Utilization of microwave and ultrasound pretreatments in the production of bioethanol from corn

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1 Clean Techn Environ Policy (2011) 13: DOI /s ORIGINAL PAPER Utilization of microwave and ultrasound pretreatments in the production of bioethanol from corn Svetlana Nikolić Ljiljana Mojović Marica Rakin Dušanka Pejin Jelena Pejin Received: 3 November 2010 / Accepted: 3 March 2011 / Published online: 11 March 2011 Ó Springer-Verlag 2011 Abstract Bioethanol production by simultaneous saccharification and fermentation (SSF) of corn meal by Saccharomyces cerevisiae var. ellipsoideus yeast in a batch system with prior ultrasound or microwave treatment was studied. The optimal duration of the pretreatments and the SSF process kinetics were assessed and determined. Also, the effect of ultrasound and microwave pretreatments on ethanol yield and productivity was investigated. An optimal duration of 5 min was determined for both pretreatments. Ultrasonic and microwave pretreatments effectively increased the glucose concentration obtained after liquefaction by 6.82 and 8.48%, respectively, compared to untreated control sample. Also, both pretreatments improved ethanol yield and productivity during the SSF process. Ultrasound and microwave pretreatments increased the maximum ethanol concentration produced in the SSF process by and 13.40% (compared to the control sample), respectively. The application of microwave pretreatment resulted in higher glucose release during liquefaction and consequently in higher ethanol concentration, compared to ultrasound pretreatment. A maximum ethanol concentration of 9.91% (w/w) and percentage of theoretical ethanol yield of 92.27% were achieved after 44 h of the SSF process of corn meal with prior microwave treatment. S. Nikolić (&) L. Mojović M. Rakin Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, Belgrade, Serbia snikolic@tmf.bg.ac.rs D. Pejin J. Pejin Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, Novi Sad, Serbia Keywords Bioethanol Corn Ultrasound Microwave Pretreatment SSF Introduction Today, world faces the complex economical and ecological problems concerning progressive energy consumption, decreased reserves and increased costs of conventional energy resources, such as fossil fuels (Caylak and Vardar Sukan 1998; Kim and Dale 2005; Siqueira et al. 2008). This has led to a major interest in expanding the use of bioenergy. Bioethanol, as an alternative bioenergy resource, is renewable and environmentally clean biofuel (Balat et al. 2008; Baras et al. 2002; Schausberger et al. 2010). Fermentation-derived ethanol can be produced from sugar, starch, or lignocellulosic biomass. In Serbia, one of the most suitable and available agricultural raw material for industrial bioethanol production is corn. In last few years, the average annual corn yield in Serbia is approximately 40% higher than the calculated domestic needs. Therefore, significant amounts of this raw material can be used for bioethanol production since there is enough corn for other purposes besides the food (Nikolić et al. 2009). Current world bioethanol research is driven by the need to reduce the costs of production. Improvement in feedstock pretreatment, shortening of fermentation time, lowering the enzyme dosages, improving the overall starch hydrolysis, and integration of SSF process could be the basis of cutting down the production costs. The application of ultrasound or microwaves in the field of biorenewables is a relatively new concept and has a potential as a pretreatment method to increase the conversion of starch materials to glucose as well as overall ethanol yield

