Renewable Energy 36 (2011) 503e509

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Renewable Energy
journal homepage: www.elsevier.com/locate/renene

Cane molasses fermentation for continuous ethanol production in an immobilized cells reactor by Saccharomyces cerevisiae
Farshid Ghorbani a, Habibollah Younesi a, *, Abbas Esmaeili Sari a, Ghasem Najafpour b
a b

Department of Environmental Science, Faculty of Natural Resources and Marine Sciences, Tarbiat Modares University, Noor, P.O. Box: 64414-356, Iran Department of Chemical Engineering, Faculty of Engineering, Noshirvani University of Technology, Babol, Iran

a r t i c l e i n f o
Article history: Received 27 May 2009 Accepted 26 July 2010 Available online 14 August 2010 Keywords: Ethanol Saccharomyces cerevisiae Cane molasses Ca-alginate Immobilized cell reactor

a b s t r a c t
Sodium-alginate immobilized yeast was employed to produce ethanol continuously using cane molasses as a carbon source in an immobilized cell reactor (ICR). The immobilization of Saccharomyces cerevisiae was performed by entrapment of the cell cultured media harvested at exponential growth phase (16 h) with 3% sodium alginate. During the initial stage of operation, the ICR was loaded with fresh beads of mean diameter of 5.01 mm. The ethanol production was affected by the concentration of the cane molasses (50, 100 and 150 g/l), dilution rates (0.064, 0.096, 0.144 and 0.192 h1) and hydraulic retention time (5.21, 6.94, 10.42 and 15.63 h) of the media. The pH of the feed medium was set at 4.5 and the fermentation was carried out at an ambient temperature. The maximum ethanol production, theoretical yield (YE/S), volumetric ethanol productivity (QP) and total sugar consumption was 19.15 g/l, 46.23%, 2.39 g l1 h1 and 96%, respectively. 2010 Elsevier Ltd. All rights reserved.

1. Introduction The use of solar energy in the form of biomass as renewable energy source is one solution, as fossil fuels are becoming depleted and the atmospheric pollution derived from combustion is becoming insupportable. The conversion of biomass into biofuels represents an important option for both the exploitation of an alternative source of energy and the reduction of polluting gases, such as CO2, NOx and SOx [1]. In the past microbial ethanol has been the focus of attention. However, agricultural wastes can be used and dependence on fossil fuels can be reduced, in particular molasses (by-product of sugar industry). Cane molasses is a low-cost source of sugar, and in contrast to other agricultural by-products, it does not require hydrolysis. Several microorganisms, including the wellknown yeast ethanol producer, Saccharomyces cerevisiae, Leuconostoc oenos and Zymomonas mobilis are suitable candidates to produce ethanol [2,3]. Various techniques for improving the production of ethanol, including continuous culturing, continuous fermentation by cell recycling, vacuum distillation by cell recycling [4] and immobilization of yeast cells [5] have been evaluated. Immobilization systems are intended to enclose the biocatalyst into a dened space in such a way that it retains its bio-activities and can be reutilized over a long period of time. Immobilization of yeast cells shows a number of advantages for industrial fermentation, such as the
* Corresponding author. Tel.: 98 0122 625 3101/2/3; fax: 98 0122 625 3499. E-mail addresses: hunesi@modares.ac.ir, hunesi@yahoo.com (H. Younesi). 0960-1481/$ e see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.renene.2010.07.016

relative ease of product separation, improved process control and reduced susceptibility of cells to contamination, an increase of yield and cellular stability and a decrease of procedural costs due to the ease of cell recovery and reutilization. Many methods namely adsorption, covalent bonding, cross-linking, entrapment, and encapsulation are widely used for immobilization. Among the different cell immobilization techniques, entrapment in calcium alginate gel has been one of the most used methods for whole cell entrapment due to its simplicity and non-toxic character [6]. Many studies were performed of continuous ethanol fermentation using Ca-alginate immobilized yeast cells in various bioreactor congurations [7e9]. In their studies, the bioreactors used were the uidized-bed bioreactor [7], continuous-ow stirred-tank bioreactor [8] and packed-bed bioreactor [9]. Packed-bed bioreactors are becoming more popular due to their low manufacturing cost, facility to operate at a low temperature and also the ease of the automation process in these reactors [10]. The purpose of the present study was to investigate the continuous process of ethanol production from sugar cane molasses in an ICR bioreactor. The effect of the initial concentration of the molasses and the hydraulic retention time (HRT) on the production of ethanol by S. cerevisiae were evaluated. The volumetric ethanol productivity and yield process parameters were examined to describe the consumption of molasses and the production of ethanol. Finally, the inner and the outer surfaces of the fresh and 25-day immobilized beads of yeast cells were scrutinized by means of scanning electronic microscopic (SEM) micrographs.

