Application of a Bubble-Column Reactor for the Production of aSingle-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 hasbeen studied using Trichosporon yeast. For this purpose, a 9-L reactor with a perforated-platedistributor was used. The effect of the aeration rate, height-to-diameter ratio (L/D), and pH ofthe medium on the efficiency of the bubble-column bioreactor was studied. Under optimizedconditions, the fermentation of the deproteinized whey in a batch system resulted in theproduction of 17.3 g L-1 biomass and 86% chemical oxygen demand reduction.

1. Introduction

Cheese whey is the liquid remaining following theprecipitation and removal of milk fats and casein duringcheese making. This byproduct represents about 85-95% of the milk volume and retains 55% of milknutrients. Among the most abundant of these nutrientsare lactose [4-5% (w/v)] and proteins [0.6-0.7% (w/v)].1,2

Cheese whey represents an important environmentalproblem: because of its high COD (60 000-80 000 ppm)and BOD (30 000-50 000 ppm),2 whey is no longerallowed to be discharged into rivers or public sewagesystems. Whey recycling poses problems because of itshigh water content, making its transport and dryinguneconomical. Because whey is highly perishable, itsprolonged storage is impossible. Cheese whey utilizationhas been the subject of much research, for example,production of biogas, ethanol, single-cell protein, proteinconcentrate, or other marketable products.2,3

There are some studies in microbial protein produc-tion from whey in continuous stirred tank reactors,4-6

but SCP production from cheese whey in a bubble-column bioreactor has not yet been reported. Bubble-column reactors are widely used in chemical andbiotechnological process industries because of theirsimple construction, lack of moving parts, high energyefficiency for mass transfer, and low shear forces.7,8

Bubble-column reactors are used in a variety ofprocesses as an apparatus to achieve mass-transfer and/or chemical reactions, usually in low-viscosity systems.In the past decade bubble columns have found wide-spread applications in biotechnological processes suchas the production of baker’s yeast, SCP, antibiotics,citric acid fermentation, and wastewater treatment.8,9

In this research the applicability and performance ofthe bubble-column bioreactor in SCP production fromcheese whey were studied and compared with those ofa stirred tank reactor. In this study not only is thevaluable product produced, but also the COD of wheyis considerably decreased.

2. Material and Methods

2.1. Microorganism. The microorganism used in thiswork was isolated in the course of our previous inves-tigation, and it was identified as Trichosporon yeast.10

It was originally isolated from dairy industry waste-water. The highest COD reduction and biomass produc-tion 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, asmuch as 70% of its solid content.

Whey from a dairy factory was clarified by heatingat 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 greenish-yellow liquid was supplemented with 0.6% (w/v) am-monium sulfate and 0.4% (w/v) dihydrogen potassiumphosphate and sterilized after adjusting the pH to 3.5.11

2.3. Inoculum Development. The inocula wereprepared by growing the yeast on potato dextrose agar(PDA) slants for 24 h at 30 °C. The cell suspensions wereprepared by washing the slants with sterilized distilledwater. Then 350 mL of a cell suspension was transferredto an Erlenmeyer and incubated at 30 °C and 200 rpmcontinuously for 24 h. The consumed inocula were about10% of the total volume of the medium.

2.4. Experimental Apparatus. The experimentswere carried out in a double-walled bubble-columnreactor of diameter 10.6 cm and height 102 cm. Thecolumn was made from Pyrex glass. The air wasdistributed by a perforated plate with 32 holes of 1.0mm diameter (triangular pitch). To compensate for thesubstrate reduction due to evaporation, a water-cooledcondenser was fitted on the top of the fermentor. Thecolumn temperature was controlled by thermo mix withwater circulation in the outer jacket of the bioreactor.The foam was controlled by the addition of an antifoamsilicon oil.

2.5. Analytical Methods. The cell concentration andCOD were determined every 3 h during 24 h of fermen-tation. The cell dry weight was determined by centri-fuging a certain volume of the culture broth at 5000 rpmfor 15 min, followed by drying of the cells for 2 h at 105°C.

