Ultrasound-assisted production of bioethanol by simultaneous ...

Food Chemistry 122 (2010) 216–222

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Ultrasound-assisted production of bioethanol by simultaneous saccharification and fermentation of corn meal Svetlana Nikolic´ a,*, Ljiljana Mojovic´ a, Marica Rakin a, Dušanka Pejin b, Jelena Pejin b a b

Faculty of Technology and Metallurgy, Department of Biochemical Engineering and Biotechnology, University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia

a r t i c l e

i n f o

Article history: Received 10 September 2009 Received in revised form 28 December 2009 Accepted 24 February 2010

Keywords: Bioethanol Ultrasound pretreatment Simultaneous saccharification and fermentation Saccharomyces cerevisiae var. ellipsoideus Corn meal

a b s t r a c t An ultrasound-assisted liquefaction as a pretreatment for bioethanol production by simultaneous saccharification and fermentation (SSF) of corn meal using Saccharomyces cerevisiae var. ellipsoideus yeast in a batch system was studied. Ultrasound pretreatment (at a frequency of 40 kHz) was performed at different sonication times and temperatures, before addition of liquefying enzyme. An optimal duration of the treatment of 5 min and sonication temperature of 60 °C were selected, taking into account glucose concentration after the liquefaction step. Under the optimum conditions an increase of glucose concentration of 6.82% over untreated control sample was achieved. Furthermore, the SSF process kinetics was assessed and determined, and the effect of ultrasound pretreatment on an increase of ethanol productivity was investigated. The obtained results indicated that the ultrasound pretreatment could increase the ethanol concentration by 11.15% (compared to the control sample) as well as other significant process parameters. In this case, the maximum ethanol concentration of 9.67% w/w (which corresponded to percentage of the theoretical ethanol yield of 88.96%) was achieved after 32 h of the SSF process. A comparison of scanning electron micrographs of the ultrasound-pretreated and untreated samples of corn meal suspensions showed that the ultrasound stimulated degradation of starch granules and release of glucose, and thereby accelerated the starch hydrolysis due to the cavitation and acoustic streaming caused by the ultrasonic action. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Bioethanol, one of the most important biofuels, is both renewable and environmentally friendly (Balat, Balat, & Öz, 2008; Baras, Gacéša, & Pejin, 2002). It can be blended with petrol (E5, E10, E85) or used as neat alcohol in dedicated engines, taking advantage of the higher octane number and higher heat of vaporisation, and it is also an excellent fuel for future advanced flexi-fuel hybrid vehicles (Chum & Overend, 2001; Kim & Dale, 2005). Fermentation-derived ethanol can be produced from sugar, starch or lignocellulosic biomass. Sugar and starch-based feedstocks are currently predominant at the industrial level and they are so far economically favourable. In Serbia, one of the most suitable and available agricultural raw materials for industrial bioethanol production is corn. It is reported that in 2009 the average corn yield in Serbia was approximately 6–7 million ton, while the calculated domestic needs for corn are only 4–4.5 million ton (http:// www.b92.net/biz/vesti/srbija.php). This means that there is enough corn for non-food uses, such as bioethanol production. * Corresponding author. Tel.: +381 113370423; fax: +381 113370387. E-mail address: [email protected] (S. Nikolic´). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.02.063

Current world bioethanol research is driven by the need to reduce the costs of production. For example, improvement in feedstock pretreatment, shortening of fermentation time, lowering the enzyme dosages, improving the overall starch hydrolysis and integration of the simultaneous saccharification and fermentation (SSF) process could be the basis of cutting down production costs. The application of ultrasound pretreatment may significantly increase the conversion of starch materials to glucose as well as overall ethanol yield (Khanal, Montalbo, Hans van Leeuwen, Srinivasan, & Grewell, 2007; Mielenz, 2001). Ultrasonication has been applied widely in various biological and chemical processes. Ultrasound (i.e. mechanical waves at a frequency above the hearing range of humans) can be divided into three frequency ranges: power ultrasound (16–100 kHz), high frequency ultrasound (100 kHz–1 MHz) and diagnostic ultrasound (1–10 MHz) (Patist & Bates, 2008). When a low frequency ultrasound (that is, power ultrasound ranging from 16 – 100 kHz) wave propagates in a medium such as a liquid or slurry, it produces cavitation and acoustic streaming. It generates large cavitation bubbles resulting in higher temperatures and pressures in the cavitation zone. The cavitation generates powerful hydro-mechanical shear forces in the bulk liquid, which disintegrate nearby particles by extreme shear forces. The main

