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Application of a Bubble-Column Reactor for the Production of a Single-Cell Protein from Cheese Whey Maryam Hosseini, Seyed A. Shojaosadati, and Jafar Towfighi* Biotechnology Group, Chemical Engineering Deptartment, Tarbiat Modarres University, P.O. Box 14155-4838, Tehran, Iran
The efficiency of a bubble-column reactor in the production of biomass from cheese whey has been studied using Trichosporon yeast. For this purpose, a 9-L reactor with a perforated-plate distributor was used. The effect of the aeration rate, height-to-diameter ratio (L/D), and pH of the medium on the efficiency of the bubble-column bioreactor was studied. Under optimized conditions, the fermentation of the deproteinized whey in a batch system resulted in the production of 17.3 g L-1 biomass and 86% chemical oxygen demand reduction. 1. Introduction Cheese whey is the liquid remaining following the precipitation and removal of milk fats and casein during cheese making. This byproduct represents about 8595% of the milk volume and retains 55% of milk nutrients. Among the most abundant of these nutrients are lactose [4-5% (w/v)] and proteins [0.6-0.7% (w/ v)].1,2 Cheese whey represents an important environmental problem: because of its high COD (60 000-80 000 ppm) and BOD (30 000-50 000 ppm),2 whey is no longer allowed to be discharged into rivers or public sewage systems. Whey recycling poses problems because of its high water content, making its transport and drying uneconomical. Because whey is highly perishable, its prolonged storage is impossible. Cheese whey utilization has been the subject of much research, for example, production of biogas, ethanol, single-cell protein, protein concentrate, or other marketable products.2,3 There are some studies in microbial protein production from whey in continuous stirred tank reactors,4-6 but SCP production from cheese whey in a bubblecolumn bioreactor has not yet been reported. Bubblecolumn reactors are widely used in chemical and biotechnological process industries because of their simple construction, lack of moving parts, high energy efficiency for mass transfer, and low shear forces.7,8 Bubble-column reactors are used in a variety of processes as an apparatus to achieve mass-transfer and/ or chemical reactions, usually in low-viscosity systems. In the past decade bubble columns have found widespread applications in biotechnological processes such as the production of baker’s yeast, SCP, antibiotics, citric acid fermentation, and wastewater treatment.8,9 In this research the applicability and performance of the bubble-column bioreactor in SCP production from cheese whey were studied and compared with those of a stirred tank reactor. In this study not only is the valuable product produced, but also the COD of whey is considerably decreased. 2. Material and Methods 2.1. Microorganism. The microorganism used in this work was isolated in the course of our previous investigation, and it was identified as Trichosporon yeast.10 * To whom correspondence should be addressed. E-mail:
[email protected].
It was originally isolated from dairy industry wastewater. The highest COD reduction and biomass production were obtained at 30 °C and pH 3.5. The specific growth rate obtained was 0.59 h-1. 2.2. Cheese Whey. The cheese whey was used as a medium for yeast growth, and it contains lactose, as much as 70% of its solid content. Whey from a dairy factory was clarified by heating at 100 °C for 15 min after adjusting the pH to 4.67 (isoelectric pH). After settling of the whey’s proteins, the supernatant was ultrafiltered and the greenishyellow liquid was supplemented with 0.6% (w/v) ammonium sulfate and 0.4% (w/v) dihydrogen potassium phosphate and sterilized after adjusting the pH to 3.5.11 2.3. Inoculum Development. The inocula were prepared by growing the yeast on potato dextrose agar (PDA) slants for 24 h at 30 °C. The cell suspensions were prepared by washing the slants with sterilized distilled water. Then 350 mL of a cell suspension was transferred to an Erlenmeyer and incubated at 30 °C and 200 rpm continuously for 24 h. The consumed inocula were about 10% of the total volume of the medium. 2.4. Experimental Apparatus. The experiments were carried out in a double-walled bubble-column reactor of diameter 10.6 cm and height 102 cm. The column was made from Pyrex glass. The air was distributed by a perforated plate with 32 holes of 1.0 mm diameter (triangular pitch). To compensate for the substrate reduction due to evaporation, a water-cooled condenser was fitted on the top of the fermentor. The column temperature was controlled by thermo mix with water circulation in the outer jacket of the bioreactor. The foam was controlled by the addition of an antifoam silicon oil. 2.5. Analytical Methods. The cell concentration and COD were determined every 3 h during 24 h of fermentation. The cell dry weight was determined by centrifuging a certain volume of the culture broth at 5000 rpm for 15 min, followed by drying of the cells for 2 h at 105 °C. The supernatant was used for COD and pH measurement. Every data point is an average of three measurements.
