Category Archives: BIOETHANOL

Vitality of the yeast cells

Fig. 8 shows the yeast cells after a glucose fermentation. A large part of the yeast cells have become inactive, even though there is still enough sugar in the substrate to sustain living cells.

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Fig. 8. Methylene blue stained yeast cells after glucose fermentation.

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Fig. 9. Methylene blue stained yeast cells after heating to 100 °С.

During the experiments with dose response on furfuryl alcohol in glucose fermentation, a relation was found between cell death and the concentration of furfuryl alcohol (Fig. 8). Yet at this writing, there still exist some inconsistencies between erioglaucine and methylene blue. Overall the results produced by methylene blue appeared slightly more consistent and matching with the data for ethanol production.

Reaction mechanism and kinetic studies

As can be seen in Section 2.7, the reaction network which would possibly occur during BESR is fairly complicated and heavily dependent on the catalyst system employed. In order to obtain maximum amount of hydrogen out of ethanol used, the side reactions should be effectively suppressed, leading to the minimization of byproducts such as methane, carbon monoxide, acetaldehyde, acetone, acetic acid and so on. For controlling the reaction proceeding along the desired pathway which will give us the highest hydrogen yield, it is critical to gain a comprehensive understanding of the reaction mechanisms involved, which will in turn guide the rational design of catalyst system. There are two approaches we can follow to achieve our final goal, that is, theoretical and experimental directions. The theoretical approach (reaction mechanism study through computational chemistry) is still at its initial stage referring to the papers published in this area and will be covered in detail in Section 6. However, the experimental route has been widely adopted to study the catalytic behaviors present during BESR.

As an interfacial phenomenon, any heterogeneous catalytic reaction takes place involving three basic steps: reactants adsorption, surface reaction, and products desorption. To be a gas-solid reaction, catalytic BESR must embroil gas composition variation and catalyst surface evolution. Therefore, in order to attain a complete view of the reaction, systematical investigation should be performed on both gas and solid phases. Gas chromatography (GC) and mass spectrometer (MS) are the two popular instruments used to monitor the gas phase composition and fourier transform infared spectroscopy (FTIR) can detect the surface species and their evolutions during BESR. In addition, using other characterization techniques including nuclear magnetic resonance (NMR) and laser Raman spectroscopy (LRS) can provide an alternative way to get better insight into the reaction mechanisms.

Based on the results reported in the literature, the dehydrogenation and dehydration reactions are the two pathways ethanol can go through first, the choice of which depends on the catalysts charged. If the catalyst has high acidity (e. g., Al2O3 and SiO2 [105, 106] supported samples), dehydration reaction is favored, resulting in the formation of C2H, a precursor of coking through polymerization. If the catalyst presents basic features (e. g., MgO and ZnO [107, 108] supported sample) instead, dehydrogenation reaction is preferred, leading to the production of acetaldehyde, an important reaction intermediate related to higher H2 yield. Acetaldehyde can then be decomposed into CH4 and CO [109] or undergoes steam reforming to generate CO and H2 relying on the catalyst employed. These single carbon containing products (CH4 and CO) can be further reformed to CO2 and H2 through methane steam reforming and water-gas shift reaction if sufficient water is supplied. Besides, two acetaldehyde molecules can react with each other to form acetone through aldol condensation reaction [35] or be oxidized to acetic acid [110]. Carbon can be formed at various stages from carbon-containing species via either cracking or Boudouard reaction [111].

