, Lewis SM, 1996). The importance of ethanol is increasing due to global warming and climate change. Bioethanol has been receiving widespread interest at the international, national and regional levels. The global market for bioethanol has entered a phase of rapid, transitional growth and many countries around the world are shifting their focus toward renewable sources for power production because of depleting crude oil reserves. The trend is extending to transport fuel as well. Ethanol has potential as a valuable replacement of gasoline in the transport fuel market. However, the cost of bioethanol production is more compared to fossil fuels.
Source: United States Energy Information Administration
Figure 1. World Ethanol Fuel Production and Consumption by Year
Table 1. World Ethanol Fuel Production and Consumption by Year
year production consumption
2000 297.37 283.15
2001 317.26 261.85
2002 362.96 303.34
2003 454.31 355.49
2004 501.94 437.80
2005 578.03 494.44
2006 701.94 623.52
2007 910.48 797.22
2008 1201.22 1092.45
2009 1296.34 1252.60
2010 1487.61 1376.94
2011 1448.46 1349.63
Current ethanol production based on corn, starch and sugar substances may not be desirable due to their food and feed value. Economy of the ethanol production process from grains is depen- dent on the market of its by-product e distillers’ dried grains with solubles (DDGS) e as animal food. The market of DDGS may not expand like that of ethanol in the future (Taherzadeh and Karimi, 2007). Cost is an important factor for large scale expansion of bioethanol production. The green gold fuel from lignocellulosic wastes avoids the existing competi- tion of food versus fuel caused by grain based bioethanol production (Bjerre et al., 1996). It has been estimated that 442 billion liters of bioethanol can be produced from lignocellulosic biomass and that total crop residues and wasted crops can produce 491 billion liters of bio- ethanol per year, about 16 times higher than the actual world bioethanol production (Kim S., 2006). Lignocellulosic materials are renewable, low cost and are abundantly available. It includes crop resi- dues, grasses, sawdust, wood chips, etc. Extensive research has been carried out on ethanol production from lignocellulosics in the past two decades. Hence bioethanol production could be the route to the effective utilization of agricultural wastes. Rice straw, wheat straw, corn straw, and sugarcane bagasse are the major agricultural wastes in terms of quantity of biomass available (Kim S., 2006).
2. Raw material
Current industrial processes for bioethanol production use sugarcane (Southern hemisphere) or cereal grain (Nothern Hemisphere) as feedstocks; but they have to compete directly with food sector (Wheals et al, 1999). Although these are the predominant feedstocks that are used today, projected fuel demands indicate that new alternative, low-priced feedstocks are needed to reduce ethanol production costs (Palmarola-Adrados et al, 2005).
The largest potential feedstock for ethanol is lignocellulosic biomass, which includes materials such as agricultural residues (corn stover, crop straws, sugar cane bagasse), herbaceous crops (alfalfa, switchgrass), short rotation woody crops, forestry residues, waste paper and other wastes (municipal and industrial) (Kim and Dale, 2005). Bioethanol production from these feedstocks could be an attractive alternative for disposal of these residues (Wymam, 2001). Importantly, lignocellulosic feedstocks do not interfere with food security.
Moreover, bioethanol is very important for both rural and urban areas in terms of energy security reason, environmental concern, employment opportunities, agricultural development, foreign exchange saving, socioeconomic issues etc.
To avoid conflicts between food use and industrial use of crops, only wasted crops are assumed to be available for producing ethanol. Wasted crops are defined as crops lost during the year at all stages between the farm and households level during handling, storage and transport. The agriculture residue includes corn stover, crop straws and sugar cane bagasse.
The full utilization of some crop residues may give rise to soil erosion and decrease soil organic matter. Therefore, a 60% ground cover by residues, instead of 30%, is recommended due to uncertainties of local situation (Kim and Dale, 2004). Most wasted biomass comes from rice, corn, and wheat.
The US, Asia and the European Union are leading producers of agricultural by-products, such as straw from rice, corn and cereal crops. Surplus straw offers an ideal feedstock for the manufacture of cellulosic ethanol, presenting no competition to the production of food or animal feed. Nor is any additional land use required to produce bioethanol based on these types of feedstock, as they are automatically created as a by-product during existing production of rice, maize and cereals.
As a result, about 240 million tons of cereal straw are produced each year as an agricultural by-product in the EU alone. Only a small part of this is currently utilized. Long term studies have shown that up to 60% could be taken of the field and are thus available for further uses. By processing this amount of straw, about 25% of the predicted EU demand for petrol could be replaced by cellulosic ethanol in 2020, solely out of surplus material. This means that cellulosic ethanol can play a key role along Europe??s path towards sustainable and climate-friendly road transport.
