Category Archives: BIOETHANOL

Properties of ethanol fermentation by F. velutipes

Because the use of basidiomycetes in bioethanol production is not common, and the ethanol fermentation abilities of basidiomycetes are not well characterized, we investigated the properties of ethanol fermentation by F. velutipes to determine its suitability for CBP (Mizuno et al., 2009b). Before the experiment, to obtain a suitable strain for CBP, 10 F. velutipes strains, culture stock of the Forest Institute of Toyama Prefectural Agricultural, Forestry, and Fisheries Research Center, were screened for cellulase production and ethanol fermentation. The Fv-1 strain was selected for further study because it not only produces high levels of cellulases, but also because its ability to ferment ethanol is superior to the other strains.

Firstly, fermentation of D-glucose was done by F. velutipes Fv-1. Figure 1A shows a conversion of 1% w/v of D-glucose to ethanol by F. velutipes. The consumption of D-glucose started gradually after incubation, and it was depleted after 6 d. Ethanol production correlated with sugar consumption, and it reached a maximum after 6 d. Thereafter, the amount of ethanol decreased gradually. Finally, F. velutipes converted 10 g/l of D-glucose to 4.5 g/l of ethanol, equivalent to a theoretical ethanol recovery rate of 88%. In the case of ethanol production from 5% w/v D-glucose, ethanol production reached a maximum, and all of the D-glucose was consumed after 18 d of incubation (Fig. 1B), and 50 g/l of D-glucose was converted to 22.4 g/l of ethanol, equivalent to a theoretical ethanol recovery rate of 87%. The conversion rate was the same as the case of 1% w/v of D-glucose. Because the incubation time to ferment 1% w/v sugar is shorter than the case of 5% w/v, we employed 1% w/v of sugar concentration in subsequent experiments.

Secondary, determination of the fermentation specificity of sugars by F. velutipes Fv-1 was done using various monosaccharides. As shown in Fig. 2, both D-mannose and D-fructose were converted to ethanol by F. velutipes. Consumption of D-mannose occurred slightly faster than that of D-glucose; it started immediately after incubation and was completely depleted after 5 d. Ethanol production from D-mannose was similar to that from D-glucose. It started during the first day of incubation and reached a maximum after 6 d. Furthermore, 4.4 g/l of ethanol was produced from 10 g/l of D-mannose, equivalent to a theoretical ethanol recovery rate of 86% (Fig. 2A). In contrast, consumption of D-fructose was slower than that of D-mannose. It started slowly after incubation and took 7 d to completely consume the D-fructose. Production of ethanol correlated with sugar consumption, and


Symbols: closed circle, sugar; closed square, ethanol. The initial D-glucose concentration was (A) 1% w/v and (B) 5% w/v. (Reproduced from Mizuno et al., 2009b)

Fig. 1. Ethanol production from fermentation of D-glucose by F. velutipes

maximum conversion of D-fructose to ethanol was observed after 6 d. Upon completion of incubation, 4.0 g/l of ethanol was obtained from 10 g/l of D-fructose (Fig. 2B), yielding a theoretical conversion rate of 77%. In contrast to these sugars, F. velutipes did not convert L — arabinose, D-xylose, or D-galactose to ethanol (Figs. 2C, 2D, and 2E). Although there was slight consumption of D-xylose and D-galactose during incubation, ethanol production was not observed. In the case of L-arabinose, little sugar consumption was observed.

