The model was compared with four different sets of anaerobic growth data on single and mixed sugars (Fig. 2.2). As a measure for the quality of model fit, coefficient of determination (also referred to as R2) is presented for each component of Figs. 2.2(a) to (d) (Table 2.3). R2 is defined as follows:

r2 = 1 _ SSn.; SSerr =X(yi, exp — yi, model )2/ SStot =X(yi, exp _ Vexp fz (5)

SStot i i

where yi exp, yi model, and yexp denote experimental data, their associated modeled value, and the mean of the observed data, respectively. R2 values are very high (i. e., over 0.9) for major components (such as glucose, xylose, biomass and ethanol). R2 values of minor components (such as glycerol and xylitol) are relatively low which is possibly due to the error introduced in data reading from literature graphs. Average R2 values are over 0.8 in all cases.

In order to appreciate if the information provided by genome analysis is pertinent, transcriptome studies should also be performed. Our purpose is not to describe the regulation of CWDE in fungi, but it is essential to determinate efficiency of CWDE transcription depending on growth conditions. The goal is of course to optimize conditions leading to high transcription of the required enzymes. This regulation is rather complex, variable and well described in reviews (as an example see Aro et al., 2005). However, global characteristics leading to transcription of hydrolases genes are interesting to point out, since they could be a rational strategy basis for ethanol production. First, and for a long time, CWDE genes were considered generally as being repressed by glucose (catabolic repression) and by other released monosaccharides upon polysaccharide hydrolysis (de Vries & Visser, 2001). On the opposite, CWDE are massively expressed when fungi are grown in presence of polysaccharides and plant material (de Vries & Visser, 2001; Foreman et al., 2003 ; Aro et al., 2005). However, the view of a strict co-regulation of all CWDE is wrong. Induction of a given hydrolase goes on as a function of the polysaccharide in contact with the fungus. An interesting illustration is found in the pea pathogen Nectria hematococca. Two pectate lyases were found to be involved in pathology (Rogers et al., 2000). The first one was induced by pectin and repressed in planta, whereas the other was induced in planta but repressed by pectin. This means that CWDE transcription could be individual and precise. In Fusarium graminearum, well known as pathogen of cereals, we performed microarray experiments to test the expression on the whole genome on glucose, cellulose, xylan and hop cell wall (Carapito et al., 2008). Methods and essential findings are summarized in Fig. 3.

First, some genes were actually found to be over-expressed on polysaccharides comparatively to their expression on glucose (Fig. 3.). Their number varies depending on carbon source. CWDE represent also a variable part of overexpressed genes. It is particularly interesting to note that the largest proportion of CWDE was observed when the fungus was grown on plant cell wall (19% of overexpressed genes) i. e. the most diverse substrate. It denotes a strong re-orientation of the metabolism towards cell wall degradation since CWDE correspond to approximately 0.5% of the genome only. Furthermore, cellulases, hemicellulases and pectinases encoding genes are quite equally represented as overexpressed ones when the fungus is grown on plant cell wall, whereas mostly cellulases were shown to be overexpressed on cellulose and mostly hemicellulases were overexpressed on xylan. This data suggest that there is no global response to the presence of plant cell wall, but that the different polysaccharides sent specific signals which are recognized by the fungus and induce various responses.

Azlin Suhaida Azmi12, Gek Cheng Ngoh1, Maizirwan Mel2 and Masitah Hasan1

1Department of Chemical Engineering, University of Malaya, Kuala Lumpur 2 Biotechnology Engineering, Kulliyah of Engineering, International Islamic University Malaysia, Kuala Lumpur

