Bioresource Technology 115 (2012) 208214

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Bioresource Technology
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Scaling up of ethanol production from sugar molasses using yeast immobilized with alginate-based MCM-41 mesoporous zeolite composite carrier
Chunming Zheng a,b, Xiaohong Sun b, Landong Li b, Naijia Guan b,
a State Key Laboratory of Hollow-ber Membrane Materials and Membrane Processes, School of Environmental and Chemical Engineering, Tianjin Polytechnic University, Tianjin 300387, PR China b Key Laboratory of Advanced Energy Materials, Ministry of Education, School of Chemistry, Nankai University, Tianjin 300071, PR China

a r t i c l e

i n f o

a b s t r a c t
Microporous and mesoporous zeolites, including ZSM-5, H-b, H-Y, and MCM-41, were modied with 3-aminopropyl-triethoxysilane (APTES), then inorganic llers, such as abovementioned zeolites or mesoporous materials, (a-AlOOH or c-Al2O3), were mixed with alginate embedded with yeast; and nally these carriers were cross-linked through the double oxirane. The alginate-based immobilized yeast with MCM-41 exhibited much shorter fermentation time and higher ethanol concentration than pure alginate and other composite carriers with the highest cell concentration of 4.8 109 cells/mL. The composite carrier maintains the highest ethanol productivity of 6.55 g/L/h for 60 days in continuous fermentation process, implying good operational durability for commercial applications. The reason for the higher bio-catalytical function of the immobilized yeast might lay in the uniformly yeast distribution in the bio-reactor and high yeast cell concentration, which contributed by the improved transmission of fermentation media and combined effects of yeast adsorption by MCM-41 and embedment by alginate. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 23 May 2011 Received in revised form 14 November 2011 Accepted 16 November 2011 Available online 25 November 2011 Keywords: Ethanol Immobilization Saccharomyces cerevisiae Alginate MCM-41 zeolite

1. Introduction The oil crisis of the last 30 years has focused research conducted in the scientic area of ethanol fermentation primarily toward increasing the ethanol productivity of bioprocesses and reduction of energy demands. In addition to providing a solution to the environmental issue arising from the disposal of sugar molasses, the production of ethanol as a fuel can also help stabilize the agricultural sector in sugar-producing countries (Kopsahelis et al., 2007; Thomas and Kwong, 2001). The fermentation of sugar molasses has continued to enhance ethanol productivity (Nahvi et al., 2002). To increase ethanol productivity and to reduce labor intensity, aspects such as bioreactor volume, energy consumption in the production of ethanol, and cell immobilization for ethanol production have been extensively studied and reviewed (Kopsahelis et al., 2009; Kourkoutas et al., 2004; Marignetti et al., 1997). Various adjuncts have been proposed by different researchers for yeast and bacteria immobilization for application in ethanol fermentation from various raw materials. These research efforts included the use of inorganic and organic adjuncts in batch and continuous processes, including organic materials such as calcium alginate, polyvinyl alcohol, and polyacrylamide (Kourkoutas et al., 2004) and inorganic materials such as alumina (Loukatos et al., 2000), silica (Gill and Ballesteros, 1998) and zeolite (Hartmann, 2005;
Corresponding author. Tel./fax: +86 022 23500319.
E-mail address: guannj@nankai.edu.cn (N. Guan). 0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.11.056

