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Biobutanol can be produced by Acetone - Butanol - Ethanol fermentation process, as shown in Figure 2.1 This process has been improved by using various strains of the bacterium either Clostridium acetobutylicum or Clostridium Beijerinckii and different substrates such as corn and molasses for many years. However, these substrates have high cost resulting in high price of butanol. Therefore, to produce butanol by using biomass as a feedstock is another choice to reduce butanol price.
Figure 2.1 ABE fermentation process (Cascone, 2008).
Butanol is a four carbon alcohol. It contains more hydrogen and carbon. Butanol has several advantages. For example butanol is easier to blend with gasoline and other hydrocarbon products and is safer to handle since butanol, is less volatile and explosive, has high flash point and low vapor pressure. It can be shipped and distributed through existing pipelines and filling stations. An 85 % butanol/gasoline blends can be used in unmodified petrol engines and it is cleaner burning than ethanol. (Nigam and Singh, 2011).
2.2 Lignocellulosic biomass
Figure 2.2 Representation of lignocellulose structure showing cellulose, hemicellulose, and lignin fractions (Mussatto et al., 2010).
Lignocellulosic biomass such as agricultural waste and crop residue resources are one of the major renewable resources for fuels and chemicals. Lignocellulosic biomass consist mainly of cellulose, hemicellulose, and lignin that are closely associated in a complex crystalline structure, as shown in Figure 2.2 These components are complex polymers that are closely associated with each other producing the cellular complex of the vegetal biomass. Basically, cellulose forms a skeleton which is surrounded by hemicellulose and lignin. The complex structure results in enzymatic hydrolysis accessibility limited. Table 2.1 shows the composition of various lignocellulosic biomass.
Table 2.1 Composition of representative lignocellulosic feedstocks (Menon et al., 2012)
Carbohydrate composition (% dry wt)
Cellulose is the major component of plant biomass including about 30-60 % of total feedstock dry matter (Balat, 2011). Cellulose is a high molecular weight linear homopolymer of repeated units of cellobiose, that is two anhydrous glucose rings joined via a Î²-1,4 glycosidic linkage. The long-chain cellulose polymers are linked together by hydrogen and van der walls bonds, which cause the cellulose to be packed into microfibrils. The microfibrils are covered by hemicelluloses and lignin. The structure of cellulose is shown in Figure 2.3. By forming these hydrogen bounds, the chains tend to arrange in parallel and form a crystalline structure. Therefore, cellulose microfibrils have both highly crystalline regions, that is around 2/3 of the total cellulose, and less-ordered amorphous regions. More ordered or crystalline cellulose is less soluble and less degradable (Taherzadeh and Karimi, 2008). The degree of cellulose crystallinity is a major factor affecting enzymatic hydrolysis of the substrate. It has been reported that a decrease in cellulose crystallinity especially influences the initial rate of cellulose hydrolysis. Physical or chemical pretreatment to disrupt the crystalline structure of cellulose is often used to promote the hydrolysis of biomass.
Figure 2.3 The structure of cellulose (http://www.plantoils.in/portal/ce/blog/ 2008_12_01_archive.html).
The main feature that differentiates hemicellulose from cellulose is that hemicellulose has branches with short lateral chains consisting of different sugars which are easy hydrolyzable polymers. Hemicellulose (20-40 % of total feedstock dry matter) is a highly branched polymer of five-carbon (pentoses) and six-carbon (hexoses) sugars, as shown in Figure 2.4. Especially, hemicelluloses contains xylose, and arabinose for five-carbon sugars and galactose, glucose, and mannose for six-carbon sugars. Hemicellulose is more readily hydrolyzed compared to cellulose because of its branched, amorphous nature (Lee et al., 2007). The dominant sugars in hemicelluloses are mannose in softwoods and xylose in hardwoods and agriculture residues (Taherzadeh and Karimi, 2008).
Figure 2.4 Monomers of hemicelluloses (Taherzadeh and Karimi, 2008).
Lignin (15-25 % of total feedstock dry matter) is an aromatic polymer. More specifically, p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol basis are the ones most commonly encountered, as shown in Figure 2.5 (Harmsen et al., 2010). The basic chemical phenylpropane units of lignin are bonded together by a set of linkages to form a very complex matrix (Demirbas, 2008). This matrix comprises a variety of functional groups, such as hydroxyl, methoxyl and carbonyl. Lignin is one of the drawbacks of using lignocellulosic biomass materials in fermentation, as it makes lignocellulose resistant to chemical and biological degradation (Taherzadeh and Karimi, 2008).
Figure 2.5 Phenyl propane units (Taherzadeh and Karimi, 2008).
2.3 Pretreatment of Lignocellulosic Biomass
Pretreatment is required to alter the structure of lignocellulosic biomass to make cellulose more accessible to the enzymes that convert the carbohydrate polymers (cellulose and hemicelluloses) into fermentable sugars. Pretreatment has great potential for improvement of efficiency and lowering of cost through research and development (Mosier et al., 2003a,b). The purpose of the pretreatment is to remove lignin and hemicellulose, 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, and (4) be cost-effective (Kumar et al., 2009). In general, pretreatment methods can be classified into three categories, including physical, chemical, and biological pretreatment.
Figure 2.6 Schematic of the role of pretreatment (Kumar et al., 2009).
2.3.1 Physical Pretreatment
Lignocellulosic biomass can be comminuted by a combination of chipping, grinding, and milling to reduce cellulose crystallinity. The size of the materials is usually 10-30 mm after chipping and 0.2-2 mm after milling or grinding
(Kumar et al., 2009, Sun and Cheng, 2002, Leustean, 2009). Power requirements of mechanical comminution depend on the final particle size and the biomass characteristics. Power requirements increase rapidly with decreasing particle size. These mechanical pretreatment techniques are time-consuming, energy intensive, or expensive to process (Balat, 2011).
2.3.2 Physico-chemical Pretreatment
18.104.22.168 Steam Explosion (Autohydrolysis)
In this method, chipped biomass is treated with high-pressure saturated steam and then the pressure is swiftly reduced, which makes the materials undergo an explosive decomposition. Steam explosion is the most commonly used method for the pretreatment of lignocellulosic biomass. Steam explosion increases crystallinity of cellulose by promoting crystallization of the amorphous portions. Moreover, steam explosion hydrolyses hemicelluloses easily. That is evidence that steam explosion promotes delignification (Jeoh, 1998).
22.214.171.124 Ammonia Fiber Explosion
Ammonia fiber explosion (AFEX) is one of the alkaline physico-chemical pretreatment processes. The material is subjected to liquid ammonia at high temperature and pressure, and a subsequent fast decompression, similar to the steam explosion, which causes a fast saccharification of lignocellulosic biomass (Abril et al., 2009). The system does not directly release any sugars but allows hemicellulose and cellulose to be attacked enzymatically and reduced to sugars (Balat, 2011).
126.96.36.199 Liquid Hot-water Pretreatment
Cooking of lignocellulosic biomass in liquid hot water (LHW) is one of the hydrothermal pretreatment methods applied for pretreatment of lignocellulosic biomass (Taherzadeh and Karimi, 2008). LHW subjects biomass to hot water in liquid state at high pressure during a fixed period and it presents elevated recovery rates for pentoses and generates low amount of inhibitors (Tomas et al., 2008). If the pH is maintained between 4 and 7, the degradation of monosaccharide sugars can be minimized (Hayes, 2009).
2.3.3 Chemical Pretreatment
Chemical pretreatments were originally developed and have been studied to date have had the primary goal of improving the biodegradability of cellulose by removing lignin and/or hemicellulose, and to a lesser degree decreasing the degree of polymerization (DP) and crystallinity of the cellulose component. Chemical pretreatment is the most studied pretreatment technique among pretreatment categories. The various commonly used chemical pretreatments includes: acid, alkali, organic acids, pH-controlled liquid hot-water, and ionic liquids.
188.8.131.52 Acid Pretreatment
Acid pretreatment normally aim for high yields of sugar from lignocellulosic biomass due to this method gives high reaction rate and significantly improves cellulose hydrolysis (Karimi et al., 2006). Acid pretreatment involves the use of concentrated and diluted acids to break the rigid structure of the lignocellulosic biomass and remove hemicellulose and expose cellulose for enzymatic digestion (Silverstein et al., 2008). The most commonly used acid is dilute sulphuric acid (H2SO4), which has been commercially used for a wide variety of biomass types such as switchgrass, corn stover, spruce (softwood), and poplar. Other acids have also been studied, such as hydrochloric acid (HCl), phosphoric acid (H3PO4), and nitric acid (HNO3). Acid pretreatments have been used as parts of overall processes in fractionating the components of lignocellulosic biomass due to its ability to remove hemicelluloses (Zhang et al., 2007). The acid addition increases hemicellulose solubilization rate in comparison with the liquid hot water or steam explosion methods; therefore, the enzymatic digestibility of cellulose is enhanced. Acid pretreatment (removal of hemicellulose) followed by alkali pretreatment (removal of lignin) results in relatively pure cellulose (Menon et al., 2012).
The potential of dilute acid pre-hydrolysis as a pretreatment method was studied for sugarcane bagasse, rice hulls, peanut shells, and cassava stalks (Martin et al., 2007). The pre-hydrolysis was performed at 122 ï‚°C during 20, 40, or 60 min using 2 % H2SO4 at a solid-to-liquid ratio of 1:10. Sugar formation increased with increasing reaction time. Xylose, glucose, arabinose, and galactose were detected in all of the pre-hydrolysates, whereas mannose was found only in the prehydrolysates of peanut shells and cassava stalks. The hemicelluloses of bagasse were hydrolyzed to a high-extent yielding concentrations of xylose and arabinose of 19.1 and 2.2 g/l, respectively, and a xylan conversion of more than 80 %. High-glucose concentrations (26-33.5 g/l) were found in the prehydrolysates of rice hulls, probably because of hydrolysis of starch of grain remains in the hulls. Peanut shells and cassava stalks rendered low amounts of sugars on pre-hydrolysis, indicating that the conditions were not severe enough to hydrolyze the hemicelluloses in these materials quantitatively.
Cara et al., (2008) studied dilute acid pretreatment of olive tree biomass. Pretreatment was performed at 0.2, 0.6, 1.0, and 1.4 % (w/w) H2SO4 and temperature range 170-210 ï‚°C. It was found that 83 % of hemicellulosic sugars in the raw material were recovered in the prehydrolysate obtained at 170 ï‚°C, 1 % H2SO4; however, the enzyme accessibility of the corresponding pretreated solid was not very high. The maximum enzymatic hydrolysis yield (76.5 %) was attained from a pretreated solid at 210 ï‚°C, 1.4 % acid concentration. Moreover, sugar recovery in the prehydrolysate was the poorest one among all the experiments performed. The maximum value (36.3 g sugar/100 g raw material) was obtained when the olive tree biomass at 180 ï‚°C and 1 % H2SO4 concentration, representing 75 % of all sugars in the raw material.
184.108.40.206 Alkaline Pretreatment
Alkali pretreatment refers to the application of alkaline solutions to remove lignin and various uronic acid substitutions on hemicellulose that lower the accessibility of enzyme to the hemicellulose and cellulose (Han et al., 2009) These processes are operated at lower temperatures and pressures compared to other pretreatment technologies. Alkali pretreatment may be carried out at ambient conditions, but pretreatment time is measured in terms of hours or days rather than minutes or seconds (Mosier et al., 2005). Sodium, potassium, calcium and ammonium hydroxide are appropriate chemicals for alkaline pretreatment. Of these four, NaOH has been studied the most (Kumar et al., 2009). Dilute NaOH treatment of lignocellulosic biomass causes swelling, leading to an increase in the internal surface area, a decrease in crystallinity, separation of structural linkages between lignin and carbohydrates, and disruption of the lignin structure.
Wang (2009) studied NaOH pretreatment of Coastal Bermuda grass. Coastal Bermuda grass was pretreated with NaOH 0.5 % to 3 % (w/v) from 15 to 90 min at 121 ï‚°C. Pretreatment time of 30 min was sufficient to achieve a significant amount of total lignin removal as long as the NaOH concentration was equal or over 1 %. On the other hand, decreasing sodium hydroxide concentration from 1 to 0.5 % significantly reduced total lignin removal, but there was no significant difference in lignin removal between 2 and 3 % NaOH. Up to 86 % lignin removal was observed. The optimal NaOH pretreatment conditions at 121 ï‚°C for total reducing sugars production as well as glucose and xylose yields were 15 min and 0.75 % NaOH. The total reducing sugars yield was about 71 % of the theoretical maximum, and the overall conversion efficiencies for glucan and xylan were 90.43 % and 65.11%, respectively.
Joshua et al. (2012) investigated the production of ABE from algae biomass. They found that the pretreatment with acid followed by alkaline produced 8.92 g/l of soluble sugars, whereas non-pretreated algae had only 0.73 g/l of soluble sugar. These data demonstrate the importance of pretreating complex substrates to produce fermentable sugars more efficiently. Additionally, pretreatment increases the surface area, or bio-availability, of the substrate for bacterial enzymes to hydrolyze the biomass more resourcefully (Kumar et al., 2009).
Ponthein and Cheirsilp (2011) studied the pretreatment of palm pressed fiber by hydrothermal, acid and base to remove lignin and obtain high cellulose content fiber. The result indicated that the pretreatment with NaOH followed by H2SO4 gave highest cellulose content and reduced the amount of hemicellulose and lignin more than pretreatment with sodium hydroxide or sulfuric acid alone. Moreover, Zhu et al. (2006) also found that the pretreatment of rice straw by alkali and acid increased cellulose content up to 75-80 %. The amount of hemicellulose and lignin content also significantly decreased to 3 and 3-5 % respectively. While the pretreatment with alkaline or acid alone gave similar lignin and hemicellulose content at 7-23 and 7-15 %, respectively.
Ozonolysis involves using ozone gas to break down the lignin and hemicellulose and increase the biodegradability of the cellulose. The pretreatment is usually carried out at room temperature and is effective at lignin removal without the formation of toxic by-products (Vidal et al., 1988). Ozonation has been widely used to reduce the lignin content of both agricultural and forestry wastes. A drawback of ozonolysis is that a large amount of ozone is required, which can make the process expensive (Kumar et al., 2009).
2.3.4 Biological Pretreatment
Biological pretreatment involves microorganisms such as brown-, white- and soft-rot fungi that are used to degrade lignin and solubilize hemicellulose. The advantages of biological pretreatment include low energy requirement and mild environmental conditions. However, the rate of hydrolysis in most biological pretreatment process is very low and requires careful control of growth conditions (Sun et al., 2002).
2.3.5 Microwave Pretreatment
Microwaves (frequencies of 0.3GHz to 300GHz and wavelengths of 1 m to 1 mm) lie between radio wave frequencies (RF) and infrared (IR) frequencies in the electromagnetic (EM) spectrum, as shown in Figure 8. Microwaves can be reflected, transmitted and/or absorbed. The absorbed microwave energy is converted into heat within the material, resulting in an increase in temperature. Gases, liquids and solids can interact with microwaves and be heated. Under certain conditions, gases can be excited by microwaves to form plasmas that also can be useful for processing (Clark and Sutton, 1996).
Figure 2.7 The electromagnetic spectrum with applications at various frequencies (https://mynasadata.larc.nasa.gov/ElectroMag.html).
Microwave pretreatment is an energy-efficient, environmentally-friendly technology. Microwave treatment seems to be similar to steam treatment. However, microwave may have new functions effective for acceleration of reactivity of cellulosic materials. In the conventional steam treatment, the cellulosic materials containing water have been heated by an external heat source, such as the electrical coils surrounding the autoclave, or high pressure steam has been supplied to the cellulosic materials externally. On the other hand, in the microwave, the cellulosic materials are heated internally; therefore, the water, cellulose, hemicelluloses, and the other low molecular compounds such as the organic acid contained in the cellulosic materials absorb the microwave as the kinetic energies when the polar molecules and their neighboring clusters are forced to orient to the specific direction. It thus appears that the microwave gives a direct serious shock to the polar molecules composing cellulosic materials (Ooshima et al., 1984).
Microwave is an alternative method for conventional heating. Compared with conduction/convection heating, which is based on superficial heat transfer; the microwave uses the ability of direct interaction between a heated object and an applied electromagnetic field to create heat. Therefore, the heating is volumetric and rapid. When microwave is used to treat lignocelluloses, it selectively heats the more polar (lossy) part and creates a "hot spot" with the inhomogeneous materials. It is hypothesized that this unique heating feature results in an "explosion" effect among the particles, and improves the disruption of the recalcitrant structures of lignocellulose. In addition, the electromagnetic field used in microwave might create non-thermal effects that also accelerate the destruction of the crystal structures (Hu et al., 2008).