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2. Methods 2.1. Microorganism and culture media Fermentation was performed by S. cerevisiae (Persian Type Culture Collection, PTCC 5010) supplied from Research and Technology of Ministry of Sciences (Iran) in the form of freeze-dried culture. The culture was maintained on a sterilized solid Potato Dextrose Agar (PDA) medium in a 20 ml-test tubes and transferred to fresh medium every six months. The culture was incubated at 30  C for 1e3 days and stored at 4  C until use. Before starting the experiment, the microorganism was inoculated under sterile conditions into glass test tubes containing the same solid culture medium. These tubes were then kept in an incubator at 30  C for 16 h in order to obtain cells at the same growth stage for every experiment. The composition of the media was (in g/l): glucose, 15; (NH4)2SO4, 9; MgSO4$7H2O, 2.5; yeast extract, 1; KH2PO4, 10; K2HPO4, 5. The media was sterilized in an autoclave (Reyhan Teb, F2000, Iran) at 121  C and 1 atm for 20 min. The pH was maintained at 4.5 by addition of either 1 N NaOH or 1 N HCl when necessary. 2.2. Characteristic of the molasses The supply of cane molasses, a by-product of sugar cane industry, used in this study and provided by Bisetoon (Kermanshah, Iran), contained 51.50 0.23% sucrose, 4.05 0.08% fructose and 9.59 2.13% glucose, determined by using a high pressure liquid chromatograph (HPLC, Waters 600, 2414, USA). The Brix degree was determined by a hand-held refractometer (Atago, N-E3, Japan). The PO3 4 was measured by the photometric method (Palintest 8000, England). The cane molasses had an initial pH of 6.2. The moisture and ash contents of molasses were determined by the method detailed in standard method [11]. The protein and nitrogen contents of the molasses were determined by the Kjeldahl determination (2300 Kjettec Analyzer Unit, Foss Tecator, Sweden). The characteristics of the molasses are given in Table 1. The composition of C and N and (NH4)2SO4 was determined in order to compute the amount of the carbon and nitrogen source in the molasses, used as resource. The concentration of carbon from glucose, fructose and sucrose derived from the molasses in the medium was xed in the culture medium and the concentration of ammonium sulfate was computed considering the required C:N ratio as well as the composition of C and N in the molasses. In this study, the C/N ratio for the growth of S. cerevisiae in the seed culture was 3. However, the concentration of glucose, fructose and sucrose accumulated in 50, 100 and 150 g/l of molasses, respectively, for ethanol production by S. cerevisiae in ICR resulted in a C/N ratio of 10 in the culture media.

2.3. Cell immobilization Prior to the immobilization step, S. cerevisiae cells were grown at an ambient temperature for 16 h, and then the culture was harvested at the exponential phase and mixed with 3% Na-alginate. A 1000 ml aliquot of alginate-cell suspension containing 1.5% (w/w) Na-alginate was added drop wise to a 2% (w/v) bath of calcium chloride with a pipette of 5 mm in diameter. The alginate drops solidied upon contact with CaCl2, forming beads and thus entrapping yeast cells. The beads were allowed to harden for 30 min and then washed with distilled water to remove excess calcium ions and cells [9]. The beads were stored at 4  C in 0.2% (w/v) yeast and 0.2% (w/v) glucose solution until use. The average diameter of the beads was 5.01 0.23 mm. 2.4. Experimental bioreactor setup The immobilized cell reactor (ICR) was a tubular column, constructed with a nominal diameter of 5 cm, ID of 4.39 cm, Plexiglas of 90 cm length. The medium was fed to the ICR column from a feed stock located beside the column. A peristaltic pump (Heighdolf 5101, Germany) was used to transfer the feed medium from a 5-L conical ask that had served as a reservoir. The efuent from the column was collected in another 5-L conical ask which served as the product reservoir. A ow breaker was installed between the column and the pump, which prevented the growth of microorganisms and contamination of the feed line and feed tank. The samples for analyses were taken from the outlet compartment of column. About 70% of the ICR column was packed with the stored beads. The extra space was anticipated for bead expansion by the fresh media. The void volume of the column was measured by distilled water pumped through the bed. A packed-bed bioreactor was used in continuous mode for fermentation of the cane molasses. The fresh feed was pumped in an up-ow manner and the total sugar and ethanol concentrations were monitored during the course of continuous fermentation. The working volume of the reactor prior to packing was 1360 ml and the column volume after packing was 750 ml. The experimental setup of ICR is shown in Fig. 1. The hydraulic retention time (HRT) or dilution rate (D 1/HRT) of continuous culture system determines the substrate uptake efciency, specic growth of the microorganism and metabolic pathway [12,13]. The hydraulic retention time (q V/Q) for the feed in the reactor column with ow

Flow breaker

Sampling Port Vent

Sampling Port

Brix (degree) Density (g/l) Reducible sugar as dextrose, g/kg molasses Total sugar, g/kg molasses Glucose (%) Fructose (%) Sucrose (%) COD (g O2/l) BOD (g O2/l) Total phosphorous (mg/l) Protein (%) Total nitrogen (%) Dry matter (%) Volatile matter (%)

79 1360 25.5 836 9.59 2.13 4.05 0.08 51.50 0.23 120 70 150 0.145 0.0232 76.7 86.3

Immobilized Cell Reactor

Parameter value

Value

Pump

Valve
Vent Fresh Media Effluent Valve

Table 1 Characteristics of cane molasses used in the fermentation experiments.

Valve

Fig. 1. Experimental setup of immobilized cell reactor.

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rates of 0.8, 1.2, 1.8 and 2.4 ml/min were 15.63, 10.42, 6.94 and 5.21 h, respectively. The major overgrowth occurred at the entrance region, at about 25 cm length of the column where the sugar concentration was very high. A hypoxic condition has been achieved along the reactor length by the consumption of oxygen gas from the culture medium. The concentrations of 50, 100 and 150 g/l of molasses were used; the corresponding sugar concentration of 50, 100 and 150 g/l were measured to be about 30, 64 and 102 g/l, respectively. In addition to carbon source (molasses), the feeding medium consisting of (in g/l): (NH4)2SO4, 9; MgSO4$7H2O, 2.5; yeast extract, 1; KH2PO4, 10; K2HPO4, 5 was pumped from the feed stock, while the ow rate was being controlled by a peristaltic pump. 2.5. Liquid analysis The total sugar concentration was determined according to the phenolesulfuric acid method [14]. The phenolesulfuric acid solution was prepared by dissolving 20 g of phenol in 80 ml distilled water. To be exact, 1 ml of the sample was reacted with 1 ml of phenol solution and 7 ml of concentrated sulfuric acid. The mixture was allowed to stand for 30 min to cool down, and then the absorbance of each sample was measured at 420 nm using a spectrophotometer. The calibration curve was prepared with 2 g/l of sucrose solution. The initial and steady state concentration of the total sugar concentration in cane molasses was also measured based on the standard calibration curve as a function of the sucrose concentration. A gas chromatograph (Philips, PU4400, US), equipped with ame ionization detector (FID) and with software (Clarity 4.2, Data Apex, Czech Republic) was used to analyze the liquid samples. The column used was PEG 20 M (glass column) 1.5 m and 1/8 mm (Philips, USA). Temperature programming was employed for the liquid analysis in GC. During the analysis, the column temperature was initially maintained at 120  C, and after 2 min the oven temperature was increased at a rate of 10  C/min until it reached to 150  C. The injector and detector temperatures were 150 and 200  C, respectively. The ow rate for carrier gas (nitrogen) was set at 30 ml/min: 2-methyl-1-butanol (1%, v/v) was used as an internal standard with concentration of 50 ml/ml per sample. The injection sample volume was 2 ml. Each set of the experiment and the data points were repeated three times. The reported value was the mean. 2.6. Scanning electronic microscopic study