Malaysia

1. Introduction

The global economic recession that began in 2008 and continued into 2009 had a profound impact on world income (as measured by GDP) and energy use. Since then the price of the energy supply by conventional crude oil and natural gas production has been fluctuating for years which has resulted in the need to explore for other alternative energy sources. One of the fastest-growing alternative energy sources is bioethanol, a renewable energy which can reduce imported oil and refined gasoline, thus creates energy security and varies energy portfolio. Global biofuel demand is projected to grow 133% by 2020 (Kosmala, 2010). However, the biofuel supply is estimated deficit by more than 32 billion liters over the same period and the deficit is worse for ethanol than biodiesel. Ethanol may serve socially desirable goals but its production cost is still remained as an issue. Extensive research has been carried out to obtain low cost raw material, efficient fermentative enzyme and organism, and optimum operating conditions for fermentation process. In addition to that, researchers have been trying to capitalize certain features of the plant equipment and facilities to increase the throughput of ethanol and other high value by products as well as to apply suitable biorefinery for the product recovery. At the same time, effort has been made to reduce utilities costs in water usage, cooling or heating, and also consumables usage via minimizing the effluent production.

Aimed to provide an alternative means for ethanol production, this book chapter introduces a single-step or direct bioconversion production in a single reactor using starch fermenting or co-culture microbes. This process not only eliminates the use of enzymes to reduce the production cost but also yield added value by-products via co-culture of strains. Before further elaboration on this single-step fermentation, we will visit the conventional process, the substrate preparation and microbe used. By this way a clear picture of the differences between conventional process and the proposed single-step fermentation with the advantages and disadvantages of both processes will be discussed.

Fig. 1 to 3 summarizes and compares average ethanol yields from sorghum grain, sweet juice and biomass. Ethanol yields vary according to variety, geography, soil fertility and temperature.

Sweet sorghums usually yield from 50 up to 120 tons of stalks after the first cut. This feedstock contains 73% moisture, 13% soluble sugars, 5.3% cellulose, 3.7% hemicelluloses and 2.7% lignin. The stalks yield up to 70% sweet juice and 15.33 ton/ha of spent bagasse (Almodares & Hadi, 2009; Prasad et al., 2007).

Water added during extraction is considered part of he sweet juice yield (Fig. 1) and the sweet juice commonly contains around 14% soluble sugars. This substrate allows the production of 3,450 L ethanol/ha with a fermentation efficiency of 95%, similar to the result reported Kim & Day (2011) (3,296 L/ha). These last researchers did not consider losses that negatively affect fermentation efficiencies. Almodares & Hadi (2009), on the other hand, reported a yield of 3,000 L ethanol/ha directly when processing juice extracted from varieties that yielded from 39 to 128 ton stalks/ha. Although Wu et al. (2010b) did not report ethanol yields per hectare, the calculated ethanol production from the amount of total fermentable sugars extracted from a high yielding M81E cultivar planted at two different locations and bioconverted with a 95% of fermentation efficiency was in the range of 4,750 to 5,220 L/ha. These potential ethanol yields are equivalent to the bioconversion of 12 to 13 tons of maize kernels.

Experimental data obtained from sweet sorghums cultivated in Central Mexico indicated that these materials are capable of yielding 6.38 tons of sugar/ha/ cut. Consequently, when are adequately bioconverted have the potential of producing 4,132 L ethanol (unpublished data). Regarding to the lignocellulosic fraction, if 15.33 ton of bagasse/ha is obtained containing 29% cellulose and hemicellulose and 5.4% of remaining unextracted soluble sugars, up to 2,400 L of ethanol can be obtained (Fig. 3). This yield represents almost half of the 4,058 L/ha described by Kim & Day (2011) as theoretical ethanol.

In central Mexico, 42.5 ton of bagasse/ha with 50% fermentable sugars are commonly obtained. This biomass is capable of yielding 6,375 L ethanol with perfect conversion efficiency. However, experimental data where the acid-treated biomass was fermented with Issatchenkia orientalis indicated only 60% fermentation efficiency (3,865 L/ha) (unpublished
data). These results indicate that there are still many areas for potential improvements especially when processing spent biomass.

Almodares & Hadi (2009) reported that a yield up to 2 ton of grain/ha can be expected from sweet sorghum. If this material is milled, hydrolyzed and fermented, a final ethanol yield of 780 L can be expected. Nevertheless, the sweet sorghum grain during optimum harvesting is not fully matured and generally collected along the vegetative parts of the plant. Thus, the immature sweet sorghum kernels are usually processed with the bagasse and not fermented using grain technologies.