Ethanol adsorption and subsequent surface reaction have been extensively studied over many different catalyst systems employing FTIR technique. Although the exact locations of the ethanol adsorption bands vary with catalysts tested, the identifications of surface species and its evolutions are well established. Ethanol can be adsorbed on the sample surface dissociatively and molecularly [112-114]. The ethoxide species is the result of ethanol dissociative adsorption. Then the surface acetate species is obtained from the oxidation of ethoxide by the lattice oxygen coming from the sample surface [115, 116]. The acetate species can then experience C-C breakage leading to the formation of single carbon fragments. Whether these fragments will be released directly from the surface or undergo further oxidation to carbonate species is closely linked to the sufficiency of oxygen stored in the sample. The adequate oxygen supplies benefit the formation of carbonate species. Finally CO2 originates from the decomposition of carbonate species. However, compared with ethanol, water adsorption and its role in the subsequent surface reaction remain unclear for BESR. Therefore, the surface features need to be investigated during water adsorption and co-adsorption of water and ethanol.

13C NMR technique has been applied into the study of ethanol adsorption behavior to track the evolution of carbon containing species over Cu/ZnO [117]. Different oxygenate species have been identified after integrating with the results obtained from MS. Unfortunately, just 1-C was labeled in the ethanol molecule, in order to get a comprehensive picture of the surface species and its evolution after ethanol adsorption, 2- C, even H and O labeled ethanol is also worth being considered. A similar approach is also applicable for water adsorption and co-adsorption of ethanol and water by choosing suitable isotopic labeled elements.

Compared with the kinetic studies focused on the steam reforming over single carbon containing reactants such as methanol [118-122] and methane [123-127] (MSR) which have been investigated intensively for tens of years, the kinetic investigations performed over ethanol steam reforming (ESR) reaction are still in their burgeoning stage, which might be due to the relatively complicated reaction networks involved originating from the increase of carbon atom. However, the knowledge accumulated during the systematic explorations of the kinetic mechanisms occurring during MSR provides a valuable starting point for ESR researchers to expand upon. In recent years, based on the observations obtained from both gas phase and sample surface, several kinetic models have been proposed to simulate the mechanistic behaviors of various catalyst systems [128-132], which will facilitate better understanding of the reaction mechanisms. If the estimated values are in good consistency with the reported experimental results, the assumed reaction pathways and rate­determining step (RDS) will uncover the actual reaction mechanisms to a certain level. Furthermore, the activation energy measured from this study provides the reference for molecular simulation. In addition, the outcomes from this kinetic analysis will benefit the reactor design which can promote mass and heat transfer during reaction.

Based on the TPD and DRIFTS results reported in [133], a possible reaction pathway for ethanol steam reforming over Co-based catalysts is proposed by our laboratories in Fig.6. In Scheme 1, the reactant molecules (EtOH and water) diffuse from gas phase to the surface of the catalyst. The ethanol molecules adsorb dissociatively on the Co sites, forming ethoxide species. Water, on the other hand, adsorbs on the support, forming hydroxyl groups. The first H abstracted from ethanol can either form OH groups with the surface oxygen species or combine with hydrogen from H2O and form H2 (Scheme 3). Ethoxide species move to the interface of metal and oxide support and be oxidized by an additional hydrogen abstraction forming aceteldehyde (Scheme 4). Acetaldehyde molecules may lead to the formation of acetone through an aldol-condensation type reaction and acetone molecules are observed only in the gas phase. Acetaldehyde species have a short surface residence time, converting readily to acetate species through further oxidation with surface oxygen or OH groups (Scheme 5). There are multiple routes for the acetate species once they are formed. In one of the routes, the metal may be involved in C-C bond cleavage leading to the formation of single carbon species (Scheme 7), leading to the formation of CH4. The carbon-oxygen surface species may desorb or further oxidize to give carbonate species, especially on supports with high oxygen storage capacity (Scheme 8), which can desorb as CO2 (Scheme 9). In a second route, especially, if oxygen accessibility is high, the CH3 fragment will undergo oxidation through H subtraction and O addition (Scheme 10) to form formate, possibly through a formaldehyde intermediate (Scheme 11), and carbonate (Scheme 12). The catalyst surface is then regenerated through CO2 desorption (Scheme 13) and ready for the next catalysis cycle regardless of the route followed.