Figure 2. Lignocellulosic feedstocks of different regions worldwide
In the US, corn stover is the main residue available for conversion into cellulosic ethanol, the second most important feedstock being cereal straw. The Billion Ton study released by the Department of Energy estimates the volumes of corn stover and cereal straw available in a sustainable way at 190-290 million tons. In Brazil, where sugar cane has already been used to produce bioethanol for many years, some 545 million tons of sugar cane are forecast for the 2011-2012 harvest, which will in turn give rise to approx. 73 million tons of bagasse. Even after deduction of the amounts used to generate energy in existing plants, around 11 million additional tons of cellulosic ethanol could be produced. This is equivalent to about 50% of Brazil??s current ethanol production (Clariant International Ltd, 1995 ‘ 2012).
The most important processing challenge in the production of biofuel is pretreatment of the biomass. Lignocellulosic biomass is composed of three main constituents namely hemicellulose, lignin and cellulose. Pretreatment methods refer to the solubilization and separation of one or more of these components of biomass. It makes the remaining solid biomass more accessible to further chemical or biological treatment (Demirbas, 2005). The lignocellulosic complex is made up of a matrix of cellulose and lignin bound by hemicellulose chains. The pretreatment is done to break the matrix in order to reduce the degree of crystallinity of the cellulose and increase the fraction of amorphous cellulose, the most suitable form for enzymatic attack. Pretreatment is undertaken to bring about a change in the macroscopic and microscopic size and structure of biomass as well as submicroscopic structure and chemical composition. It makes the lignocellulosic biomass susceptible to quick hydrolysis with increased yields of monomeric sugars (Mosier et al, 2005). Goals of an effective pretreatment process are formation of sugars directly or subsequently by hydrolysis to avoid loss and/ or degradation of sugars formed to limit formation of inhibitory products to reduce energy demands and to minimize costs. Physical, chemical, physicochemical and biological treatments are the four fundamental types of pretreatment techniques employed. In general a combination of these processes is used in the pretreatment step.
3.1. Physical pretreatment
Physical pretreatment can increase the accessible surface area and size of pores, and decrease the crystallinity and degrees of polymerization of cellulose. Different types of physical processes such as milling (e.g. ball milling, two-roll milling, hammer milling, colloid milling, and vibro energy milling) and irradiation (e.g. by gamma rays, electron beam or microwaves) can be used to improve the enzymatic hydrolysis or biodegradability of lignocellulosic waste materials.
Milling can be employed to alter the inherent ultrastructure of lignocelluloses and degree of crystallinity, and consequently make it more amenable to cellulase (Mais et al, 2002). Milling and size reduction have been applied prior to enzymatic hydrolysis, or even other pretreatment processes with dilute acid, steam or ammonia, on several lignocellulosic waste materials, MSW and activated sludge (Muller et al, 2007). Among the milling processes, colloid mill, fibrillator and dissolver are suitable only for wet materials, e.g. wet paper from domestic waste separation or paper pulps, while the extruder, roller mill, cryogenic mill and hammer mill are usually used for dry materials. The ball mill can be used for either dry or wet materials. Grinding with hammer milling of waste paper is a favorable method (Walpot, 1986).
Milling can improve susceptibility to enzymatic hydrolysis by reducing the size of the materials, and degree of crystallinity of lignocelluloses (Fan et al, 1980) , which improves enzymatic degradation of these materials toward ethanol or biogas. Without any pretreatment, corn stover with sizes of 53’75 ??m was 1.5 times more productive than larger corn stover particles of 425’710 ??m (Zeng et al 2007).
Irradiation by e.g. gamma rays, electron beam and microwaves can improve enzymatic hydrolysis of lignocelluloses. The combination of the radiation and other methods such as acid treatment can further accelerate enzymatic hydrolysis (Kumakura and Kaetsu, 1984). Irradiation has enhanced enzymatic degradation of cellulose into glucose. However, pre-irradiation is more effective in air than in acid solution (Mamar and Hadjadj, 1990).
3.2. Physico-chemical pretreatment
Pretreatments that combine both chemical and physical processes are referred to as physico-chemical processes (Chandra et al, 2007).
3.2.1. Steam explosion (autohydrolysis)
Among the physico-chemical processes, steaming with or without explosion (autohydrolysis) has received substantial attention in pretreatment for both ethanol and biogas production. The pretreatment removes most of the hemicellulose, thus improving the enzymatic digestion. In steam explosion, the pressure is suddenly reduced and makes the materials undergo an explosive decompression. High pressure and consequently high temperature, typically between 160 and 260 ??C, for a few seconds (e.g. 30 s) to several minutes (e.g. 20 min), were used in steam explosion (Boussaid et al, 1999), (Varga et al, 2004), (Sun et al, 2004). The steam explosion process is well documented and was tested in lab-and pilot processes by several research groups and companies. Its energy cost is relatively moderate, and it satisfies all the requirements of the pretreatment process.
The process of steam explosion was demonstrated on a commercial scale at the Masonite plants. Increase in temperature up to a certain level can effectively release hemicellulosic sugars. However, the sugars loss steadily increases by further increasing the temperature, resulting in a decrease in total sugar recovery (Ruiz et al, 2008).
Special care should be taken in selecting the steam explosion conditions in order to avoid excessive degradation of the physical and chemical properties of the cellulose. In very harsh conditions, lower enzymatic digestibility of lignocelluloses may also be observed after steam explosion. For instance, generation of condensation substances between the polymers in steam explosion of wheat straw may lead to a more recalcitrant residue (Sun et al, 2005).
3.2.2. Steam explosion with addition of SO2
Steam pretreatment can be performed with addition of sulfur dioxide (SO2), while the aim of adding this chemical is to improve recovering both cellulose and hemicellulose fractions. The treatment can be carried out by 1’4% SO2 (w/w substrate) at elevated temperatures, e.g. 160’230 ??C, for a period of e.g. 10 min (Eklund et al, 1995).
3.2.3. Ammonia fiber explosion (AFEX)
AFEX is one of the alkaline physico-chemical pretreatment processes. Here the biomass is exposed to liquid ammonia at relatively high temperature (e.g. 90’100 ??C) for a period of e.g. 30 min, followed by immediate reduction of pressure. The effective parameters in the AFEX process are ammonia loading, temperature, water loading, blowdown pressure, time, and number of treatments (Holtzapple et al, 1991). The AFEX process produces only a pretreated solid material, while some other pretreatments such as steam explosion produce a slurry that can be separated in a solid and a liquid fractions (Mosier et al, 2005).
The AFEX process can either modify or effectively reduce the lignin fraction of the lignocellulosic materials, while the hemicellulose and cellulose fractions may remain intact. At optimum conditions, AFEX can significantly improve the enzymatic hydrolysis. The optimum conditions for AFEX depend on the lignocellulosic materials. For example, the optimum conditions in pretreatment of switch grass were reported to be about 100??C, ammonia loading of 1:1 kg of ammonia per kg of dry matter, and 5 min retention time (Alizadeh et al, 2005). One of the major advantages of AFEX pretreatment is no formation of some types of inhibitory by-products, which are produced during the other pretreatment methods, such as furans in dilute-acid and steam explosion pretreatment. However, part of phenolic fragments of lignin and other cell wall extractives may remain on the cellulosic surface. Therefore, washing with water might be necessary to remove part of these inhibitory components, although increasing the amount of wastewater from the process (Chundawat et al, 2007. However, there are some disadvantages in using the AFEX process compared to some other processes. AFEX is more effective on the biomass that contains less lignin, and the AFEX pretreatment does not significantly solubilize hemicellulose compared to other pretreatment processes such as dilute-acid pretreatment. Furthermore, ammonia must be recycled after the pretreatment to reduce the cost and protect the environment (Eggeman and Elander, 2005), (Sun and Cheng, 2002).
3.2.4. CO2 explosion
Supercritical carbon dioxide has been considered as an extraction solvent for non-extractive purposes, due to several advantages such as availability at relatively low cost, non-toxicity, non-flammability, easy recovery after extraction, and environmental acceptability (Zheng and Tsao, 1996). Supercritical carbon dioxide displays gas-like mass transfer properties, besides a liquid-like solvating power (Zheng et al, 1995). It was shown that in the presence of water, supercritical CO2 can efficiently improve the enzymatic digestibility of aspen (hardwood) and southern yellow pine (softwood) (Kim and Hong, 2001). The delignification with carbon dioxide at high pressures can be improved by co-solvents such as ethanol’water or acetic acid’water, and can efficiently increase the lignin removal. Carbon dioxide molecules should be comparable in size to those of water and ammonia, and should be able to penetrate small pores accessible to water and ammonia molecules.
3.2.5. Liquid hot-water pretreatment
Cooking of lignocellulosic materials in liquid hot water (LHW) is one of the hydrothermal pretreatment methods applied for pretreatment of lignocellulosic materials since several decades ago in e.g. pulp industries. Water under high pressure can penetrate into the biomass, hydrate cellulose, and remove hemicellulose and part of lignin. The major advantages are no addition of chemicals and no requirement of corrosion-resistant materials for hydrolysis reactors in this process. The feedstock size reduction is a highly energy-demanding operation for the huge bulk of materials on a commercial scale; there could be no need for size reduction in LHW pretreatment. In addition, the process has a much lower need of chemicals for neutralization of the produced hydrolyzate, and produces lower amounts of neutralization residues compared to many processes such as dilute-acid pretreatment. Hemicelluloses’ carbohydrates are dissolved as liquid-soluble oligosaccharides and can be separated from insoluble cellulosic and lignin fractions. LHW can enlarge the accessible and susceptible surface area of the cellulose and make it more accessible to hydrolytic enzymes (Zeng et al, 2007).
