Category Archives: BIOETHANOL

Effect of sugar composition

Подпись: Fig. 4.1. Productivities with different initial sugar concentration in (a) batch and (b) continuous reactors. Adapted from Song and Ramkrishna (2010).

We examine the effect of increasing the portion of xylose in the culture medium on ethanol productivity (Fig. 4.1). The total conversion of mixed sugars is set to 0.99 as before. Additional xylose is assumed obtainable by collecting an unconverted sugar from fermentation systems using wild-type yeast which converts glucose only.

First, the change of ethanol productivity with initial glucose concentration in a batch reactor is given in Fig. 4.1 (a). Ethanol productivity may or may not increase with the ratio of xylose to glucose concentration depending on initial glucose concentrations. If, for example, the upper limit of Xxyl, o/xgLC, o is 1.0, xylose addition results in increase (or decrease) of productivity when xgLC,0 is below (or above) about 50 g/L. If the ratio of initial sugar concentration is allowed to vary up to 2.0, such threshold is extended to xgLC, o = 58 g/L. Higher improvement of ethanol productivity is expected for lower initial concentrations of glucose (e. g., 45% up at xgLC, o = 20 g/L, but 5.4% up at xgLC, o = 50 g/L). Optimal operating conditions correspond to segments of curves above other ones. In Fig. 4.1(a), for example, optimal operating conditions imply that xxyL/o/xgLc/0 = 2 when 20 < xGLC/o < 58, and xxyl, o/xglc, o = 0.5 when 58 < xglc, o < 80.

Next, operating curves in a continuous reactor are presented in Fig. 4.1(b). It is shown that, unlike the batch case, it is always recommendable to increase xylose level in the feed to increase the ethanol productivity. The best productivity is obtained when xxyL, in/ xgLC, in = 2.0, which increases the productivity by 56%, 26%, and 12% at xgLC, in = 20, 50, and 80 g/L, respectively.

Enzymatic hydrolysis of lignocellulose

In native cellulolytic organisms, enzymes needed for cellulose hydrolysis — xylanase, endoglucanase, exoglucanase, and ^-glucosidase — are expressed either separately or in complexes called cellulosomes (Fig 2). Noncomplexed cellulase systems are characteristic of cellulolytic aerobic bacteria (such as Bacillus spp.) and fungi (such as Trichoderma spp.) (Lynd et al. 2002). Endoglucanase hydrolyzes amorphous cellulose randomly, leading to the formation of cello-oligosaccharides of varying chain length. Exoglucanases are highly selective enzymes and act on either the reducing or the nonreducing end of cello — oligosaccharides to liberate glucose or cellobiose, respectively. ^-Glucosidase hydrolyzes cellobiose into its glucose monomers (Lynd et al. 2002). Cellobiose inhibits both exoglucanase and endoglucanase. Hence, ^-glucosidase plays an important role in the overall process, because it prevents the accumulation of cellobiose (Shewale 1982).


Fig. 2. (A) A model of cellulosome. (B) Synthetic scaffoldin favors arrangement of cellulases with higher activity in close proximity and hence would favor a proper synergy.

Reproduced with a permission from Annals of New York Academy of Science (Doi 2008).

Anaerobic bacteria such as Clostridium spp. usually produce complexed cellulases called cellulosomes. In cellulosomes, individual enzymes attach to a scaffoldin with their dockerin domains, while exposing the cellulose-binding domain. This complex enables proper synergy among endoglucanase, exoglucanase, and ^-glucosidase. (Bayer et al. 1998). Several chimeric scaffoldins have been engineered to position enzymes of higher activity together, and thereby increase the overall hydrolysis efficiency (Fig 2B) (Wen et al. 2009). Even though the large size of the cellulosomes restricts them to only the most readily accessible regions of cellulose, cellulosomes can hydrolyze cellulose more efficiently than free cellulases can (Wilson 2009).