industry has high COD (120 g O2/l) and BOD (70 g O2/l). Also, the high total sugar content (836 g/kg) can cause eutrophication of aquatic ecosystems. Some of these problems may be overcome by the bioconversion of sugars to ethanol as a promising alternative, which would not only reduce the environmental impact of molasses [16], but it also represents an alternative way of producing ethanol as a valuable fuel resource [15]. Thus, due to the decrease of sugars (about 90%) in the fermentation process and biosorption of some nutrients to the yeast biomass [17], by utilizing them for ethanol production, we can prevent these contaminants from polluting nature. On the other hand, the yield of this process is clean fuel for transportation that will produce only CO2 and H2O during combustion. Moreover, the distillation of cane molasses for the production of ethanol generates a dark brown efuent, known as vinasse, which can be stored and easily used as recycling media for dilution of molasses. 3.1. System stability The immobilized cell reactor was conducted using different molasses concentration and HRT for 25 days. The pH of the feed medium was 4.5 and the reactor was located in ambient temperature. Within this period, the molasses consumption and ethanol production and immobilized cell capability were examined. The factors affecting the bead penetration were the Ca-alginate content, the ratio of cells to alginate and the size of the bead particles that inuence the hardness of the resulted beads and the pore size of the microlattice which were optimized in our previous study [9]. The amount of the bead particles when packed in a reactor, determination of activity, stability and the pressure drop have also been examined in our previous study [9]. However, phosphate buffer containing potassium cations was employed in the liquid media in order to reduce denaturing of the resulting beads and the rate of cell leakage. In comparison with the traditional batch fermentation, immobilized cells by entrapment in pertinent gel achieved considerably higher productivity due to high cell density and induced cellular immobilization. The economics of an immobilized cell process depends on the life time of the viable cells, mass transfer limitations and a continued level of clean product delivered by the xed cells. It is important to eliminate the free cells from the down stream product without the use of any units such as ltration or centrifuge processes [9]. 3.2. Molasses consumption and ethanol production

For electronic microscopic scanning (SEM) micrographs, samples were taken from fresh and 25-day beads from the ICR reactor. The samples were freeze-dried for 10 h in the Freeze Drier (Operon OPN-FDU-7012, Korea). The sample was xed on a stub and coated with gold palladium. Finally, the samples were observed under SEM at various magnications (Phillips XL30, Holland). 2.7. Statistical analysis Continuous fermentation experiments with different concentrations of molasses and HRT were carried out and then the mean value of samples taken every 8 h from the efuent were reported. The statistical analysis of the obtained data was carried out on a spreadsheet of commercial software (Microsoft Excel 2005). 3. Results and discussion The sugar cane industry generates a dark brown efuent known as molasses, with annual production of about 3 103 Mg in Iran. This efuent can create major environmental problems (groundwater contamination, strong smells, appearance of insects and other nuisances) [15]. Table 1 shows the efuent molasses from sugar cane

S. cerevisiae have different behavior for consumption of glucose, fructose and sucrose [18]. It appears that glucose and fructose have a similar course; the slight difference observed is that the fructose consumption is slower than the glucose consumption [18]. After 17 h of fermentation, start up of sucrose consumption and its consumption rate was slower and depended on initial sugar concentration. We also observed that the fructose level is lower than the glucose level and glucose level is lower than sucrose level in the cane molasses (Table 1). Different sugars consumption can cause limitation of ethanol production by cane molasses. The sucrose may not be consumed completely in all of the HRT. Fig. 2 shows mean values of total residual sugar concentration with respect to dened HRT for period of 25 days. The mean values for the concentration of total residual sugar increased with the increase of the HRT. The different trend of the experimental data was observed for the mean values of concentration of 50, 100 and 150 g/l of cane molasses in liquid media, while same trend was observed with the regression (Fig. 2). The corresponding initial total sugar concentration was 30 1.4, 64 2.1 and 101 1.7 g/l, respectively. However, there was a remarkable consumption of 96 2, 89 3 and 85 2.5% in initial concentration of total sugar