Compared with conventional heating techniques, microwave heating has the following additional advantages (Jones et al., 2002).
Higher heating rates
No direct contact between the heating source and the heated material
Selective heating may be achieved
Greater control of the heating or drying process
Reduced equipment size and waste
However, the microwave technology has been shown possibilities to be an energy efficient technique for chemical processing. The advantages and challenges of microwave processing are summarized in Table 2.2
Table 2.2 Benefits and challenges of microwave processing (Clark and Sutton, 1996)
Cost savings (time and energy, reduced floor space)
Rapid heating of thermal insulators (most ceramics and polymers)
Precise and controlled heating (instantaneous on/off heating)
Volumetric and uniform heating (due to deep energy penetration)
Short processing times
Improved quality and properties
Synthesis of new materials
Processing not possible with conventional means
Reduction of hazardous emissions
Increased product yields
Environmentally friendly (clean and quiet)
Self-limiting heating in some materials
Power supply can be remote
Clean power and process conditions
Heating low-loss poorly absorbing materials
Controlling accelerated heating (thermal runaway)
Exploiting inverted temperature profiles
Eliminating arcing and controlling plasmas
Efficient transfer of microwave energy to work piece
Compatibility of the microwave process with the rest of the process line
Reluctance to abandon proven technologies
Antonio et al. (2005) studied thermal effect of microwave irradiation. Microwave irradiation is rapid and volumetric, with the whole material heated simultaneously. In contrast, conventional heating was slow and introduced into the sample from the surface. The temperature profile as shown in Figure 2.8
Figure 2.8 The temperature profile after 60 sec as affected by microwave radiation (left) compared to treatment in oil bath (right).
Microwave irradiation raises the temperature of the whole reaction volume simultaneously, whereas in the oil heated tube, the reaction mixture in contact with the vessel wall is heated.
Hu and Wen (2008) studied microwave-based heating pretreated switchgrass, which was then hydrolyzed by cellulase enzymes. When switchgrass was soaked in water and treated by microwave, total sugar (xylose + glucose) yield from the combined treatment and hydrolysis was 34.5 g/100 g biomass, equivalent to 58.5 % of the maximum potential sugars released. This yield was 53 % higher than that obtained from conventional heating of switchgrass. With alkali loading from 0.05 to 0.3 g alkali/g biomass, microwave pretreatment resulted in a higher sugar yield than from conventional heating, with the highest yield (90 % of maximum potential sugars) being achieved at 0.1 g/g of alkali loading. Scanning electron microscope (SEM) images revealed that the advantage of microwave over conventional heating was due to the disruption of recalcitrant structures. At optimal conditions of 190 ï‚°C, 50 g/l solid content, and 30 min treatment time, the sugar yield from the combined pretreatment and hydrolysis was 58.7 g/100 g biomass, equivalent 99 % of potential maximum sugars. The results demonstrate that microwave-assisted alkali treatment is an efficient way to improve the enzymatic digestibility of switchgrass.
Zhu et al. (2005) investigated microwave-assisted alkali pretreatment of wheat straw and its enzymatic hydrolysis and compared with the conventional alkali pretreatment process. The results show that the higher microwave power with shorter pretreatment time and the lower microwave power with longer pretreatment time had the same effect on the weight loss and composition at the same energy consumption. It was found that the wheat straw had a weight loss of 48.4 % and a composition of cellulose 79.6 %, lignin 5.7 %, and hemicellulose 7.8 % after 25 min microwave assisted alkali pretreatment at 700 W, compared with a weight loss of 44.7 % and a composition of cellulose 73.5 %, lignin 7.2 %, and hemicellulose 11.2 % after 60 min conventional alkali pretreatment. The microwave assisted alkali pretreatment removed more lignin and hemicellulose from wheat straw with shorter pretreatment time compared with the conventional alkali one. Finally, the enzymatic hydrolysis of pretreated wheat straw (substrate concentration 50 g/l, enzyme loading 20 mg/g substrate) was also investigated and the results indicated that the microwave-assisted alkali pre-treated wheat straw had higher hydrolysis rate, reducing sugar concentration and glucose content in the hydrolysate than the conventional alkali pretreated one. Microwave-assisted alkali pretreatment is a potential alternative of wheat straw pre-treatment for it enzymatic hydrolysis.