The biomass production per cultivated surface (Fig. 3) is the key and most important factor that affects ethanol yields indicating the importance of both plant breeding for the generation of new improved cultivars and the agronomic conditions mainly affected by soil fertility and water availability. The new biomass cultivars should adapt to marginal lands in order to minimize competition with basic grain production. The potential to obtain ethanol yields of 6630, 7000 and 10000 L/ha (with 95% of extraction and fermentation efficiency) can be achieved because yields of 50 to 120 tons of biomass/ha are reported. Comparatively Kim & Day (2011) indicated that the theoretical yield of maize kernels can be as high as 5,100 L/ha and up to 8,625 L/ha when the whole plant is bioconverted into ethanol (grain + corn stover). One of the most important factors to be addressed during yield calculation is indeed the energy required for ethanol production. Biomass and starch require more energy for hydrolysis compared to sweet sorghum juice. The technologies for starchy kernels and sweet juice are matured but the conversion and estimation of energy balances when processing lignocellulosic material will be critically important for the evaluation of economic advisability.

With the depletion of petroleum resources and increasing demand on energy, lignocellulose derived ethanol seems to be the future of transportation fuels. Also, it is noticeable that the integrated biorefineries, which generate chemicals, materials, fuels and energy from the biomass, would replace the current petroleum refineries, moving the world toward a carbohydrate-based economy (Gnansounou 2009).

By-products like HSSL cannot be discharged into natural basins due to environmental concerns (211 g COD. L-1) and must be processed (Evtuguin et al. 2010). The biochemical processing of HSSL is a well-known approach to produce value-added products such as SCP and ethanol, among others (Busch et al. 2006).

As seen previously, biological detoxification of HSSL by P. variotti was possible and the fungal biomass obtained (2.0 g biomass/g substrate consumed) can be sold as SCP, for animal nutrition. For process optimization a Sequential Batch Reactor (SBR) was chosen and the same inoculum was used during three batches to treat fresh HSSL. Each cycle was ended when the acetic acid reached a non-inhibitory concentration for P. stipitis and this operating strategy provided high volumes of detoxified HSSL, for subsequent bioethanol fermentation (Pereira et al. 2011). With this detoxification process as well as with the described ion — exchange process (Xavier et al. 2010) HSSL can be further bioprocessed by P. stipitis, as reviewed before. The maximum concentration of ethanol attained was 8.1 g. L-1 with a yield of 0.49 g ethanol. g sugars-1 (Xavier et al. 2010). The bioethanol produced from HSSL, regarding the aforementioned fermentation results, may be estimated as high as 100 litters per one ton of pulp (Evtuguin et al. 2010).

Biopolymers are also important value-added products that can be produced within a biorefinery concept, being capable to replace fossil-fuels based polymers. Microbial mixed cultures (MMC) under aerobic dynamic feeding conditions (ADF) in HSSL, can utilize acetic acid for polyhydroxyalkanoates (PHAs) production. PHAs are biodegradable plastics that can be stored intracellularly by bacteria from renewable resources. A MMC culture was selected in a SBR under

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1.1 Use of whole crop sorghums as a raw material in consolidated bioprocessing bioethanol production using Flammulina velutipes