The supernatant was used for COD and pH measure-ment.

Every data point is an average of three measure-ments.

* To whom correspondence should be addressed. E-mail:Towfighi@modares.ac.ir.

764 Ind. Eng. Chem. Res. 2003, 42, 764-766

10.1021/ie020254o CCC: $25.00 © 2003 American Chemical SocietyPublished on Web 01/22/2003

3. Results and Discussion

In our previous study, the most efficient microorgan-ism in SCP production from cheese whey was isolatedand identified as Trichosporon.10

SCP production had been studied in batch and stirredtank reactors during 24 h of fermentation. The workingvolume in both batch and continuous systems was 1 L.

In batch experiments, the maximum biomass produc-tion and COD removal were obtained as 8.73 g L-1 and52%, respectively, at 35 °C, pH ) 3.5, an aeration rateof 2 vvm, and a stirrer speed of 800 rpm.

In continuous experiments, under the optimal condi-tions at 34 °C, pH ) 4.2, an aeration rate of 2 vvm, astirrer speed of 800 rpm, and a dilution rate of 0.42 h-1,the amounts of SCP production and COD reduction wereobtained as 8.1 g L-1 and 53.21%, respectively.10,11

The scope of this work was to investigate the ef-ficiency of the bubble column in the production of SCPand COD reduction. For this purpose the effect of thegas flow rate and L/D ratio on biomass production andCOD reduction was studied.

3.1. Effect of the Gas Flow Rate on BiomassProduction and COD Reduction. The aeration in abubble-column reactor provides the required oxygen foran aerobic microorganism and also mixing. Properaeration provides suitable gas holdup, a higher resi-dence time of the gas in the liquid, and a high gas-liquid interaction area available for mass transfer.

The variation of the biomass production and CODreduction with gas velocity in the bubble column fol-lowed 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 produc-tion and COD reduction after reaching the maximum(about 7.5-8 vvm) begins to decrease. The initialincrease is due to a more cellular growth. After a certaintime, changing occurs in the flow regime from bubbleto churn-turbulent flow and the amount of biomassproduction and COD reduction will decrease. Otherreasons are high shear forces and the addition of excessantifoam to the medium because the increased gas flowrate will impose an inverse effect on the mass-transfercoefficient.

3.2. Effect of the L/D Ratio on Biomass Produc-tion and COD Reduction. The size of the bioreactor

is one of the effective factors on the initial cost,productivity, and economy of the system, which affectsthe hydrodynamics, reaction rate, and useful volume ofthe reactor.

At this stage the effect of the liquid height on biomassproduction and COD reduction in the bubble-columnreactor was studied. The results of biomass productionand COD reduction at different flow rates and varyingheights are shown in Figures 3 and 4.

As pointed out before, the L/D increase will decreasethe gas holdup. At a constant aeration rate of 4.5 vvm,biomass production increases up to 28% by increasingL/D from 2.5 to 4.7, and by increasing the aeration rateto 7.5 vvm, biomass production does not show a sensiblechange with an increase of the liquid height.

Generally, by increasing L/D, the gas holdup de-creases, and so the mass-transfer coefficient also de-creases, but in order to keep aeration at 4.5 vvm bychanging L/D from 2.5 to 4.7, the gas superficial velocityshould be increased from 1.99 to 3.74 cm s-1. Becausethe aeration rate is in the range of the homogeneous

Figure 1. Biomass production and COD reduction versus aerationrate 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).

Figure 3. Biomass production and COD reduction versus disper-sion height at 4.5 vvm during 24 h (T ) 30 °C and pH ) 3.5).

Ind. Eng. Chem. Res., Vol. 42, No. 4, 2003 765

regime, the increase of the superficial gas velocity hasa positive effect on the gas holdup, mass-transfercoefficient, and, consequently, biomass production andCOD reduction; this positive effect is higher than thenegative effect of increasing L/D on the mass-transfercoefficient, and overall the amount of biomass produc-tion will be increased.