Next, we examined the fermentation specificity of F. velutipes Fv-1 toward various disaccharides. As shown in Fig. 3, F. velutipes possibly converted these sugars to ethanol and produced high yields. The theoretical conversion rates of these sugars were 83% and 77% from sucrose and maltose respectively. Degradation of sucrose was observed immediately after the incubation to import the sugar. The amount of reducing sugars was maximum on day 3 and was completely consumed after 7 d of incubation. Ethanol production was observed 1 d after incubation, and the amount of ethanol reached a maximum after 6 d. Finally, 4.5 g/l of ethanol was produced from 10 g/l of sucrose (Fig. 3A). In the case of maltose, degradation was observed on the first day of incubation, and the amount of reducing sugars reached a maximum after 2 d. Furthermore, the reducing sugars were completely depleted after 7 d of incubation. Ethanol production started during the first day of incubation and reached a maximum after 7 d. At the end of incubation, 10 g/l of maltose was converted to 3.8 g/l of ethanol (Fig. 3B). No conversion of xylobiose to ethanol was detected (data not shown), but a significant amount of ethanol production was observed when cellobiose was used as the carbon source (Fig. 4A). Cellobiose began degrading during the first day of incubation, and both D-glucose and cellobiose were completely depleted after 8 d. p-Glucosidase activity increased gradually during incubation. Ethanol production started after 1 d of incubation, and the amount of ethanol reached a maximum after 8 d. Upon completion of incubation, 10 g/l of cellobiose was converted to 4.5 g/l of ethanol (Fig. 4A). The theoretical conversion rate was 83%, a value similar to that of glucose and significantly higher than that of maltose. A high yield of ethanol was observed also in the higher concentration of cellobiose (Fig. 4D). Finally, 25 g/l of ethanol was produced from 50 g/l of D-glucose, and the theoretical conversion rate was 91%.

Since cellobiose was converted to ethanol at a relatively high rate, the conversions of cello- oligosaccharides to ethanol by F. velutipes were also investigated. Figures 4B and 4C show



Symbols: closed circle, sugar; closed square, ethanol. The initial sugar concentration was 1% w/v. (Reproduced from Mizuno et al., 2009b)

Fig. 2. Ethanol fermentation from (A) D-mannose, (B) D-fructose, (C) L-arabinose, (D) D- xylose and (E) D-galactose by F. velutipes

the results of the conversion of cellotriose and cellotetraose to ethanol. Both cello- oligosaccharides were effectively converted to ethanol by F. velutipes. During incubation, cellotriose was initially hydrolyzed to D-glucose and cellobiose, and almost 80% of the initial amount of cellotriose was hydrolyzed by 2 d. Cellotriose was not detected after 5 d of incubation, and D-glucose and cellobiose were completely depleted after 7 d. p-Glucosidase


Symbols: closed circle, reducing sugar; closed square, ethanol. The initial sugar concentration was 1% w/v. (Reproduced from Mizuno et al., 2009b)

Fig. 3. Ethanol fermentation from (A) sucrose and (B) maltose by F. velutipes

was slightly induced by 6 d, and the activity gradually increased after 6 d. The amount of ethanol increased during incubation and reached a maximum after 7 d of incubation. F. velutipes produced 4.2 g/l of ethanol from 10 g/l of cellotriose, equivalent to a theoretical conversion rate of 76% (Fig. 4B). In the case of cellotetraose, it was initially hydrolyzed to cellotriose, cellobiose, and D-glucose, and more than 90% of the cellotetraose was hydrolyzed by 2 d. Cellotetraose was not detected after 3 d of incubation, and cellotriose, cellobiose, and D-glucose were completely depleted after 4, 6 and 7 d respectively. p — Glucosidase activity increased rapidly over 2 d then decreased gradually from 2 d to 5 d, and stabilized at an activity level of about 30 mU / ml. The amount of ethanol increased after incubation, and 4.4 g/l of ethanol was produced from 10 g/l of cellotetraose after 7 d of incubation (Fig. 4C). The ethanol recovery for the theoretical conversion value was 78%.

To date, many microorganisms, including Saccharomyces cerevisiae, Zymmonas mobilis, Pichia stipitis, Rhizopus oryzae, and Clostridium thermocellum, have been reported to produce ethanol (DeMoss & Gibb, 1951; Maas et al., 2006; Ng et al., 1981; Parekh & Wayman, 1986; Weimer & Zeikus, 1977). In general, S. cerevisiae is the most widely used microorganism in the industry and is popular in bioethanol production, because it has high efficiency of ethanol production and high ethanol tolerance. However, we focused on basidiomycetes to develop CBP because these microorganisms have both lignocellulose degradation and ethanol fermentation abilities.