S. Nikolic´ et al. / Food Chemistry 122 (2010) 216–222

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 with short processing time (Khanal et al., 2007; Nitayavardhana, Rakshit, Grewell, (Hans) van Leeuwen, & Khanal, 2008; Patist & Bates, 2008). Besides the pretreatment, the process mode used for saccharification and fermentation is also an important factor affecting the production costs. There are many reports that the simultaneous saccharification and fermentation (SSF) is an economically more favourable process than the traditional separate saccharification 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 (Marques, Alves, Roseiro, & Gírio, 2008; Mojovic´, Nikolic´, Rakin, & Vukašinovic´, 2006; Nikolic´, Mojovic´, Rakin, & Pejin, 2009; Öhgren, Bura, Lesnicki, Saddler, & Zacchi, 2007). On the other hand, the critical problem with SSF is different optimum temperatures of hydrolysing enzymes and fermenting organisms. Saccharomyces strains are well known ethanol-producing microorganisms but they require operating temperature of about 30–35 °C, which differs from the optimal temperature for the saccharification step, i.e., 55–60 °C in the case of using glucoamylase in starch conversion to glucose (Karimi, Emtiazi, & Taherzadeh, 2006; Nikolic´ et al., 2009). The aim of this study was to investigate the possibilities of improving glucose yield and ethanol productivity by applying an ultrasound pretreatment in the bioethanol production by simultaneous saccharification and fermentation of corn meal with Saccharomyces cerevisiae var. ellipsoideus yeast in a batch system. The efficiency of ultrasound pretreatment to disintegrate corn starch granules and improve glucose release at different sonication parameters was studied. Additionally, changes in physical properties of corn meal suspensions before and after sonication were examined at the microscopic level by SEM studies. The kinetics of the SSF process, as well as bioethanol yield and productivity, were also assessed. 2. Materials and methods 2.1. 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 0.2–1.7 mm (95% or more particles pass through a 1.70 mm sieve). The content of the main components in the corn meal, determined by chemical analysis, was the following: starch 76.75% (w/w), proteins 6.35% (w/w), lipids 4.50% (w/w), fibres 1.36%, ash 0.70% (w/w) and water 10.34% (w/w). 2.2. Enzymes and microorganisms Termamyl SC, a heat-stable a-amylase from Bacillus licheniformis was used for corn meal liquefaction. The enzyme activity was 133 KNU/g (KNU, kilo novo units a-amylases – the amount of enzyme which breaks down 5.26 g of starch per hour according to Novozyme’s standard method for the determination of a-amylase). SAN Extra L, Aspergillus niger glucoamylase, activity 437 AGU/g (AGU 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 a gift from Novozymes, Denmark.