If the surface is highly acidic, ethanol dehydration may dominate the reaction pathway and result in the formation of H2O and C2H4 which is the major precursor to coke due to polymerization, as described in Scheme 2 and 6. If the oxygen mobility in the catalyst is not high enough, the acetate species may remain on the surface and lead to coke formation, as reported earlier [34, 134].

Briefly speaking, dissociative adsorption of ethanol and water leads to ethoxide species and hydroxyl groups, respectively. The active metal catalyzes the C-C bond cleavage and formation of single carbon species. BESR reaction could happen at the interface of the active metal and the oxide support, which could participate by providing oxygen from the lattice to facilitate the oxidation of carbon species. The resulting oxygen vacancies can be filled by the oxygen in the hydroxyl species formed from water adsorption. Therefore, it is necessary to have rapid oxygen delivery mechanism throughout the oxide support to prevent carbon deposition on the surface due to deficient oxidation of carbon species. High metal dispersion

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Fig. 6. Proposed Reaction Mechanism for Ethanol Steam Reforming over supported Co catalysts

will favor the ethanol adsorption and formation of more accessible metal/oxide interfaces as well as C-C cleavage. High oxygen storage capability and mobility will facilitate the oxygen delivery through the support and suppress coke deposition. The Co-based systems that incorporate oxides with high oxygen storage and oxygen mobility could deliver the required characteristics needed for active and stable BESR catalysts.

Cassava bioethanol production

As described previously, the ethanol production from cassava feedstock involves 5 main steps (Figure 6a) which are

— Feedstock preparation: the main purpose of this step is to make cassava feedstock be physically suitable for downstream processing, i. e. cooking, starch hydrolysis, fermentation and distillation & dehydration. Details are different regarding to types of feedstock and milling process. In general, the preparation includes impurity removal (washing and peel removal of fresh roots, metal detector, sand and soil removal of slurry by hydrocyclone), size reduction by milling or rasping and fiber separation.

— Cooking: The starch is cooked to rupture the granular structure and hence improve its susceptibility to enzyme hydrolysis. Cooking is achieved at a temperature greater than a gelatinization temperature. During cooking, the high viscosity of the slurry is developed due to starch gelatinization and swelling of some particles. Cooking is, therefore, commonly performed in a presence of liquefying enzymes, i. e. a-amylase to liquefy cooked slurry.

— Starch hydrolysis: Starch is enzymatically hydrolyzed to glucose by a-amylase and subsequently by glucoamylase. The liquefaction by a-amylase is usually conducted at high temperatures at which the starch become gelatinized. After liquefaction, the liquefied slurry is cooled down to an optimum temperature for glucoamylase hydrolysis which is about 50-55°C, depending on enzyme types.

— Yeast fermentation: Glucose is then fermented by yeast. By the end of fermentation, the obtained beer contains approximately 10%v/v ethanol, depending on solid loading during fermentation.

— Distillation and dehydration: The beer is subjected to distillation to concentrate the ethanol to 95% and then dehydration to remove water, yielding anhydrous ethanol (99.5%).

Nowadays, the production process of bioethanol from starch feedstock is developed to significantly reduce processing time and energy consumption by conducting saccharification and fermentation in a same step; this process is called "Simultaneous Saccharification Fermentation", or SSF process (Figure 6b). In this SSF process, the liquefied slurry is cooled down to 32°, afterward glucoamylase and yeast are added together. While glucoamylase produces glucose, yeast can use glucose to produce ethanol immediately. No glucose is accumulated throughout the fermentation period (Figure 7) (Rojanaridpiched et al, 2003).