The ethanol fermentation abilities of basidiomycetes have not been well characterized, we evaluated the ability of F. velutipes in CBP. Preliminary fermentation experiments indicate that F. velutipes convert sugars to ethanol much more under the high concentration of biomass which close to solid state cultivation than liquid cultivation condition. Therefore, we employed solid state cultivation which usually performed in artificial cultivation of mushrooms for the coversion of biomass to produce bioethanol. Sorghum is selected as a possible raw material to produce bioethanol by CBP using F. velutipes. Sorghum is a C4 crop of the grass family belonging to the genus Sorghum bicolor L. It is well adapted to temperate climates and can be cultured from Kyushu to Tohoku area in Japan. The plant grows to a height from about 120 to above 400 cm, depending on the variety and growing conditions, and can be an annual or a short perennial crop. Sorghum is considered to be one of the most drought resistant agricultural crops, as it is able to remain dormant during the driest periods (Xu et al., 2000). These properties of sorghum are suitable as raw material for the ethanol production. We evaluated the ability of F. velutipes in CBP using sorghum strains as a raw material, and solid-state CBP of ground sorghum strains (SIL-05 and Kyushukou No. 4) using F. velutipes was investigated. The possibility of sorghum strains as a raw material in the CBP ethanol production by F. velutipes is also discussed below.

We selected grinding for the pretreatment of sorghum strains. This can be used on both dry and wet materials, and the cost of grinding is one of the cheapest compared to other methods used for milling biomass. The grinding of sorghum was carried out with an ultra­fine friction grinder. Grinding was performed at room temperature, and was repeated twice. To examine the efficiency of grinding as a pretreatment, the degree of saccharification was tested using commercially available enzymes Celluclast 1.5L (Sigma, St. Louis, MO), Novozyme 188 (Sigma) and Multifect xylanase (Genencor Kyowa, Tokyo).

The saccharification yields of SIL-05 and Kyushukou No. 4 by the enzymes were 30.1% and 51.7% respectively (Fig. 5A). Kyushukou No. 4 is one of the sorghum brown mid-rib (bmr) mutants in which cafferic acid O-methyltransferase (COMT), a lignin biosynthetic enzyme, activity is reduced as compared to the wild type (Bout & Vermerris, 2003). This property of bmr significantly affected the hydrolysis of polysaccharides in the biomass, but there were no significant differences in the proportions of hydrolysis of the components such as cellulose and hemicellulose (Fig. 5). When the saccharification yields of cellulose and hemicelluloses were compared, degradation of hemicelluloses was slightly higher than for cellulose in both types of sorghum.

Next, solid-state ethanol fermentation by F. velutipes was performed for both sorghum strains. Solid-state fermentation is advantageous because it carries a low ethanol production cost. Generally, sorghums contain 70-80% v/ v water, corresponding to 43-25% w/ v. These concentrations are necessary to obtain relatively high final ethanol concentrations. Furthermore, it is possible to reduce the costs of many procedures, such as amount of water, concentration of biomass, treatment of waste water, and so forth, if the water concentration of the raw materials in the all ethanol production procedures is retained. 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. Mycelia of Fv-1 were harvested in the late exponential growth phase by centrifugation at 3,000 x g and washed with sterile water. The prepared wet mycelia (20 mg of dry weight) were mixed with 100 mg of ground sorghum for solid-state fermentation.

A larger amount of ethanol was produced from SIL-05 than from Kyushukou No. 4 (Fig. 6). Because SIL-05 contained a larger amount of soluble sugars than Kyushukou No. 4 (Table 1), it should be advantageous for total ethanol fermentation. The ethanol conversion rates for the soluble sugars contained in SIL-05 and Kyushukou No. 4 were 57.2% and 38.9% respectively. The addition of saccharification enzymes was not effective for SIL-05 (Fig. 6A). This corresponded with the results of enzymatic hydrolysis (SIL-05 just hydrolyzed almost 30%) (Fig. 6). However, the ethanol conversion rate for the degraded cellulose was 85.6%, significantly higher than that for soluble sugars. In contrast, although total ethanol production was not high, ethanol production from Kyushukou No. 4 significantly increased when saccharification enzymes were added to the culture (Fig. 6B). Because the cellulose and hemicellulose in Kyushukou No. 4 were more easily hydrolyzed than SIL-05 by cellulases, significantly more ethanol was produced by the addition of the saccharification enzymes. The ethanol conversion rate for the degraded cellulose of Kyushukou No. 4 (98.3%) was much higher than that of SIL-05 (85.6%). Thus, the bmr mutation appears to be useful for CBP because it gives a high yield of glucose from biomass without acid or alkali pretreatment. However, the results indicate that the production of cellulases by F. velutipes is not sufficient for CBP, or that the saccharification enzymes are suppressed by carbon

catabolites due to the existence of soluble sugars. Therefore, an effective saccharification enzyme inducing method for F. velutipes in the CBP is required.