217

S. cerevisiae var. ellipsoideus was used for the fermentation of hydrolysed corn meal. The culture originated from the collection of the Department of Biochemical Engineering and Biotechnology, Faculty of Technology and Metallurgy, Belgrade (BIB-TMFB), and was maintained on a malt agar slant. The agar slant consisted of malt extract (3 g/l), yeast extract (3 g/l), peptone (5 g/l), agar (20 g/l) 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), peptone (3.5 g/l), KH2PO4 (2.0 g/l), MgSO47H2O (1.0 g/l), (NH4)2SO4 (1.0 g/l), glucose (10 g/l) and distilled water. 2.3. Ultrasound pretreatment Samples of the mixture of corn meal and water at a weight ratio (hidromodul) of 1:3, placed in glass flasks, were subjected to ultrasound pretreatment before the addition of liquefying enzyme Termamyl SC. The ultrasound pretreatment was carried out in a sonicator (Model: USK 28, power 600 W, EI Niš, Niš, Serbia) at a frequency of 40 kHz. Parameters such as sonication temperature (room temperature, 60 and 80 °C) and the duration of ultrasound pretreatment (0.5, 1, 3, 5, 10, 20 and 30 min) were investigated. The control samples were not subjected to the 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. All sonication tests were conducted in triplicate. 2.4. Liquefaction and SSF experiments Corn meal (100 g) was mixed with water at a weight ratio (hidromodul) of 1:3, and 60 ppm of Ca2+ (as CaCl2) ions were added. The liquefaction was carried out at 85 °C and pH 6.0 for 1 h by adding 0.026% (v/w of starch) enzyme Termamyl SC. The liquefaction and SSF process were performed in flasks in a thermostated water bath with shaking (100 rpm), as described by Mojovic´ et al. (2006). The liquefied mash was cooled, pH was adjusted to 5.0 using 2 M HCl, and KH2PO4 (4.0 g/l), MgSO47H2O (0.4 g/l) and (NH4)2SO4 (2.0 g/l) were added. The SSF process was initiated by adding 0.156% (v/w of starch) enzyme SAN Extra L and 2% (v/v) of inoculum of S. cerevisiae var. ellipsoideus to the liquefied mash, and carried out for up to 48 h at 30 °C. The fermentation volume was 380 ml. Initial viable cell number was 106 CFU/ml. It was considered that the pasteurisation of the substrate achieved during the enzymatic liquefaction (85 °C for 1 h) was sufficient thermal treatment, and thus no additional sterilisation prior to SSF process was performed. 2.5. Scanning electron microscopy (SEM) The surface structure of the control (without ultrasound pretreatment) and ultrasound-pretreated samples of corn meal suspensions were observed by scanning electron microscopy. 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 an acceleration voltage of 20 kV. 2.6. Analytical methods The starch content was determined by Ewers polarimetric method (International Standards: ISO 10520, 1997). The water content in the corn meal was determined by the standard drying method in an oven at 105 °C to a constant mass (Official Methods


218

of Analysis, 2000). Lipid concentration was determined according to the Soxhlet method (Official Methods of Analysis, 2000). The protein content was estimated as the total nitrogen by the Kjeldahl method multiplied by 6.25 (Official Methods of Analysis, 2000), the ash content was determined by slow combustion of the sample at 650 °C for 2 h (Official Methods of Analysis, 2000), and the fibre content was determined by the Scharrer–Kürschner method (Official Methods of Analysis, 2000). 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 as % 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. At least three measurements were made for each condition and the data given were averages. 3. Results and discussion 3.1. Effect of ultrasound pretreatment on the liquefaction of corn starch The first set of experiments was conducted in order to investigate the influence of the duration of ultrasound pretreatment on the glucose concentration achieved after the liquefaction of corn meal suspensions. The ultrasound pretreatment was performed at room temperature and at different sonication times. The pretreatment was followed by the addition of enzyme Termamyl SC, and then the liquefaction was performed under optimal conditions, as described in our previous study (Nikolic´ et al., 2009). We tested seven different sonication times: 0.5, 1, 3, 5, 10, 20 and 30 min. The control sample was not subjected to the ultrasound pretreatment. The results of glucose concentration achieved are presented in Fig. 1.

103

3.30%

102

2.14%

glucose concentration, g/l

101 100

2.59% 2.22%

0.36% 0.71%

99

1.62%

98 97 96 95 94 93 92 91 90

control

0.5

1 3 5 10 sonication time, min

20

30

Fig. 1. Effect of time of ultrasound pretreatment on glucose concentration obtained after the liquefaction of samples of corn meal suspensions. The ultrasound pretreatment was performed at room temperature with frequency of 40 kHz, before addition of the liquefying enzyme. Experimental conditions for liquefaction: hidromodul 1:3, pH 6.0, 85 °C, 1 h, 100 rpm, enzyme Termamyl SC was added in concentration of 0.026% (v/w of starch). The numbers above the bars represent the percentage of the increase in glucose concentration compared to the control sample.