Kourkoutas et al., 2004). The reduction of costs involved in ethanol production from bioprocesses employing immobilized cells systems are associated with aspects such as the cost of raw materials, the use of cheap, abundant and stable immobilization adjuncts, the high cell concentration in the bioreactors, the simplicity and low cost of immobilization techniques, the stability of the immobilized biocatalyst in its operational state, ease of regeneration and the design and development of a suitable bioreactor system (Kourkoutas et al., 2004; Wang et al., 2011). Alginate, as a carrier, has a high immobilized yeast concentration; however, its biochemical stability and mechanical strength is poor, and, therefore, the industrial application of organic materials is restricted. The MCM-41, a type of ordered mesoporous inorganic materials, has been applied successfully as a carrier in immobilizing cells processes (Tope et al., 2001). It has good mechanical property, permeability and renewability, all of which increase the mechanical strength of immobilized yeast, and enhance the transportation of fermentation products; however, it has low immobilized yeast concentration for relying on weak physical or chemical adsorption with microorganisms. Therefore, the preparation of organicinorganic composite materials for yeast immobilization appears very attractive as a prospective method because it combines features of organic and inorganic materials. These organicinorganic carriers not only utilize their good embedded afnity to organic materials and fast propagation of yeast cells, but also possess good mechanical properties and permeability to inorganic materials; these traits prolong the operating life of carriers, and effectively improve the

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fermentation efciency of immobilized yeast in production of ethanol as a fuel. The published literature (Chen et al., 2007) reports mesopores and macropores as being among carriers those are vital to the high density proliferation of the yeast as well as transportation of substances and products between carriers and the medium. In this study, micro- and mesoporous zeolites such as ZSM-5, H-b, H-Y, and MCM-41 were modied with 3-aminopropyl-triethoxysilane (APTES); inorganic llers, including the abovementioned zeolites and other mesoporous materials such as a-AlOOH and c-Al2O3, were mixed with alginate embedded with yeast and then cross-linked through the double oxirane. The alginate-based immobilized yeast with MCM-41 has the highest biology capacity of 4.8 109 cells/mL, and can be utilized repeatedly in batch and continuous fermentation applications. The aim of this study was to evaluate and compare the suitability of the alginate-based MCM-41 composite carrier, for utilization in yeast immobilization when applied in ethanol fermentation of sugar molasses, with regard to operational stability, ethanol productivity, and costeffectiveness of the proposed process. Validated by the scanning electron microscopy (SEM) images and associated fermentation experiments, the possible mechanism underlying the higher biocatalytic function was also studied. 2. Methods 2.1. Microorganism and media Dry yeast cells (Saccharomyces cerevisiae, provided by the Danbaoli Brewery, Guangdong, PR China) were initially cultivated in a sterile growth medium at 30 C in shaken asks. Cells were harvested in the early exponential phase by centrifugation at 8000 rpm. The chemical reagents utilized in this study include sodium alginate, sucrose, glucose, calcium chloride, monopotassium phosphate, ammonium sulfate, magnesium sulfate and 3-aminopropyl-triethoxysilane (APTES), a cationic surfactant. All chemical reagents were of analytical grade and were obtained from SigmaAldrich (China). ZSM-5 (SiO2:Al2O3 = 100), H-b (SiO2: Al2O3 = 12.5), and H-Y (SiO2:Al2O3 = 3) were provided by the Fuchen Catalyst Co. Ltd., Tianjin. Pseudo-boehmite (short for a-AlOOH, average pore diameter of 10 nm, BET surface area is 380 m2/g) and c-Al2O3 (average pore diameter of 4 nm and BET surface area of 313 m2/g) were provided by Tianjin Chemical Research and Design Institute. Sugar molasses were obtained from the Yunxin Sugar Factory (Yunnan, China). The growth medium for immobilized yeast comprised sucrose 150 g/L, (NH4)2SO4 10 g/L, KH2PO4 10 g/L, MgSO4 5 g/L, and yeast extract 10 g/L (pH 4.8). The fermentation medium comprised molasses of 12 Bx or 30 Brix densities (corresponding to sugar content of approximately 70 or 170 g/L, respectively), (NH4)2SO4 10 g/L, and KH2PO4 10 g/L (pH 5.6). All media were sterilized by autoclaving at 116 C for 20 min prior to use. 2.2. Reactor and immobilization Taken hexadecylpyridinium bromide as a template agent, MCM-41 zeolite was synthesized in HCl medium, and the molar ratio was TEOS:CPTE:CPBr:HCl:H2O = 1:0.1:0.3:6:120. The mixture was crystallized at a temperature of 50 C for 24 h, and following ltration, the solid was reuxed in EtOH/HCl (150 mL/g solid) for 2 h. This process was repeated 3 times, and the solid was vacuumly dried at 80 C for 12 h; the product obtained was designated as MCM-41 zeolite. To functionalize micro- and mesoporous zeolites such as ZSM-5, H-b, H-Y and MCM-41 with an amino-functional group, 5 g of a single type of zeolite, any of ZSM-5, H-b, H-Y or MCM-41, was heated