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Residual total sugar concentration, g/l

20

The Fig. 3 shows the mean values of ethanol produced with respect to HRT. The maximum ethanol production was obtained at 6.32, 12.55 and 19.15 g/l using a feed containing 50, 100 and 150 g/l of molasses, respectively, and at an HRT of 15.63 h (0.064 h1). This means that a stable continuous operation was possible even at high

15

10

50 g/l molasses 100 g/l molasses 150 g/l molasses Regresion

4 6 8 10 12 14 16 18

Hydraulic retention time, h

Fig. 2. Effect of initial molasses concentration in a continuous immobilized cells reactor by S. cerevisiae with different HRT.

with the increase in HRT. In the 50 and 100 g/l molasses, further increase in HRT from 10.42 to 15.36 h led to a decrease in residual total sugar concentration (Fig. 2). The molasses consumption at lower feed concentration showed to be more sensitive to HRT. However, further increase in total sugar concentration from 100 to 150 g/l led to an increase in residual total sugar concentration (Fig. 2). This might be due to homogeneous distribution of sugar concentration and higher transfer rate of sugar substrate resulting from high HRT (low dilution rate) at 150 g/l of total sugar concentration. This result indicates that high molasses concentrations at high HRT favor the metabolic ux through fermentative pathway. Interestingly, total sugar concentration at 150 g/l affected positively the ethanol production in the liquid media (Fig. 3). This seems that fermentations of high concentration of total sugar produced high levels of ethanol in the liquid media. On the other hand, better fermentation results can be obtained working with high concentration of total sugar in the inuent liquid medium and at high HRT. Such results are possibly due to high sugar concentration throughout immobilized cells reactor, which permitted a better mass transfer rate.

25
50 g/l molasses 100 g/l molasses 150 g/l molasses

20

Regresion

Ethanol concentration, g/l

15

10

0 4 6 8 10 12 14 16 18

Hydraulic retention time, h

Fig. 3. Effect of initial molasses concentration on ethanol production in a continuous immobilized cells reactor with different HRT. Fig. 4. Effect of different molasses concentrations in a continuous ethanol production by immobilized cells of S. cerevisiae with different HRT; (a) total sugar consumption, (b) ethanol production yield, and (c) volumetric ethanol productivity (QP).

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dilution rate in an immobilized cell bioreactor system, leading to maximum production of ethanol from cane molasses. In this experiment, the initial total sugar concentration was 63.66 g/l, of which 85.35% was shown to have been consumed. Finally, in 150 g/l molasses, the maximum ethanol produced was 19.15 g/l, which occurred at an HRT of 15.63 h (0.064 h1 dilution rate). This fact is attributed to an improvement of mass transfer between liquid medium and immobilized cells, thus leading to a conclusion that the use of Ca-alginate immobilized yeast cells at high HRT and high concentration of total sugar in the inuent liquid medium resulted in high ethanol concentration and high sugar consumption. In this experiment, the initial total sugar concentration was 101.4 g/l, which means that 89.93% of it was consumed. 3.3. Effect of HRT on yield and productivity Fig. 4a, b, and c shows that the total sugar consumption, ethanol production yield per total sugar and volumetric ethanol productivity (QP) were affected by different concentrations of molasses in the process of a continuous ethanol production by immobilized cells of S. cerevisiae with respect to varying of the HRT. The Fig. 4a shows that the increase in sugar concentration on molasses from 50 to 150 g/l was accompanied by linear decrease of HRT (increase of dilution rate). This indicated that the high concentration of molasses in the efuent liquid medium did not negatively affect the activities of the immobilized cells. It is to be noted that the high concentration of molasses affected negatively the growth of S. cerevisiae at low HRT (high dilution rate). This observation reveals that S. cerevisiae seems to be able to maintain its growth as the concentration of molasses increases and a higher portion of sugar is converted to ethanol production. The actual value of the theoretical yield (YE/S) of ethanol production from glucose is about 0.51 g/g. The ethanol product yield per total sugar (g/g) in all of the concentrations of molasses increased with the increase of HRT, and the maximum yield was about 0.23 g/g (Fig. 4b). The fermentative capacity of S. cerevisiae was conrmed in