Pretreatments with steam and LHW are both hydrothermal pretreatments. Higher pentosan recovery and lower formation of inhibitory components are the main advantages of LHW pretreatment compared to steam explosion. For instance, treating of de-starched corn fiber with hot water at 160 ??C for 20 min dissolved 75% of the xylan (Dien et al, 2006). At higher temperatures, e.g. 220 ??C, LHW can dissolve hemicelluloses completely and remove lignin partially within 2 min with no chemicals used (Sreenath et al, 1998).
Xylan removal via percolation reactor, or by base addition (adjusting the pH) during the process, has been suggested to reduce the formation of inhibitors such as furfural and degradation of xylose(Laser et al, 2002) . The pH, processing temperature, and time should be controlled in order to optimize the enzymatic digestibility by LHW pretreatment. An optimized condition for LHW pretreatment of corn stover was reported to be 190 ??C for 15 min, in which 90% of the cellulose conversion was observed by subsequent enzymatic hydrolysis . LHW pretreatment at 160 ??C and a pH above 4.0 can dissolve 50% of the fibers from corn fibers in 20 min (Mosier, 2005).
3.2.6. Microwave oven and electron beam irradiation pretreatment
Pretreatment of lignocellulosic biomass in a microwave oven is also a feasible method which uses the high heating ef’ciency of a microwave oven and it is also easy to operate (Bjerre et al, 1996). Microwave treatment utilizes thermal and non-thermal effects generated by microwaves in aqueous environments. In the thermal method, internal heat is generated in the biomass by microwave radiation, resulting from the vibrations of the polar bonds in the biomass and the surrounding aqueous medium. Thus a hot spot is created within the inhomogeneous material. This unique heating feature results in an explosion effect among the particles and improves the disrup- tion of recalcitrant structures of lignocellulose (Hu and Wen, 2008). Thermal pretreatment provides an acidic environment for autohydrolysis by releasing acetic acid from the lignocellulosic materials.
In the non-thermal method, i.e., the electron beam irradiation method, polar bonds vibrate, as they are aligned with a continu- ously changing magnetic ‘eld and the disruption and shock to the polar bonds accelerates chemical, biological and physical processes (Sridar, 1998). High energy radiation results in more changes in cellulosic biomass including increase of speci’c surface area, decrease of degree of polymerization and crystallinity of cellulose, hydrolysis of hemicellulose and partial depolymerization of lignin.
3.3. Chemical pretreatment
3.3.1. Alkaline hydrolysis
Alkaline pretreatment refers to the application of alkaline solutions such as NaOH, Ca(OH)2 (lime) or ammonia to remove lignin and a part of the hemicellulose, and efficiently increase the accessibility of enzyme to the cellulose. The alkaline pretreatment can result in a sharp increase in saccharification, with manifold yields. Pretreatment can be performed at low temperatures but with a relatively long time and high concentration of the base. For instance, when soybean straw was soaked in ammonia liquor (10%) for 24 h at room temperature, the hemicellulose and lignin decreased by 41.45% and 30.16% respectively (Xu et al, 2007). However, alkaline pretreatment was shown to be more effective on agricultural residues than on wood materials.
3.3.2. Alkaline peroxide
Alkaline peroxide is an effective method for pretreatment of biomass. In this method, the lignocelluloses are soaked in pH-adjusted water (e.g. to pH 11’12 using NaOH) containing H2O2 at room temperatures for a period of time (e.g. 6’24 h). The process can improve the enzymatic hydrolysis by delignification.
3.3.3. Organosolv process
Organosolv can be used to provide treated cellulose suitable for enzymatic hydrolysis, using an organic or aqueous organic solvent to remove or decompose the network of lignin and possibly a part of the hemicellulose (Curreli et al, 1997). In this process, lignocellulose is mixed with organic liquid and water and heated to dissolve the lignin and part of the hemicellulose, leaving reactive cellulose in the solid phase. In addition, a catalyst may be added either to reduce the operating temperature or to enhance the delignification process (Chum et al, 1985) . Lignin in the biomass can be extracted from the solvent for e.g. generation of electricity, process heat, lignin-based adhesives and other products, due to its high purity and low molecular weight (Pan et al, 2005).