In this work, we demonstrated CBP ethanol fermentation of sorghum strains by F. velutipes Fv-1. The procedure is quite simple and cost effective, and can reduce energy consumption, because the raw material is simply ground and then mixed with mycelia. We demonstrated the merit of high concentrations of soluble sugars and the bmr mutation in sorghums. Both sorghum strains can be used in CBP. The bmr mutation is only found in sorghums, corn, and pearl millet, giving sorghum an advantage over many other crops for ethanol production. Future studies should focus on the improvement of CBP using F. velutipes and the selective breeding of novel types of sorghums with high concentrations of soluble sugars and the bmr mutation.

An Ideal Biofuel Producing Microorganism (IBPM) should possess four important traits: it should be able to carry out (1) biomass degradation and (2) product formation; (3) it should show tolerance to solvents, and (4) it should serve as a chassis organism for rapid growth in the bioreactor (French 2009). Chassis organisms, such as yeast and E. coli, are well characterized. Commercial bioethanol has been produced from sugarcane by yeast. In addition, E. coli and Z. mobilis are progressing as efficient ethanol producers. A current challenge is to engineer biomass degradation (cellulolytic) ability. Further, investigators seek to enhance tolerance to harsh conditions that arise during cellulose fermentation, such as substrate and product toxicity. In particular, the chassis organism should have enhanced tolerance to toxic compounds of lignin. Classical strain improvement through long-term adaptation and mutagenesis may be an effective way to increase the tolerance to harsh environments, such as ethanol or lignin, because the mechanisms of toxicity and tolerance are largely unknown (Fischer et al. 2008).

Engineering cellulolytic ability into recombinant hosts has long been a challenge. The number of cellulase genes that should be cloned into the recombinant host remains unclear (Vinuselvi et al. 2011). The main obstacle to developing a recombinant cellulolytic host is the inability of hosts to support expression and secretion of a sufficient quantity of cellulases. Although cellulase expression is well established in yeast, there is no known study demonstrating direct conversion of plant biomass into ethanol. Despite the characterization of several cellulases in E. coli, a cellulolytic cassette containing all three cellulases has not been established for E. coli. Furthermore, efficient genetic tools are still lacking for Zymomonas, limiting its potential to be engineered with a heterologous gene.

One way to address the problems associated with heterologous cellulase expression and to reduce the metabolic burden imposed by the expression of cellulolytic enzymes in recombinant hosts would be the development of a well-defined synthetic consortium with two efficient players — native cellulolytic and solventogenic organisms —acting together. A high level of expression of multiple heterologous proteins would impose a heavy metabolic burden on the host. With a synthetic consortium, this burden could be shared by different species or by different strains of the same species. A co-culture of these strains to produce a cellulase cocktail would, therefore, reduces the overall metabolic burden and increase the ethanol yield (Brenner et al. 2008). Synthetic biology also offers superior inducible systems, such as light-inducible promoters and the fim inversion system, which are capable of providing spatiotemporal changes in gene expression (Levskaya et al. 2005; Ham et al. 2006). With such systems, it is possible to regulate the expression of genes with time and, potentially, to help reduce the metabolic burden imposed on the recombinant host (Drepper et al. 2011). Using metabolic engineering and synthetic biology, Steen et al. (2010) have developed a promising way of causing E. coli to produce more complex biofuels — fatty esters and fatty alcohols — directly from hemicellulose, a major component of plant-derived biomass (Fig 6). This study is representative of the recent progress in cellulosic fuel production. However, the possibility of increasing the productivity of such advanced biofuels remains a significant challenge.