2 588 S. Nikolić et al. (Khanal et al. 2007; Mielenz 2001; Palav and Seetharaman 2007). Besides being considered as a crucial step in the biological conversion to ethanol, biomass pretreatment represents one of the main economic costs in the process (Alvira et al. 2010). When low frequency ultrasound (power of ultrasound ranging from 16 to 100 khz) wave propagates in a liquid or slurry, it produces a hydrodynamic shear force due to the rapid collapse of microbubbles formed during cavitation and acoustic streaming (Patist and Bates 2008). The main benefit of streaming in corn slurry processing is mixing, which facilitates the uniform distribution of ultrasound energy within the slurry mass, better mass transfer of enzymes, convection of the liquid and dissipation of any heating that occurs. As a result, ultrasound facilitates the disintegration of corn starch granules, thereby exposing a much larger surface area to enzymes and enhancing enzyme activity during the hydrolysis. By integrating an ultrasound pretreatment in bioethanol production, the overall ethanol yield could be significantly increased within a short processing time (Khanal et al. 2007; Nitayavardhana et al. 2010; Patist and Bates 2008). Some previous studies have shown that the microwave heating influences the process swelling and gelatinization of starch granules and thus could be very efficient in destroying the starch crystalline arrangement and obtaining a soft gel (Mielenz 2001; Palav and Seetharaman 2007). These phenomena could also enhance the enzyme susceptibility needed for efficient hydrolysis which may later on improve the outcome of the fermentation (Palav and Seetharaman 2007; Zhang et al. 2008). Besides the pretreatment, the process mode used for saccharification and fermentation (SSF) is also an important factor affecting the production costs. There are many reports that the SSF is economically more favorable process to the traditional separate hydrolysis and fermentation (SHF) for bioethanol production primarily due to a lower energy consumption, higher ethanol yield, decreased substrate inhibition of yeast and reduced process time (Mojović et al. 2006; Nikolić et al. 2009; Öhgren et al. 2007). The aim of this study was to investigate the possibilities of improving glucose and ethanol yield by applying an ultrasound or microwave pretreatment of corn meal suspensions in bioethanol production by SSF process with Saccharomyces cerevisiae var. ellipsoideus yeast in a batch system. The efficiency of these pretreatments to disintegrate corn starch granules and improve glucose release at different processing time was studied. In addition, changes in physical properties of corn meal suspensions before and after ultrasound and microwave pretreatments were examined at microscopic level by scanning electron microscopy (SEM) studies. The kinetics of SSF process as well as bioethanol yield and productivity were also assessed and compared. Materials and methods Starch Corn meal obtained by dry milling process was a product of corn processing factory RJ Corn Product, Sremska Mitrovica, Serbia. The corn meal consisted of particles with diameter mm (95% or more particles pass through a 1.70 mm sieve). The content of the main components in the corn meal was the following: starch 76.75% (w/w), proteins 6.35% (w/w), lipids 4.50% (w/w), fibers 1.36% (w/w), ash 0.70% (w/w), and water 10.34% (w/w), as determined by chemical analysis in our previous study (Nikolić et al. 2009). Enzymes and microorganisms Termamyl Ò SC, a heat-stable a-amylase from Bacillus licheniformis was used for corn meal liquefaction. The enzyme activity was 133 KNU g -1 (KNU, kilo novo unit is the amount of enzyme a-amylase which breaks down 5.26 g of starch per hour according to Novozyme s standard method). SAN Extra Ò L, Aspergillus niger glucoamylase, activity 437 AGU g -1 (AGU, amyloglucosidase unit is the amount of enzyme which hydrolyses 1 lmol of maltose per minute under specified conditions) was used for corn meal saccharification. The enzymes were gift from Novozymes, Denmark. S. cerevisiae var. ellipsoideus was used for the fermentation of hydrolyzed corn meal. The culture originated from the collection of Department of Biochemical Engineering and Biotechnology, Faculty of Technology and Metallurgy, Belgrade, and was maintained on a malt agar slant. The agar slant consisted of malt extract (3 g l -1 ), yeast extract (3 g l -1 ), peptone (5 g l -1 ), agar (20 g l -1 ), and distilled water (up to 1 l). Before use as an inoculum for the fermentation, the culture was aerobically propagated in 500 ml flasks in a shaking bath at 30 C for 48 h and then separated by centrifugation. The liquid media consisted of yeast extract (3 g l -1 ), peptone (3.5 g l -1 ), KH 2 PO 4 (2.0 g l -1 ), MgSO 4 7H 2 O (1.0 g l -1 ), (NH 4 ) 2 SO 4 (1.0 g l -1 ), glucose (10 g l -1 ), and distilled water. Ultrasound pretreatment Samples of the mixture of corn meal and water at the weight ratio (hidromodul) 1:3 placed in glass flasks were subjected to ultrasound pretreatment before addition of liquefying enzyme Termamyl Ò SC. The ultrasound pretreatment was carried out in a sonicator (Model: USK 28, power 600 W, EI Niš, Niš, Serbia) at temperature of 60 C and frequency of 40 khz. The duration of ultrasound pretreatment (1 10 min) was investigated. The control