Here, we characterized properties of ethanol fermentation by F. velutipes Fv-1. The strain converted D-glucose to ethanol at a theoretical conversion rate of 88%, comparable to those of S. cerevisiae and Zymomonas (Swings & DeLey, 1977). On the other hand, F. velutipes scarcely converted pentose and D-galactose to ethanol (Fig. 2). These properties of F. velutipes are similar to those of S. cerevisiae (Barnett, 1976). Moreover, F. velutipes demonstrated the preferable features for CBP when oligosaccharides were used as starting materials (Figs. 3 and 4). The tested oligosaccharides were converted to ethanol at almost the same rate as that of D-glucose, and p-glucosidase activity increased during fermentation. These features are indispensable in CBP, which requires saccharification and fermentation of cellulose contained in the cell wall. It has been reported that C. thermocellum and P. stipitis can ferment cellobiose (Parekh & Wayman, 1986). Furthermore, C. thermocellum can also convert cellulose to ethanol directly (Ng et al., 1981; Lynd et al., 1989; Weimer & Zeikus,


Symbols: open square, D-glucose; open diamond, cellobiose; open triangle, cellotriose; open circle, cellotetraose; closed circle, reducing sugar; closed square, ethanol; closed triangle, P-glucosidase activity. The initial sugar concentration was 1% w/v (A, B, and C) or 5% w/v (D). (Reproduced from Mizuno et al., 2009b)

Fig. 4. Ethanol fermentation from (A) cellobiose, (B) cellotriose, (C) cellotetraose and (D) 5% cellobiose by F. velutipes

1997). However, this species cannot be used at the scene of ethanol production because fermentation of C. thermocellum is strongly inhibited at relatively low ethanol concentrations (5 g/l) (Herrero & Gomez, 1980). In contrast, it has been reported that basidiomycetes have tolerance of up to 120 g/l of ethanol (Okamura et al., 2001), and therefore basidiomycetes are more suitable for CBP than Clostridium strains. From these results, we concluded that F. velutipes possesses advantageous characteristics for use in CBP.

Z. mobilis

Zymomonas is an efficient ethanol producer, together with a higher tolerance to ethanol and to several inhibitory substances of lignin. Zymomonas species also possess a gene that codes for cellulase (Rajnish et al. 2008). While protein secretion is not a hurdle in Zymomonas spp., a major difficulty arises with the lack of amenable genetic tools for the introduction or modification of a gene (Linger et al. 2010). Two cellulases from Acidothermus cellulolyticus have been expressed in Z. mobilis, and a significant amount of secretion was observed when they were fused with predicted N-terminal signal peptides of Z. mobilis (Linger et al. 2010). Endoglucanases from different cellulolytic organisms such as Cellulomonas spp., Enterobacter cloacae, Pseudomonas fluorescens, and Erwinia spp., have been expressed in Z. mobilis. However, none of these cellulases were secreted efficiently (Lejeune et al. 1988; Misawa et al. 1988; Brestic-Goachet et al. 1989; Thirumalai Vasan et al. 2011).

Botanical features and agronomic characteristics

Sorghum is a member of Poaceae family, a high-efficient photosynthetic crop, well adapted to tropical and arid climates. As a result, sorghum is extremely efficient in the use of water, carbon dioxide, nutrients and solar light (Kundiyana, 1996; Serna-Saldivar, 2010). This crop is considered one of the most drought resistant, making it one of the most successful in semi-desert regions from Africa and Asia (Woods, 2000). This resistance is due mainly to its photosynthetic C4 metabolism that allows sorghum to accumulate CO2 during the night, to lower the photorespiration rate in presence of light, to reduce the loss of water across the stoma and the waste of carbon (Keeley & Rundel, 2003).

The leaves of sorghum and maize are similar but in the case of sorghum they are covered by a waxy coat that protects the plant from prolonged droughts. The sorghum grain is grouped in panicles and the plant height ranges from 120 to 400 cm depending on type of cultivar and growing conditions. An advantage of sorghum compared to maize is that it has a comparatively lower seed requirement because only 10 to 15 kg/ha are used compared with 40 kg/ha required by other cereals (Kundiyana, 1996). In some regions is possible to produce multiple crops per year, either from seed (replanting) or from ratoon (Saballos, 2008; Turhollow et al., 2010).