for 3 h at 250 C in vacuum. This calcined and activated solid was placed in 200 mL of anhydrous toluene and stirred for 30 min; then, a suspension of APTES (4.56 g) and calcined zeolite was heated to reux with stirring in an inert atmosphere for 24 h. After cooling to 2530 C and ltering with dry toluene and diethyl ether, the resulting mass was subjected to Soxhlet extraction with dry dichloromethane for 24 h. Finally, the resulting solids were dried at 5055 C in vacuum for 8 h. This processed zeolite was marked as H2NZSM-5, H2NH-b, H2NH-Y or H2NMCM-41, separately, based on the type of zeolite used initially. Then 2 g of alginate was dissolved in 100 mL of sterile water in which 1.2 g of one type of inorganic ller had been added. The mixture was stirred and blended with 4 g of dry yeast cells, which were initially cultivated over a sterile growth medium at 30 C in shaken asks. Sterile water was added to the mixture to make the volume up to 200 mL. Then, 0.01% of the total volume of double oxirane was added for cross-linking, and the mixture thus obtained was trickled down into 4% calcium chloride solution with continuous stirring (80 rpm) for 4 h to induce granulation. The immobilized yeast of alginate-based H2NMCM-41 mesoporous zeolite compound carrier was marked by the abbreviated term alginate-based MCM-41 immobilized yeast. Immobilized yeasts prepared from other inorganic llers were denoted by the same method.

2.3. Pre-cultivation and ethanol production from sugar molasses by various methods The growth of immobilized yeast was investigated by cultivating yeast cells in 500 mL shaken asks, which contained 200 mL of growth medium and 25 mL of either immobilized yeast or yeast suspension (control sample), on an orbital shaker at 115 rpm and a temperature of 30 C. The abovementioned systems were allowed to ferment for 1050 h until the density of the fermented liquids was reduced to a stable value (00.5 Bx). The liquids were then washed twice with sterilized molasses medium and were used for repeated fermentation batches. The initial overall cell concentration in the free cells system was approximately 1 107 cells/ mL. The immobilized yeast and medium were sampled at timed intervals, and analyzed for cell viability and concentration. A series of batch fermentations was performed in 500 mL asks, which contained 200 mL of fermentation medium (12 Bx) and 25 mL of immobilized yeast with various carriers, on an orbital shaker at 115 rpm and 30 C for 24 days each. The fermented medium was decanted at the end of each batch, and the carriers were washed with fresh molasses medium; then, a fresh medium was added for the next fermentation batch. Samples of the fermented medium were collected and analyzed for ethanol and residual sugar. The initial concentration of each kind of immobilized yeast was approximately 2 108 cells/mL. These experiments were carried out in triplicate. Continuous fermentation was performed in a water-jacketed bioreactor with a working volume of 30 L (including the inoculum). The bioreactor comprised of a plug ow plexiglass tubular column (300 mm i.d., 800 mm height, 5 mm wall thickness) and contained 3.6 L of alginate-based MCM-41 immobilized yeast and 26.4 L of fermentation medium without nutrients (packing ratio is 12 v/ v%). The above medium was continuously fed into the lower unit of the bioreactor and fermented at 30 C. The exhaust gas and efuent solution were removed via an outlet located at the top of the bioreactor. Oxygen was introduced through a glass sparger at the bottom of the reactor every 5 min. The ethanol productivity was calculated as the grams of ethanol per liter of liquid volume produced per hour (g/L/h). The initial concentration of alginate-based MCM-41 immobilized yeast was 2 108 cells/mL.