3 Utilization of microwave and ultrasound pretreatments in the production of bioethanol from corn 589 samples were not subjected to sonication. After the ultrasound pretreatment, the flasks were kept in a thermostated water bath at 85 C for up to 1 h to facilitate enzymatic liquefaction and then subjected to SSF. Microwave pretreatment Samples of the mixture of corn meal and water at the weight ratio (hidromodul) 1:3 placed in glass flasks were subjected to microwave pretreatment in a microwave oven (Model: R-677, Sharp Electronics, UK) at heat power 80 W and temperature of 96 C. The duration of microwave pretreatment (1 10 min) was investigated. The control samples were not subjected to the microwave pretreatment. After the pretreatment, the flasks were kept in a thermostated water bath at 85 C for up to 1 h to facilitate enzymatic liquefaction and then subjected to SSF. Liquefaction and SSF experiments A 100 g of corn meal was mixed with water at the weight ratio (hidromodul) 1:3, and 60 ppm of Ca 2? (as CaCl 2 ) ions was added. After the pretreatment, liquefaction was carried out at 85 C and ph 6.0 for 1 h by adding 0.026% (v/w, volume of enzyme per weight of starch) enzyme Termamyl Ò SC. The liquefaction and SSF process were performed in flasks in a thermostated water bath with shaking (100 rpm) as described by Mojović et al. (2006). The liquefied mash was cooled, ph was adjusted to 5.0 using 2 M HCl, and KH 2 PO 4 (4.0 g l -1 ), MgSO 4 7H 2 O (0.4 g l -1 ), and (NH 4 ) 2 SO 4 (2.0 g l -1 ) were added. The SSF process was initiated by adding 0.156% (v/w, volume of enzyme per weight of starch) enzyme SAN Extra Ò L and 2% (v/v) of inoculum of S. cerevisiae var. ellipsoideus to the liquefied mash, and carried out up to 48 h at 30 C. Initial viable cell number was *10 6 CFU ml -1. It was considered that the pasteurization of the substrate achieved during the enzymatic liquefaction (85 C for 1 h) was sufficient thermal treatment, and thus no additional sterilization prior to SSF process was performed. Scanning electron microscopy The surface structure of the control (without pretreatment) and ultrasound and microwave pretreated samples of corn meal suspensions were observed by SEM. A thin layer of the sample was mounted on the copper sample holder, using a double-sided carbon tape and coated with gold of 10 nm thicknesses to make the samples conductive. SEM studies were carried out using a scanning electron microscope (JSM5800, JEOL, Tokyo, Japan) at acceleration voltage of 20 kv. Analytical methods During the liquefaction and SSF process, the content of reducing sugars, calculated as glucose, was determined by 3,5-dinitrosalicylic acid (Miller 1959). A standard curve was drawn by measuring the absorbance of known concentrations of glucose solutions at 570 nm. The ethanol concentration was determined based on the density of the alcohol distillate at 20 C and expressed in weight % (w/w) (Official Methods of Analysis 2000). Indirect counting method, i.e., pour plate technique was used to determine the number of viable cells. Serial dilutions of the samples were performed, and after the incubation time at 30 C, colonies grown in Petri dishes were used to count the number of viable cells. Statistical analysis The experiments were done in triplicates. The results are expressed as means ± standard deviations. The means of treatments were compared by Student s t test. A p value as a decreasing index of the reliability of results was \0.05 indicating statistically significant differences. Results and discussion Effect of time of ultrasound and microwave pretreatments on the liquefaction of corn starch The first set of experiments was conducted in order to investigate the influence