5. Conclusions

Cellulosic bioethanol is gaining importance to circumvent the oil crisis and climate change. However, two major problems remain to be solved, in order to produce cellulosic ethanol economically. One problem is the high price of the cellulolytic enzymes used in the saccharification of lignocelluloses. The other problem is that the traditional saccharification and fermentation for bioethanol requires huge initial capital investment and operational cost. Consolidated bioprocessing presents a desirable way to produce bioethanol economically from lignocellulose. Microorganisms such as Trichoderma spp. and C. thermocellum effectively challenge the recalcitrance of lignocellulose, whereas microbes such as yeast and Z. mobilis can produce ethanol more efficiently. Several attempts have been made to combine these two abilities into a single organism, but with little success. Recent progress in synthetic biology, metabolic engineering, and protein engineering gives hope that the goal of generating cellulosic ethanol with a single organism may not be far from reality.

6. Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) through grants funded by the Ministry of Education, Science and Technology (NRF-2009- C1AAA001-2009-0093479, NRF-2009-0076912, NRF-2010-0006436) and UNIST (Ulsan National Institute of Science and Technology) research grant.

1.1 Juice from sweet sorghum

The mature stems of sweet sorghum contain about 73% moisture and the solids are divided in structural and non-structural carbohydrates. Approximately 13% are non-structural carbohydrates composed of sucrose, glucose and fructose, in variable amounts according to cultivar, harvesting season, maturity stage, among other agronomic factors (Mamma et al., 1996; Phowchinda et al., 1997). Anglani (1998) suggests a classification of sweet sorghums based on proportion of soluble sugars in the juice. The first group with a high content of sucrose (sugary type) and the second with more monosaccharides (glucose and fructose) compared to other soluble carbohydrates (syrup type). Smith et al. (1987) in their evaluation of six sweet sorghum varieties throughout four years in nine different locations did not find significant differences in sugar content or composition. The typical composition indicates that around 70% was sucrose and the rest glucose and fructose in equal parts. In stem dry basis, Woods (2000) reported fermentable sugars content between 41 to 44% in Keller and Wray varieties with 80 and 63% represented by sucrose and the rest by glucose and fructose. A fiber variety analyzed by the same author (H173) reached only 20% fermentable sugars based on the dry stem weight; sucrose, glucose and fructose were found in equivalent

amounts (around 7% for each sugar). Compared to sugar cane, the main difference is that the sucrose content in cane is significantly higher compared to glucose and fructose (90, 4 and 6%respectively) and the total content sugar is 49% of the dry stem weight. In general terms, composition of simple sugars in sweet sorghum juice is 53-85, 9-33 and 6-21% for sucrose, glucose and fructose, respectively (Gnansounou et al., 2005; Mamma et al., 1996; Phowchinda et al., 1997; Prasad et al., 2007).

Beyond the proportion of soluble sugars in sweet sorghum plants, the yield of total sugars per harvested area is a better guide in the analysis for fuel production. Woods (2000) reported for sweet sorghum cultivars (Keller, Wray and H173) an average of 7, 10 and 4 ton of fermentable sugars/ha respectively, significantly lower compared to the 17 ton/ha for sugarcane indicated by the same author. The varieties studied by Davila-Gomez et al. (2011) yielded an average of 1.85 to 2.03 ton of sugar/ha, whereas Smith et al. (1987) in a extensive study performed in several locations of continental United States and Hawaii, obtained from 4.5 to 10.6 ton/ha. In other varieties evaluated in China, the best yields reached 18 ton/ha (Zhang et al., 2010).

