Mass transfer studies in batch fermentation: Mixing characteristics

JournalofFood Engineering 23 (1994) 145-158 0 1994 Elsevier Science Limited Printed in Great Britain. All rights reserved 0260~8774/94/$7.00 ELSEVIER

Mass Transfer Studies in Batch Fermentation: Mixing Characteristics M. N. Ahmad, C. R. Holland & G. McKay Department

of Chemical Engineering, The Queen’s University of Belfast, David Keir Building, Stranmillis Road, Belfast BT9 5AG, UK

(Received 2 February

1992; revised version received 8 April 1993; accepted 13 April 1993)

ABSTRACT Studies have been undertaken to investigatethe mass transferparameters due to mixing effects during fermentation using Candida utilis in a 2*0dm3 batch fermentor. Mechanical agitation and mixing via airflow have been studied and each varied independently of the other. Oxygen transfer rates werefound to increase up to a limiting value when both airflow rate and agitationwere increased. Aerobic fermentation is characterised by the specific oxygen rate which increased with agitation and air flow rate. It was found, however, that agitation and aeration had no efect on the carbon dioxide production rate but the mass transfer coeficient increased markedly with increasing agitation. Valuesfor all the mass transfer terms were determined. The mass transfer coejjicient, k,a, has been correlated withthe impeller agitationspeed, N, asfollows: k,a = 1.079 x lo--’ N’,%

NOTATION a

CI_ C: Cm

CPR FN Hu kc

Interfacial area per unit volume of liquid ( m2/m3) Dissolved oxygen concentration in bulk liquid (mmol/litre) Equilibrium or saturation concentration of dissolved oxygen (mmol/litre) Cell mass concentration (g/litre) Carbon dioxide production rate (mmol/litre/h) Molar flow rate of inert (N2) gas (h- ‘) Fractional gas hold-up Specific carbon dioxide production rate (mmol/g/h) 145

146

k kl_a kr

OTR OUR P PA PC PO pw RI

M. N. Ahmad, C. R. Holland, G. McKay

Liquid mass transfer coefficient (m/h) Volumetric mass transfer coefficient (h- ‘) Specific oxygen uptake rate (mmol/g/g) Oxygen transfer rate (mmol/litre/h) Oxygen uptake rate (mmol/litre/h) Agitator power dissipation (W ) Total pressure (atm) Partial pressure of carbon dioxide (atm) Partial pressure of oxygen in air (atm) Partial pressure of water in the gas phase (atm) Empirical constant Liquid volume ( m3) Superficial gas velocity (m/s)

INTRODUCTION Considerable attention has been given to the oxygen transfer characteristics of an agitated bubble fermentation system because of their widespread use in intensive industrial fermentation processes. Dissolved oxygen levels in culture media are affected by gas flow rate, degree of agitation and oxygen partial pressure in the aerating gas. Attempts to control dissolved oxygen levels by varying agitator speed (Phillips & Johnson, 1961) or gas flow rate (Terui et aZ., 1960) have been reported. In conventional aerobic culture systems, changes in mechanical agitation and air flow rate interact with each other simultaneously affecting dissolved oxygen levels. It is, therefore, difficult to isolate the effects of bubbling oxygen from those which may result from changes in fluid turbulence by mechanical means. Since the intensity of agitation is usually expressed in terms of the power dissipation per unit volume of the liquid, most correlations relate this parameter to k,a. The earliest was proposed by Cooper et al. (Winkler, 1981) in the form k,a = R,(P/VL)0’95 ( V,)*13 where: k,a = volumetric mass transfer coefficient, R, = empirical constant, P= agitator power dissipation, V, = volume of the fermentation medium, V, = superficial gas velocity.

(1)

Mass transfer studies in batch fermentation

147

The above equation indicates that k,_a is related to power dissipation and superficial gas velocity, highlighting the advantage of using higher air flow rates. The main purpose of this paper is to study the effects of agitation and air flow rate variation on the system mass transfer characteristics in terms of oxygen transfer rate (OTR), carbon dioxide production rate (CPR), specific oxygen uptake rate (kr), specific carbon dioxide production rate (kc), and the volumetric mass transfer coefficient (k,a).

EQUIPMENT The fermentation equipment contains a fully controlled 2-litre fermenter (500 series, 1985 Model, I_H Fermentation Ltd) as shown in Fig. 1. The reactor vessel is made of Pyrex with a stainless steel top plate. The vessel is fitted with four baffles evenly spaced around the perimeter to improve mixing and has a working volume of 1.5 litres. The exhaust gas is monitored for oxygen and carbon dioxide contents.