In organosolv pretreatment of lignocellulosic materials, a large number of organic or aqueous-organic solvents at temperatures of 150’200 ??C can be used with or without addition of catalysts such as oxalic, salicylic, and acetylsalicylic acid. Furthermore, the solvent may accompany acetic acid released from acetyl groups developed by hydrolysis of hemicelluloses. A variety of organic solvents such as alcohols, esters, ketones, glycols, organic acids, phenols, and ethers have been used. However, the price of solvent and simplicity in recovery of solvent should also be considered. The applied solvents should be separated by e.g. evaporation and condensation, and recycled to reduce the operational costs of the process. Removal of solvents from the pretreated cellulose is usually necessary because the solvents might be inhibitors to the enzymatic hydrolysis and fermentation or digestion of hydrolyzate (Sun and Cheng, 2002).
3.3.4. Wet oxidation
Wet oxidation has been applied as pretreatment for both ethanol and biogas production. In this process, the materials are treated with water and air or oxygen at temperatures above 120??C (e.g. 148’200??C) for a period of e.g. 30 min (Palonen et al, 2004). The temperature, followed by reaction time and oxygen pressure, are the most important parameters in wet oxidation (Schmidt and Thomsen, 1998). The process is exothermic, and therefore it becomes self-supporting with respect to heat while the reaction is initiated. Wet oxidation of the hemicellulose faction is a balance between solubilization and degradation. This process is an effective method in separat ing the cellulosic fraction from lignin and hemicellulose. Oxygen participates in the degradation reactions and allows operation at comparatively reduced temperatures by enhancing generation of organic acids. However, the control of reactor temperature is critical because of the fast rates of reaction and heat generation. The main reactions in wet oxidation pretreatment are the formation of acids from hydrolytic processes, as well as oxidative reactions. All three fractions of lignocellulosic materials are affected in this process. The hemicelluloses are extensively cleaved to monomeric sugars; the lignins undergo both cleavage and oxidation; and cellulose is partly degraded. The cellulose becomes highly susceptible to enzymatic hydrolysis (Schultz et al, 1984).
Similar to many other delignification methods, the lignin produced by wet oxidation cannot be used as a fuel, since a major part of the lignin undergoes both cleavage and oxidation. This phenomenon considerably reduces the income from this by-product at industrial scale for ethanol production from lignocellulosic materials (Galbe and Zacchi, 2002) .
Wet oxidation can also be performed by oxidation agents such as hydrogen peroxide (H2O2). Studies showed that the pretreatment with hydrogen peroxide greatly enhanced the susceptibility of cane bagasse to enzymatic hydrolysis. About 50% of the lignin and most hemicellulose were solubilized by treating the biomass with 2% H2O2 at 30??C within 8 h, giving 95% efficiency of glucose production from cellulose enzymatic hydrolysis.
3.3.5. Ozonolysis pretreatment
Pretreatment of lignocellulosic materials can be performed by treatment with ozone, referred to as ‘ozonolysis’ pretreatment. This method can effectively degrade lignin and part of hemicellulose. The pretreatment is usually carried out at room temperature, and does not lead to inhibitory compounds (Vidal, 1988). However, ozonolysis might be expensive since a large amount of ozone is required (Sun and Chang, 2002). The main parameters in ozonolysis pretreatment are moisture content of the sample, particle size, and ozone concentration in the gas flow. Among these parameters, an essential factor is the percentage of water in the feed, and it has the most significant effect on the solubilization. The optimum water content was found to be around 30%, corresponding to the saturation point of the fibers. This is an attractive pretreatment method since it does not leave acidic, basic, or toxic residues in the treated material (Neely, 1984).
3.3.6. Acid hydrolysis pretreatment
Treatment of lignocellulosic materials with acid at a high temperature can efficiently improve the enzymatic hydrolysis. Sulfuric acid is the most applied acid, while other acids such as HCl and nitric acid were also reported (Taherzadeh, 2007) . The acid pretreatment can operate either under a high temperature and low acid concentration (dilute-acid pretreatment) or under a low temperature and high acid concentration (concentrated-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. Therefore, this process requires either specialized non-metallic constructions or expensive alloys. The acid recovery, which is necessary in the concentrated-acid process for economical reasons, is an energy-demanding process. On the other hand, the neutralization process produces large amounts of gypsum. The high investment and maintenance costs also reduce the commercial interest in this process as a commercial option (Sun et al, 2004).
Dilute-acid hydrolysis is probably the most commonly applied method among the chemical pretreatment methods. It can be used either as a pretreatment of lignocellulose for enzymatic hydrolysis, or as the actual method of hydrolyzing to fermentable sugars. Different types of reactors such as batch, percolation, plug flow, countercurrent, and shrinking-bed reactors, for either pretreatment or hydrolysis of lignocellulosic materials by the dilute-acid processes, have been applied.
The major drawback of some pretreatment methods, particularly at low pH is the formation of different types of inhibitors such as carboxylic acids, furans and phenolic compounds (Taherzadeh and Karimi, 2007). These chemicals may not affect the enzymatic hydrolyses, but they usually inhibit the microbial growth and fermentation, which results in less yield and productivity of ethanol or biogas. Therefore, the pretreatments at low pH should be selected properly in order to avoid or at least reduce the formation of these inhibitors.
