Starch/sugar-based crop

Starch/sugar-based crops can be utilized by organisms directly or after liquification or gelatinization, such as sugarcane and corn. The technologies for converting starch/sugar containing energy crops into ABE products are well-established. Table 2-5 shows several researches that focus on starch-based crops as ABE fermentation substrates. The productivity of C. acetobutylicum was 0.26 g/L/h when cassava used as substrate (Gu et al., 2009) and sago starch (Madihah et al., 2001). The addition of ammonia acetate elevates the solvent productivity of C. acetobutylicum to 0.4 g/L/h in cassava medium. Ezeji research group made a series study of using corn starch as a medium, the productivity were 0.15-0.29 g/L/h for C. beijerinckii (Ezeji et al., 2007b; Ezeji et al., 2005; Ezeji et al., 2007d).

Table 2-4 Comparisons of three different kinds of fermentation substrates.

Type Monosaccharide Starch/sugar-based crops

Lignocellulosic biomass

Example Glucose Corn

Starch Advantages Easily and directly

utilize by organisms.

Disadvantages Costly Costly

Edible parts of plants, competition with the food and feed supplies.

Difficult to utilize Reference (Ezeji et al., 2005;

Qureshi et al., 2007)

(Ezeji et al., 2007a; Guo et al., 2009)

(Qureshi et al., 2007; Qureshi et al.,

2010a)

Table 2-5 Performances of different Starch/sugar-based crops being medium for ABE fermentation.

Substrate

(Initial conc. ^a) Microorganism

Temp.

(40.8 g/L) C. beijerinckii

BA101 36/ND 37.2 Batch 20.0

(72 h) 0.28 - 14.3 1.7 (Ezeji et

al., 2005)

a Initial concentration indicate starch concentration at t=0 if there is no further explanation.

b The concentration of starch consumed by microorganism during ABE fermentation if no specific explanation.

c Productivity = Total ABE concentration/Fermentation time

d Yield = The weight of ABE solvent/The weight of sugar utilized by microorganism

e Acetic acid and butyric acid

Table 2-5 Different Starch/sugar-based crops for ABE fermentation (continuous).

Substrate

(Initial conc. ^a) Microorganism

Temp.

a Initial concentration indicate starch concentration at t=0 if there is no further explanation.

b The concentration of starch consumed by microorganism during ABE fermentation if no specific explanation.

c Productivity = Total ABE concentration/ fermentation time

d Yield = The weight of ABE solvent/ The weight of sugar utilized by microorganism

e Acetic acid and butyric acid

2.4.3 Lignocellulosic biomass

The production of biofuel from edible parts of plants has been increasing dramatically, which results in competition with the food and feed supplies. Lignocellulosic biomass is the most abundant renewable resource on the planet which offers an attractive alternative as ABE fermentation substrate. Wheat straw (Qureshi et al., 2007; Qureshi et al., 2008b), corn stover, switchgrass (Qureshi et al., 2010b), barley straw (Qureshi et al., 2010a), corn fiber xylan (Ezeji et al., 2007a; Qureshi et al., 2006), bagasse, silvergrass (Guo et al., 2009), and rice straw (Ko et al., 2009) are commonly used lignocellulosic biomass for the production of biofuels through ABE fermentation or ethanol fermentation in recent studies.

Rice straw is considered to account for the largest portion of available biomass feedstock in the world and Asia is responsible for 90% of the annual global production (Kim and Dale, 2003). In Taiwan, rice is one of the main food crop. According to Concil of Agriculture, Executive Yuan of Taiwan ―Agricultural Statistics Yearbook 2009‖, the crop area of rice in Taiwan was 254590 ha and every hectare of crop land could produce 6199 kilograms of rice in 2009. The great amount of rice straw residues left over after cropping would be excellent substrates for boifuel production. Currently, half of the agricultural residues of the world is burned, which cause health and environmental problems (Demain, 2009).

The main components of lignocellulose are cellulose, hemicellulose, and lignin.

Depends on the sorts of plant and material, the compositions are different in proportion.

Lignin and hemicellulose formed matrix and covered cellulose, which is naturally resistant to enzymatic attack (Sheehan John, 1994). Among the three main compositions of lignocelluloses, cellulose and hemicellulose are belongs to polysaccharides. Cellulose is the major components of plant’s cell wall, which plays a role of structural support. It consists of a linear chain of β (1-4) linked glucose monomers. Cellulose is tightly packed and highly crystalline structures make it water insoluble and resistant to depolymerization.