terms of the theoretical yield value of ethanol. This value was obtained at 0.21 and 0.23 g/g for 50 and 150 g/l concentration of cane molasses, respectively, and at HRT 15.63 h. Finally, this was revealed that a high bioconversion of cane molasses under hypoxic conditions and high HRT could allow maximum continuous ethanol production in an immobilized cells reactor by use of S. cerevisiae. The volumetric ethanol productivity (g l1 h1) (QP) with respect to HRT and different concentrations of molasses are shown in Fig. 4c. The volumetric ethanol productivity (QP) increased with increase of the concentrations of molasses and HRT, its maximum being 2.39 g l1 h1. In the fermentation process, it is important that the capacity of the yeast cells should be supported against the toxic effects of the produced ethanol. The major overgrowth along 25 cm length at the entrance part of the column was due to the high sugar concentration and presence of oxygen gas. However, the ethanol productivity of S. cerevisiae was practically not affected by the high ethanol concentration. 3.4. Electronic microscopic scanning of immobilized cells Figs. 5 and 6 show electronic micrographs of outer and inner surfaces of the beads right after immobilization and on the 25th day (at 1500 h) of the fermentation process of ethanol. The outer surface of fresh beads at magnication of 448 and 2000 mm are shown in Fig. 5, and the outer surface of 25-day beads at magnication of 250 and 2000 mm are shown in Fig. 6. A visual comparison between them indicates that the cells on the surface grow without signicant contaminations. The inner surfaces of the beads before and after use were compared. The cells were initially entrapped inside the beads, and after 25 days the cells apparently migrated from the inner side to the surface. The inner surface of the fresh beads at magnication of 1000 and 5000 mm are shown in Fig. 5, and the inner surface of 25-day beads at magnication of 500 and 2000 mm are shown in Fig. 6. It seems that after 25 days, new colonies were present among the alginates layer formed.

Fig. 5. Electronic photomicroscope of the fresh beads: (a) outer surface of the fresh beads; (b) outer surface of the fresh beads; (c) inner surface of the fresh beads; (d) inner surface of the fresh beads.

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Fig. 6. Electronic photomicroscope of the used beads: (a) outer surface of the used beads after 25 days; (b) outer surface of the used beads after 25 days; (c) inner surface of the used beads after 25 days; (d) inner surface of the used beads after 25 days.

These micrographs were used as a comparative indicator of yeast growth on the surface and inside of the calcium alginate. The cell immobilization using alginate polysaccharide has an advantage due to cell immobilization and polymerization and good stability of the gel matrix under mild conditions at ambient temperature [19]. Fig. 5 shows the cell entrapment in the alginate matrix, which included cells within a rigid network. As a rule, this kind of gel should prevent the cells from diffusing into the liquid medium while still allowing penetration of the substrate. In this case, there was no apparent leakage of cells from the beads into the bulk of the liquid medium. It is also obvious that the yeast cells were grown on the outer and the inner surfaces of the Ca-alginate beads after the 25 day of operation (Fig. 6). The maintenance of high cell density without washout even at low HRT (high dilution rate) was a relevant advantage of the immobilized cell bioreactor. Therefore, the metabolically active cells were potentially available for ethanol production without diffusion problems. However, large amount of CO2 was produced in the ethanol fermentation where the bead surface may disintegrate due to CO2 pressure in the bioreactor bed. This phenomenon did not prevail due to the physiological conditions of the bead particles, i.e. due to the high strength of the gel particles, stability, porosity and the high cell density of the outer and the inner parts of the beads. 4. Conclusion Continuous ethanol production in an immobilized cell reactor (ICR), with sugar cane molasses as a low-cost source, was successfully carried out. In this system, the maximum ethanol production, the theoretical yield and volumetric productivity (QP) were 19.15 g/l, 46.23% and 2.39 g l1 h1, respectively. The total sugar consumption in the ICR column was between 85 and 96%. The results indicated that the immobilization of S. cerevisiae possesses the capacity not only to utilize a high concentration of sugar but also to yield higher ethanol productivities during the course of