Sugars in sweet sorghum are very sensitive to microbial contamination especially after crushing stalks for juice production. In data reported by Davila-Gomez et al. (2011), the percentage of sugars, as °Brix before fermentation, was lower (11 to 24% lower) than the obtained immediately after harvest in summer time, when temperatures easily reached 32°C in Northeast Mexico. The microbial contamination was the most obvious explanation of this phenomenon. Besides, the sucrose proportion in the fermented juices was lower in relation to glucose and fructose (0 to 10% of total). This can be related to invertase activity of contaminating wild yeasts that hydrolyzed sucrose into glucose and fructose. These monomers are quickly metabolized by means of facilitated diffusion into the yeast cell. Wu et al. (2010b), working with cultivars with 16 to 18% of fermentable sugars, found that as much as 20% of substrate can be lost in 3 days at 25°C. This loss corresponds to approximately 700 L ethanol/ha when a yield of 50 ton of sorghum stems/ha is considered. Daeschel et al. (1981) reported that juices can be preserved during 14 days at 4°C without detectable changes or deterioration (sour odor and foaming). These authors also reported that the dominant spoilage microorganisms were Leuconostoc mesenteroides and Lactobacillus plantarum at 25 and 32°C, respectively and recommended to process the juice within five hours after extraction.

Distillation and molecular — sieve absorption are used to recover ethanol from the raw fermentation beer. The flow sheet of this section is presented in Figure 12 and figure 13. Distillation itself is a two-way progress include heating and cooling. That could be possible to save much steam and cooling water if we take good advantage of the heat exchange in the system. Due to its energy-saving, so far negative pressure distillation system has been popular in China. Take molasses alcohol as an example, compare to air distillation system, negative pressure distillation system could save approximately 2t steam per ton 95% (v/ v) alcohol. The system showed in figure contains 3 columns, which is .fractioning column 1, fractioning column 2, and separating methanol column respectively. Making use of the different boiling points the alcohol in the fermented wine is separated from the main resting solid components. The remaining product is hydrated ethanol with a concentration of 95% (v/v). Further dehydration is normally done by molecular-sieve absorption, up to the specified 99.7°GL in order to produce anhydrous ethanol which is used for blending with pure gasoline to obtain the country’s E10 mandatory blend. The fermented mash which contains 10~13 %(v/ v) alcohol is preheated by the alcohol gas from the top of the first column and gas is cooled simultaneously. Then the gas stream is cooled by 3 heat exchangers, the cooler is water. Subsequently the liquid distillate which contains 30% (v/ v) alcohol is feeding on the middle tray of column 2. Wastewater of column 1 is heated by the alcohol gas from the top of column 2 in the reboiler, meanwhile the steam flash evaporated in the vacuum bottom. The waste goes to anaerobic jar and then aeration tank. Cooled alcohol is pumped back to the top trays of column 2. Fusel oil is extracted from the middle trays of the column 2. Liquid distillate contains 95 %( v/v) alcohol and exceeded methanol amount. In order to decrease the concentration of aldehyde and methanol, one more column is needed. The 96%v/v alcohol with 4% water is feeding on the molecular-sieve absorption systemFinally 99.5%v/v ethanol which could be added to the gas to make gasohol is achieved.

Fig. 13. Ethanol dehydration with molecular sieve bed

1.3.2 Description of the experiments

Bioethanol production from wheat straw was investigated. Several improvements, particularly one washing step and the recirculation strategy, were made. The washed wheat straw was named inhibitor-controlled wheat straw. These improvements increase both the sugar concentration and the bioethanol yield by up to 7%(vol). Also, the lignocellulose — containing substrate corn stover was tested for its potential in bioethanol production. Furthermore, recirculation of bioethanol was performed to ultimately raise the end concentration of bioethanol. Therefore, ethanol was added during the pretreatment process and a possible effect on the hydrolysis and fermentation steps was examined.

The enzyme mixture Accellerase TM1000 from Genencor® was used with enzyme activities of 775 IU cellulase (CMC)/g solids and 138 IU beta-glucosidase/g solids. Suspensions with various dry substances (10-20%) were produced with the pretreated substrate in citrate buffer (50 mM, pH 5.0) and incubated at 50°C for 96 hours in a shaking incubator (100 rpm). The hydrolysis of pretreated substrate was repeated three times in a recirculation process. Sample analysis was performed with HPLC. Diverse salts were added to the straw hydrolysate for fermentation. A wild-type strain of Saccharomyces cerevisiae was used exclusively for all experiments. The fermentation process was conducted at 30°C in a shaking incubator for one week (110 rpm).