of duration of ultrasound and microwave pretreatments on the glucose concentration achieved after liquefaction of corn meal suspensions. Five different pretreatment times 1, 3, 5, 7, and 10 min were tested. The pretreatments were followed by the addition of enzyme Termamyl Ò SC, and then the liquefaction was performed under optimal conditions as described previously (Mojović et al. 2006). The control sample was not subjected to the pretreatment. The results of glucose concentration achieved are presented in Fig. 1. As shown in Fig. 1, a maximum increase in glucose concentration was achieved after 5 min of both pretreatments. In this case, ultrasound and microwave pretreatments effectively increased the glucose concentration obtained after the liquefaction of corn meal suspensions by 6.82 and 8.48%, respectively (compared to untreated control sample). In addition, an increase of time over 5 min did not cause further increase in glucose concentration after both pretreatments. This was probably due to the fact that after a longer duration of the pretreatment the concentration of glucose released from starch increased, but at the end of liquefaction step the final glucose concentration

4 590 S. Nikolić et al. Glucose concentration, g l ultrasound microwave control Pretreatment time, min Fig. 1 Effect of time of ultrasound and microwave pretreatments on glucose concentration obtained after the liquefaction of samples of corn meal suspensions. The pretreatments were performed before addition of the liquefying enzyme. Data points and error bars represent the mean and standard deviation, respectively, from three independent experiments decreased due to enzyme inhibition caused by glucose accumulation. Similarly, Kolusheva and Marinova (2007) concluded that elevated concentration of glucose affected the a-amylase inhibition and significantly decreased the starch hydrolysis rate. Huang et al. (2007) reported that the degree of corn starch hydrolysis increased sharply when the sonication time was increased from 3 to 9 min, but continuing ultrasound pretreatment had little effect on the degree of hydrolysis. This phenomenon was attributed to the effect of ultrasound on starch structure and its crystalline arrangement. Namely, the ultrasound pretreatment may be effective in the amorphous regions during 3 9 min, while the compact crystalline regions cannot be easily degraded even by longer ultrasound treatment. Khanal et al. (2007) reported that ultrasound pretreatment enhanced glucose release during enzymatic hydrolysis of corn meal mainly due to reduction in particle size, better mixing and the release of starch that was bound to lipids and did not have access to the hydrolyzing enzyme. Alvira et al. (2010) reported that cavitation effects caused by introduction of ultrasound field into the enzyme processing solution greatly enhanced the transport of enzyme macromolecules toward the substrate surface, and thus increased enzymatic hydrolysis yields. Hu and Wen (2008) and Palav and Seetharaman (2007) reported that microwave pretreatment also enhanced glucose release during enzymatic hydrolysis. Yadav and Lathi (2007) also reported that the rate of enzymatic reaction was much higher under microwave irradiation compared to conventional heating. From the economic viewpoint it is recommended to keep the pretreatment time low since both ultrasound or microwaves consume a significant amount of energy. Taking into account that fact and also the obtained results, 5 min was selected as optimal duration of the ultrasound and microwave pretreatments in further experiments. Similarly, relatively short duration of both pretreatments was selected by other investigators as appropriate for destroying the starch crystalline arrangement of various substrates and enhancing the glucose yield (Huang et al. 2007; Khanal et al. 2007; Kunlan et al. 2001; Nitayavardhana et al. 2008, 2010; Palav and Seetharaman 2007; Shewale and Pandit 2009). Future work is needed to scale-up system designs to large batch or continuous processes in order to fully realize the potential benefits of ultrasound or microwave pretreatment. However, it should be noted that a critical assessment of the costs and benefit analysis are needed before an industrial application which should take into account the initial capital investment and operation cost of these treatments as well as the benefits achieved by the treatments. Scanning electron microscopy examinations The changes in physical structure of control (without pretreatment) and pretreated samples of corn meal suspensions, before and after liquefaction, were imaged by scanning electron microscope, as presented in Fig. 2. SEM images in Fig. 2a c show that both ultrasound and microwaves affected the decomposition of starch granules even before the liquefaction started. As shown in Fig. 2d f, after the liquefaction the sizes of starch granules were smaller in pretreated samples than in the control sample because ultrasound and microwaves stimulated starch granules degradation, but microwaves in higher level. Therefore, both pretreatments were very effective in enhancing the enzymatic hydrolysis as discussed earlier (Fig. 1). In contrast, conventional heating observed in the control sample caused less change in structure. Effect of ultrasound and microwave pretreatments on SSF process of liquefied corn meal Further experiments were conducted in order to investigate the improvement of ethanol production in SSF process of corn meal by using ultrasound or microwave pretreatment. The SSF process was found to be economically favorable compared to the conventional SHF process in our previous studies (Mojović et al. 2006; Nikolić et al. 2009). Figure 3 shows the time course of ethanol production and glucose consumption in SSF process of liquefied corn meal

5 Utilization of microwave and ultrasound pretreatments in the production of bioethanol from corn 591 Fig. 2 SEM images of samples of corn meal suspensions a control sample (without pretreatment) before liquefaction, b ultrasound pretreated sample before liquefaction, c microwave pretreated sample before liquefaction, d control sample after liquefaction, e ultrasound pretreated sample after liquefaction, and f microwave pretreated sample after liquefaction. Both pretreatments were performed within 5 min before addition of the liquefying enzyme. The length of the scale bar is equivalent to 20 lm in a c (magnification 2,0009), and 500 lm in d f (magnification 1009) suspension by S. cerevisiae var. ellipsoideus, with and without the ultrasound or microwave pretreatment. As shown in Fig. 3, during SSF process the ethanol concentrations obtained in pretreated samples were higher than in the control sample. This was in accordance with results showed earlier (Fig. 1) since both ultrasound and microwaves increased the glucose concentration and consequently enhanced the ethanol production. As shown in Fig. 3, the ultrasound and microwave pretreatments Ethanol concentration, % (w/w) control ultrasound microwave Time, h Fig. 3 Time course of ethanol production and glucose consumption in the SSF process of corn meal hydrolyzates by Saccharomyces cerevisiae var. ellipsoideus with and without the ultrasound and microwave pretreatments. Both pretreatments were performed within 5 min before addition of the liquefying enzyme. Solid lines pretreated sample, dashed lines control sample. Data points and error bars represent the mean and standard deviation, respectively, from three independent experiments Glucose concentration, g l -1 increased the maximum ethanol concentration by and 13.40% (compared to the control sample), respectively, after 32 h of SSF process. However, the application of microwave pretreatment resulted in higher increase in ethanol concentrations compared to the ultrasound pretreatment. This was probably due to the fact that a temperature of 96 C reached during the microwave treatment at a power of 80 W was higher compared to a temperature of sonication of 60 C. Also, the mechanism of microwave action on swelling and gelatinization of starch granules and destroying the starch crystalline arrangement was probably different from the action of ultrasound. The product inhibition was noticed in both pretreated samples, but in ultrasound pretreated sample it occurred much earlier (after 32 h). This phenomenon can also be noticed in Table 1 which shows a decrease in the number of viable yeast cells at the end of the SSF process. Thatipamala