Sugarcane pieces as yeast supports for alcohol production from sugarcane juice and molasses

A limitation to continuous fermentation is the difficulty of maintaining high cell concentration in the fermenter. The use of immobilized cells circumvents this difficulty. Immobilization by adhesion to a surface (electrostatic or covalent), entrapment in polymeric matrices or retention by membranes has been successful for ethanol production (Godia et al., 1987). The applications of immobilized cells have made a significant advance in fuel ethanol production technology. Immobilized cells offer rapid fermentation rates with high productivity — that is, large fermenter volumes of mash put through per day, without risk of cell washout. In continuous fermentation, the direct immobilization of intact cells helps to retain cells during transfer of broth into collecting vessel. Moreover, the loss of intracellular enzyme activity can be kept to a minimum level by avoiding the removal of cells from downstream products (Najafpour, 1990). Immobilization of microbial cells for fermentation has been developed to eliminate inhibition caused by high concentration of substrate and product and also to enhance ethanol productivity and yield. Neelakantam (2004) demonstrated that a high yeast inoculation at the start of the sugarcane juice fermentation allows the yeast outgrow the contaminant bacteria and inhibit its growth and metabolism. Varies immobilization supports for variety of products have been reported such as polyvinyl alcohol (PVA, see Fig10), alginates (Kiran Sree, 2000; Corton et al., 2000), Apple pieces (Kourkoutas et al., 2006), orange peel (S. plessas, 2007), and delignified cellulosic residues (Kopsahelis, 2006; Bardi & Koutinas, 1994). We applied sugarcane pieces as yeast supports for alcohol production from sugarcane juice and molasses(Fig 11).The results(Liang et al.,2008) showed ethanol concentrations (about 77g/l or 89.76g/l in average value) , and ethanol productivities (about 62.76 g/l. d or 59.55g/l. d in average value)were high and stable, and residual sugar concentrations were low in all fermentations(0.3- 3.6g/l)with conversions ranging from 97.7-99.8%, showing efficiency(90.2-94.2%) and operational stability of the biocatalyst for ethanol fermentation. the results presented in this paper (see table 3), according to initial concentration of sugars in the must, showed that the


Fig. 10. Yeast immobilized in Polyvinyl Alcohol


Fig. 11. Scanning electron micrographs of the middle part of the support after yeast immobilization.

sugarcane supported biocatalyst was equally efficient to that described in the literature for ethanol fermentation. Sugarcane pieces were found suitable as support for yeast cell immobilization in fuel ethanol industry. The sugarcane immobilized biocatalysts showed high fermentation activity. The immobilized yeast would dominate in the fermentation broth due to its high populations and lower fermentation time, that in relation with low price of the support and its abundance in nature, reuse availability make this biocatalyst attractive in the ethanol production as well as in wine making and beer production. After a long period of using, spent immobilized supports can be used as protein-enriched( SCP production) animal feeds.







Ferm. time









(g/l. d)


Apple pieces (Y. Kourkoutas et

Grape must









figs (Bekatorou et al., 2002)

Spent grains








(Kopsahelis et al.,2006)








Orange peel















(S. plessas et










Sugarcane pieces present study
















Table 3. Fermentation parameters (average value) obtained in batch fermentation with Saccharomyces cerevisiae, immobilized on various carriers, at 30°C

Recycling of low ethanol concentration solutions into the steam explosion reactor

The outcome of an economic study shows that the most important factor for economic bioethanol production is maximum ethanol output (von Sivers & Zacchi, 1996). A possibility to increase the ethanol output would be the recycling of effluents with low ethanol concentration, e. g. the stillage from the distillation, which contains about 1% ethanol (Cortella & Da Porto, 2003) or low concentration effluents from membrane separation steps via the steam explosion reactor. In this case, the added water would be replaced by the effluent to be recycled. During the steam treatment, vapour-liquid equilibrium of the ethanol-water system will be reached. Due to the fact that ethanol is more volatile than water, the concentration of ethanol in the vapour phase will be much higher than in the liquid phase. The vapour-liquid equilibrium of the ethanol-water system at 1.5 MPa is shown in Fig. 6.