The material balance of ethanol process with a production capacity of 150,000 liters/day, a recommended size of ethanol plants for optimum production costs, feasible feedstock management and effective waste water treatment, from dried cassava chips by Simultaneous Saccharification and Fermentation (SSF) process is estimated from production data collected during pilot trials (at 2,500 L working volume) and factory survey (Figure 8) (Sriroth et al., 2006). The conversion ratio of feedstock (kg) to ethanol (liter, L) is about 2.5:1 for dried chips or 6:1 for fresh roots, this conversion factors are starch-quantity dependent. Based on the pilot production data, the estimated production cost, excluding the feedstock cost, of ethanol from cassava chips by SSF process is about 0.259 USD/L (Rojanaridpiched et al., 2003; Sriroth et al., 2006) which is close to a value reported by FO Licht to be 0.24 USD/L (FO Licht, 2006). The estimated production cost of cassava chips are detailed in Table 7 (Sriroth et al., 2010a).

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Note: The temperatures are enzyme — and yeast-type dependent.

Fig. 6. (a) Conventional, (b) SSF, Simultaneous Saccharification and Fermentation and (c) SLSF, Simultaneous Liquefaction, Saccharification and Fermentation process of ethanol production from cassava feedstock.

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Fig. 7. Changes of total soluble solid (TSS, °Brix by a refractometer), cell counts, glucose and ethanol contents (by High Performance Liquid Chromatography using Bio-Rad Aminex HPX-87H column), during ethanol fermentation from cassava chips by conventional fermentation (CF) and Simultaneous Saccharification and Fermentation (SSF) process. (Experimental condition for CF; a slurry of 25% dry solid was initially liquefied by a — amylase at 95°C, saccharified by glucoamylase at 60°C and then fermented by yeast (Saccharomyces cerevisiae) at 32°C) and for SSF; a slurry of 25% dry solid was liquefied in a similar manner and then subjected to SSF by adding a mixture of glucoamylase enzyme and yeast at 32°C).

In SSF process, the starch in cassava material has to be initially cooked and liquefied prior to SSF process. Recently, a granular starch hydrolyzing enzyme has been developed to produce fermentable sugars from native or uncooked corn starch and is then applied to cassava chips (Piyachomkwan et al., 2007; Sriroth et al., 2008). This enzyme can attack directly to uncooked starch granules (Figure 9), allowing liquefaction, saccharification and, in the presence of yeast, fermentation to occur simultaneously in one step at the ambient temperature without cooking; this process is Simultaneous Liquefaction, Saccharification Fermentation or SLSF (Figure 6c). Figure 10 demonstrates the ethanol production from cassava chips using conventional, SSF and SLSF process. It is interesting to note that by SLSF process, the total soluble solid and glucose content do not change over fermentation as starch is used in a native, granular insoluble form. The fermentation efficiency of SLSF process is reported to be comparable with cooked process (Table 8). SLSF process is energy­saving, easy to operate and can be applied economically to produce sustainable energy, at a small scale, for community.

ra«49va Chin ‘Moisture 15 %

v>uip starch content 65o/o (wet basis)

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Fuel Ethanol

118.35 T/D or

150,0 L/D

Fig. 8. Mass balance of ethanol production from cassava chip by SSF (Simultaneous Saccharification and Fermentation) process; T/D = Tons/Day, TS = Total Solid,

L/D =Liter/day (Fermentation efficiency 90%, Distillation efficiency 98.5%) Source: Sriroth et al., 2006

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O hr 12 hr 24 hr 48hr

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ethanol production from cassava chips by Simultaneous Saccharification and Fermentation (SSF; the slurry of 25% dry solid was liquefied by 0.1% Termamyl 120L (Novozymes) at 95- 100°C, 2 hr followed by simultaneous saccharification and fermentation with 0.1% AMG (Novozymes) and Saccharomyces cirivisiae yeast at 32°C for 48 hrs) and Simultaneous Liquefaction, Saccharification and Fermentation (SLSF; the slurry of 25% dry solid was liquefied, saccharified and fermented with 0.25% granular starch hydrolyzing enzyme (Stargen™, Danisco-Genencor, USA) and Saccharomyces cirivisiae yeast at 32°C, 60 hr.