As shown in Fig. 1, a maximum increase in glucose concentration (3.30% compared to the control sample) was achieved after 5 min of the ultrasound pretreatment. In this case, the glucose concentration of 102 g/l was achieved. Additionally, an increase of the sonication time over 5 min did not cause further increase in glucose concentration. This was probably due to the fact that after longer duration of ultrasound pretreatment the concentration of glucose released from starch increased, but at the end of liquefaction step the final glucose concentration decreased, due to enzyme inhibition by glucose accumulation. Similarly, Kolusheva and Marinova (2007) concluded that elevated concentration of glucose significantly decreased the starch hydrolysis rate and affected the enzyme inhibition. This phenomenon was also reported in the work of Yankov, Dobreva, Beschkov, and Emanuilova (1986). Huang, Li, and Fu (2007) investigated the effect of ultrasound on the structure and chemical reactivity of corn starch granules. They reported that the degree of hydrolysis increased sharply when the sonication time was increased from 3 to 9 min, but continuing treatment had little effect on the degree of hydrolysis of corn starch. This phenomenon was attributed to the effect of ultrasound on the structure of starch and its crystalline arrangement. Namely, starch granules contain both ordered crystalline regions and amorphous regions, in which polymer chains are less well ordered and more susceptible to attack by amylase action. Thereby, 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. In the work of Shewale and Pandit (2009) an application of ultrasound pretreatment of grain sorghum increased the dextrose equivalent of liquefact by 10–25% depending on sonication time and ultrasound intensity. This was attributed to the availability of additional starch for hydrolysis, due to ultrasound-assisted disruption of the protein matrix (surrounding starch granules) and the amylase-lipid complex. The sonication time of 1 min was considered as the optimum time for ultrasound treatment in their study, because it gave a maximum increase in DE of the liquefact. From the economic viewpoint it is recommended to keep sonication time low, since ultrasound treatment consumes a large amount of energy. Taking into account that fact and also the obtained results, 5 min was selected as the optimal duration of the ultrasound pretreatment in further experiments. Similarly, relatively short duration of the ultrasound pretreatment was also 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; Nitayavardhana, Rakshit, Grewell, (Hans) van Leeuwen, & Khanal, 2008; Shewale & Pandit, 2009). The effect of the temperature of the ultrasound pretreatment on liquefaction of corn meal suspensions was investigated by maintaining the pretreatment duration at 5 min for different sonication temperatures (room temperature, 60 and 80 °C). The control sample was not subjected to the ultrasound pretreatment. The results of glucose concentration achieved are presented in Fig. 2. As shown in Fig. 2, a maximum glucose concentration of 105 g/l was achieved at 60 °C. In this case an increase in glucose concentration of 6.82% over untreated control sample was observed. For this reason, the temperature of 60 °C was found to be optimum for the ultrasound pretreatment. Even though these results show a modest increase in the glucose concentration, the significant effect of ultrasound pretreatment on enhanced ethanol yield has been observed in the SSF process (Table 1). Khanal et al. (2007) reported that ultrasound pretreatment (20 kHz; 20 and 40 s) enhanced glucose release during enzymatic hydrolysis of corn meal, mainly due to reduction in particle size and better mixing. They also pointed out that increased sugar yield of ultrasound-treated samples could also be due to the release of

glucose concentration, g/l


107 106 105 104 103 102 101 100 99 98 97 96 95 94 93 92 91 90

6.82%

5.90%

3.30%

control

room temp.

60 Temperature, ºC

80

Fig. 2. Effect of sonication temperature on glucose concentration obtained after the liquefaction of samples of corn meal suspensions. The ultrasound pretreatment was performed within 5 min before addition of the liquefying enzyme with a frequency of 40 kHz. Experimental conditions for liquefaction were as in Fig. 1. The numbers above the bars represent the percentage of the increase in glucose concentration compared to the control sample.

Table 1 Values of the significant parameters obtained after 32 h of the SSF processing of corn meal hydrolysates by S. cerevisiae var. ellipsoideus with and without ultrasound pretreatment. Process parameter

SSFa process without ultrasound pretreatment (control sample)

SSFa process with ultrasound pretreatment

Ethanol concentration (% w/w) Ethanol yield YP/S (g ethanol/g starch) Percentage of the theoretical ethanol yield (%) Volumetric productivity P (g/lh) Utilised glucoseb (%)

8.70 ± 0.09 0.45 ± 0.004 80.04 ± 1.42

9.67 ± 0.11 0.50 ± 0.003 88.96 ± 1.46

2.72 ± 0.03 77.33 ± 0.04

3.02 ± 0.02 85.22 ± 0.05

a

Process conditions are the same as in Fig. 4. Utilised glucose (%) was calculated as the ratio of the consumed mass of glucose to initial mass of glucose. b

starch that was bound to lipids and did not have access to the hydrolysing enzyme. Investigating the effect of sonication temperature, they obtained high glucose yields at temperatures in the range of 30–40 °C. However, the results obtained even with the same feedstock could significantly differ depending on its type, origin and chemical composition, as well as other process parameters employed. In the work of Sun, Sun, and Ma (2002) the optimum sonication temperature was 60 °C as in our work, but they investigated the effect of ultrasound pretreatment (20 kHz; 20 min) on lignocellulosic material (wheat straw). Future work is needed to scale up system designs to large batch or continuous processes, in order to fully realise the potential benefits of ultrasound pretreatment. However, it should be noted that a critical assessment of the costs and benefits are needed because the initial capital investment and operation cost of ultrasound treatment are not cheap. 3.2. Scanning electron microscopy examinations The changes in physical structure of control (without ultrasound pretreatment) and ultrasound-pretreated samples of corn meal suspensions, before and after liquefaction, were imaged by

219

scanning electron microscope, and presented in Fig. 3. The ultrasound pretreatment of presented samples was performed under optimal conditions (5 min, 60 °C) determined in the previous experiments. SEM images in Fig. 3a and b show that ultrasound affected the decomposition of the starch granules even before the liquefaction started. As shown in Fig. 3c and d, after liquefaction the sizes of starch granules were smaller in the pretreated sample than in the control sample because the ultrasound stimulated starch granule degradation and glucose release. In contrast, conventional heating observed in the control sample caused less change in structure. Therefore, the ultrasound pretreatment was more effective in enhancing the enzymatic hydrolysis as discussed earlier (Figs. 1 and 2).

3.3. SSF of liquefied corn meal after the ultrasound pretreatment Further experiments were conducted in order to investigate the improvement of ethanol production in SSF processing of corn meal by using ultrasound. The ultrasound pretreatment was performed under optimal conditions (5 min, 60 °C) determined in the previous experiments. The SSF process was found to be economically favourable, compared to the conventional separate hydrolysis and fermentation (SHF) process in our previous studies (Mojovic´ et al., 2006; Nikolic´, Mojovic´, Rakin, Pejin, & Savic´, 2008; Nikolic´ et al., 2009). Figs. 4 and 5 present the time course of ethanol production, glucose consumption and the number of viable yeast cells in the SSF processing of liquefied corn meal suspension by S. cerevisiae var. ellipsoideus, with and without the ultrasound pretreatment. As shown in Fig. 4, the ethanol production profiles in both ultrasound-pretreated and untreated samples were similar. During the SSF process the ethanol concentrations obtained in ultrasoundpretreated sample were higher than in the control sample. This was in accordance with earlier results (Figs. 1 and 2), since ultrasound increased glucose concentration and consequently enhanced the ethanol production. In the ultrasound-pretreated sample, the maximum ethanol concentration of 9.67% (w/w), the ethanol yield of 0.50 g/g, percentage of the theoretical ethanol yield of 88.96% and the volumetric productivity of 3.02 g/l/h were achieved after 32 h of the SSF process (Fig. 4, Table 1). In this case, the increase in ethanol concentration was 11.15% compared to the control sample (Table 1). After 32 h, product inhibition was noticed. The highest ethanol concentration of 9.67% (w/w) was achieved after 32 h, and the final ethanol concentration was lower – 9.13% (w/w) after 48 h. This phenomenon can also be seen in Fig. 5, which shows a decrease in the number of viable yeast cells after 32 h of the SSF process. Thatipamala, Rohani, and Hill (1992) also reported that the product inhibition significantly affect the ethanol and biomass yield during the ethanol batch fermentation. In their study, S. cerevisiae biomass yield decreased from 0.156 to 0.026 when ethanol concentration increased from 0 to 107 g/l. As shown in Fig. 4, the glucose consumption was in accordance with the results of ethanol concentration, since the glucose was consumed as a carbon source by the yeast. 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 no significant ethanol production (Figs. 4 and 5). This phenomenon was also observed by other authors (Choi, Moon, Kang, Min, & Chung, 2008; Montesinos & Navarro, 2000; Zhu et al., 2005). At the end of the SSF process, in the ultrasound-pretreated sample the glucose concentration was 0.98 g/l


220

Fig. 3. SEM images of samples of corn meal suspensions: (a) control sample (without ultrasound pretreatment) before liquefaction, (b) ultrasound-pretreated sample before liquefaction, (c) control sample after liquefaction, (d) ultrasound-pretreated sample after liquefaction. The ultrasound pretreatment was performed under optimal conditions: 60 °C, 5 min, 40 kHz. Experimental conditions for liquefaction were as in Fig. 1. The length of the scale bar is equivalent to 20 lm in Fig. 3a and b (magnification 2000), and 500 lm in Fig. 3c and d (magnification 100).

180 160

8

140

7

120

6

100

5 4

ethanol glucose

3

80 60

2

40

1

20 0

5

10

15

20

25

30

35

40

45

0 50

Time, h Fig. 4. Time course of ethanol production and glucose consumption in the SSF processing of corn meal hydrolysates by S. cerevisiae var. ellipsoideus with and without the ultrasound pretreatment. The ultrasound pretreatment was performed under optimal conditions: 60 °C, 5 min, 40 kHz. Experimental conditions for liquefaction were as in Fig. 1. Experimental conditions for SSF process: 30 °C, pH 5.0, 48 h, 100 rpm, enzyme SAN Extra L was at 0.156% (v/w of starch), inoculum concentration 2% (v/v). Solid lines – ultrasound-pretreated sample, dashed lines – control sample.

(the total amount of utilised glucose was 99.07%) indicating the end of fermentation (Fig. 4).

8.5 8.0

log (CFU/ml)

9

0

9.0

200

10

Glucose concentration, g/l

Ethanol concentration, % (w/w)

11

7.5 7.0 6.5 6.0 0

5

10

15

20

25

30

35

40

45

50

Time, h Fig. 5. The number of viable cells during the SSF processing of corn meal hydrolysates by S. cerevisiae var. ellipsoideus with and without ultrasound pretreatment. Process conditions are the same as in Fig. 4. Solid lines – ultrasound-pretreated sample, dashed lines – control sample.

As shown in Fig. 5, the number of viable cells slowly increased during the first 8 h of the SSF in both pretreated and untreated samples, since the yeast cells were still in the adaptation (lag) phase of their growth. During the logarithmic phase the numbers of viable cells obtained in ultrasound-pretreated samples were


higher than in the control sample. Maximum number of viable cells (5.89 108 CFU/ml) in the ultrasound-pretreated sample was achieved after 32 h of the process, which corresponds well to the fact that the maximum ethanol concentration was obtained at that point of the SSF. In the control sample, the maximum number of viable cells (2.04 108 CFU/ml) was achieved also after 32 h of the process but it was much less than in the pretreated sample. After that the decline phase of the yeast growth was observed, suggesting the inhibition effect of the high ethanol level. The decrease in the yeast viability, observed in the work of Torija, Rozès, Poblet, Guillamón, and Mas (2003), was also due to the greater accumulaˇ alver, Moreno, and Lagunas tion of intracellular ethanol. Lucero, Pen (2000) reported that the toxic concentration of intracellular ethanol altered the structure of the membrane, decreasing its functionality. The comparison of the significant process parameters achieved after 32 h of the batch SSF of corn meal by S. cerevisiae var. ellipsoideus with and without ultrasound pretreatment is presented in Table 1. The results indicate that the ultrasound pretreatment increased the maximum ethanol concentration to 11.15% (as mentioned earlier), and consequently increased the ethanol yield as well as the other process parameters. This improvement could be attributed primarily to the effect of ultrasound on the disintegration of corn starch granules, the acceleration of the starch hydrolysis, the enhanced release of fermentable sugars and thereby the increased ethanol productivity (Huang et al., 2007; Khanal et al., 2007; Nitayavardhana et al., 2008; Shewale & Pandit, 2009). In our previous study (Nikolic´ et al., 2008) we investigated SSF processing of corn meal by S. cerevisiae var. ellipsoideus with a prior microwave treatment and reported significant process parameters. When we compared the results obtained in the SSF with microwave (Nikolic´ et al., 2008) and ultrasound pretreatment (this study) to the control sample, the ethanol concentration was increased by 13.4% by microwave and 11.15% by ultrasound pretreatment (Table 1). This was probably due to the fact that the temperature of 96 °C during microwave treatment (Nikolic´ et al., 2008) was higher compared to the optimal sonication temperature of 60 °C, and also the mechanism of microwave action on swelling and gelatinisation of starch granules and destruction of the starch crystalline arrangement was probably different compared to the ultrasound. Based on the obtained results the time of the SSF processing of corn meal with ultrasound pretreatment may be reduced to 32 h. In this way, integration of the ultrasound treatment and the SSF processing mode in the production of bioethanol could reduce the processing time and attain higher ethanol concentrations. Additionally, in the SSF process energy savings could be attained compared to the conventional SHF process because: (a) the SSF ends at least 4 h before the SHF process (4 h is required for saccharification when carried out separately) and, (b) the overall process is effectively performed at temperature of 30 °C, which is lower than the optimal temperature for the action of glucoamylase enzyme itself (55 °C). Other advantages of the SSF process compared to the SHF process are a lower end-product inhibition of the enzymes because the yeast immediately consumes the released glucose, and lower capital investments as the total reactor volume is decreased due to higher ethanol productivity. These advantages are also noticed and reported previously by us (Mojovic´ et al., 2006; Nikolic´ et al., 2008, 2009) and by other researchers (Balat et al., 2008; Marques 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 pretreatment in the SSF process may be energy-consuming (especially since the optimal sonication temperature was 60 °C), suggesting that the obtained benefits should be considered and compared with the increased production costs.