Zhu et al. (2006) also examined three microwave/chemical processes for pretreatment of rice straw that are microwave/alkali, microwave/acid/alkali, and microwave/acid/alkali/H2O2 for its enzymatic hydrolysis and for xylose recovery from the pretreatment liquid. They found that xylose could not be recovered during the microwave/alkali pretreatment process, but could be recovered as crystalline xylose during the microwave/acid/alkali and microwave/acid/alkali/H2O2 pretreatment. The enzymatic hydrolysis of pretreated rice straw showed that the pretreatment by microwave/acid/alkali/H2O2 had the highest hydrolysis rate and glucose content in the hydrolysate.
In our group, Ploypradith P. (2010) studied the NaOH pretreatment with microwave on corncobs. The optimum conditions were found at 2 % NaOH at 100 Â°C for 30 min which could reduce lignin by 66.27 % and increse in surface area by 38.31 %. And the highest glucose concentration can reach up to 32.53 g/l and total sugar of 42.93 g/l was released. Moreover, microwave assists NaOH can produce total sugar concentration at shorter pretreatment time and lower pretreatment temperature compared with autoclave. In addition, total sugar concentration of microwave was higher than that of conventional heating.
Wanitwattanarumlug B. (2011) also studied the pretreatment of corncobs using microwave and potassium hydroxide. The highest sugar yield of 34.79 g/l was obtained from the corn cobs pretreated by microwave and 2 % KOH at 120 Â°C for 25 min. The results indicated that microwave-assisted alkali treatment was an efficient way to improve the enzymatic hydrolysis accessibility.
There are many research work related with pretreatment methods to improve its conversion. Among them, the microwave-assisted chemical pretreatment is a more effective to enhance the enzymatic hydrolysis by accelerating the reaction. In this study, The combined pretreatment of corncobs with microwave was conducted. A two-stage pretreatment using 2 % NaOH at 100 Â°C for 30 min the optimal condition of NaOH from Ploypradith P. (2010) and followed by H2SO4 pretreatment. In this work NaOH was used to separate lignin in the first stage and the effect of temperature, residence time and solid loading were determined in the second stage of two-stage pretreatment.
2.4 Inhibitors from Biomass Pretreatment
The pretreatment process generates numerous by-products that inhibit the growth of microorganism and fermentation. However, the generation of by-product depends on feedstock and pretreatment method (Jonsson et al., 2013). Especially, acid pretreatment that solubilizes hemicellulose leading to the formation of pentoses, hexoses, and inhibitors such as feruric acid, acetic acid, 2-furaldehyde (furfural), formic acid, and furoic acid. Furthermore, cellulose also degrades hexoses to 5-hydroxymethylfurfural (HMF), and levulinic acid. Other aldehydes and phenol can be formed by degration of lignin. Figure 2.9 showed the inhibitors that are generated during pretreatment (Liu and Blaschek, 2010). Although more than 100 compounds were detected as inhibitors, many have not been well studied (Liu et al, 2004).
Figure 2.9 The degradation product of lignocellulosic biomass during pretreatment (Liu and Blaschek, 2010).
Inhibitors can be classified base on chemical functional groups into 4 groups as aldehydes, ketones, phenols, and organic acids. Some studies have investigated that the low molecular weight compounds have more toxic to microbes than high molecular weight due to easier to transport (Sierra et al, 1991).
2.4.1 Aldehyde inhibitors
Aldehyde inhibitors are compounds with one or more aldehyde functional groups with a furan ring, a benzene ring or a phenol structure. For example, furfural and HMF which contain a furan ring and an aldehyde functional group. Other aldehyde inhibitors include 4-hydroxyÂbenzaldehyde, vanillin (Klinke et ai., 2002), syringaldehyde, and other compounds having a benzene ring or a phenol-based structure including isovanillin, ortho-vanillin, and coniferylaldehyde (Liu and Blaschek, 2010). The structure of aldehyde inhibitors are showed in Figure 2.10.
Figure 2.10 The structure of aldehyde inhibitors (Liu and Blaschek, 2010).
2.4.2 Ketone inhibitors
Ketone inhibitors include 4-hydroxyacetopheone and the closely related compounds acetovanillone and acetocsyringone. These compounds all share a common ketone functional group (Klinke et al., 2003). The structure of ketone inhibitors are showed in Figure 2.11.
Figure 2.11 The structure of ketones inhibitors (Liu and Blaschek, 2010).
2.4.3 Phenol-based inhibitors
Phenol-based inhibitors are grouped together including phenol, benzene-l,2-diol (catechol), benzene-1,4-diol (hydroquinone), 4-ethylbenzene-l,2-diol (ethy1catechol), 2-methylphenol, 3-methylbenzene-l,2-diol (methy1catechol), 2-methoxyphenol (guaiacol), 4-(hydroxymethyl)Â2-methoxyphenol (vanillyl alcohol), and 2,6-dimethoxybenzene-1,4-diol (Klinke et al., 2002). The structure of phonols inhibitors are showed in Figure 2.12.
Figure 2.12 The structure of phenols inhibitors (Liu and Blaschek, 2010).
2.4.4 Organic acid inhibitors
Organic acid inhibitors include simple acids as well as furoic acid with a furan ring that was considered as being a furan inhibitor. Moreover, many previously recognized phenolic compounds are now grouped as members of the organic acid inhibitor class based on their functional structure. Inhibitory compounds of this class all contain a carboxyl functional group and include acetic acid, formic acid, levulinic acid, caproic acid, furoic acid, 4-hydroxybenzoic acid, 3-hydroxybenzoic acid, 2-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, protocatechic acid, vanillic acid, gallic acid, syringic acid ,4Âhydroxycinnarnic acid, ferulic acid, homovanillic acid, guaiaclyglycolic acid, and sinapic acid. These inhibitors are thought to be exert their inhibitory actions via their carboxyl functional groups. The structure of organic acid inhibitors are showed in Figure 2.13.
Figure 2.13 The structure of organic acid inhibitors (Liu and Blaschek, 2010).
2.5 Butanol Fermentation Inhibitors
Pretreatment has been seen as a preferred method that make the enzyme in enzymatic hydrolysis step highly digests biomass in order to produce high amount of reducing sugar for further ABE fermentation. However, the toxic compounds including weak acids, furan derivatives, phenolic compounds, vanillic aldehyde, and tannin are generated during pretreatment (Eva and Bärbel, 2000); therefore, no microorganism can efï¬ciently produce butanol from lignocellulosic biomass (Weber et al., 2010) due to the inhibitors affect cell growth and ABE production.
Ezeji et al. (2007) studied the impact of inhibitors that generated from H2SO4 pretreatment on ABE concentrations. The results showed that syringaldehyde, ferulic, and Ï-coumaric acids were potent inhibitors of ABE production by Clostridium beijerinckii BA101 as shown in Figure 2.14. In general, ferulic and coumaric acids inhibit microorganism by damaging the hydrophobic sites of the bacteria cells because ferulic and coumaric acids are phenolic acids and phenolic compounds that affect membrane permeability (Heipieper et al., 1994). Furthermore, the authors observed that furfural and HMF (3 g/l) were not inhitory to Clostridium beijerinckii BA101. However, the combination of furfural and HMF affects the culture negatively. In addition, the production of salt, sulfate, which is result of sulfuric acid used for pretreatment was also toxic to Clostridium beijerinckii BA101.
Figure 2.14 The effect of inhibitors generated during 0.5 % H2SO4 pretreatment of corn fiber on ABE concentrations (Ezeji et al., 2007).
2.6 Detoxification Method (Chandel et al., 2011)
Since inhibitors from pretreatment process can be problematic for fermentation, the removal of inhibitors from hydrolysates is necessary to enhance microbial growth and fermentation efficiency. Nevertheless, inhibitors depend on type of pretreatment and feedstock. The most detoxification methods are physical, chemical, and biological (Chandel et al., 2011).
2.6.1 Physical Methods
The evaporation under vacuum can remove volatile compounds for example, furfural, acetic acid, and vanillin from hydrolysate of lignocellulosic biomass. However, evaporation retains the non-volatile toxic compounds such as lignin derivatives and extractives in the hydrolysates. A study by Wilson et al. (1989) found a decrease in the concentration of furfural, vanillin, and acetic acid by 100 %, 29 % and 54 %, respectively, compared with the concentrations in the hydrolysate. Likewise, Larsson et al. (1999) studied the removal of furfural and HMF using vacuum evaporation from wood hydrolysate. The results showed that furfural and HMF were reduced 90 %, 4 %, respectively.
220.127.116.11 Membrane separations
Adsorptive micro porous membranes have surface functional groups attached to their internal pores, that remove the cell wall derived inhibitors from acid hydrolysates. Grzenia et al. (2010) applied the membrane extraction for inhibitors removal from sulfuric acid hydrolysate of corn stover. The results showed that acetic acid, formic acid, levulinic acid, HMF, and furfural was eliminated.
2.6.2 Chemical Methods
The neutralization of acid hydrolysates is required step before fermentation because of low pH. Alkali (Ca(OH)2 or NaOH) is used for hydrolysates neutralization (pH in the range of 6-7). Phenolics and furfural may be removed by precipitation.
It was reported that overliming is the most cost effective method for detoxifying soft wood hydrolysates. Detoxification after pretreatment and enzymatic hydrolysis or before fermentation by alkali treatment begins by adding lime (NaOH or Ca(OH)2) to adjust the pH of the hydrolysate to a high value (in the range of 9-11) followed by pH readjustment to 6.6 with H2SO4. Adjustment of pH with Ca(OH)2 has been reported to increase the fermentability more than that with NaOH. The total amount of phenolic compounds was more efficiently decreased by Ca(OH)2. However, it has been shown that monovalent ions such as Na+ affect the ethanol productivity negatively, whereas Ca2+ does not. However, acetic acid and sugars were not removed by treatment process with NaOH or Ca(OH)2. Moreover, a heating step in the overliming procedure (leading to some evaporation) improves fermentability (Larsson, 1999). Furthermore, Ethanol productivity was more than twice as high after treatment with Ca(OH)2 compared with NaOH. The total concentration of phenolic compounds was affected by overliming detoxification due to phenolics were most efficiently removed with this method (Larsson, 1999).
18.104.22.168 Activated Charcoal Treatment
Activated charcoal is a cost effective method with high capacity to absorb compounds without affecting the amount of sugar in hydrolysate (Chandel et al., 2007). The activated charcoal treatment efficiency depends on pH, temperature, contact time, and the activated charcoal taken and the liquid hydrolysate volume ratio (Prakasham et al., 2009).
22.214.171.124 Ion Exchange Resins
Ion exchange resins treatment was applied to remove lignin-derived inhibitors, acetic acid and furfurals. The ion-exchange resins based separation of fermentative inhibitors may not be cost effective (Lee et al., 1999).
2.6.3 Biological Methods
The biological methods for detoxification are more feasible, environmental friendly, with fewer side-reactions and less energy requirements (Parawira and Tekere, 2011). The microorganisms and/or the enzymes have potential to alter the chemical nature of inhibitors. However, the slow reaction time and the loss of fermentable sugars make this methods unattractive (Yang and Wyman, 2008).
2.7 Response Surface Methodology (RSM) (Carley et. al., 2004)
Response Surface Methodology (RSM) is a statistical and mathematical techniques beneficial for developing, improving, and optimizing processes. A low-order polynomial is appropriate to use. In many cases, either a first-order or a second-order model is used. The first-order model is suitable when the experimenter is interested in approximating the true response surface over a relatively small region of the independent variable space.
In general, the first-order model is expressed as following equation
Where Yi is the response; xi is the input variables, which influence the response variable Yi; a0 is the offset term; ai is the ith linear coefficient.
The curvature in the true response surface is often strong enough that the first-order model is inadequate. A second-order model will be required. The following equation was used to correlate the dependent and independent variables of second-order model.
Where Yi is the response; xi, xj are the input variables, which influence the response variable Yi; a0 is the offset term; ai is the ith linear coefficient; aii is the quadratic coefficient and aij is the ijth interaction coefficient.
The second-order model is widely used in response surface methodology for several reasons:
1. The second-order model is very flexible due to a wide variety of functional forms; therefore it will often work well as an approximation to the true response surface.
2. It is easy to estimate the parameters in the second-order model. The method of least squares can be used for this purpose.
3. There is considerable practical experience indicating that second-order models work well in solving real response surface problems.