Engineering efforts to increase the efficiency of cellulases and to enhance their kinetic properties have focused mainly on improving the specific activity by improving the thermal or the pH stability of the enzymes (Wen et al. 2009). However, a more important parameter to consider is the efficiency of access to the cellulose interior. While the active — site plays an essential role in other hydrolytic enzymes, the cellulose-binding domain constitutes the key module for cellulases (Bayer et al. 1998). In fact, the cellulose-binding domain determines the type of cellulase. Several efforts to establish a kinetic model for cellulose hydrolysis have failed because of the heterogeneous nature of the cellulosic substrate and the need for multiple enzyme activities (Kadam et al. 2004). In addition to enzyme-substrate proximity, enzyme-enzyme synergy should be considered as a factor for the efficient hydrolysis of cellulose. Whether any relationship or correlation between the crystallinity of lignocellulose and the rate of enzymatic hydrolysis exists remains unclear (Zhang and Lynd 2004). Moreover, the mechanism of cellulose hydrolysis remains incompletely understood, because some groups of cellulases have both exoglucanase and endoglucanase activities.

The low processivity of cellulases demands that the enzymes be replenished several times during the saccharification process. The economic feasibility of enzymatic hydrolysis of lignocellulose to simple sugars is limited by the poor kinetic properties of the enzymes. The use of cellulase-secreting microbes could be an economical alternative to the enzymatic saccharification process. With microbes, the enzymes can be continuously produced, secreted, and used to hydrolyze cellulose into simple sugars that could be directly fermented to ethanol (Fig 3). Thus, microbial fermentation of lignocellulose offers greater promise for economical bioethanol production.

Preparation method

The first step in the pre-separation process of starchy root or cassava tuber is to remove the adherent soil from roots by washing in order to prevent any problem later caused by the soil and sand. The process is followed by disintegration of cell structure to break down the size mechanically (i. e. milling) or thermally (i. e. boiling or steaming) or by combination of both processes. Slurry will be produced from the disintegration process which contains a mixture of pulp (cell walls), fruit juice and starch. This slurry can be cooked directly to gelatinized starch. When it is required, it can also be separated to produce flour by exploiting the difference in density using hydrocyclone and/or centrifuge separators as presented in Table 1.


Density g/ml



Cell walls (fibers)




Soil, sand

above 2

Table 1. Density of root components, water and soil (International Starch Institute, 2010).

For direct fermentation from starch to ethanol, there are two techniques normally employed in preparing starch medium which are non-cooking and low-temperature cooking fermentation. In non-cooking technique no heating is required however an aseptic chemical or method may be required to avoid contamination. Since it is uncooked, some aeration or agitation may also be required to avoid sedimentation of the starch particle. In low-cooking temperature fermentation, the medium is either semi or completely gelatinized first prior to inoculation of fermenting microorganism. Gelatinized starch forms a very viscous and complex fermentation media. It contains nutrients that required by microorganisms to grow and to produce different fermentation products. During fermentation, various physical, biochemical and physical reactions take place in the media. The nature and composition of the fermentation media will also affect the efficiency of the fermentation process. Many difficulties in designing and managing biological processes are due to the rheologically complicated behavior of fermentation media. Due to that, a pseudoplastic of a non­Newtonian behavior of starch solution is essential for cooked or gelatinized starch. This pseudoplastic property of gelatinized starch is important because it has suspending properties at low shear rates and its viscosity becomes sufficiently low when it is processed at higher rates of shear. Any fermentation medium which does not apply any viscosity reduction agent such as enzyme, its viscous nature combined with non-Newtonian flow will affect the mass heat transfer, dissolved oxygen homogeneity, mixing intensity, cell growth rate and eventually, the product accumulation state. Thus, it is imperative to minimize the viscosity to eliminate these problems. Starch slurry or flour concentration, temperature, agitation speed and cooking/gelatinization time are the major factors affecting media preparation. Optimization study of these conditions is useful prior to single-step fermentation of consortium or co-culture microorganisms. Table 2 gives the gelatinization temperature for different sources of starch. This information is helpful to prepare cooked or gelatinized starch for direct bioconversion at low temperature cooking.


Gelatinization Temperature Range (oC)


59-68a, b,c

Cassava/ tapioca

58.5-70a, c


62-80a, b

Paddy, rice and brown rice




Waxy corn



52-85a, d

aTurhan and Sagol (2004), b Whistler and Daniel (2006), cTulyathan et al. (2006), d Sagol et al. (2006) Table 2. Starch gelatinization temperature range

Simultaneous production of sugar and ethanol from sugarcane

As the major raw material, most of sugarcanes are refined into sugar in China now. Also the international sugar price is running in high level, and it needs to balance the domestic sugar supply and demand through imports, so it is impossible to produce large amounts of ethanol by sugarcane. However, it is unfavorable to sugar price stability and its healthy development if only refining sugar. To achieve more economic benefits, a viable option is to explore the "Simultaneous production of sugar and ethanol " mode. In recent years, we have made some progress on the sugarcane breeding, ethanol production technologies and process optimization for simultaneous production of sugar and ethanol.


With conventional distillation at atmospheric pressure, the maximum achievable ethanol concentration is 90-95%, because in the system ethanol-water there is an azeotrope at 95.6% (w/w) ethanol, boiling at a temperature of 78.2°C. For the production of anhydrous ethanol further dehydration of the concentrated ethanol is required. This can be achieved by employing azeotropic distillation, extractive distillation, liquid-liquid extraction, adsorption, membrane separation or molecular sieves (Hatti-Kaul, 2010; Huang et al., 2008).

Separation of ethanol from water is an energy-intensive process. The energy required for production of concentrated ethanol by distillation also depends very much on the feed concentration (Zacchi & Axelsson, 1989). The search for solutions for the reduction of the energy required is a field of intensive research. Membrane separation processes need much less energy for ethanol separation but are not in operation on an industrial scale. First results from a pilot plant using the SiftekTM membrane technology show a reduction of the energy required for dehydration of about 50% (Cote et al., 2010). Process and heat integration techniques also play an important role in energy saving in the bioethanol process (Alzate & Toro, 2006; Wingren et al., 2008). Maximum energy saving in the distillation of about 40% is possible by applying mechanical vapour recompression (Xiao-Ping et al., 2008). Solar distillation of ethanol is under investigation for distillation of bioethanol in smaller plants (Vorayos et al., 2006). The production of solid biofuel or biogas for thermal energy supply also reduces the net energy requirement of bioethanol production (Eriksson & Kjellstrom, 2010; Santek et al., 2010).

Wet oxidation of rape straw

Rape straw was produced after harvest during cultivation of Brassica napus Linnaeus commonly known as rape in Denmark in 2007. The straw was milled to a particle size of 2 mm using a knife mill. The milled straw was then soaked in 80°C hot water for 20 min before wet oxidation in a 2 L loop autoclave (Bjerre et al., 1996). The autoclave setup includes 1 L water, 60 g dry milled rape straw with the canister pressurized to 12 bar oxygen during the reaction. The mixing of oxygen and liquid is obtained by a pumping wheel. The wet oxidation was performed at 205°C for 3 min (Arvaniti et. al 2011). Following the wet oxidation, pressure is released and the prehydrolysate is cooled to room temperature. The filter cake and filtrate are separated and stored at -18° C (Thygesen et. al. 2003).

During wet oxidation, oxygen is introduced in the pre-treatment phase at high pressure and temperature. This causes 50 % (w/w) of the lignin to oxidize into CO2, H2O, carboxylic acids and phenolic compounds. It is important that the lignin fraction is low since lignin can denaturize the enzymes involved in the subsequent hydrolysis of cellulose. The majority of the hemicellulose (80 %) is dissolved while 10 % is oxidized to CO2, H2O and carboxylic acids. During this process, carboxylic acids, phenolic compounds, and furans are produced, which act as inhibitors in the fermentation process. However, the concentrations are too low to fully hinder microbial growth as wet oxidation also degrades the toxic intermediate reaction products (Thomsen et. al. 2009).

CO2 dry reforming

In addition to H2O, CO2 can also acts as oxidant to reform ethanol to generate gaseous products. The reaction involved in this process is depicted in Reaction (2).

C2HsOH(l) + CO2 3 H2 + 3 CO (AHr,298K = 338 kJ/mol) (2)

Compared to Reaction (1), although only 3 moles of hydrogen are produced per mole of ethanol by using dry reforming process, it is still a valuable approach to utilize CO2 for hydrogen or syngas production beneficial for reducing greenhouse gas emission. The process feasibility and optimal operation parameters have been investigated by W. Wang, et al. thermodynamically, which is valuable for desirable product yield maximization. According to the calculations performed in [22], higher temperature, lower pressure, addition of inert gas, and lower CO2 to ethanol ratio benefit the improvement of hydrogen yield. Several catalysts such as Ni/ Al2O3 [23] and Rh/ CeO2 [24] have been developed in recent years for hydrogen or syngas production. Generally speaking, CO2 is less active than water in oxidizing ethanol. Therefore, more active catalysts are critical for making ethanol dry reforming more attractive to industrial investors. Similarly to methane dry reforming, coke can be formed with high possbility at certain reaction conditions on the catalyst surface, resulting in catalyst deactivation. Carbon tends to form at low temperature and low CO2/ethanol ratio based on thermodynamic prediction, which should be avoided to prevent catalyst deactivation. However, sometimes as a preferable byproducts, production of various types of carbon nanofilaments is desired by following Reaction (3), which has been found to be effectively catalyzed by stainless steel or carbon steel catalysts [25, 26].

C2H5OH(i) + CO2 2 H2 + 2 CO + 2 C +H2O(i) (AH^98K = 163 kJ/mol) (3)

Cassava agronomy and plantation

Cassava is well recognized for its excellent tolerance to drought and capability to grow in impoverished soils. The plant can grow in all soil types even in infertile soil or acid soil (pH

4.2-4.5), but not in alkaline soil (pH > 8). Despite of that, cassava prefers loosen-structured soil such as light sandy loams and loamy sands for its root formation. As the drought — tolerant crop, cassava can be planted in the lands having the rainfall less than 1,000 mm or unpredictable rainfall. Rather than seeding, the plants are propagated vegetatively from stem cuttings or stakes, having 20-cm in length and at least 4 nodes. To ensure good propagation, good-quality stakes obtained from mature plants with 9-12 months old should be used. The appropriate time of planting is usually at an early period of rainy seasons when the soil has adequate moisture for stake germination. When planted, the stakes are pushed into the soil horizontally, vertically or slanted; depending on soil structure. For loosen and friable soil, the stakes are planted by pushing vertically ("standing"), or slanted approximately 10 cm in depth below the soil surface with the buds facing upward. This planting method gives higher root yields, better plant survival rates and is easy for plant cultivation and root harvest (Howeler, 2007). The horizontal planting is suited for heavy clay soils. Planting with 100 x 100 cm spacing (or 10,000 plants/hectare) is typical, however, less spacing (100 x 80 cm or 80 x 80 cm) and larger spacing (100 x 120 cm or 120 x 120 cm) are recommended for infertile sandy soil and fertile soil, respectively. At maturity stage with 8­18 months after planting, the plants with two big branches (i. e. dichotomous branching) or three branches (i. e. trichotomous branching) are 1-5 m in height with the starch — accumulating roots extending radially 1 m into the soil. Mature roots are different in shapes (as conical, conical-cylindrical, cylindrical and fusiforms), in sizes (ranging from 3 to 15 cm in diameter, as influenced by variety, age and growth conditions) and in peel colors (including white, dark brown and light brown). Although the roots can be harvested at any time between 6-18 months, it is typically to be harvested on average at 10-12 months after planting. Early or late harvesting may lower root yields and root starch contents. Still, the actual practice of farmers is depending on economic factors, i. e. market demand and root prices. Root harvesting can be accomplished manually by cutting the stem at a height of 40 — 60 cm above the ground and roots are then pulled out by using the iron or woody stalk with a fulcrum point in between the branches of the plant. Plant tops are cut into pieces for replanting, leaves are used for making animal fodder and roots are delivered to the market for direct consumption or to processing areas for subsequent conversion to primary products as flour, chips and starch.

Exogenous enzymes

As explained before, protein digestibility is related to ethanol production and this digestibility in turn is related to the tendency of sorghum proteins to form web-like structures during mashing which reduces the possibility of enzymes to access starch. Protein solubility should decrease with the increase of protein cross-linking; thus, this parameter can be used as a quality indicator in sorghum biorefineries (Zhao et al., 2008).

The utilization of proteases before conventional starch liquefaction can be used as an alternative method to improve rate of starch hydrolysis and yield hydrolyzates with high FAN concentration (Perez-Carrillo & Serna-Saldivar, 2007).

Perez-Carrillo et al. (2008) proposed the use of protease before starch gelatinization and liquefaction of both decorticated and whole sorghum meals. The use of decortication to remove the sorghum outer layers and the exogenous protease had a positive synergic effect in terms of ethanol yield and energy savings because mashes required about half of the fermentation time compared to conventionally processed sorghum. Decorticated meals with more starch were more susceptible to alpha-amylase during liquefaction and produced more ethanol during fermentation (Alvarez et al., 2010). This technology produced similar ethanol yields compared to soft yellow dent maize and 44% more ethanol compared to the whole sorghum control treatment. The other advantage of mechanical decortication is that the bran, separated beforehand, is shelf-stable and can be directly channeled for production of animal feeds and consequently the yield of wet distilled grains from decorticated sorghum is significantly lower compared to the obtained after processing whole sorghum meals. Thus, if dried distilled grains are produced, the biorefinery plant will spend less energy when processing decorticated sorghum.

Acid hydrolysis

The main advantage of the acid hydrolysis is that acids can penetrate lignin without any preliminary pretreatment of biomass, thus breaking down the cellulose and hemicellulose polymers to form individual sugar molecules. Several types of acids, concentrated or diluted, can be used, such as sulphurous, sulphuric, hydrocloric, hydrofluoric, phosphoric, nitric and formic acid (Galbe & Zacchi, 2002). Sulphuric and hydrochloric acids are the most commonly used catalysts for hydrolysis of lignocellulosic biomass (Lenihan et al., 2010).

The acid concentration used in the concentrated acid hydrolysis process is in the range of 10-30%. The process occurs at low temperatures, producing high hydrolysis yields of cellulose (i. e. 90% of theoretical glucose yield) (Iranmahboob et al., 2002).


Fig. 3. Process for production ethanol from lignocellulosic biomass. The circle in the scheme indicates two alternative process routes: simultaneous hydrolysis and fermentation (SSF); separate hydrolysis and fermentation (SHF).

However, this process requires large amounts of acids causing corrosion problems to the equipments. The main advantage of the dilute hydrolysis process is the low amount of acid required (2-5%). However this process is carried out at high temperatures to achieve acceptable rates of cellulose conversion. The high temperature increases the rates of hemicellulose sugars decomposition thus causing the formation of toxic compounds such as furfural and 5-hydroxymethyl-furfural (HMF). These compounds inhibit yeast cells and the subsequent fermentation stage, causing a lower ethanol production rate (Larsson et al., 1999; kootstra et al., 2009). In addition, these compounds lead to reduction of fermentable sugars (Kootstra et al., 2009). In addition, high temperatures increase the equipment corrosion (Jones & Semrau, 1984).

In 1999, the BC International (BCI) of United States has marketed a technology based on two-step dilute acid hydrolysis: the first hydrolysis stage at mild conditions (170-190°C) to hydrolyze hemicellulose; the second step at more severe conditions to hydrolyze cellulose 200-230°C (Wyman, 1999).

In 1991, the Swedish Ethanol Development Foundation developed the CASH process. This is a two-stage dilute acid process that provides the impregnation of biomass with sulphur dioxide followed by a second step in which diluted hydrochloric acid is used. In 1995, this foundation has focused researches on the conversion of softwoods using sulphuric acid (Galbe & Zacchi, 2002).