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2.4. Analytical methods The size of immobilized cells with various carriers was estimated using a microscope with an accuracy of 10 mm. Cell concentrations and viabilities of immobilized yeast were determined after dissolution by heating and mixing by means of methylene blue staining technique. Immobilized yeast (10 g each) were blended with 90 mL of quartet strength sterilized ringer solution and submitted to serial dilutions; thereafter, yeast cells were counted by plating on Malt Agar Plates and incubation after 48 h at 30 C under aerobic conditions (Collins et al., 1989). Liquid samples from both the growth and fermentation media were collected aseptically and analyzed for specic gravity by a method described previously (Bezbradica et al., 2007; Nedovic et al., 2001). 2.5. Characterization of materials The alginate-based MCM-41 carriers and immobilized cells were washed with deionized water and soaked in 3.5% glutaraldehyde for 6 h, then dried by treatment with 50%, 70%, 90%, 95% and 100% ethanol, followed by overnight retention of the samples in a desiccator for the removal of moisture. The samples were coated with gold and examined in a Shimadzu SS-550 (Shimadzu, Japan) eld emission scanning electron microscope. 3. Results and discussion 3.1. Selection of suitable porous inorganic llers used in the composite carriers for yeast immobilization In this study, yeast cells were immobilized with the carriers modied by various inorganic llers. The parameters of the carriers with various inorganic llers, which are crucial in the selection of suitable carriers for yeast immobilization and fermentation of sugar molasses, are summarized in Table 1. Composite carriers with three microporous inorganic llers, i.e. H2NZSM-5, H2NH-b, and H2NH-Y, were observed to get low cell densities, and the fermentation time was relatively long with these microporous inorganic llers. With the other three mesoporous inorganic llers, i.e. aAlOOH, c-Al2O3, and H2NMCM-41, composite carriers were found to achieve high cell densities for immobilizing yeast cells. Further, these carriers with mesoporous inorganic llers had a much shorter fermentation time and better ethanol concentration in the batch fermentation containing the sugar molasses fermentation medium. The reason for the different fermentation results could be attributed to the diversity of pore microstructure of inorganic llers (Hartmann, 2005) and volume content of the composite carriers (Bottcher et al., 2004). The size and distribution of the mesoscopic pores determine the velocity of mass transport. One necessary precondition for germination or colonization of microorganisms in

immobilization processes is sufcient macroscopic porosity (Rooke et al., 2008a). Similarly, the volume content of the biocomponent is a relevant parameter. The embedded cells are mutually separated in pure alginate with small volume fractions; however, they come into contact at higher concentrations to interact and eventually form colonies in composite carriers with the improved transmission of fermentation media (Plessas et al., 2007). From the fermentation results of immobilized yeast with above inorganic llers, the H2NMCM-41 was observed to be mechanically stable and had good fermentation effects. Therefore, H2NMCM-41 was selected as the immobilizing material, based on its good properties, for further experiments.

3.2. Characterization of immobilized yeast of H2NMCM-41, alginate, and alginate-based MCM-41 composite carrier To study the proliferation mechanism of yeast immobilized in alginate-based MCM-41 composite carrier, the yeast cell concentration and surviving fraction of the immobilized yeast was compared with pure H2NMCM-41 and alginate (Table 2). From Table 2, a maximum cell concentration of 4.8 109 cells/mL was obtained by using the composite carrier. The surviving fraction of yeast in the composite carrier (84%) was also much higher than that for H2NMCM-41. To further investigate the growth and proliferation of yeast cells immobilized in the different carriers, pure alginate, H2NMCM-41 and alginate-based MCM-41 composite carriers were characterized by SEM technology (gures not shown). From SEM investigation of three immobilized yeast cells, large numbers of yeast cells were observed to proliferate in alginatebased MCM-41 composite carrier; the concentration of yeast was much higher than that in the H2NMCM-41 and pure alginate carrier. The H2NMCM-41 carrier was identied to possess some yeast cells, which were adsorbed on the carrier conrmed by SEM characterization; more numbers of yeast cells were observed to be embedded in pure alginate (Wang et al., 2011). SEM characterization also conrmed that large numbers of yeast cells were adherent on all sides of the composite carrier; meanwhile, the vacuum of the composite carrier was lled with yeast cells as a result of either natural entrapment into the porous material of composite carrier or physical adsorption by electrostatic forces or covalent binding between the cell membrane and the carrier (Tope et al., 2001). Thus, the combined effects of adsorption of pure H2NMCM-41 zeolite and investment of pure alginate contributed to the maximum cell concentration and productivity (Hartmann, 2005). The alginate-based MCM-41 composite carrier is vacuous, porous and has good cell-retention capacity. The advantages of the composite carrier as an yeast cell carrier include: (1) combined features of organic materials that include good embedded afnity, fast propagation of yeast cells and of inorganic materials, which includes good mechanical properties; these properties prolong the operating life of carriers and contributed to the maximum cell concentration and productivity. (2) For there are a lot of pores within the composite carrier conrmed by SEM characterization (gures not shown); this makes the composite carrier porous and

Table 1 Fermentation activity of composite carriers with different inorganic llers. Inorganic llers None H2NZSM5 H2NH-b H2NHY a-AlOOH c-Al2O3 H2NMCM41 Initial sugar (g/L) 172 170 170 170 168 170 170 Ferm. time (h) 42 28 37 16 18 30 12 Residual sugar (g/L) 28.6 19.5 28.3 15.2 13.6 22 12.5 Ethanol (g/L) 70.4 73.6 67.8 76.9 75.2 73.8 78.6 Conversion (%) 79.0 83.5 76.9 86.5 90.4 83.8 89.2

Table 2 Effect of carriers on the immobilized yeast cells concentration and surviving fraction. Carrier H2NMCM41 Immobilized cell concentration (108 cells/mL) Surviving fraction (%) 0.8 67 Alginate 26 86 Alginate-based MCM-41 48 84

Fermentation conditions: sugar molasses 30 Bx, fermentation temperature 32 C, pH 4.0, packing ratio 12%.

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facilitates the transmission of substrates and products between carriers and medium more easily. Both of these properties could solve the problem of mass transfer which occurs in the immobilization systems; thus, they effectively increase operational stability, ethanol productivity, and also decrease the cost of the proposed ethanol fermentation process (Rooke et al., 2008b).

3.3. The kinetics of immobilization yeast of alginate-based MCM-41 composite carrier Effects of immobilization procedure on viability and proliferation potential of yeast cells were studied in comparative cultivations of cells immobilized in alginate-based MCM-41 composite carrier and freely suspended cells. Kinetics of cell concentrations in the alginate-based MCM-41 composite carrier and those released in the medium are presented in Fig. 1(a). Released cells detected in the medium only after 18 h of cultivation, which a lag phase of 20 h and an exponential growth phase of 44 h with a specic growth rate of 0.39 h1 can be observed on the growth curve obtained for immobilized cells. The results coincide with the commencement of intensive proliferation of immobilized cells. The combined effects of cell proliferation in the medium and cell leakage from the carriers could be conducted since the apparent specic growth rate of cell concentration in the medium was 0.73 h1. And the tolerance of immobilized cells to inhibitor metabolites was higher than free yeast cells for the exponential growth phase of immobilized cells was almost double that of released cells (44 vs. 22 h). The comparison of the kinetics of overall cell concentration in the immobilized system (in carriers and medium) and in the free cell suspension are shown in Fig. 1(b). The lag and exponential growth phases of yeast cells in immobilized system were signicantly longer than these of free cells (18 and 39 h vs. 6 and 22 h, respectively). And the yeast cells in immobilized system also exhibited dramatically slower apparent specic growth rate than free cells (0.55 vs. 0.49 h1, respectively). For the prolonged, although slower, growth in the immobilized system, the nal overall cell concentration in the immobilized system was slightly higher than that in the free cell suspension. The concentration of immobilized cells increased rapidly during the fermentation time; after this, the concentration appeared to decrease more slowly than that of free cells. This could be attributed to the difference in sugar concentration between the fermentation medium and the inner aspect of the composite carrier (Yu et al., 2007).

Therefore, the cell viability and growth were greatly affected with alginate-based MCM-41 immobilizing process. Similar study also reported a prolonged lag phase for yeast cells entrapped in polymer matrices (Bezbradica et al., 2007). However, the apparent specic growth rate of cells released in the medium was comparable to that of free cells, this results indicates cell physiology was preserved in the present work. In addition, the nal cell concentration achieved in the alginate-based MCM-41 immobilized yeast was much higher than the range of previously reported concentrations of yeast in MCM-41 or alginate. Following replacement of sugar molasses medium by a fresh one, the yeast cell concentration remained constant thereafter. This was considered the steady state and alginate-based MCM-41 immobilized yeast could be utilized for further fermentation studies.

3.4. The comparison of the fermentation kinetics between immobilized yeast embedded by different composite carriers and free cells system Immobilized cell technology has several advantages over conventional systems. One advantage is that cell immobilization increases fermentation productivity to two or three times of that seen conventionally (Najafpour et al., 2004) by substantially increasing the population density. As is shown in Fig. 2, experiments were carried out with the same initial sugar concentration of 30 Bx (170 g/L). Eighty three percentage of the total sugar was consumed within 42 h and the ethanol productivity was 1.68 g/L/h in the free cells system. The ethanol concentration and yield were 70.4 g/L and 0.49 g of ethanol per gram of consumed sugar, respectively. In sharp contrast, 92.6% of the total sugar was consumed in the immobilized cells system within 12 h and an ethanol productivity of 6.55 g/L/h, which is 3.90 times higher than that of the free cells system, was obtained. The ethanol concentration and yield were 78.6 g/L and 0.50 g of ethanol per gram of consumed sugar, respectively. In general, the ethanol yield by alginate-based MCM-41 composite carrier for immobilized yeast is approximately 95% of the theoretical value, demonstrating the efciency of the biocatalyst for ethanol fermentation. With the assistance of adsorption and covalent binding of MCM-41 zeolite (Tope et al., 2001) and embedding of alginate provided to yeast cells, cells in the alginate-based MCM-41 composite carrier grew even better than those in pure carriers, especially the concentration of yeast cells. Therefore, the fermentation time of composite carrier system appears to be the shortest, and the ethanol productivity increased substantially (Fig. 2). Meanwhile, the regular

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Fig. 1. Growth curves for cells in alginate-based MCM-41 immobilized yeast system, and free cells: (a) cell concentration in immobilized carrier and released in growth medium of alginate-based MCM-41immobilized yeast system; (b) comparison of total cell concentration (cells in immobilized carrier + cells released in growth medium) in alginate-based MCM-41 immobilized yeast system, and free cells.

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and, therefore, greater survival and productivity in subsequent cycles as compared with free yeast cells (Krisch and Szajani, 1997). In addition, the carrier remained intact after experimental runs, thus conrming its chemical and mechanical stability (Ogbonna et al., 2001).