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Figure 1. Biomass production and COD reduction versus aeration rate at L/D ) 2.5 during 24 h (T ) 30 °C and pH ) 3.5).
Figure 2. Biomass production and COD reduction versus aeration rate at L/D ) 3.5 during 24 h (T ) 30 °C and pH ) 3.5).
3. Results and Discussion In our previous study, the most efficient microorganism in SCP production from cheese whey was isolated and identified as Trichosporon.10 SCP production had been studied in batch and stirred tank reactors during 24 h of fermentation. The working volume in both batch and continuous systems was 1 L. In batch experiments, the maximum biomass production and COD removal were obtained as 8.73 g L-1 and 52%, respectively, at 35 °C, pH ) 3.5, an aeration rate of 2 vvm, and a stirrer speed of 800 rpm. In continuous experiments, under the optimal conditions at 34 °C, pH ) 4.2, an aeration rate of 2 vvm, a stirrer speed of 800 rpm, and a dilution rate of 0.42 h-1, the amounts of SCP production and COD reduction were obtained as 8.1 g L-1 and 53.21%, respectively.10,11 The scope of this work was to investigate the efficiency of the bubble column in the production of SCP and COD reduction. For this purpose the effect of the gas flow rate and L/D ratio on biomass production and COD reduction was studied. 3.1. Effect of the Gas Flow Rate on Biomass Production and COD Reduction. The aeration in a bubble-column reactor provides the required oxygen for an aerobic microorganism and also mixing. Proper aeration provides suitable gas holdup, a higher residence time of the gas in the liquid, and a high gasliquid interaction area available for mass transfer. The variation of the biomass production and COD reduction with gas velocity in the bubble column followed the pattern depicted in Figures 1 and 2 for L/D ) 2.5 (4.5-12 vvm) and 3.5 (4.5-8.25 vvm), respectively. When the aeration rate is increased, biomass production and COD reduction after reaching the maximum (about 7.5-8 vvm) begins to decrease. The initial increase is due to a more cellular growth. After a certain time, changing occurs in the flow regime from bubble to churn-turbulent flow and the amount of biomass production and COD reduction will decrease. Other reasons are high shear forces and the addition of excess antifoam to the medium because the increased gas flow rate will impose an inverse effect on the mass-transfer coefficient. 3.2. Effect of the L/D Ratio on Biomass Production and COD Reduction. The size of the bioreactor
Figure 3. Biomass production and COD reduction versus dispersion height at 4.5 vvm during 24 h (T ) 30 °C and pH ) 3.5).
is one of the effective factors on the initial cost, productivity, and economy of the system, which affects the hydrodynamics, reaction rate, and useful volume of the reactor. At this stage the effect of the liquid height on biomass production and COD reduction in the bubble-column reactor was studied. The results of biomass production and COD reduction at different flow rates and varying heights are shown in Figures 3 and 4. As pointed out before, the L/D increase will decrease the gas holdup. At a constant aeration rate of 4.5 vvm, biomass production increases up to 28% by increasing L/D from 2.5 to 4.7, and by increasing the aeration rate to 7.5 vvm, biomass production does not show a sensible change with an increase of the liquid height. Generally, by increasing L/D, the gas holdup decreases, and so the mass-transfer coefficient also decreases, but in order to keep aeration at 4.5 vvm by changing L/D from 2.5 to 4.7, the gas superficial velocity should be increased from 1.99 to 3.74 cm s-1. Because the aeration rate is in the range of the homogeneous
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4. Conclusion In this research, the effect of the aeration rate, L/D ratio, and primary pH of the medium on biomass production and COD reduction was investigated. The optimum values for operational conditions were obtained as follows: aeration, 7.5 vvm; L/D ratio, 3.5; pH, 3.5. Under optimum conditions, the fermentation of deproteinized cheese whey resulted in 17.3 g L-1 biomass and 86% COD reduction. The result of biomass production and COD reduction compared with previous work in a stirred tank bioreactor shows the superiority of a bubble-column bioreactor for this purpose. The major reason for that is that in the bubble-column reactor cells are not exposed to large variations in shear forces and thus are able to grow in a more stable physical environment. In contrast, in stirred tank reactors, high shear conditions will arise near the impeller, causing cell damage or cell stress and thus lowering productivity. Figure 4. Biomass production and COD reduction versus dispersion height at 7.5 vvm during 24 h (T ) 30 °C and pH ) 3.5).
Acknowledgment The financial support of this project by Tarbiat Modarres University is highly appreciated. Nomenclature BOD ) biological oxygen demand COD ) chemical oxygen demand SCP ) single-cell protein L ) length D ) diameter
Literature Cited
Figure 5. Biomass production and COD reduction versus pH during 24 h (7.5 vvm, L/D ) 3.5, T ) 30 °C, and pH ) 3.5).
regime, the increase of the superficial gas velocity has a positive effect on the gas holdup, mass-transfer coefficient, and, consequently, biomass production and COD reduction; this positive effect is higher than the negative effect of increasing L/D on the mass-transfer coefficient, and overall the amount of biomass production will be increased. At an aeration rate of 7.5 vvm by changing L/D from 2.5 to 4, the superficial gas velocity increases from 3.31 to 5.3 cm s-1, which is in the heterogeneous regime, and it has a less positive effect on the holdup to overcome a negative effect of L/D; therefore, biomass production remains almost constant. 3.3. pH Effect. pH is one of the most important factors in cellular growth. pH has a pronounced effect on enzyme kinetics, and the reaction rate is maximum at some optimum pH and moved to either side of the optimum value. Biomass production and COD reduction as a function of pH are shown in Figure 5. The maximum biomass production and COD reduction were obtained at pH ) 3.5-4.
(1) Zadow, J. G. Whey and lactose processing; Elsevier Applied Science: New York, 1992. (2) Gonza´lez Siso, M. I. The biotechnological utilization of cheese whey: a review. Bioresour. Technol. 1996, 57, 1-11. (3) Tahoun, M. K.; El-Merheb, Z.; Salam, A.; Youssef, A. Biomass and lipids from lactose or whey by Trichosporon beigelii. Biotechnol. Bioeng. 1987, 29, 358-360. (4) Blanc, P.; Goma, G. Propionic acid and biomass production using continuous ultrafiltration fementation of whey. Biotechnol. Lett. 1989, 11, 189-194. (5) Garcia Garibay, M.; Gomez Ruiz, L.; Barzana, E. Studies on the simultaneous production of singel cell proein and polygalactosidase from Kluyveromyces fragilis. Biotechnol. Lett. 1987, 9, 411-416. (6) Zalashko, L. S.; Shmgin, V. K. Synthesis of microbial protein and vitamins in concentrated whey. Brief Communication of the 29th Internatinal Dairy Congress, Paris, 1987. (7) Parasu Veera, U.; Joshi, J. B. Measurement of gas hold-up profiles by gamma ray tomography: effect of sparger design and hight of dispersion in bubble columns. Trans. Inst. Chem. Eng. 1999, 77, 303-317. (8) Deckwer & Wolf-Dieter. Bubble column reactors; Wiley: New York, 1992. (9) Blanch, H. W.; Clark, D. S. Biochemical engineering; Dekker: New York, 1996. (10) Shojaosadati, S. A.; Rasouli, B. Isolation, evaluation and selection of microorganism suitable for singel cell protein production from cheese whey. Res. J. Esfahan 2000, 11, 44-48. (11) Shojaosadati, S. A.; Rezaei, M. R.; Rasouli, B. Optimization of singel cell protein production from cheese whey under batch and continuous cultivation. J. Estaghlal 1999, 18, 33-39.
Received for review April 5, 2002 Revised manuscript received November 4, 2002 Accepted November 8, 2002 IE020254O