Fig. 6. Vapour-liquid equilibrium of the ethanol-water system at 1.5 MPa, calculated with the Wilson equation (Gmehling & Brehm, 1996)

When the reactor is vented, the exploded biomass is separated from the vapour phase in a cyclonic separator. In the separator secondary vapour is also produced by evaporation cooling of the wet biomass. The vapour phase has to be condensed by cooling at the separator outlet to recapture the ethanol. The collected condensate can be added to the feed of the distillation column.

In a first series of experiments on the recycling of ethanol-containing effluent, the added water in the feed to the steam explosion reactor was replaced by a solution containing 10% (w/w) ethanol. Analyses of the pretreated wet straw are shown in Table 2. The samples were taken from the treated straw heap in the separator immediately after the explosion step and transferred into a gastight bottle. With the exception of ethanol no significant differences were found when 10% ethanol (w/w) solution was used. The ethanol content of 31.6 g/kg feed straw (d. b.) in the treated straw from the experiment with the addition of 10% ethanol solution (w/w) is equivalent to 31.6% of the added ethanol; the remaining 68.4% is expected to be in the condensate. It was not possible to verify this due to limitations in the drainage of such small amounts of condensate from the installed regenerative cooler.

Treated straw samples taken from the separator about five minutes after the explosion step showed a significantly lower ethanol content. The average ethanol content in these samples was 13.5 g/kg of feed straw (d. b.), whereas the concentrations of the other components were

Added water 1 kg/kg wheat straw


Formic acid

Acetic acid









10% ethanol (w/w)






Table 2. Analyses of steam-exploded wheat straw (pretreatment conditions: 200°C, 10 min); all values in g/kg feed straw (d. b.); averages of two pretreatment experiments; wet straw samples were leached with deionised water, analysis of the filtrate by HPLC

very much the same. This can be explained by the evaporation of ethanol during the cooling of the treated straw. For example, the recycling of a 1% ethanol (w/w) solution would result in a condensate with about 5% ethanol (w/w) considering also the dilution of the liquid phase in the reactor by condensation of steam.

However, recycling of low ethanol concentration effluents could be limited by inhibitors contained in the effluent. Further tests with real effluents are therefore required.

. Results and discussion2

1.2 Effect of temperature on ethanol fermentation from glucose

Подпись: Fig. 2. The effect of temperature on Turbo yeast in ethanol fermentation with glucose.

Temperature optimizations for glucose fermentation are shown in Fig. 2. The highest rate was obtained at 32°C this fermentation is not limited by hydrolysis of cellulose. A challenge is that the optimal temperature for the yeast is 20°C lower than for the cellulase enzymes.

By further fermentation of the rape straw in which hydrolysis of cellulose is needed it is therefore an advantage to use a higher temperature at which the yeast survives while the enzymes work at a higher reaction rate (Arvaniti et. al 2011).

Catalyst overview

In order to achieve equilibrated or even higher hydrogen yield especially at lower temperatures, catalytic bio-ethanol steam reforming (BESR) has been studied increasingly in recent years. More than three hundreds papers have been devoted to this field within the last two decades. The catalyst systems developed in these studies can be generally classified into two categories, i. e., supported noble and non-noble metal catalysts [32, 33]. However, based on the results reported in the literature, there is no commonly accepted optimal catalyst system which has excellent performance as well as low cost.

The noble metal catalysts such as Rh, Ru, Pd, Pt, Re, Au, and Ir [34-39] have been extensively investigated for BESR, which exhibit promising catalytic activity within a wide range of temperatures (350 oC~800 oC) and gas hourly space velocities (GHSV: 5,000~300,000 h-1). The outstanding catalytic performance experienced by noble metal catalysts might be closely related to its remarkable capability in C-C bond cleavage [40]. Among the noble metal catalysts reported so far, it is evidenced [41-44] that Rh is generally more effective than other noble metals in terms of ethanol conversion and hydrogen production. Diagne et al. [45] claimed that up to 5.7 mol H2 can be produced per mol ethanol (equal to 95 % H2 yield) at 350 oC-450 oC over CeO2-ZrO2 supported Rh catalyst. However, although the metal loading is relatively low (1~5 wt.%) compared with its non-noble counterparts (10~15 wt.%), the extremely high unit price still limits its wide-scale industrial applications.