At an aeration rate of 7.5 vvm by changing L/D from2.5 to 4, the superficial gas velocity increases from 3.31to 5.3 cm s-1, which is in the heterogeneous regime, andit has a less positive effect on the holdup to overcome anegative effect of L/D; therefore, biomass productionremains almost constant.

3.3. pH Effect. pH is one of the most importantfactors in cellular growth. pH has a pronounced effecton enzyme kinetics, and the reaction rate is maximumat some optimum pH and moved to either side of theoptimum value.

Biomass production and COD reduction as a functionof pH are shown in Figure 5. The maximum biomassproduction and COD reduction were obtained at pH )3.5-4.

4. Conclusion

In this research, the effect of the aeration rate, L/Dratio, and primary pH of the medium on biomassproduction and COD reduction was investigated. Theoptimum values for operational conditions were ob-tained as follows: aeration, 7.5 vvm; L/D ratio, 3.5; pH,3.5. Under optimum conditions, the fermentation ofdeproteinized cheese whey resulted in 17.3 g L-1 bio-mass and 86% COD reduction.

The result of biomass production and COD reductioncompared with previous work in a stirred tank bio-reactor shows the superiority of a bubble-column bio-reactor for this purpose. The major reason for that isthat in the bubble-column reactor cells are not exposedto large variations in shear forces and thus are able togrow in a more stable physical environment. In contrast,in stirred tank reactors, high shear conditions will arisenear the impeller, causing cell damage or cell stress andthus lowering productivity.

Acknowledgment

The financial support of this project by TarbiatModarres University is highly appreciated.

Nomenclature

BOD ) biological oxygen demandCOD ) chemical oxygen demandSCP ) single-cell proteinL ) lengthD ) diameter

Literature Cited

(1) Zadow, J. G. Whey and lactose processing; Elsevier AppliedScience: New York, 1992.

(2) Gonzalez Siso, M. I. The biotechnological utilization ofcheese 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 productionusing continuous ultrafiltration fementation of whey. Biotechnol.Lett. 1989, 11, 189-194.

(5) Garcia Garibay, M.; Gomez Ruiz, L.; Barzana, E. Studieson the simultaneous production of singel cell proein and polyga-lactosidase from Kluyveromyces fragilis. Biotechnol. Lett. 1987, 9,411-416.

(6) Zalashko, L. S.; Shmgin, V. K. Synthesis of microbial proteinand vitamins in concentrated whey. Brief Communication of the29th Internatinal Dairy Congress, Paris, 1987.

(7) Parasu Veera, U.; Joshi, J. B. Measurement of gas hold-upprofiles by gamma ray tomography: effect of sparger design andhight of dispersion in bubble columns. Trans. Inst. Chem. Eng.1999, 77, 303-317.

(8) Deckwer & Wolf-Dieter. Bubble column reactors; Wiley: NewYork, 1992.

(9) Blanch, H. W.; Clark, D. S. Biochemical engineering; Dek-ker: New York, 1996.

(10) Shojaosadati, S. A.; Rasouli, B. Isolation, evaluation andselection of microorganism suitable for singel cell protein produc-tion from cheese whey. Res. J. Esfahan 2000, 11, 44-48.

(11) Shojaosadati, S. A.; Rezaei, M. R.; Rasouli, B. Optimizationof singel cell protein production from cheese whey under batchand continuous cultivation. J. Estaghlal 1999, 18, 33-39.

Received for review April 5, 2002Revised manuscript received November 4, 2002

Accepted November 8, 2002

IE020254O

Figure 4. Biomass production and COD reduction versus disper-sion height at 7.5 vvm during 24 h (T ) 30 °C and pH ) 3.5).

Figure 5. Biomass production and COD reduction versus pHduring 24 h (7.5 vvm, L/D ) 3.5, T ) 30 °C, and pH ) 3.5).

766 Ind. Eng. Chem. Res., Vol. 42, No. 4, 2003