Exhaust

Oxygen meter

gas

Air flow meter

CO2 analyser Drying column it i- f oan!j AgZ!jol

peratukAontro1 B-

w

Cooling water

Fig. 1.

Dissolved oxygen

Ill1 ---

Schematicdiagramof the fermenter.

hf. N. Ahmad, C. R. ~o~~~d~ G. McKay

148

EFFECT OF AGITAnON SPEED Agitation speeds varied from 200 to 800 rpm. In this range the various degrees of mixing effects on mass transfer terms are now discussed. The effect of agitation speed on oxygen transfer rates during the growth cycle is shown in Fig. 2. An increase in OTR from 8.94 to 3863 mmol/litre h is obtained as the agitation speed is increased from 300 to 600 rpm. Further increasing the agitation speed to 800 t-pm decreases the oxygen transfer rate to 31.4 mmolflitre h. The initial increase can be explained by the surface renewal theories of Danckwerts and others (Danckwerts, 1951). It has been reported that an increase in turbulence will result in ~creas~g the renewal rate of the thin film around a gas bubble, and accordingly an increase in the rate of exposure of fresh surface per unit total surface area of the gas phase. This would result in an increase in the oxygen transfer. At higher agitation speeds (800 rpm), the cells are damaged especially during the reproduction when the buds may be removed from the mother-cell before they have reached the required

0 0

2

4

6

8 Tliw m

l0

12

l4

16

Agitatton ftpal -

800

-e-

400

-f3- 600

+eOO

-e-800

Fig. 2. OTR versus time at different agitation speeds. The constant values of the process parameters were: air flow rate = 1.26 litres/min; glucose concentration = 1% (w/v); agitation speed = 500 rpm; inoculum dosage = 1% (v/v~; temperature = 32°C and pi5 = 5.6. These parameters also apply for Figs 3-l 1.

Mass transfer studies in batch fermentation

149

size. This reduces the respiration rate, resulting also in an increase in the dissolved oxygen concentration and a reduction in the driving force ( Ct - C,_), resulting in lower OTR values (OTR = k,a( Ct - C,_)). The reduction of the dissolved oxygen concentration at higher agitation speed is shown in Table 1. A slight increase in CPR values is observed with agitation (Fig. 3) before the maximum values are obtained. This is mainly because partial TABLE 1 Time Dependence of Dissolved Oxygen Dissolved oxygen % saturation

Time (h)

0 2 4 6 8 10 12

300 i-pm

400 rpm

500 rpm

600 rpm

700 rpm

800 rpm

1 oo*oo 95-76 87.7 1 54.60 1.36 7.22 2.65

100~00 94.28 91.60 79.61 37.05 15.09 12.92

100~00 96.96 95.55 81.24 48.15 11.29 12.17

100~00 98.45 97.40 94.09 X6.70 60.96 27.19

100~00 98.90 98.90 94.32 85.90 61.82 35.30

100~00 98.97 98.81 97.56 94.32 85.38 70.9 1

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A2ltotlon (rpm) .

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200

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0

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A

200

Fig. 3.

0

200

Variation of CPR with the degree of agitation.

150

M. N. A~~~

C. R. comic,

G. McKay

anaerobic conditions which might exist during the growth produce CO2 as well as aerobic conditions. At higher agitation speeds damage to the cells occurs, reducing activities and hence production rates, as indicated at speeds greater than 700 rpm. The effect of the different degrees of mixing on the specific oxygen uptake rate, kr, is shown in Fig. 4. An increase in kr values is noticed by ~cr~s~g agitation up to 500 rpm due to the higher rates of OTR (kr = OTFt/Cm). Due to the similar effects on both cell mass and OTR, a constant value of kr of around 21 (mmol/g h) is observed during the exponential growth phase. That is equivalent to the maximum specific growth rate, p,,. Lower kr values are obtained at higher agitation speeds due to the adverse effects on cell mass and oxygen transfer rates. Figure 5 shows little effect of agitation on the specific carbon dioxide production rate, kc. This is because of the similar effects of agitation on both CPR and cell mass concentration. The drop in kc values towards the end of the growth cycle is due to the reduction in the CPR while the total cell population which consists of active cells, inactive cells and dead cells is still increasing. The magnitude of this reduction is higher at lower agitation speeds. It was shown in Table 1 that the driving force f CE - CL} is reduced by agitation, This is mainly because of the increase in the surface renewal

Af#tatton bpm) . x

900

0

400

0

100

$00

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700

A

(Loo

Fig. 4.