3.6. Continuous ethanol fermentation in a lled-bed reactor with alginate-based MCM-41 immobilized yeast Continuous ethanol fermentation using an abovementioned lled-bed reactor with fermentation medium under the following conditions: void volume ratio of carriers of 12%, temperature at 30 C, and dilution rates of 0.063, 0.083, 0.100, and 0.125 h1; a start-up procedure was required to establish a steady-state phase. Initially, the medium was fed into the lled-bed reactor at the dilution rate of 0.063 h1. After 4 days, a steady state was achieved. The average ethanol concentration of the efuent was 74.1 g/L. At a regular interval of 10 days, the dilution rate was changed to 0.083, 0.100, and 0.125 h1 and the average ethanol concentration of the efuent decreased to 70.3, 63.2, and 41.9 g/L, respectively. As shown in Fig. 4(a), the system was maintained for 40 days and the alginate-based MCM-41 immobilized yeast was not damaged during the course of the entire experiment. Under steady-state conditions, the biomass concentration leaving the reactor remained nearly constant at a given dilution rate and decreased slightly with increase in the dilution rates. In addition, the immobilized cells continued to grow such that the overall cell concentration in the reactor increased during repeated fermentations. Reactor ethanol productivity (based on valid volume), efuent ethanol concentration and residual sugar concentration are shown as a function of dilution rate in Fig. 4(b). The maximum ethanol productivity of 6.32 g/L/h was obtained at a dilution rate of 0.100 h1. However, this was at the substantial expense of unfermented residual sugar in the efuent. Complete utilization of the total sugar was obtained at a dilution rate of 0.056 h1 with the maximum ethanol concentration of 75.1 g/L. Ethanol productivity decreased after the dilution rate reached approximately 0.100 h1. During the continuous ethanol fermentation, cell leakage was especially prominent during intensive formation of carbon dioxide, probably due to the creation of pores in the carrier. However, bursts of carbon dioxide, which are inevitable during the brewing process, do not impair the alginate-based MCM-41 composite carrier structure extensively, since the overall cell concentration in the particles was not affected by the cell release. Larger volumes of CO2 could be produced when increasing the initial sugar concentrations, and high pressure caused by produced CO2 was overcome by using reactors with a special conguration (Converti et al., 1987; Hamamci and

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Fig. 2. The comparison of the fermentation kinetics between immobilized cells, which were immobilized by H2NMCM-41, alginate, and alginate-based MCM-41 composite carriers, and free cells.

aperture and the high specic surface area of MCM-41 ensures that the transmission of substrates and products among the yeast cells is carried out more easily, these features may have facilitated mass transportation and may be responsible for maximum cell immobilization (Rooke et al., 2008a). Both these properties could solve the problem of mass transfer that is usually seen in the immobilization systems. 3.5. Reuse of immobilized cells in a batch process for production of ethanol Twenty-ve milliliters alginate-based MCM-41 immobilized yeast was incubated in 200 mL of fermentation medium at 30 C and fermented with abovementioned methods in Section 2.3. The batch fermentation results are depicted in Fig. 3. In this system, the overall ethanol concentration remained nearly constant at 75 g/L during 30 repeated batch fermentations, and, thereafter, increased to 78 g/L. The immobilized cells could be reused for at least 60 days and retained approximately 95% of its original activity (Fig. 3 illustrates the rst 30 batches). The alginate-based MCM41 immobilized yeast retained their activity for more than 2 months. High rates of biomass formation exhibited by the immobilized yeast could be crucial for batch mode processes that provide a stable source of yeast supply and improved proliferating condition beyond the free yeast cells system. This could be due to the fact that immobilized yeast contain signicantly higher percentages of saturated fatty acids as compared with free yeast cells; this leads to greater ethanol tolerance in the immobilized yeast,

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Fig. 4. Continuous ethanol fermentation in xed-bed reactor with immobilized yeast embedded by alginate-based MCM-41 composite carrier: (a) fermentation results of immobilized yeast in alginate-based MCM-41 composite carrier in 40 days; (b) effect of dilution rate on reactor productivity and ethanol concentration of immobilized yeast in alginate-based MCM-41 composite carrier.

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Fig. 5. Pilot-scale continuous ethanol fermentation in xed-bed reactor with immobilized yeast embedded by alginate-based MCM-41 composite carrier: (a) pilot-scale fermentation results of immobilized yeast in alginate-based MCM-41 composite carrier in 40 days; (b) pilot-scale effect of dilution rate on reactor productivity and ethanol concentration of immobilized yeast in alginate-based MCM-41 composite carrier.