Source: Rojanaridpiched et al., 2003 ; Sriroth et al., 2007

Ethanol from sorghum bagasse and straw

4.2 Raw material conditioning

After extraction of juice or grain harvesting, the lignocellulosic residue is chopped, milled, and dried at 50-60 °C to reduce the moisture content to about 10 to 15% (Herrera et al., 2003; Sipos et al., 2009). There are many options to reduce particle size; the most commonly used are hammer or rotary mills. Grinding can be used on both dry and wet materials, and the cost is one of the lowest compared with others methods used for milling biomass. The grinder reduces the particle size to a fine powder by mechanical shearing and this operation can also be made with rotating and stationary abrasive stones (Mizuno et al., 2009).

Process strategies for the hydrolysis and fermentation of lignocellulosics

After the pretreatment step, the bioconversion of lignocellulosic materials includes the biopolymers hydrolysis and the sugar fermentation. These two steps can be performed separately (SHF, separate hydrolysis and fermentation) or simultaneously (SSF, simultaneous saccharification and fermentation). SSF technology is generally considered more advantageous than SHF technology, for several reasons:

• reduced number of the process steps (Koon Ong, 2004)

• reduced end product inhibition because of the rapid conversion of glucose into ethanol by yeast (Viikari et al., 2007)

• reduced contamination by unwanted microorganisms thanks to the presence of ethanol (Elumalai & Thangavelu, 2010).

However, the optimum temperature for the enzymatic hydrolysis is typically higher than that of fermentation. Therefore, in SHF process, the temperature for the enzymatic hydrolysis can be optimized independently from the fermentation temperature, whereas a compromise must be found in SSF process (Olofsson et al., 2008). Another obstacle of the SSF process is the difficulty to carry out continuous fermentation by recirculating and reusing the yeast due to the presence of the solid residues from the hydrolysis.

High solids loadings are usually required to obtain high ethanol levels in the fermentation broths (high gravity fermentation). In particular, solids loadings of pretreated biomass up to 30% (w/w) could be necessary to reach an ethanol concentration of 4-5 wt% that is considered a threshold level for a sustainable distillation process. However, increasing the amount of the solids content in a bioreactor, the hydrolytic performances of the enzymes mixture tends to worsen. In particular, the high initial substrate consistency causes a viscosity increase (Sassner et al., 2006) that is an obstacle toward the homogeneous and effective distribution of the enzymes in the bioreactor. This problem could be partly overcome by using thermostable enzymes. In particular, the hydrolysis could be carried out in two steps: a former step at elevated temperatures with thermostable hydrolytic enzymes producing the liquefaction of biomass (SHF); the latter step, aimed at completing the biomass saccharification, could be carried out at milder temperatures by using the SSF approach (Olofsson et al., 2008).

2. Innovative bioreactor geometries and process strategies

A major requirement in cost-efficient lignocellulosics-to-ethanol process is to employ reactor systems yielding the maximal conversion of the cellulose with the minimal enzyme dosage. As consequence, one of the most important parameter for the design and operation of bioreactors for lignocellulosic conversion is the effective use of the biocatalysts to obtain high specific rates of cellulose conversion (namely the yield of glucose obtained per amount of enzymes). The maximization of the product concentration, i. e. the amount of glucose obtained per liquid volume, is also an important parameter as well as the optimization of the volumetric productivity, in this case the rate of glucose formation per reactor volume. When the hydrolysis is carried out with high dry matter contents, hence high cellulose levels, the product concentration will drive up. For this reason, some recent researches have been finalized into attempting the enzymatic biomass conversion at high-solids loads (Jorgensen et al., 2007; Tolan, 2002). The most important problem of high solid loadings is related to the fact that the viscosity of the reaction mixture is very high and the rheology of the mixture has to be well studied: normal stress might become very significant during bioconversion. In particular, mixing and mass transfer limitations, and, presumably increased inhibition by intermediates come into play. Various fed-batch strategies have been attempted with the scope of supplying the substrate without reaching excessive viscosities and unproductive enzyme binding to the substrate (Rosgaard et al., 2007a; Rudolf et al., 2005).