As a less expensive alternative way to address the cost issue, increasing attention has been focused on the development of non-noble metal catalysts. According to the publications documented so far, the efforts are mainly focused on the Cu, Ni, and Co based catalyst systems, especially supported Ni catalysts. As typical transition metals, the active outer layer electrons and associated valence states determine their identities as the candidates for BESR. Similar with noble metals, Ni also works well as it favors C-C rupture. Based on the observations reported by several authors [38, 43, 46], the non-precious metals are less reactive than noble metal supported samples. Specifically, Rh sites resulted to be 3.7 and 5.8 times more active than Co and Ni, respectively, supported by MgO under the reaction conditions used in [43]. For obtaining the same reactivity (H2 yield > 95 %), much higher temperatures (650 oC) have to be employed [43, 47] over Ni catalysts. Furthermore, the non­noble metals are more prone to be deactivated due to sintering and coking compared with Rh. In order to achieve the comparable catalytic performance with noble metals, the formulation modifications of non-noble metal catalyst systems are worth studying for future commercialization. After summarizing the papers dedicated to investigation of various supports, ZnO and La2O3 seem more promising than MgO, Y2O3, and Al2O3 in terms of activity and stability [48, 49]. The basicity of sample surface has been evidenced crucial to improve its stability by adding La2O3 into the Al2O3 support aiming to neutralize the acidic sites present on the Al2O3 surface [50]. The addition of alkali metals (e. g., Na, K) to Ni/MgO has been observed to depress the deactivation occurrence by preventing Ni sintering [51]. It is worth noting that the recent interests on Ni catalysts seem to be transferred to CeO2 and ZrO2 supported samples, which could be ascribed to its well-known oxygen mobility, oxygen storage capability (OSC), and thermal stability [52-55], in turn improving coke — resistance. In addition, the synergetic effects become notable leading to better catalytic performance (activity, selectivity, and stability) when the second component (Cu) is incorporated into the Ni catalysts indicated by the work performed by Fierro et al., Marino et al., and Velu et al. [56-58]. They believe that the introduction of Cu might favor the dehydrogenation of ethanol to acetaldehyde, one of the important surface reaction intermediates during BESR. Compared with Ni based catalysts, cobalt samples have been less studied as catalysts for BESR. However, recent years have witnessed a significant increase in publications focusing on the development of Co-based catalysts, among which is the pioneering work by Haga et al. [59, 60]. Then Llorca et al. reported the promising results that 5.1 mol of H2 can be produced per mol of reacted ethanol over Co/ZnO sample [61]. Although the reaction condition is slightly unrealistic for industrial applications, this study proved that cobalt is also efficient in C-C bond breakage [62]. Neither copper nor nickel alone supported on zinc oxide appears to have as good reactivity and stability as that of its Co counterpart for hydrogen production under the same reaction conditions [63, 64]. After thorough investigation of the product distribution at various temperatures, it was indicated that the copper sample prefers dehydrogenation of ethanol into acetaldehyde but the reforming reaction does not further progress significantly into H2 and COx. On the other hand, the nickel sample favors the decomposition reaction of ethanol to CH4 and COx, accounting for the lower H2 yield at lower temperatures. Only at high temperatures can the methane production be lowered through steam-reforming. Moreover, Co catalysts have been applied in the Fischer-Tropsch to generate liquid hydrocarbons for more than 80 years. The knowledge accumulated during the study of Co based catalyst systems provides a good starting point. With these encouraging initial data, cobalt catalysts merit to be studied extensively as an alternative solution for reducing the cost from usage of noble metals.