221

4. Conclusions This study examined the feasibility of ultrasound pretreatment to enhance glucose release from corn meal during enzymatic hydrolysis and consequently increase ethanol yield in the SSF process by S. cerevisiae var. ellipsoideus yeast. Ultrasonic pretreatment (at frequency of 40 kHz) effectively increased the glucose concentration after liquefaction of corn meal suspension (by 6.82% compared to the untreated control sample) under determined optimal conditions of sonication (5 min, 60 °C). The present investigation also shows that the ultrasound pretreatment consequently improved ethanol production during SSF processing since the ethanol concentration was increased by 11.15% (compared to the control sample) as well as other process parameters. Taking into consideration significant process parameters obtained during the SSF process of corn meal with ultrasound pretreatment, the process time may be reduced to 32 h. At that point of the SSF, the maximum ethanol concentration of 9.67% (w/w), the ethanol yield of 0.50 g/g, percentage of the theoretical ethanol yield of 88.96%, the volumetric productivity of 3.02 g/l/h, the utilised glucose of 85.22% and maximum number of viable cells of 5.89 108 CFU/ml were achieved. SEM images showed that cavitation and acoustic streaming caused by ultrasound-stimulated disruption of corn starch structure, either before or after the liquefaction step, enhanced glucose concentration and consequently ethanol productivity in the SSF process. It is needed to scale up system designs to large batch or continuous processes in order to fully realise the potential benefits of ultrasound pretreatment, which is a part of our future research. Acknowledgement This work was funded by the Serbian Ministry of Science and Environmental Protection (TR 18002). References Balat, M., Balat, H., & Öz, C. (2008). Progress in bioethanol processing. Progress in Energy and Combustion Science, 34(5), 551–573. Baras, J., Gacéša, S., & Pejin, D. (2002). Ethanol is a strategic raw material. Chemical Industry, 56(3), 89–105. Choi, G.-W., Moon, S.-K., Kang, H.-W., Min, J., & Chung, B.-W. (2008). Simultaneous saccharification and fermentation of sludge-containing cassava mash for batch and repeated batch production of bioethanol by Saccharomyces cerevisiae CHFY0321. Journal of Chemical Technology and Biotechnology, 84(4), 547–553. Chum, H. L., & Overend, R. P. (2001). Biomass and renewable fuels. Fuel Processing Technology, 71(1–3), 187–195. . Huang, Q., Li, L., & Fu, X. (2007). Ultrasound effects on the structure and chemical reactivity of cornstarch granules. Starch/Stärke, 59(8), 371–378. International Standards: ISO 10520, 1997. Determination of starch content – Ewers polarimetric method. Karimi, K., Emtiazi, G., & Taherzadeh, M. J. (2006). Ethanol production from diluteacid pretreated rice straw by simultaneous saccharification and fermentation with Mucor indicus, Rhizopus oryzae, and Saccharomyces cerevisiae. Enzyme and Microbial Technology, 40(1), 138–144. Khanal, S. K., Montalbo, M., Hans van Leeuwen, J., Srinivasan, G., & Grewell, D. (2007). Ultrasound enhanced glucose release from corn in ethanol plants. Biotechnology and Bioengineering, 98(5), 978–985. Kim, S., & Dale, B. E. (2005). Environmental aspects of ethanol derived from no-tilled corn grain: Nonrenewable energy consumption and greenhouse gas emissions. Biomass and Bioenergy, 28(5), 475–489. Kolusheva, T., & Marinova, A. (2007). A study of the optimal conditions for starch hydrolysis through thermostable a-amylase. Journal of the University of Chemical Technology and Metallurgy, 42(1), 93–96. ˇ alver, E., Moreno, E., & Lagunas, R. (2000). Internal trehalose protects Lucero, P., Pen endocytosis from inhibition by ethanol in Saccharomyces cerevisiae. Applied and Environmental Microbiology, 66(10), 4456–4461. Marques, S., Alves, L., Roseiro, J. C., & Gírio, F. M. (2008). Conversion of recycled paper sludge to ethanol by SHF and SSF using Pichia stipitis. Biomass and Bioenergy, 32(5), 400–406. Mielenz, J. (2001). Ethanol production from biomass: Technology and commercialization status. Current Opinion in Microbiology, 4(3), 324–329. Miller, G. L. (1959). Use of dinitrosalycilic acid for determining reducing sugars. Analytical Chemistry, 31(3), 426–428.