As said, the currently employed cellulolytic enzyme systems, that include the widely studied T. reesei enzymes, are significantly inhibited by the hydrolysis products cellobiose and glucose. This inhibition retards the overall conversion rate of lignocellulosics-to glucose (Gan et al., 2002; Katz and Reese, 1968). Product inhibition is particularly significant during processing at high substrate loadings mainly because the glucose concentration is higher than that obtained in diluted biomass suspensions. (Kristensen et al., 2009; Rosgaard et al., 2007a). As consequence, both the conversion rate and the glucose yields achievable in batch processing of lignocellulose are reduced (Rosgaard et al., 2007b; Tengborg et al., 2001). General criteria in the bioreactor design and in the selection of the operating conditions could be: use of reactors or reaction regimes that allow a rapid reduction of the glucose concentration; running of the reactions at low to medium substrate concentrations in order to maintain higher conversion rates and hence obtain higher volumetric productivity of the reactor (AndriC et al. 2010, a).

The integration of the bioreactor with a separation unit (reaction-separation hybrids) has shown promising results with product inhibited or equilibrium limited enzyme-catalyzed conversions, because it is possible to remove the products as they are formed (Ahmed et al., 2001; Gan et al., 2002). In this regard, membrane (bio) reactors could be a viable process configuration. Unlike the SSF approach in which the glucose consumption is carried out by the microrganisms simultaneously available in the hydrolyzate, the use of membrane bioreactors would accomplish the same function without any compromise in the reaction temperature. A membrane (bio-) reactor is a multifunction reactor that combines the reaction with a separation, namely in this case product removal by membrane separation, in one integrated unit, i. e. in-situ removal, or alternatively in two or more separate units. The membrane bioreactors hitherto used for the separation in enzymatic processes have been mainly ultra — and nanofiltration (Pinelo et al., 2009). However, the use of this technology is limited by the bank-up of unreacted lignocellulosics (lignin and particularly recalcitrant cellulose) in large — scale and/ or continuous processing (Andric et al. 2010, b). Already in the past, some authors improved the efficiency of the continuous stirred tank bioreactor (CSTR) by incorporating separation membranes in the reactor design. In particular, Henley et al (1980) incorporated an UF membrane (UF) or hollow-fiber cartridge (HFC) into the CSTR-UF and CSTR-HFC system, respectively (Henley et Al., 1980). Ishihara et al. (1991) accomplished a semi-continuous hydrolysis reaction by using a continuously stirred reservoir tank, connected to a suction filter unit for the removal of the lignin-rich residue and an ultra-filtration membrane unit (tubular module), through which the filtrate was pumped in order to separate the hydrolysis products from cellulases. The concentration of the lignocellulosic substrate in the reactor was maintained almost constant by the addition of fresh substrate at appropriate intervals. The filter and ultrafiltration units were operated intermittently, while the enzymes were added at the start, recovered in the UF module, and recycled back into the reactor (Ishihara et al., 1991). More recently, Yang et al. (2006) designed the removal of reducing sugars during the cellulose enzymatic hydrolysis through a system consisting in a tubular reactor, in which the substrate was retained with a porous filter at the bottom and buffer entered at the top through a distributor. The hollow-fiber ultrafiltration module with polysulfone membrane enabled the permeation and the separation of the sugars. To keep the volume constant in the tubular reactor, all the remaining buffer was recycled back from the UF membrane and the make-up buffer was continuously supplied from the reservoir (Yang et al., 2006). In some applications an additional microfiltration unit has exceptionally been used to retain the unconverted lignin — rich solid fraction due to the presence of tightly bound enzymes (Knutsen and Davis, 2004) or has been employed to remove the unconverted substrate from the reactor. These set-ups result in slightly complex process layouts for the hydrolysis.