3.4. Biological pretreatment
Microorganisms can also be used to treat the lignocelluloses and enhance enzymatic hydrolysis. The applied microorganisms usually degrade lignin and hemicellulose but very little part of cellulose, since cellulose is more resistance than the other parts of lignocelluloses to the biological attack. Several fungi, e.g. brown-, white- and soft-rot fungi, have been used for this purpose. White-rot fungi are among the most effective microorganisms for biological pretreatment of lignocelluloses (Sun and Cheng, 2002).
Biological treatments with microorganisms or enzymes are also investigated to improve digestion in biogas production. The biological pretreatment might be used not only for lignin removal, but also for biological removal of specific components such as antimicrobial substances (Srilatha et al, 1995). Solid-state fermentation of orange peels by fungal strains of Sporotrichum, Aspergillus, Fusarium and Penicillum enhanced the availability of feed constituents and reduced the level of the antimicrobial substances. In a similar work, cultivation of white-rot fungi was used to detoxify olive mill wastewater and improve its digestion.
Low energy requirement, no chemical requirement, and mild environmental conditions are the main advantages of biological pretreatment. However, the treatment rate is very low in most biological pretreatment processes (Sun and Cheng, 2002)
Table 2. Summary of some bio-deligni’cation processes.
Substrate Microorganism for lignin degradation Time of pretreatment % of substrate converted to reducing sugars
Sugarcane bagasse Pleurotus ostreatus
Phanerochaete sordida; Pycnoporus cinnabarinus 115
Phlebia sp. MG-60 (A marine fungus) 5 weeks
60 days 35%
92% deligni’cation; total reducing sugar
yield 11.26 0.73 mg/g
71% deligni’cation; total reducing sugar
yield 11.56 0.51 mg/g
73.6% deligni’cation; Total Reducing sugar
yield 11.16 0.64
41% lignin degraded
4. Enzymatic hydrolysis
Sacchari’cation is the critical step for bioethanol production where complex carbohydrates are converted to simple monomers. Compared to acid hydrolysis, enzymatic hydrolysis requires less energy and mild environment conditions (Ferreira et al, 2009)). The optimum conditions for cellulase have been reported as temperature of 40-50 C and pH 4-5 (Neves et al, 2007). Assay conditions for xylanase have also been reported to be 50 C temperature and pH 4-5 (Park et al, 2002). Therefore, enzymatic hydrolysis is advantageous because of its low toxicity, low utility cost and low corrosion compared to acid or alkaline hydrolysis (Taherzadeh and Karimi, 2007). Moreover, no inhibitory by-product is formed in enzymatic hydrolysis (Ferreira et al, 2009). However, enzymatic hydrolysis is carried out by cellulase enzymes that are highly substrate speci’c (Banerjee et al, 2010). Here cellulase and hemicellulase enzymes cleave the bonds of cellulose and hemicellulose respectively. Cellulose contains glucan and hemicellulose contains different sugar units such as mannan, xylan, glucan, galactan and arabinan. Cellulase enzymes involve endo and exoglucanase and b-glucosidases. Endoglucanase (endo 1,4-D glucanhydrolase or E.C. 220.127.116.11) attacks the low crystallinity regions of the cellulose ‘ber, exoglucanase (1,4-b-D glucan cellobiohydrolase or E.C. 18.104.22.168) removes the cellobiase units from the free chain ends and ‘nally cellobiose units are hydrolysed to glucose by b-glucosidase (E.C. 22.214.171.124). Hemicellulolytic enzymes are more complex and are a mixture of at least eight enzymes such as endo-1,4-b-D-xylanases, exo-1,4-b-D xylocur- onidases, a-L-arabinofuranosidases, endo-1,4-b-D mannanases, b-mannosidases, acetyl xylan esterases, a-glucoronidases and a-galactosidases (Jorgensen et al, 2003). Cellulose is hydrolysed to glucose whereas hemicellulose gives rise to several pentoses and hexoses. Several species of Clostridium, Cellulomonas, Thermonospora, Bacillus, Bac- teriodes, Ruminococcus, Erwinia, Acetovibrio, Microbispora, Strepto- myces are able to produce cellulase enzyme. Many fungi such as Trichoderma, Penicillium, Fusarium, Phanerochaete, Humicola, Schizophillum sp. also have been reported for cellulase production. Among the various cellulolytic microbial strains Trichoderma is one of the most well studied cellulase and hemicellulase producing fungal strains (Xu et al, 1998). Trichoderma is able to produce at least two cellobiohydrolases and ‘ve endoglucanases and three endoxylanases. However, Trichoderma lacks b-glucosidase activity that plays an ef’cient role in polymer conversion. On the other hand, Aspergillus is a very ef’cient b-glucosidase producer. Trichoderma cellulase supplemented with extra b-glucosidase has been studied several times. Combina- tion of Trichoderma reesei ZU-02 cellulase and cellobiase from Aspergillus niger ZU-07 improved the hydrolysis yield to 81.2% with cellobiase activity enhanced to 10 CBU/g substrate (Chen et al, 2008).
Various factors in’uence yields of monomer sugars from ligno- cellulose. Temperature, pH and mixing rate are the main factors of enzymatic hydrolysis of lignocellulosic material (Olsson et al, 1996). Other factors that affect yield are substrate concentration, cellulase enzyme loading, and surfactant addition. High substrate concentration may lead to substrate inhibition. Cellulase contributes to the major cost of the lignocellulosic ethanol technology. Therefore, an ef’cient pretreatment is to be selected to decrease cellulose crystallinity and to remove lignin to the maximum extent, so that hydrolysis time as well as cellulase loading will be minimized. Surfactants modify the cellulose surface by adsorbing lignin onto surfactant and thus the surfactant prevents the enzyme from unproductive binding with lignin and lowers enzyme loading (Eriksson et al, 2002).
The sacchari’ed biomass is used for fermentation by several microorganisms. But the industrial utilization of lignocelluloses for bioethanol production is hindered by the lack of ideal microor- ganisms which can ef’ciently ferment both pentose and hexose sugars (Talebnia et al, 2010). For a commercially viable ethanol production method, an ideal microorganism should have broad substrate utilization, high ethanol yield and productivity, should have the ability to withstand high concentrations of ethanol and high temperature, should be tolerant to inhibitors present in hydrolysate and have cellulolytic activity. Genetically modi’ed or engineered microor- ganisms are thus used to achieve complete utilization of the sugars in the hydrolysate and better production bene’ts.
The processes usually employed in the fermentation of ligno- cellulosic hydrolysate are simultaneous sacchari’cation and fermentation (SSF) and separate hydrolysis and fermentation (SHF). Conventionally or traditionally the SHF process has been employed but SSF is superior for ethanol production since it can improve ethanol yields by removing end product inhibition and eliminate the need for separate reactors. It is also cost effective but difference in optimum temperature conditions of enzyme for hydrolysis and fermentation poses some limitations (Neves et al, 2007) . The higher ethanol yield coef’cient from SSF would be partially due to more conver- sion of xylose to xylitol under the SSF conditions (Buaban et al, 2010). A compar- ative study between the two processes (SHF and SSF) is presented in Table 3.
Table 3. Comparison between the two main fermentation techniques.
Fermentation process Features and advantages Limitations
and fermentation ‘ Low costs
‘ Higher ethanol yields due to removal of end product inhibition of sacchari’cation step
‘ Reduces the number of reactors required Each step can be processed at its optimal operating conditions
‘ Separate steps minimize interaction between the steps ‘ Difference in optimum temperature conditions of enzyme for hydrolysis and fermentation.
‘ End product inhibition minimizes the yield of ethanol. Chance of contamination due to long period process
Separate hydrolysis and fermentation
Studies have shown that SSF is a better alternative to SHF. The slow xylose consumption during fermentation in SHF may be due to the presence of toxic compounds which inhibit the growth and fermentation activity of the microorganism. The drawback of SSF can be removed by using thermo-tolerant micro- organisms like Kluyveromyces marxianus which has been developed to withstand the higher temperatures needed for enzymatic hydrolysis.
Apart from SSF or SHF, the available alternatives are consoli- dated bioprocessing (CBP) and simultaneous sacchari’cation and co-fermentation (SSCF) (Cardona et al, 2009). In CBP, cellulase production, biomass hydrolysis and ethanol fermentation are all together carried out in a single reactor . The process is also known as direct microbial conversion (DMC). Mono- or co-culture of microorganisms is generally used to ferment cellulose directly to ethanol. Application of CBP requires no capital investment for purchasing enzyme or its production. Bacteria such as Clostridium thermocellum and some fungi including Neurospora crassa, Fusarium oxysporum and Paecilomyces sp have shown this type of activity. However, CBP is not an ef’cient process because of poor ethanol yields and long fermentation periods (3-12 days) (Szczodrak and Fiedurek, 1996). In SSCF the co-fermenting microorganisms need to be compatible in terms of operating pH and temperature . A combination of Candida shehatae and Saccharomyces cerevisiae was reported as suitable for the SSCF process (Neves et al, 2007). Sequential fermentation with two different microor- ganisms in different time periods of the fermentation process for better utilization of sugar has also been employed using S. cerevisiae in the ‘rst phase for hexose utilization and C. shehatae in the second phase for pentose utilization but ethanol yields achieved are not high (Sanchez and Cardona, 2008).
Some native or wild type microorganisms used in the fermen- tation are S. cerevisiae, Escherichia coli, Zymomonas mobilis, Pachy- solen tannophilus, C. shehatae, Pichia stipitis, Candida brassicae, Mucor indicus etc. Among all the best known yeast and bacteria employed in ethanol production from hexoses are S. cerevisiae and Z. mobilis respectively . But S. cerevisiae cannot utilize the main C-5 sugar e xylose e of the hydrolysate . Native organisms such as Pichia and Candida species can be used in place of S. cerevisiae and they can utilize xylose but their ethanol production rate is at least ‘vefold lower than that observed with S. cerevisiae . Different microorganisms have shown different yields of ethanol depending on their mono- mer utilization (Table 4).
Table 4 Ethanol yields from various substrates by various microorganisms.
Substrate Fermenting microbe Yield of ethanol Feature of the employed microorganism
Can ferment both glucose and xylose
Utilizes xylose and glucose present
Adapted at increased concentration
Co-ferment glucose and xylose and
utilizes ethanol in absence of sugar
Ferment only glucose
Ferment glucose ‘rst and then
xylose from the mixture
Sugar cane bagasse
Rice straw Pichia stipitis BCC15191
Recombinant Escherichia coli KO11
Pichia stipitis NRRL Y-7124
Pichia stipitis A
Candida shehatae NCL-3501
Pichia stipitis NRRL Y-7124 0.29 0.02 g ethanol/g available fermentable sugars (glucose and xylose) after 24 h
31.50 g of ethanol/L in 48 h equivalent to a theoretical maximum yield of 91.5%
0.35 gp/gs Calculated as ‘nal ethanol
0.41 gp/gs concentration divided by total sugar in the fermentation medium
0.45 g/g and 0.5 g/g of sugar utilized produced from autohydrolysate by free and immobilized cells in 48 h
0.37 g/g and 0.47 g/g of sugar utilized produced from acid hydrolysate by free and immobilized cells in 48 h
Maximum ethanol production achieved 4 g/L
Maximum ethanol production achieved 6 g/L (78% of theoretical maximum) Can ferment both glucose and xylose
Utilizes xylose and glucose present in hydrolysates
Adapted at increased concentration of hydrolysate
Co-ferment glucose and xylose and
utilizes ethanol in absence of sugar
Ferment only glucose
Ferment glucose ‘rst and then xylose from the mixture
Genetic engineering has been employed to develop the various aspects of fermentation from higher yield to better and wide substrate utilization to increased recovery rates. A number of genetically modi’ed microorganisms such as P. stipitis BCC15191 (Buaban et al, 2010) , P. stipitis NRRL Y-7124, recombinant E. coli KO11, C. shehatae NCL-3501 S. cerevisiae ATCC 26603 have been developed. Strict anaerobic hemophilic bacteria such as Clostridium sp. and Thermoanaerobacter sp. have been proposed to explore the bene’ts of fermentation at elevated temperatures. Some other thermo-tolerant microorganisms developed are K. marxianus, Candida lusitanieae and Z. mobilis (Bjerre et al, 1996).
Lignocellulosic biomass has been projected to be one of the main resources for economically attractive bioethanol production. Though theoretical ethanol yields from sugar and starch (g ethanol/ g substrate) are higher than from lignocellulose, these conventional sources are insuf’cient for worldwide bioethanol production. In that aspect agricultural wastes are renewable, less costly and abundantly available in nature. Agricultural wastes do not demand separate land, water, and energy requirements. They do not have food value as well. For economically feasible bioethanol production, several hindrances are to be overcome. These refer to the four major aspects which are feedstock, conversion technology, hydrolysis process, and fermentation con’guration. With regard to feedstock major obstacles are cost, supply, harvesting, and handling. As regards conversion technology the hindrances are biomass pro- cessing, proper and cost effective pretreatment technology to liberate cellulose and hemicellulose from their complex with lignin. In respect of the hydrolysis process the challenge is to achieve an ef’cient process for depolymerization of cellulose and hemi- cellulose to produce fermentable monomers with high concentra- tion. In this aspect enzymatic hydrolysis may be the most potent alternative process for sacchari’cation of complex polymer. Several efforts have been made to reduce the cost of cellulase enzyme to optimize the enzymatic hydrolysis process. Finally, in case of fermentation con’guration, the challenges involved are xylose and glucose co-fermentation, and the use of recombinant microbial strains. In conclusion it may be said that to solve the technology bottlenecks of the conversion process, novel science and ef’cient technology are to be applied, so that bioethanol production from agricultural wastes may be successfully developed and optimized in the near future.
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