Ryu, 1987). In the case of immobilized cells during the fermentation process, no contamination of the substrate by other microorganisms occurred. This may be attributed to the high amount of the inoculum dominating the existing microora, and the ethanol produced inhibited the growth of contaminating microorganisms. The cells growing free in the medium exhibited higher growth rates since the growth medium was readily accessible. Cell growth and release rates conrm the potential of alginate-based MCM-41 composite carrier as a permanent source of cells that allows stable continuous operation. 3.7. Pilot-scale continuous ethanol fermentation in a lled-bed reactor with alginate-based MCM-41 immobilized yeast To compare the fermentation results between different scale with alginate-based MCM-41 immobilized yeast, pilot-scale continuous ethanol fermentation was carried out using a 10 m3 lled-bed reactor with the fermentation conditions aforementioned in Section 2.3 (Fig. 5). From Fig. 5(a), a start-up procedure was also required to establish a steady-state phase as 30 L fermentation process. The ethanol production rate in the early and stable phase of fermentation was relatively same as 30 L small-scale results. After 4 days, a steady state was achieved and the average

concentration of ethanol was 72.3 g/L. At every 10-day interval, the dilution rate was changed to 0.083, 0.100, and 0.125 h1 and the average ethanol concentration of the efuent decreased to 68.1, 66.7, and 35.7 g/L, respectively. The ethanol yields from the consumed sugar and ethanol productivities in the 30 L and 10 m3 bioreactors were nearly the same. Therefore, there were no signicant differences in the data obtained with the two different bioreactor scales. This indicates that, if the immobilized cells are uniformly distributed in the packed bed, the data obtained for the experiment in a small bioreactor can be reproduced when the process is scaled up. By applying the method described here, immobilization of cells uniformly in a larger scale reactor can also be achieved. The key to enlarge these fermentation results would involve uniform distribution of immobilized cells among the upper, middle and lower beds. The more uniform cell distribution obtained with alginate-based MCM-41 immobilized yeast is probably due to better mixing within the bioreactor (mainly by oxygen was introduced through a sparger at the bottom of the reactor at time intervals of 5 min). Optimal cell circulation between the top and bottom of the bed is necessary for efcient and uniform cell immobilization when the alginate-based MCM-41 immobilized yeast bed is very high. The reason might be that the mixing time was shorter while the volumetric oxygen

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transfer coefcient (Hideno et al., 2007) was higher when the immobilized yeast was used for bed construction. Thus, alginatebased MCM-41 immobilized yeast is preferred for construction of beds in large scale bioreactors. The fermentations results achieved for sugar molasses medium and cell proliferation imply that the approach based on the use of alginate-based MCM-41 immobilization yeast can provide operation with efcient mass transport without signicant internal and external mass transfer limitations. Another noteworthy point was that no nutrients, such as KH2PO4, urea, penicillin, or (NH4)2SO4, were added in the molasses solutions as reported in other studies (Bakoyianis and Koutinas, 1996; Nahvi et al., 2002). 4. Conclusions In the present study, alginate-based micro- and mesoporous composite materials, such as zeolites (including ZSM-5, H-b, H-Y, and MCM-41) modied by APTES, a-AlOOH and c-Al2O3, as carriers of yeast cells were successfully synthesized and evaluated for ethanol fermentation of sugar molasses. Immobilization yeast of alginate-based MCM-41 composite carriers demonstrated the highest concentration of 4.8 109 cells/mL and shortest batch fermentation time of 12 h after repeated use across 60 days. No signicant differences in continuous fermentation results with 30 L and 10 m3 bioreactor scales were observed when the cells are uniformly immobilized in the packed bed. Acknowledgements Our deepest gratitude goes to Prof. Minglin Guo and Prof. Junfu Wei for their help in writing and in-depth discussions. This research work was nancially supported by National Basic Research Program of China (also called 973, Grant No. 2009CB623502), the National Natural Science Foundation of China (Grant Nos. 20777039 and 51072129), and the Yunnan Province-University Cooperation Foundation (Grant No. 2005YX39). References
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