It is evident that the optimization of the reactor designs will permit to overcome both the rheological and inhibition limit of the bioconversion and maximize the enzymatic conversion. Therefore, the reactor design become strong relevant for large-scale processing of cellulosic biomass (Lynd et al., 2008; Wyman, 2008).

3. Conclusion

In this chapter an overview of the current knowledge on the hydrolysis of lignocellulosics for bioethanol production has been presented. In the last years several important breakthroughs have been made either on the biochemical and technological sides. This is confirmed by several industrial initiatives spread over the world. Among these, in recent days, the first brick of the lignocellulosic bioethanol demo plant (40 kton/y) has been layed in Northern Italy by the Mossi and Ghisolfi Group. Some cooperation agreements were strengthen with Novozymes for improving the efficiency of the hydrolysis step. This event represents an important stage for all the Europe making the production of lignocellulosic ethanol closer to the industrialization and opening the way to new lignocellulosic biorefineries.

Effect of amplifying the flux of the target pathway

The effect of amplifying EM6 flux on ethanol productivity is more clearly presented in batch fermentation profiles (Fig. 3.2).

Obviously, overexpression of enzymes has a limit due to the finite internal resources and other reasons. Although it is difficult to estimate the upper limit to overexpression level, we constrain the constitutive synthesis rates of enzymes to be less than a certain threshold, i. e., the total increase of «м/s is less than or equal to 0.4.

Fig. 3.2 shows that xylose consumption rate is accelerated, while glucose consumption rate is reduced. The decrease of glucose consumption rate can be attributed to a combined effect of metabolic burden and cellular regulation. Consequently, simultaneous consumption of glucose and xylose is facilitated, leading to the substantial increase of ethanol productivity from 1.5 to 2.07 g/L/h (i. e., increase of volumetric productivity by 38%), while the ethanol yield is slightly decreased from 0.402 to 0.392. The total conversion of mixed sugars is fixed to 0.99 in this calculation.

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Fig. 3.2. Dynamic fermentation curves in a batch reactor before (black lines) and after (red lines) overexpressing the target mode (EM6). (a) Ethanol, (b) glucose (GLC), xylose (XYL) and biomass (BIOM).

Synergy

A relevant feature of CWDE activities is the synergy observed between them. Typically an endo-enzyme acts randomly on the polysaccharide to yield oligomers. These oligomers are numerous for a single starting polysaccharide and their extremities are hydrolyzed to di — or monosaccharides by exo-acting enzymes. Accessory enzymes (debranching or desubstituting) proceed if necessary and all three kinds of enzymes work together. Synergy leads to the concept of "Minimal enzyme cocktail", i. e. a few selected enzymes (Sorensen et al., 2007), supposed to be sufficient for the complete digestion of plant cell wall. This paper described efforts performed to digest the more efficiently wheat arabinoxylan to arabinose and xylose. Starting from an "enzymatic base" (a P-xylosidase, and three endo-P-xylanases), they screened three different arabinofuranosidases to enhance substrates digestion. AbfIII was found to be the best enzyme when used alone (Fig. 5). Addition of Abfl significantly increased hydrolysis yields, although in different extent depending on the substrate used. Addition of the three arabinofuranosidases together did not improve the yields further. The strategy used and the results obtained clearly support the view of the necessity of a rational design of the process leading to ethanol. In other words, such a study has to be repeated when another biomass is concerned. Would the same enzyme be as efficient with the other substrate? Only experiments could address this question. Nevertheless, this study indicates that the starting biomass influence the choice of the enzymes to be used. For the complete

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Fig. 5. Yields of arabinose and xylose released from different substrates (substrates are indicated below each pyramid). Base mix of enzyme (top of each pyramid) is constituted of a xylosidase and three endo-P-xylanases. ArfI, AfrI and ArfIII are three arabinofuranosidases belonging to GH (glycosyl hydrolase) families 51, 43 and 51, respectively. As described in the top left pyramid, the more different enzymes were added, the more the result is shown at the bottom of the pyramid. Yield superior to 100% was due to pretreatment. Adapted from Sorensen et al., 2007.

digestion of a plant biomass, minimal enzyme cocktails for all kind of polysaccharide are required. Note that Section2 of this chapter concludes to a great diversity of cell wall fine structures leading to design of much larger and much diverse cocktails.

Microbes

Many investigators offer direct fermentation of starch using amylolytic microorganisms as an alternative to the conventional multistage that employs commercial amylases for liquefaction and saccharification, and followed by yeast fermentation. By using the amylolytic microorganism, ethanol production cost can be reduced via recycling some of the microorganism back to fermentors, thereby maintaining a high cell density, which facilitates rapid conversion of sugar into ethanol. However, there are very few types of amylolytic yeasts that are capable of efficiently hydrolyzing starch. Recombinant microbes and mix of amylolytic microorganism with glucose fermenting yeast in co-culture fermentation can resolve this setback. To further minimize contaminations and process handling cost, a single step or direct bioconversion of cassava or tapioca starch to bioethanol in one reactor (i. e. simultaneously saccharification and fermentation) in place of separate multistage processes will be focused upon in this chapter.

Sugarcane for bioethanol — A new highlight of sugar industry development

The technology of producing fuel ethanol using sugarcane, which has a characteristic of high rate of energy conversion, wide adaptability, and strong resistance, etc, has received extensive attention (Watanabe, 2009). Brazil, Australia and other countries have made breakthroughs in the sugarcane improvement, ethanol fermentation process and its application (Goldemberg et al., 2008; International Energy Agency (IEA), 2004). Brazil is the world’s largest sugar producer and exporter of fuel ethanol, which is expected that annual

output of 65 billion liters by 2020(Walter et al., 2008). Energy security and environmental stress force China to seek and develop biofuels as a substitute of fossil energy. Meanwhile, China has also introduced policies that encourage the development of fuel ethanol using sugarcane and other non-food crop, to ease pressure on energy demand. Recently, the study and the industrial-scale production of biofuels, particularly, fuel ethanol and biodiesel, have progressed remarkably in China as a result of government preferential policies and funding supports (Zhong et al., 2010).

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Fig. 1. Highlight of sugarcane for bioethanol

Potential of second generation bioethanol

The world-wide availibility of feedstock has to be taken into account if bioethanol from lignocelluloses is to contribute significantly to the world fuel market. A report by Bentsen & Felby (2010) shows existing agricultural residue of 1.6 Gt/year cellulose and 0.8 Gt/year hemicelluloses (figures do not include Africa and Australia). This gives a theoretical quantity of 1.24 Gm3 bioethanol from cellulose (690 l/1 using Saccharomyces spec.) and 0.480 Gm3 (600 l/t using Zymomonas spec.) from hemicelluloses. For comparison: the worldwide production of crude oil is estimated to reach not more than 4.8 Gm3 pa (83 million bbl/day) and is supposed/predicted to decline to under 2.4 Gm3 pa (41.5 million bbl/day) by 2040 (Zittel, 2010). The potential of bioethanol from agricultural residues seems to be high. But not all residues will be available and the conversion rates will not be 100%. Therefore, it is thought that lignocellulose-containing materials have to be produced on agricultural land possibly in combination with the production of feedstocks like wheat, corn or sugar cane. These crops would serve as feedstocks for bioethanol production of the first generation.

The yield per hectare is conservatively estimated at 3000 l and 1500 l per hectare of agricultural land for bioethanol of the first and second generation, respectively. A replacement of 41.5 million bbl/day of crude oil would require an area of land of around 5 million km2.

Using DDGS (distillers dried grain solubles) as protein-rich animal feed, taking into account an increase in productivity in agriculture and using intermediate crops as feedstock, the required area could be reduced to under 2.5 million km2. This represents approximately 3% of the world’s land (Bentsen & Felby, 2010).