Cassava feedstock

Cassava roots can be used as the feedstock for bioethanol production. During the harvest season, roots are plenty and cheap. However, roots contain very high moisture contents and


Fig. 1. Schematic diagram of bioethanol production by fermentation process of sugar, starch and lignocellulosic feedstock.

are prone to spoilage over the storage time. Mostly, roots are transformed readily to a dried form called cassava chips nearby the plantation areas. To produce chips, harvested roots are cut into pieces manually or by small machine and then sun-dried. The dried chips contain low moisture contents (< 14%), are less bulky, less costly for transportation and can be stored for a year in the warehouse. In addition, dried cassava chips have comparable characteristics as corn grains and can be processed by adopting conversion technology of corn grains. Cares must be taken when storing dried chips as heat can be generated and accumulated inside the heap. Therefore, the warehouse should have a good air ventilation system to prevent overheating and burning of chips. When used, the chips have to be transferred, using the rule of first-in and first-out, to the process line. Dusts are produced, resulting in starch loss as well as severe air pollution. The major concern of using chips is soil and sand contamination, being introduced from roots and during drying on the floor. Sand and soil can cause machine corrosion and result in shorter machine shelf life. They have to be removed in the production process. In Thailand where chips are used for many applications including animal feed and bioethanol production, farmers are encouraged to produce a premium quality of chips that meets with the standard regulation announced by Ministry of Commerce (Table 5).



Starch content

— by Polarimetric method

Not less than 70%by weight

— by Nitrogen Free Extract, NFE

Not less than 75% by weight


Not greater than 4% by weight


Not greater than 13% by weight

Sand and soil

Not greater than 2% by weight

Unusual color and odor

Not detected

Spoilage and molds

Not detected

Living insects

Not detected

Table 5. Standard quality of premium grade of cassava chips, announced by Ministry of Commerce, Thailand.

When cassava is used for bioethanol production, different forms including fresh roots, chips and starch can be used. Table 6 summarizes advantages and disadvantages of using different forms of cassava feedstock. The factory has to make and manage an effective feedstock plan as the feedstock cost can account upto 70%of total ethanol production cost. Types of feedstock used for bioethanol plants depend on many concerns including plant production capacity, plant location, nearby cassava growing areas, amount of feedstock available and processing technology. Ethanol plants that are not close to cassava farms prefer to use dried chips to reduce costs of transportation and storage, while those locating next to cassava fields can use chips and roots. When using both feedstocks, the plants have to somewhat adjust the process in particular feedstock preparation.


According to Rooney & Serna-Saldivar (2000) pericarp, testa, aleurone and mainly peripheral endosperm are grain tissues directly related to the lower nutrient digestibility of sorghum. These layers can be removed through decortication or pearling, an abrasive process used on a regular basis for production of refined flours or grits (Serna-Saldivar, 2010). Commercial mills are typically batch type and are equipped with a set of abrasive disks or carborundum stones to mechanically remove from 10 to 30% of the grain weight. The resulting mixture of bran and decorticated sorghum is separated via air aspiration or sifting (Serna-Saldivar, 2010). The classified pearled grain is then conventionally milled into a meal or flour. This technology requires little capital investment or alteration of existing facilities (Wang et al., 1999). The mechanical removal of the sorghum outer layers increases starch concentration and decreases fiber, fat and phenolics. The ground decorticated sorghum kernels are more susceptible to thermoresistant alpha-amylase hydrolysis (Perez — Carrillo & Serna-Saldivar, 2007). Furthermore, the removal of the sorghum outer layers allows greater starch loading and results in improved ethanol yields.

Enzymes for the cellulose liquefaction: Thermophilic enzymes

The thermophilic microrganisms can be grouped in thermophiles (growth up to 60 °C), extreme thermophiles (65-80 °C) and hyperthermophiles (85-110 °C). The unique stability of the enzymes produced by these microrganisms at elevated temperatures, extreme pH and high pressure (up to 1000 bar) makes them a valuable resource for the industrial











Bio-feed beta L




T. longibrachiatum T. reesei









T. longibrachiatum A. niger


Cellulase 2000L




T. longibrachiatum T. reesei

Rodhia — Danisco (Vinay, France)

Cellulyve 50L




T. longibrachiatum T. reesei

Lyven (Colombelles France)

Energex L




T. longibrachiatum T. reesei






T. longibrachiatum T. reesei

Genencor-Danisco (Rochester, USA)





T. longibrachiatum T. reesei






T. longibrachiatum T. reesei


Novozymes 188




A. niger


Rohament CL




T. longibrachiatum T. reesei

Rhom-AB Enzymes (Rajamaki, Finland)

Spezyme CP




T. longibrachiatum T. reesei


Ultraflo L




T. longibrachiatum T. reesei


Viscozyme L




T. longibrachiatum T. reesei


Viscostar 150L




T. longibrachiatum T. reesei

Dyadic (Jupiter, Usa)

A) One FPU (filter paper unit) is the amount of enzyme that forms 1 pmol of reducing sugars/min during the hydrolysis reaction of filter paper Whatman No.1

B) One CBU (cellobiase unit) corresponds to the amount of enzyme which forms 2 pmol of glucose/min from cellobiose

Table 4. Commercial cellulases

Commercial mixture




Temperature (°C)



Biocellulase A




A. niger

Quest Intl. (Sarasota, Fl)

Cellulase AP 30 K




A. niger

Amano Enzyme Inc.

Table 5. Commercial cellulases able to work at temperature ranging from 50 to 60°C.

bioprocesses that run at harsh conditions (Demain et al., 2005). Of special interest is the thermoactivity and thermostability of these enzymes in the presence of high concentrations of organic solvents, detergents and alcohols. On the whole, thermophilic enzymes have an increased resistance to many denaturing conditions such as the use of detergents which can be often the unique efficient mean to obviate the irreversible adsorption of cellulases on the substrates. Furthermore, the utilization of high operation temperatures, which cause a decrease in viscosity and an increase in the diffusion coefficients of substrates, have a significant influence on the cellulose solubilization. It is worth noting that, differently from the mesophilic enzymes, most thermophilic cellulases did not show inhibition at high level of reaction products (e. g. cellobiose and glucose). As consequence, higher reaction rates and higher process yields are expected (Bergquist et al., 2004). The high process temperature also reduces any contamination of the fermentation medium.

Several cellulose degrading enzymes from various thermophilic organisms have been investigated. These include cellulases mainly isolated from anaerobic bacteria such as Anaerocellum thermophilum (Zverlov et al., 1998), Clostridium thermocellum (Romaniec et al., 1992), Clostridium stercorarium (Bronnenmeier et al., 1991; Bronnenmeier & Staudenbauer, 1990) and Caldocellum saccharolyticum (Te’o V et l., 1995), Pyrococcus furiosus (Ma & Adams,

1994) , Pyrococcus horikoshi (Rahman et al.,1998), Rhodothermus strains (Hreggvidsson et al., 1996), Thermotoga sp., (Ruttersmith et al., 1991), Thermotoga marittima (Bronnenmeier et al.,

1995) , Thermotoga neapolitana (Bok et al., 1998).

Xylanase have been detected in Acidothermus cellulolyticus in different Thermus, Bacillus, Geobacillus, Alicyclobacillus and Sulfolobales species (Sakon et al., 1996).

Although many cellulolytic anaerobic bacteria such as Clostridium thermocellum produce cellulases with high specific activity, they do not produce high enzymes quantities. Since the anaerobes show limited growth, most researches on thermostable cellulases production have been addressed to aerobic species. Several mesophilic or moderately thermophilic fungal strains are also known to produce enzymes stable and active at high temperatures. These enzymes are produced from species such as Chaetomium thermophila (Venturi et al., 2002), Talaromyces emersonii (Grassick et al., 2004), Thermoascus aurantiacus (Parry et al., 2002). They may be stable at temperatures around 70 °C for prolonged periods. Table 6 summarizes some of thermostable enzymes isolated from Archea, Bacteria and Fungi. During the last decade several efforts have been devoted to develop different mixtures of selected thermostable enzymes. In 2007, mixtures of thermostable enzymes, including cellulases from Thermoascus auranticus, Thrichoderma reseei, Acremonium thermophilum and Thermoascus auranticus, have been produced by ROAL, Finland (Viikari et al., 2007). Multienzyme mixtures were also reconstituted using purified Chrysosporium lucknowense enzymes (Gusakov et al., 2005).

Despite the noticeable advantages of thermostable enzymes, cultivation of thermophiles and hyperthermophyles requires special and expensive media, and it is hampered by the low specific growth rates and product inhibition (Krahe et al., 1996; Schiraldi et al., 2002;Turner et al., 2007). Large scale commercial production of thermostable enzymes still remains a challenge also dependent on the optimization of their production from mesophilic microorganisms.