et al. (1992) also reported that product inhibition significantly affect the ethanol and biomass yield during batch fermentation. As shown in Fig. 3, the glucose consumption was in accordance with the results of ethanol concentration since glucose was consumed as a carbon source by the yeast and fermented to ethanol. An increase in glucose concentration was observed in both pretreated and untreated samples at the beginning of the SSF. This indicates that the hydrolysis was much faster than the ethanol fermentation, since the yeast cells were still in the adaptation phase and during this time there was not a significant ethanol production. This phenomenon was also observed by other authors (Choi et al. 2008; Zhu et al. 2005). At the end of SSF process, in ultrasound and microwave pretreated samples a glucose concentration was 0.98 and 0.87 g l -1, respectively,

6 592 S. Nikolić et al. Table 1 Number of viable yeast cells obtained during the SSF process of corn meal hydrolyzates by Saccharomyces cerevisiae var. ellipsoideus with and without the ultrasound and microwave pretreatments Time (h) Number of viable cells (CFU ml -1 ) SSF process without pretreatment (control sample) SSF process with ultrasound pretreatment SSF process with microwave pretreatment ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.04 Both pretreatments were performed within 5 min before addition of the liquefying enzyme. Presented data are expressed as the mean value and standard deviation from three independent experiments indicating the end of fermentation. Thus, in microwave pretreated sample a total amount of utilized glucose of 99.19% was higher than in ultrasound pretreated sample. This was in accordance with the obtained ethanol concentrations. Table 1 shows the number of viable yeast cells obtained during the SSF process of the liquefied corn meal suspension by S. cerevisiae var. ellipsoideus, with and without the pretreatments. As shown in Table 1, the number of viable cells slowly increased during the SSF process in both pretreated and untreated samples. A maximum number of viable cells ( CFU ml -1 ) was achieved after 32 h of the process in the microwave pretreated sample which corresponds well to the fact that the maximum ethanol concentration was obtained at that point of the SSF. After that, a decline phase of yeast growth was observed suggesting the inhibition with high ethanol level, as mentioned before. Similarly, in the work of Torija et al. (2003) the decrease observed in yeast viability was also due to greater accumulation of intracellular ethanol. The comparison of significant process parameters achieved after 32 h of the batch SSF of corn meal by S. cerevisiae var. ellipsoideus with and without ultrasound and microwave pretreatments is presented in Table 2. The results indicate that the ultrasound and microwave pretreatments increased maximum ethanol concentration by and 13.40% (compared to the control sample), respectively, as mentioned above, and consequently increased ethanol yield as well as other process parameters. An ethanol concentration of 9.87% (w/w), ethanol yield of 0.52 g g -1, percentage of theoretical ethanol yield of 90.80% and volumetric productivity of 3.08 g l -1 h -1 were achieved after 32 h of SSF process on corn meal with prior microwave treatment (Fig. 3, Table 2). The improvement in ethanol production obtained in pretreated samples could be attributed primarily to the effect of ultrasound and microwaves on disintegration of corn starch granules, acceleration of starch hydrolysis, enhanced release of fermentable sugars and thereby increased ethanol productivity (Hu and Wen 2008; Huang et al. 2007; Khanal et al. 2007; Nitayavardhana et al. 2008, 2010; Shewale and Pandit 2009). Based on the obtained results (Fig. 3, Tables 1 and 2), the time of SSF process of pretreated samples may be reduced to 32 h. In this way, by implementation of the ultrasound or microwave pretreatment in SSF production of bioethanol, the process time could be reduced and higher Table 2 Values of the significant parameters obtained after 32 h of the SSF process of corn meal hydrolyzates by Saccharomyces cerevisiae var. ellipsoideus with and without the ultrasound and microwave pretreatments Process parameter SSF process without pretreatment (control sample) SSF process with ultrasound pretreatment SSF process with microwave pretreatment Ethanol concentration (% w/w) 8.70 ± ± ± 0.10 Ethanol yield Y P/S (g ethanol g -1 starch) ± ± ± Percentage of the theoretical ethanol yield (%) ± ± ± 0.92 Volumetric productivity P (g l -1 h -1 ) 2.72 ± ± ± 0.03 Utilized glucose a (%) ± ± ± 0.48 Both pretreatments were performed within 5 min before addition of the liquefying enzyme. Presented data are expressed as the mean value and standard deviation from three independent experiments a Utilized glucose (%) was calculated as the ratio of the consumed mass of glucose to initial mass of glucose

7 Utilization of microwave and ultrasound pretreatments in the production of bioethanol from corn 593 ethanol concentrations may be attained which leads to significant improvement in production economy. From the economic viewpoint, it is desired to attain high ethanol concentration in order to decrease the costs of subsequent ethanol distillation which affects the economy of overall process (Kim and Dale 2005). Similarly, in the work of Nitayavardhana et al. (2010) application of ultrasonic pretreatment not only enhanced the ethanol yield, but also reduced the fermentation time. In addition, the SSF process is more energy efficient compared to the conventional SHF process because (a) SSF ends at least 4 h before SHF process (4 h is required for saccharification when carried out separately) and (b) overall process is effectively performed at temperature of 30 C which is lower than the optimal temperature for the action of enzyme glucoamylase itself (55 C). These advantages are also noticed and reported previously by us (Nikolić et al. 2009) and by other researchers (Balat et al. 2008; Öhgren et al. 2007; Zhu et al. 2005). Besides the positive effects on improved ethanol yield and process productivity, an implementation of the ultrasound or microwave pretreatment in the SSF process may be energy consuming. Therefore, the obtained benefits should be considered and compared with the increased production costs. Conclusions Bioethanol production by SSF process of corn meal by S. cerevisiae var. ellipsoideus yeast in a batch system with prior ultrasound or microwave treatment was studied. Ultrasonic and microwave pretreatments effectively increased the glucose concentration obtained after liquefaction, and consequently improved the ethanol yield and productivity during the SSF process. An optimal duration of 5 min was determined for both pretreatments. The ultrasound and microwave pretreatments increased maximum ethanol concentration obtained after 32 h of SSF process by and 13.40% (compared to the control sample), respectively. The application of microwave pretreatment resulted in higher increase in glucose and ethanol concentration, compared to the ultrasound pretreatment. A maximum ethanol concentration of 9.91% (w/w) and percentage of theoretical ethanol yield of 92.27% were achieved after 44 h of SSF process on corn meal with prior microwave treatment. The application of both pretreatments can not only enhance the ethanol yield, but also may reduce the SSF time to 32 h and thus efficiently diminish the production costs. SEM images showed that ultrasound and microwaves stimulated disruption of corn starch structure either before or after liquefaction step, and thereby enhanced the glucose concentration and consequently the ethanol productivity in the SSF process. It is needed to scale-up the system to large batch or continuous processes in order to fully realize the potential benefits of ultrasound or microwave pretreatment, which is a part of our further research. Acknowledgment This work was funded by the Serbian Ministry of Science and Environmental Protection (TR 31017). References Alvira P, Tomás-Pejó E, Ballesteros M, Negro MJ (2010) Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresource Technol 101: Balat M, Balat H, Oz C (2008) Progress in bioethanol processing. Prog Energy Combust Sci 34: Baras J, Gaćeša S, Pejin D (2002) Ethanol is a strategic raw material. Chem Ind 56: Caylak B, Vardar Sukan F (1998) Comparison of different production processes for bioethanol. Turk J Chem 22: Choi GW, Moon SK, Kang HW, Min J, Chung BW (2008) Simultaneous saccharification and fermentation of sludge-containing cassava mash for batch and repeated batch production of bioethanol by Saccharomyces cerevisiae CHFY0321. J Chem Technol Biotechnol 84: Hu Z, Wen Z (2008) Enhancing enzymatic digestibility of switchgrass by microwave-assisted alkali pretreatment. Biochem Eng J 38: Huang Q, Li L, Fu X (2007) Ultrasound effects on the structure and chemical reactivity of cornstarch granules. Starch/Stärke 59: Khanal SK, Montalbo M, Hans van Leeuwen J, Srinivasan G, Grewell D (2007) Ultrasound enhanced glucose release from corn in ethanol plants. Biotechnol Bioeng 98: Kim S, Dale BE (2005) Environmental aspects of ethanol derived from no-tilled corn grain: nonrenewable energy consumption and greenhouse gas emissions. Biomass Bioenergy 28: Kolusheva T, Marinova A (2007) A study of the optimal conditions for starch hydrolysis through thermostable a-amylase. J Univ Chem Technol Metall 42:93 96 Kunlan L, Lixin X, Jun L, Jun P, Guoyoing C, Zuwei X (2001) Saltassisted acid hydrolysis of starch to D-glucose under microwave irradiation. Carbohydr Res 331:9 12 Mielenz J (2001) Ethanol production from biomass: technology and commercialization status. Curr Opin Microbiol 4: Miller GL (1959) Use of dinitrosalycilic acid for determining reducing sugars. Anal Chem 31: Mojović L, Nikolić S, Rakin M, Vukašinović M (2006) Production of bioethanol from corn meal hydrolyzates. Fuel 85: Nikolić S, Mojović L, Rakin M, Pejin D (2009) Bioethanol production from corn meal by simultaneous enzymatic saccharification and fermentation with immobilized cells of Saccharomyces cerevisiae var. ellipsoideus. Fuel 88: Nitayavardhana S, Rakshit SK, Grewell D, van Leeuwen JH, Khanal SK (2008) Ultrasound pretreatment of cassava chip slurry to enhance sugar release for subsequent ethanol production. Biotechnol Bioeng 101: Nitayavardhana S, Shrestha P, Rasmussen ML, Lamsal BP, van Leeuwen JH, Khanal SK (2010) Ultrasound improved ethanol fermentation from cassava chips in cassava-based ethanol plants. Bioresource Technol 101: Official Method (2000) Official methods of analysis of AOAC international, 17th edn. AOAC International, Gaithersburg

8 594 S. Nikolić et al. Öhgren K, Bura R, Lesnicki G, Saddler J, Zacchi G (2007) A comparison between simultaneous saccharification and fermentation and separate hydrolysis and fermentation using steampretreatment corn stover. Process Biochem 42: Palav T, Seetharaman K (2007) Impact of microwave heating on the physico-chemical properties of a starch water model system. Carbohydr Polym 67: Patist A, Bates D (2008) Ultrasonic innovations in the food industry: from the laboratory to commercial production. Innov Food Sci Emerg Technol 9: Schausberger P, Bösch P, Friedl A (2010) Modeling and simulation of coupled ethanol and biogas production. Clean Technol Environ Policy 12: Shewale SD, Pandit AB (2009) Enzymatic production of glucose from different qualities of grain sorghum and application of ultrasound to enhance the yield. Carbohydr Res 344:52 60 Siqueira PF, Karp SG, Carvalho JC, Sturm W, Rodríguez-León JA, Tholozan JL, Singhania RR, Pandey A, Soccol CR (2008) Production of bio-ethanol from soybean molasses by Saccharomyces cerevisiae at laboratory, pilot and industrial scales. Bioresource Technol 99: Thatipamala R, Rohani S, Hill GA (1992) Effects of high product and substrate inhibitions on the kinetics and biomass and product yields during ethanol batch fermentation. Biotechnol Bioeng 40: Torija MJ, Rozès N, Poblet M, Guillamón JM, Mas A (2003) Effects of fermentation temperature on the strain population of Saccharomyces cerevisiae. Int J Food Microbiol 80:47 53 Yadav GD, Lathi PS (2007) Microwave assisted enzyme catalysis for synthesis of n-butyl diphenyl methyl mercapto acetate in nonaqueous media. Clean Technol Environ Policy 9: Zhang Y, Fu E, Liang J (2008) Effect of ultrasonic waves on the saccharification processes of lignocellulose. Chem Eng Technol 31: Zhu S, Wu Y, Yu Z, Zhang X, Wang C, Yu F, Jin S, Zhao Y, Tu S, Xue Y (2005) Simultaneous saccharification and fermentation of microwave/alkali pre-treated rice straw to ethanol. Biosyst Eng 92:

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