The effect of agitation speed on kr.

Mass transfer studies in batch fermentation

151

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Variation of kc with the degree of a@tation.

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Variation of k,a with the degree of agitation.

152

M. N. Ahmad, C. R. Holland, G. McKay

rate (Danckwerts, 1951), and the reduction of the boundary layer surrounding the air bubble (Whitman, 1923), and the decrease in the bubble size by the increased shear. Similarly, through the work of Koetsier (Koetsier et al., 1973), Calderbank (Calderbank et al. ) and others, it has been shown that the mass transfer coefficient, kL, the inter-facial area, a, and the gas holdup, Hu, increase with increasing agitation rate, and consequently will produce an increase in the oxygen transfer coefficient, k,a. This is seen to be true in Fig. 6. k,a increases from 75 h-l at 300 r-pm to 630 h-l at 800 rpm. This effect is due to (i) the increased shear created by the agitator, which breaks the larger bubbles coalescence; (ii) the greater turbulence which improves mixing; and (iii) the increased residence time the bubbles have in the fermenter. Similar values were obtained by other researchers as shown in Table 2. The data are correlated by eqn (2) which has a correlation, r*, of o-994. kL a = l-079 x 10e3 N1’96 (2) where: k,a = oxygen transfer coefficient (h- *), N= agitation speed (r-pm). THE EFFECT

OF AIR FLOW RATE

Air flow rate which characterises aerobic fermenters, was varied between O-21 litres/min to l-26 litres/min and its effect on the oxygen

Comparison

TABLE 2 of k,a Values Obtained in the Present Work with Other Researchers k,a values (h-l)

Present work ( Candida u tilis ) Mukhopadhay and Ghose (McAleavy, 1987) (Saccharomyces cerevisiae) Kaeppeli and Fiechter (1980) ( Trichosporon cutaneum) Heineken ( 197 1) (Bacillus subtilis )

Fermenter volume (litres)

300

500

700

75

232

385

1.50

90

169

216

3-00

100

250

400

5.00

78

168

330

2.10

Mass transferstudies in batch fermentation

153

transfer rate at the different stages of the growth cycle is shown in Fig. 7. The OTR increases from 57 mmol/litre h at 0.21 litres/min air rate to 205 mmol/litre h at l-05 litres/min air flow rate. Based on the fact that oxygen transfer rates were evaluated from the equation reco~ended by Cooney et al. (1977). P O(1) -pw(li

pOC21 -&Cl,

PT -f&21

-&2)

(3) -&(a

where OUR = oxygen uptake rate (mm01 O~/~tre/h), FN = molar flow rate of inert (NJ gas per hour, V, = liquid volume (litres), PT = total pressure (atm), PO{, ) = partial pressure of oxygen in the inlet gas (atm), P = partial pressure of water in the inlet gas (atm), Pz:‘= partial pressure of the carbon dioxide in the inlet gas (atm), Pot 2j = partial pressure of oxygen in the outlet gas (atm), PwC2)= partial pressure of water in the outlet gas (atm), PCczj= partial pressure of carbon dioxide in the outlet gas (atm).

Air ’

0.41

Fig. 7.

0

0.m

tmta(Wmhnln) 0

0.84

v

1.08

Q I.P(I

Variation of OTR with air flow rate.

154

M. N. Ahmd,

C. R. Holhmd, G. McKay

The oxygen transfer rate (OTR) is nearly equal to the oxygen uptake rate, OUR, especiaily at the limiting conditions. According to eqn (3), it will be expected that an increase in the air flow rate will increase the OTR. There is also an indication that there is a limiting air flow rate above which no major increases in OTR are obtained. This phenomenon was observed when the higher air flow rates had little effect on the biomass production after su~cient amounts of air were pumped into the system (Ahmad et af, 1991). Kaeppeli and Fiechter ( 1980) reported similar findings. Carbon dioxide production rates were calculated from a similar equation recommended by Cooney et al. (1977).

&

-&cl,

(4)

According to the equation, it would be expected that the variation in air flow rates on CPR has a similar efefct to that on OTR. This effect is shown in Fig. 8, where a slight increase in CPR values occurs with increasing air flow rate. The dissolved oxygen concentration, CL, is maintained at higher levels at higher air flow rates. This promotes aerobic respiration with CO2 being produced. However, with the reduction in air Bow rate, the concentration of the dissolved oxygen tends to oxygen limitation quicker, increasing the time the fermentation operates under partial anaerobic conditions. Consequently, due to the Pasteur effect, the concentration of CO, present in the exhaust stream increases and overcomes the drop in air flow rates to produce CPR values as high as those of aerobic growth conditions. The effect of air flow rate on kr is shown in Fig. 9. An increase in kr from 6.93 mmol/g h at air rate of @21 ~tres/~ to 22.4 mmol/g h at 1.26 litres/min is observed. This is due to the rapid increase in the OTR (Fig. 7) with increasing air flow rate, while the air flow rate has little effect on the biomass production, especially at high flow rates. This makes the OTR a major factor in the evaluation of kr

where Cm is the total cell mass concentration (active, inactive and dead cells). The increase in OTR with increasing air flow rate is essentially due to the increase in the total inter-facial area between the dispersed gas and the liquid phase.

155

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6 mm tiu

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ii?

l4

18

Alr rata(IitrUmln)

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Fig. 8.

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0

0.84

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tlte

The effect of air flow rate on CPR.

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Fig. 9.

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The effect of air flow rate on kr.

1

156

M. N. Ahmad, C. R. Holland, G. McKay

rate, kc, is evaluated

Since the specific carbon dioxide production according to the equation:

it would be expected that both CPR and Cm control the values of kc. It was shown that air flow rate had little effect on both Cm and CPR, especially at higher flow rates. For this reason, the air flow rate should have little effect on kc values. This is borne out in Fig. 10. An average value of approximately 10 mmol/g h is obtained at the different air flow rates. Values of kc drop towards the end of the growth cycle due to a decrease in CPR values, while the total cell concentration (active cells, inactive cells and dead cells) is increasing. The OTR increases with air flow rate (Fig. 7), and so does the mass transfer coefficient, k,a. This increase is observed in Fig. 11. An increase in k,_a from 59 to 232 h-l is observed by increasing the air flow rate from O-42 to l-36 litres/min. Similar findings were reported by Figueiredo and Calderbank ( 1979), who demonstrated that k,a is strongly influenced by the superficial gas velocity. However, Bjurstrom et

100-i

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0.1

0

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2

4

6

8

10

12

14

Tim. Air

*I

mta (litm/mln)

.

0.21

v

0.42

0

0-m

0

0.24

X

1.06

0

1.22

Fig. 10.

The effect of air flow rate on kc.

l8

Mass transfer studies in batch fermentation

157

0.6 t 01 0

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4

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8 rm* inl

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10

12

14

16

Air rate (litmdmin) 0

0.42

Fig. 11.

0

0.84

A

1.06

0

1.26

The effect of air flow rate on k,a.

al. (1985) have shown that k,a is only dependent on the aeration rate on a large scale, and largely independent on a small scale system. The results of this study agree strongly with McAleavy ( 1987) and the work of other researchers (Heineken, 1971; Sobotka & Vortruba, 1979; Kaeppeli & Fiechter, 1980; Pareilleux & Vinas, 1983).

CONCLUSIONS The effect of the intensity of mixing and the degree of aeration on mass transfer parameters were investigated. It was found that the oxygen transfer rates are increased by increasing agitation and air flow rates up to limiting values of both agitation and aeration. The specific oxygen uptake rate, kr, which characterises the aerobic fermentation, was found to increase by agitation and the increase in air flow rates. It was also concluded that agitation and aeration has negligible effects on CPR and consequently kc, which were found to be dependent on the substrate concentration only. Turbulent conditions created by higher agitation speeds, and the increase in the interfacial area at higher air flow rates, have remarkably increased the mass transfer coefficients, k,a.

158

M. N. Ahmad, C. R. Holland, G. McKay

REFERENCES Ahmad, M. N., Holland, C. R. & McKay, G. (199 1). Effect of system variables on biomass production by fermentation using C. utilis. J. Chem. Technol. Biotechnol. (submitted). Bjurstrom, E. E., Just, J. & Swartz, J. R. (1985). Analysis of the behaviours of some industrial microbes towards oxygen. In Biotech. ‘85 USA. Online, Pirme, UK, pp. 527-44. Calderbank, P, H., Johnson, D. S. L. & Loudin, J. ( 1970). Mechanics and mass transfer of single bubbles in free rise through some Newtonian and nonNewtonian liquids. Chem. Engng Sci., 25,235-56. