INTERNATIONAL JOURNAL Of ACADEMIC RESEARCH

Vol. 2. No. 6. November, 2010, Part I

ETHANOL PRODUCTION FROM MOLASSES USING IMMOBILIZED CELLS CA-ALGINATE AND K-CARRAGEENAN BY MUTATION ZYMOMONAS MOBILIS IN A PACKED BED BIOREACTOR
Tri Widjaja, Soeprijanto, Ali Altway, Setiyo Gunawan, R. Darmawan Department of Chemical Engineering, Sepuluh Nopember Institute of Technology (INDONESIA) ABSTRACT The aim of this experiment was to study the effect of total sugar concentration using Ca-Alginate and K Carrageenan immobilized cells of mutation Zymomonas mobilis on packed-bed and batch bioreactor performance for ethanol production. This experiment was carried out with total sugar concentrations of 14, 16, and 18% (v/v) and immobilized cells of Ca-Alginate and K-Carrageenan at a concentration of 2% (w/v). The results revealed that the total sugar concentration influenced the concentration and productivity of ethanol. Immobilized Ca-Alginate cells produced the maximum ethanol concentration, yield and productivity at 60.83 g/L, 43.94% and 24.33 g/L.hr, respectively. Meanwhile, immobilized K-Carrageenan cells produced the maximum ethanol concentration, yield and productivity at 60.54 g/L.hr, 41.85% and 24.22 g/L.hr, respectively. In a batch fermentation process showed that the maximum ethanol concentration, yield and productivity were 63.47 g/L, 43.79%, 0.88 g/L.hr, respectively. Key words: Ca-Alginate; ethanol; immobilized cells; K-Carrageenan; packed-bed bioreactor 1. INTRODUCTION The well-known conventional processes of ethanol were some weaknesses. Firstly, it commonly uses batch process to easily control the fermentation process from microorganism contamination. Secondly, the ethanol produced was so low because the accumulation of ethanol brought about poison towards microorganism while fermenting. The accumulation of dissolved product halted microorganism growth [1]. There was a process limitation in conventional process such as ethanol inhibition, at ethanol concentration of 12% (v/v) fermented broth, specific microorganism growth and specific rate production will decrease and cell density in a packed bed column will be low, therefore the sugar solution is not completely fermented. In order to increase ethanol productivity, the inhibiting contents in the system have to be removed during fermentation. Ethanol is more volatile than water and therefore, there was a concept to implement ethanol water separation technique using ethanol vacuum distillation during fermentation. This technique has a purpose to remove the ethanol inhibition. Other authors Minier and Goma [1] have conducted experiments using fermentation technique in vacuum fermentation circumstances with cell recycle. The results showed that ethanol production increased up to 82 g/L.hr, if the fermentation was carried out without cell recycle, ethanol produced became 40 g/L.hr. When compared to conventional process, the ethanol produced was only 29 g/L.hr. Unfortunately, the cost to produce azeotrope ethanol is 1.05 times energy cost required for conventional batch process. It is well known that immobilizing cells is a process that is used to halt the action of enzymes and catalysts. Immobilization is performed due to microorganisms density is closed to that of water, and therefore, there is the possibility they could bond in supporting matrice while in the product stream. The advantages in using immobilized cell than those of the other free cells are the ease to separate the product, a highly volumetric productivity, an increase in the process control and a decrease in the contamination, a lessen separation cost, and prevention of wash out occurring in product stream. Of the various immobilized techniques, trapping cell in calcium-alginate gel system is the simplest method and it does not have poisonous property. Then immobilized cell technique with Ca-alginate was developed by Goksungur and Zorlu [2] which involved drop-wise cell suspension in sodium alginate that hardened. The immobilized cell technique with Ca-Alginate and -Carrageenan as supporting matrice was also learned by Grote et al [3] who fermented sugar cane juice to high concentrations of ethanol. It was more ethanol tolerant and had faster specific rates of glucose uptake and ethanol production than the other strains of zymomonas mobilis which had been conducted by Skotnicki et al [4]. This method was also easy to apply with various cells for example bacteria, cynobacteria, algae, and yeast fungi. A variety of bioreactors, such as agitated bioreactor (continuously stirred tank reactor), fluidized bed reactor, and packed bed reactor could be used in this experiment, but the authors recommended that the packed bed should be used as a result of a low cost, easier operability compared to others, and automatic industrial operations. Previous studies on strain of zymomonas mobilis have known that these microorganisms can convert glucose efficiently and rapidly to ethanol with significantly higher specific rates of glucose uptake and ethanol production than useful yeasts[5].The microorganisms used to produce ethanol in this study is mutation Zymomonas mobilis, the mutated bacteria Zymomonas mobilis using hydroxylamine mutagen selected in acid medium to form a characteristic resistant to acid. A3 is the excess of Zymomonas mobilis, has a high temperature tolerance, ability to attain a faster conversion, more resistant to high levels of ethanol produced in the fermentation process when compared Zymomonas mobilis is not mutated [6].

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INTERNATIONAL JOURNAL Of ACADEMIC RESEARCH

Vol. 2. No. 6. November, 2010, Part I

The aim of this experiment was to study the effect of total sugar concentration using Ca-Alginate and K Carrageenan immobilized cells of Zymomonas mobilis mutation on packed-bed and batch bioreactor performance for ethanol production. 2. MATERIALS AND METHODS Processing conditions Reactor type used in this experiment was a packed-bed bioreactor with K-Carrageenan and Ca-Alginate o beads of 2% (w/v). During fermentation process, it was operated on temperature of 30 C and pH 4 5. Pretreatment molasses The incubated mutation Zymomonas mobilis from a sloping breeding media was taken using a sterile wire ose, was embedded as much as 6 ose into 100 ml nutrition media (yeast extract 10 g, (NH4)2SO4 1,0 g, KH2PO4 1,0 g and MgSO4.7H2O 0,5g) with a certain total sugar concentration. These organisms were then cultivated in an o incubator shaker on temperature of 30 C for 36 hours. Also, the observation of total bacteria found in the media was counted using haemacytometer neubaeur and the logarithmic phase of bacterial growth was detected using a microscope. Immobilized cells preparation K-Carrageenan: 1 gram of -Carrageenan was dissolved in 45 ml of aquadest, then heated on temperature o of 70 C for 15 minutes until it began to form gel. A solution of -Carrageenan was then chilled up to temperature of o 40 C. 5 ml nutrient media was mixed with 45 ml solution K-Carrageenan, so that this mixture achieved 2%. 50 ml of this mixture solution was injected in 1000 ml solution of 3.5% KCL until a desirable bead formed. The bead was ossified for 15 minutes. After 15 minutes solid formed was washed with 0.85% NaCl to decrease excessive ions To increase the cell growth, the bead was put into production of media containing (NH4)2SO4 5,19 gram, KH2PO4 1,53 gram, MgSO4.7H2O 0,55 gram and yeast extract 1 gram + breeding was put into incubator shaker for 24 hours. o Then, the bead was kept on temperature 4 C until cell was used. Ca-Alginate: A nutrient media containing (NH4)2SO4 5,19 gram, KH2PO4 1,53 gram, MgSO4.7H2O 0,55 gram and yeast extract 1 gram + breeding was put into incubator shaker for 24 hours. Then 50 ml of this nutrient media was mixed with 50 ml of alginate solution. Then 100 ml of alginate-cell mixture was added in 1000 ml solution of 2% CaCl2 until a solid solution formed. After 30 minutes solid formed was washed with 0.85% NaCl to decrease excessive ions. To increase cell growth, immobilized cell in production media was incubated in an incubator shaker for 24 hours. A production media was made by mixing 1 litre of molasses with (NH4)2SO4 5,19 gram, KH2PO4 1,53 gram and MgSO4.7H2O 0,55 gram, then this solution was added with H2SO4 until pH 4.6. if not used directly, the solid in 2% of yeast extract was kept in temperature of 4C. Fermentation process Certain total sugar concentrations of immobilized cell beads produced was put into a packed bed bioreactor tray. Then the sterilized molasses containing a total sugar concentration according to the variables (14% ; 16% ; -1 18% ) (v/v) was fed into packed bed bioreactor with a volumetric rate of 0.06 L/hr and dilution rate of 0.4 hr . A schematic diagram of a continuously packed bed bioreactor is shown in Figure 1.

7
3 6 2

1. 2. 3. 4. 5. 6. 7. 8.

Information: Molasses Peristaltic pump Fermentor Ethanol Product Outlet gas Immobilized cells Statif Thermometer

4 5
1

Fig. 1. Continuously Packed Bed Bioreactor

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Vol. 2. No. 6. November, 2010, Part I

Analytical Methods Concentration of glucose was determined using a high performance liquid chromatography with cosmosil sugar-D column and the UV detector at wavelength of 540 nm, using the reagent 3,5 - dinitrosalicylic acid [7,8]. Counting cells were carried out using haemocytometer. The product ethanol concentration was analyzed using a Gas Chromatography. 3. RESULTS AND DISCUSSION At the beginning of experiment for continuous fermentation, a packed column was filled with Ca-Alginate or K-Carrageenan beads until they were fully filled up. Furthermore, molasses was pumped down by a peristaltic -1 pump through silicon tube to the packed bed bioreactor with a flow rate of 0.06 L/hr and a dilution rate of 0.4 hr , and ethanol as a product was flowing out from the liquid effluent stream on the upper of the bioreactor. Meanwhile, a batch fermentation was conducted using a batch stirred tank reactor equipped with jacket and agitator. The agitators speed rotation was kept constant. During the experiments, the continuous and batch fermentation pH of 4-5 was adjusted. A Gas Chromatography method was used to analyze the ethanol products and the Optical Density method was used to analyze the free cells from batch fermentation. Table 1 shows parameters and conditions for continuous fermentation and batch fermentation. Table 1. Parameters and conditions of the experiments
Information Bioreactor Reactor Volume (ml) Bead Bead weight (g) Residence time (hr) Continuous Fermentation Packed bed 510 Ca-Alginate/ K-Carrageenan 250 2,5 Batch Fermentation Batch Stirer 1800 Free Cells 72

The Effect Of Total Sugar Concentration On Ethanol Production The Ratio between average ethanol concentrations and various total sugar concentrations with 2% bead Ca-Alginate and 2% K-Carrageenan is shown in Figure 2.

66

Ethanol Concentration (g/L)

64 62 60 58 56 54 52 50 48 178
60,57

63,47 60,83 57,23 61,7 60,54 57,53 58,66

53,39

186

205

Total Sugar Concentration (g/L) Ca-Alginate 2% K-Carrageenan 2%

Fig. 2. Effect of total sugar concentration on ethanol production The concentration of ethanol concentrations produced for 2% of Ca-Alginate bead were 60.57 g/L; 60.83 g/L; and 57.53 g/L, respectively; while the ethanol concentrations produced for 2% of K-Carrageenan beads were 57.23 g/L; 60.54 g/L; and 53.39 g/L, respectively. The ethanol concentrations produced for batch fermentation process were 63.47 g/L; 61.7 g/L; and 58.66 g/L, respectively. The highest ethanol concentration obtained for 2% of Ca-Alginate bead and 16% of total sugar concentration was 60.83 g/L and the highest ethanol concentration obtained for 2% of K-Carrageenan and 16% of total sugar concentration was 60.54 g/L. In 2% of Ca-Alginate and 2% K-Carrageenan beads, total sugar concentration (16%) gave the higher ethanol production. This was caused by a larger amount of substrates of sugar converted into ethanol as product. But in 18% concentration of total sugar gave decrease ethanol concentration. This was caused substrate inhibition.

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INTERNATIONAL JOURNAL Of ACADEMIC RESEARCH

Vol. 2. No. 6. November, 2010, Part I

While for batch fermentation process, 63.47 g/L was the highest ethanol concentration achieved for 14% of total sugar concentration. The Effect Of Total Sugar Concentration On Ethanol Yield Yield is defined as the ratio between ethanol produced and total sugar consumed during fermentation. Figure 3 shows the relationship between ethanol yields (%) and total sugar concentration in molasses (%).

46

Ethanol Yield (%)

44 42 40 38 36 34 178 41,19

43,79

43,94
41,85

40,20

39,53 38,07

38,79

37,54

186

205

Ca-Alginate 2%

Total Sugar Concentration (g/L) K-Carrageenan 2% Batch (Free Cells)

Fig. 3. The relationship between ethanol yield and Total sugar Concentration According to figure 3 the ethanol yield for 2% bead Ca-Alginate are: 41.19%; 43.94%; and 38.07% while the ethanol yield for 2% bead K-Carrageenan are: 40.20%; 41.85%; and 37.54 %. The ethanol concentrations produced for batch fermentation process are 43.79%; 39.53%; and 38.79%. The highest ethanol yield achieved for 2% of Ca-Alginate bead and 16% of total sugar was 43.94% and then the highest ethanol yield reached for 2% bead K-Carrageenan and 16% of total sugar was 41.85%. While for batch fermentation process, it was obtained for 14% concentration of substrate. The results showed that the increasing of total sugar concentration does not affect the amount of ethanol yield, using immobilized cells of Ca-Alginate bead. The higher concentration of total sugar used, the less the yield of ethanol produced. However, for immobilized cells of K-Carrageenan bead, the higher concentration of total sugar used, the more yield of ethanol produced. While for batch fermentation process, the higher concentration of total sugar used, the less ethanol yield produced. The Effect Of Total Sugar Concentration On Ethanol Productivity In fermentation, productivity is defined as grams ethanol product/liter/hours. Figure 4 shows the relationship between ethanol productivity (g/L.hr) and concentration of total sugar in molasses (%).

Ethanol Productivity (g/L.hr)

30 25 20 15 10 5 0

24,23

22,89

24,33 24,22

23,01

21,36

0,88 178 186

0,86 205

0,81

Total Sugar Concentration (g/L) Ca-Alginate 2% K-Carrageenan 2%

Fig. 4. The relationship between ethanol productivity concentration of total sugar The results from the experiments showed that the ethanol productivities produced for 14%; 16%; 18% of total sugar concentrations and 2% of Ca-Alginate bead are 24.23 g/L.hr; 24.33 g/L.hr; and 23.01 g/L.hr, then the ethanol productivities produced for 14%; 16%; 18% of total sugar concentrations and 2% K-Carrageenan bead are 22.89 g/L.hr; 24.22 g/L.hr; and 21.36 g/L.hr. While in batch fermentation, the ethanol productivities produced for 14%; 16%; and 18% of total sugar concentrations are 0.88 g/L.hr; 0.86 g/L.hr; and 0.81 g/L.hr. Ethanol productivity is directly proportional to ethanol concentration because ethanol productivity is ethanol concentration divided by residence time.

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Vol. 2. No. 6. November, 2010, Part I

According to these results, the immobilized cells with Ca-Alginate supporting matrices were found to have larger to yield and ethanol productivity than the immobilized cells of K-Carrageenan supporting matrices. This was because of the differences in the generation treatment for those Ca-Alginate and K-Carrageenan bead. In the o generation of Ca-Alginate bead, heating process was not required (only room temperature 30 C needed), while Ko o Carrageenan bead needed 70 C and it would decrease up to 40 C for heating process in the generation treatment of K-Carrageenan. It was found that productivity of ethanol using a continuous fermentation method was better than that in a batch. Immobilized cells Ca-Alginate and K-Carrageenan are used in continuous fermentation as supporting matrices, where mutation Zymomonas mobilis bacteria was trapped in the beads. It made gradient of sugar, that following the lower total sugar concentrations which will become non inhibitor [9].While in a batch fermentation process, the mutation Zymomonas mobilis was free (free cells), thus, causing the membrane plasma to break out of its cell wall membrane so that the substrates, inhibitor characterized to cell, caused the decreasing rate of fermentation [2]. 4. CONCLUSION Immobilized cells of Ca-Alginat resulted in a higher concentration, yield and productivity of ethanol than those of immobilized cells of K-Carrageenan. It was found that the yield and productivity of ethanol using a continuous fermentation method was significantly better than using a batch one. ACKNOWLEDGMENT This project was granted by Hibah Bersaing Research Project 2010 by Directorate Penelitian dan Pengabdian Kepada Masyarakat (DP2M), General Director of Higher Education National Education 2010. The authors would like to thanks Nur Fauziah Arini, Winda Savitri, Natalia Hariani, Emmanuel Topan, for their contribution to this experiment. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. Minier, M, dan Goma, G, (1982). Ethanol Production by Extractive Fermentation, Biotechnology and Bioengineering, 24: 1565-1579. Goksungur, Y. and Zorlu,N., (2001). Production of Ethanol From Beet Molasses by Ca-Alginate Immobilized Yeast Cells in a Packed-Bed Bioreactor, Turk J. Biol., 25: 265-275. Grote, W. ,K.J Lee dan P.L Rogers, (1980). Continuous Ethanol Production By Immobilized Sels of Zymomonas Mobilis, Biotechnology Letters, 11: 481-486. Skotnicki, M.L.,Tribe,D.E. and Rogers,P.L. (1980). Appl.&Env.Microbiol., 40: 7 12. K.J.Lee, Skotnicki, M.L.,Tribe,D.E. and Rogers,P.L., (1981). The effect of temperature on The Kinetics of Ethanol Production by Strains of Zymomonas mobilis, Biotechnology Letters, 3 (6): 291-296. Putra, S,R. and Chrisnawati, A., (2008). Produksi Etanol Menggunakan Zymomonas mobilis yang Dimutasi dengan Hydroxylamin, Chemistry Department, Faculty of Mathematic and Science, ITS Surabaya, Indonesia. Adney,B. and J.Baker, (1996). Chemical Analysis and Testing Task Laboratory Analitical Procedur, LAP-006. Miller, G.L., (1959). Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar, Anal. Chem., 31: 426. Barros, M.R.A., J.M.S Cabra dan J.M.Novais, (1987). Production Ethanol by Immobilized Saccharomyces Bayanus in an extractive Fermentation System, Biotechnology and Bioengineering, 29: 1097-1104.

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