222


Mojovic´, L., Nikolic´, S., Rakin, M., & Vukašinovic´, M. (2006). Production of bioethanol from corn meal hydrolyzates. Fuel, 85(12–13), 1750–1755. Montesinos, T., & Navarro, J. M. (2000). Production of alcohol from raw wheat flour by amyloglucosidase and Saccharomyces cerevisiae. Enzyme and Microbial Technology, 27(6), 362–370. Nikolic´, S., Mojovic´, 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(9), 1602–1607. Nikolic´, S., Mojovic´, L., Rakin, M., Pejin, D., & Savic´, D. (2008). A microwave-assisted liquefaction as a pretreatment for bioethanol production by the simultaneous saccharification and fermentation of corn meal. Chemical Industry and Chemical Engineering Quarterly, 14(4), 231–234. Nitayavardhana, S., Rakshit, S. K., Grewell, D., (Hans) van Leeuwen, J., & Khanal, S. K. (2008). Ultrasound pretreatment of cassava chip slurry to enhance sugar release for subsequent ethanol production. Biotechnology and Bioengineering, 101(3), 487–496. Official Methods of Analysis 923.03, 925.09, 930.15, 955.04, 960.39 (2000). Official Methods of Analysis of AOAC International (17th ed.). Gaithersburg, MD, USA: AOAC International. Öhgren, K., Bura, R., Lesnicki, G., Saddler, J., & Zacchi, G. (2007). A comparison between simultaneous saccharification and fermentation and separate hydrolysis and fermentation using steam-pretreatment corn stover. Process Biochemistry, 42(5), 834–839.

Patist, A., & Bates, D. (2008). Ultrasonic innovations in the food industry: From the laboratory to commercial production. Innovative Food Science and Emerging Technologies, 9(2), 147–154. Shewale, S. D., & Pandit, A. B. (2009). Enzymatic production of glucose from different qualities of grain sorghum and application of ultrasound to enhance the yield. Carbohydrate Research, 344(1), 52–60. Sun, R. C., Sun, X. F., & Ma, X. H. (2002). Effect of ultrasound on the structural and physiochemical properties of organosolv soluble hemicellulose from wheat straw. Ultrasonics Sonochemistry, 9(2), 95–101. Thatipamala, R., Rohani, S., & Hill, G. A. (1992). Effects of high product and substrate inhibitions on the kinetics and biomass and product yields during ethanol batch fermentation. Biotechnology and Bioengineering, 40(2), 289–297. Torija, M. J., Rozès, N., Poblet, M., Guillamón, J. M., & Mas, A. (2003). Effects of fermentation temperature on the strain population of Saccharomyces cerevisiae. International Journal of Food Microbiology, 80(1), 47–53. Yankov, D., Dobreva, E., Beschkov, V., & Emanuilova, E. (1986). Study of optimum conditions and kinetics of starch hydrolysis by means of thermostable aamylase. Enzyme and Microbial Technology, 8(11), 665–667. Zhu, S., Wu, Y., Yu, Z., Zhang, X., Wang, C., Yu, F., et al. (2005). Simultaneous saccharification and fermentation of microwave/alkali pre-treated rice straw to ethanol. Biosystems Engineering, 92(2), 229–235.