The structure of cellulose is shown in Figure 2-4. Unlike cellulose, hemicelulose is a heterogeneous compound that contains not only hexose (glucose and/or galactose) but also pentose (xylose, arabinose, mannose, etc.) monomers. Because of the branched structure, hemicellulose can be attached and broken easier by enzyme than cellulose. In addition, hemicellulose is soluble in acid solution. The structure of hemicellulose is shown in Figure 2-5. On the other hand, lignin is a three dimensional, net structural and non-crystalline polymer, which mainly consist of aromatic compound. However, the actual structure is still unclear. Overall, hemicellulose hydrogen-bonds to cellulose microfibrils and form a network that provides the structural backbones of cell wall. And lignin further strengthens the cell walls and provides resistance against diseases and pests (Mosier et al., 2005). As a result, lignocellulosic biomass is difficult to utilize directly by fermenting microorganisms due to their complex structure. To utilize the valuable resources, potential sugar monomers, contained in lignocellulose for fermentation processes, appropriate pretreatment and hydrolysis steps are required for lignocellulosic materials before fermentation by microorganisms. Figure 2-6 shows the general scheme for lignocellulosic biomass to produce ABE biofuels.

Figure 2-4 The structure of cellulose.

Figure 2-5 The structure of hemicellulose (arabinoxylan).

Figure 2-6 The general scheme of lignocellulosic biomass used for ABE fermentation.

2.4.2.1 Pretreatments

The main functions of pretreatments are to reduce the size of feedstock, open up the hemicelluloses-lignin matrix surrounds cellulose, and to break cellulose crystal structure (Sheehan John, 1994). The structure of lignocelluloses is altered to make cellulose and hemicellulose more accessible to the enzymes that saccharified the carbohydrate polymer into fermentable sugars (Mosier et al., 2005). A variety of pretreatment technologies with different characteristics have been developed. Pretreatments are mostly carried out under high temperature and pressure. However, a good pretreatment process is the one with a

high yield of carbohydrates combined with a low production of fermentation inhibitors.

Also good pretreatments need to minimize energy demand and limit cost.

There are four common categories of pretreatment technologies, biological, physical, chemical, and physio-chemical pretreatments. Biological pretreatments utilize wood degrading fungi, brown-, white-, and soft-fungi, to modify the chemical composition of lignocellulosic biomass. Brown rots mainly attack cellulose, while white and soft fungi attack both cellulose and lignin. The advantages of biological pretreatments are low energy requirement and mild environmental conditions. However, biological pretreatment processes need careful control of growth conditions control, large operation space, and long residence time (10-14 days) (Chandra et al., 2007), and throughout biological pretreatments are considered to be less attractive commercially.

Physical pretreatments include comminution, and pyrolysis. Comminution is a method to mechanically reduce biomass into particulate size by chipping, grinding, and milling. The enzymatic conversion yield of wet disk milling and ball milling pretreated rice straw were reported to be 0.79 and 0.89 of glucose, and 0.42 and 0.54 of xylose, respectively (Hideno et al., 2009). Pyrolysis decomposes the lignocelluloses through high temperature. Overall, physical methods break the crystaline structure, decrease the size, and increase the surface area of lignocellulosic biomass through mechanical power or heat.

Chemical methods, on the other hand, mainly break structure by chemical reactions, such as bond breaking. The example of chemical pretreatment methods are ozonolysis, acid or base hydrolysis, oxidative delignification, organosolv process, etc. In ozonolysis, ozone mainly attack lignin. Hemicellulose is slightly attacked and cellulose is hardly affected. Ozonlysis pretreatment does not produce toxic residues. In addition, it performs under room temperature and pressure. However, this process is expensive

solution gets high treatment efficiency in pretreatment process. However, it also gets corrosive feature and safety issues. Many researchers use dilute acid and dilute base solution instead of concentrated acid and base for pretreatment, which is efficient, safe, and economical (Cara et al., 2008). Dilute acid pretreatment can significantly improve cellulose hydrolysis (Guo et al., 2009; Karimi et al., 2006). Dilute base pretreatment of lignocelluloses caused swelling, leading to an increase in internal surface area and a decrease of polymerization and crystallinity, and disruption of the lignin structure (Mosier et al., 2005). Both acid and base pretreatments need to neutralize pH for the following enzymatic saccharification or fermentation processes. Soaking in aqueous-ammonia (SAA) is a new method of alkaline pretreatments which is highly selective for lignin removal and shows significant swelling effect on lignocelluloses. And ammonia is easily recoverable due to its high volatility. Ko et al. reported that rice straw pretreated by SAA could reach the maximum enzymatic digestibility of 71.1% at 69 ℃ for 10 h with an ammonia concentration of 21% (w/w) (Ko et al., 2009). Organosolv process use mix solution of inorganic acid (HCl or H2SO4) and organic solvent (methanol, ethanol, acetone, ethylene glycol, etc.) as reagent to break lignocellulose structure. The inorganic acids play a catalyst role in organosolv process. It is necessary to remove the solvent from the system after pretreatment because the solvent may inhibit microorganisms in enzymatic and fermentation processes.

Physio-chemical pretreatment methods are combination of both chemical and physical processes. Steam explosion, ammonia fiber explosion (AFEX) (Dale et al., 1996), and CO₂ steam explosion are the most well-kown and common methods. Steam explosion process is tipically treated with high pressure (0.69-4.83 Mpa) and high temperature (160-260℃), and then reduce pressure in a few seconds or munites. The materials undergo an explosive decompression (Sun and Cheng, 2002). The major effect is attributed to the removal of hemicellulose which improve the acessibility of enzymes to

cellulose fibrils. Steam explosion is a cost-effective methods compared to mechanical comminution. However, degradation products formed in this process are kown to inhibit the microorganism activity in the following processes, and therefore water washing step needs to be performed after pretreatment. The washing step remove not only inhibitors but also soluble sugars which cause the decrease of overall saccharification yields. The concept of AFEX and CO₂ explosion is similar to steam explosion. They are performed at high temperature and pressure for a period of time, and then the pressure reduced swiftly.

The major difference is that the materials are exposed in water, ammonia, and CO₂ for steam explosion, AFEX, and CO2 explosion, respectively. Unlike steam explosion, both AFEX and CO₂ explosion processes do not produce inhibitors (Sun and Cheng, 2002).

Still there are other novel technologies, such as Teramoto research group examined a sulfuric acid-free ethanol cooking pretreatment (SFEC) to pretreat lignocellulosic biomass.

This process exposes cut-milled lignocellulosic flours to an ethanol/water/acetic acid mixture in an autoclave. SFEC does not intensively delignified, instead it improves the accessibility of enzyme to cellulosic component (Teramoto et al., 2009).

Table 2-7 indicates the composition of lignocellulosic biomass before and after various pretreatment methods when rice straw represent as lignocellulosic biomass. Compared to other agriculture residues, rice straw primarily consists of cellulose, hemicellulose and lignin (Chandra et al., 2007), and 10-28% soft carbohydrates (starch, sucrose, glucose, fructose, and β-1,3-1,4-glucan.) (Park et al., 2009). It contains significantly larger amounts of starch than other cereal straws, and in some cases, the amount of starch in the rice straw reaches over 20% of the dry weight. Unlike wheat straw, high silica content and low digestibility prevents rice straw from being suitable cattle feed.

Table 2-6 Comparison of rice straw composition.

Cultivar Crop time

Original composition of rice straw

Table 2-6 Comparison of rice straw composition (continuous).

Cultivar Crop time

Original composition of rice straw

Lignin (acid soluble) 3%

Lignin (acid insoluble) 13%

Ash 15%

Shimane Bioethanol -

Holocellulose 57%

α-Cellulose 27%

Hemicellulose 30%

Cooking - - (Teramoto et al.,

2.4.2.2 Enzymatic saccharification

After pretreatment, it is followed by an enzymatic saccharification step for conversion of cellulose and hemicellulose polysaccharides to fermentable monosaccharides (hexoses and pentoses). Enzymatic saccharification needs three categories of enzyme, cellulase, hemicellulase, and cellobiase. Commercial cellulase usually contains at least three different enzymes, which are endoglucanase, exoglucanase, and β-glucosidase. Three types of reaction are involved in the reaction of cellulase. First, breakage of the non-covalent interactions present in the crystalline structure of cellulose by endoglucanase. Second, hydrolysis of the individual cellulose fibers to break it into smaller sugar compounds by endoglucanase. Third, hydrolysis of disaccharides and tetrasaccharides to break them into glucose by β-glucosidase. Hemicellulase hydrolyzes hemicellulose, which releases pentose (xylose, arabinose, mannose, etc.) and hexose (glucose and galactose). Cellobiase has same function as β-glucosidase in cellulase, which adds to assist the enzyme activity. Enzymes reach their maximum activity when pH is 5 at 50℃ (Abedinifar et al., 2009).

Many studies reported that hydrolysates produced by dilute acid pretreatment coupled with enzymatic saccharification of lignocellulosic biomass are potential feedstocks for ABE fermentation. Table 2-7 shows the results of dilute acid pretreatment and enzymatic saccharification of lignocellulose in several previous studies.

Table 2-7 Dilute acid pretreatment and enzymatic hydrolysis of lignocellulosic biomass.

Buffer Enzymes Incubation condition

Wheat straw Viscostar 150 L (xylanase)

45℃, 80 rpm, 72 h (Qureshi et al.,

2.5 Alternative operation strategies of ABE fermentation to reduce the