continuous fermentation. The difference between the fresh and the 25-day Ca-alginate immobilized cell was shown by scanning electronic micrographs of beads taken from their outer and inner surfaces. Acknowledgements The present research was made possible through a university grant, sponsored by the Ministry of Science, Iran and Tarbiat Modares University (TMU). The authors wish to thank Mrs. Haghdoust (Technical assistant of Environmental Libratory) for her assistance and Ellen Vuosalo Tavakoli (University of Mazandaran) for nal editing of the English text. References
[1] Belgiorno V, De Feo G, Della Rocca C, Napoli RMA. Energy from gasication of solid wastes. Waste Manag 2003;23(1):1e15. [2] Sitton OC, Gaddy JL. Ethanol production in an immobilized-cell reactor. Biotechnol Bioeng 1980;22(8):1735e48. [3] Spettoli P, Bottacin A, Nuti MP, Zamorani A. Immobilization of Leuconostoc oenos Ml 34 in calcium alginate gels and its application to wine technology. Am J Enol Vitic 1982;33(1):1e5. [4] Gerald RC, Charles RW. Process design and economic studies of alternative fermentation methods for the production of ethanol. Biotechnol Bioeng 1978;20(9):1421e44. [5] Kolot FB. New trends in yeast technology: immobilized cells. Process Biochem 1980;15:2e8. [6] Alan R. Immobilised biocatalysts: a critical review. J Chem Technol Biotechnol 1984;34(3):127e50. [7] Bravo P, Gonzalez G. Continuous ethanol fermentation by immobilized yeast cells in a uidized-bed reactor. J Chem Technol Biotechnol 1991;52(1):127e34. [8] Matteau PP. Enhanced performance of immobilized bioreactors using variable surface bioreactors, In: International conference on bioreactors and biotransformations, Scotland; 1987. [9] Najafpour G, Younesi H, Ku Ismail KS. Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae. Bioresour Technol 2004;92 (3):251e60. [10] Bdalo-Santoyo A, Gmez-Carrasco JL, Gmez-Gmez E, Bastida-Rodriquez J, Mximo-Martn MF, Hidalgo-Montesinos AM. Production of optically pure L-valine

F. Ghorbani et al. / Renewable Energy 36 (2011) 503e509 in uidized and packed bed reactors with immobilized L-aminoacylase. J Chem Technol Biotechnol 1999;74(5):403e8. American Public Health Association (APHA). Standard methods for the examination of water and wastewater. 20th ed. Washington, DC: APHA; 1999. Judd S, Stephenson T. Process science and engineering for water and wastewater treatment. 1st ed. Padstow, Cornwall: IWA; 2002. Kim S-H, Han S-K, Shin H- S. Optimization of continuous hydrogen fermentation of food waste as a function of solids retention time independent of hydraulic retention time. Process Biochem 2008;43(2):213e8. DuBois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956;28 (3):350e6.

509

[11] [12] [13]

[14]

[15] Nissen TL, Kielland-Brandt MC, Nielsen J, Villadsen J. Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation. Metab Eng 2000;2(1):69e77. [16] Madejn E, Lpez R, Murillo JM, Cabrera F. Agricultural use of three (sugar-beet) vinasse composts: effect on crops and chemical properties of a Cambisol soil in the Guadalquivir river valley (SW Spain). Agric Ecosyst Environ 2001;84(1):55e65. [17] Han R, Li H, Li Y, Zhang J, Xiao H, Shi J. Biosorption of copper and lead ions by waste beer yeast. J Hazard Mater 2006;137(3):1569e76. [18] Oranut Phowchinda PS. Utilization of mixed sugars for alcoholic fermentation by Saccharomyces cerevisiae. Thammasat Int J Sci Tech 1999;4(2):23e31. [19] Johann R. Cell trapping in microuidic chips. Anal Bioanal Chem 2006;385 (3):408e12.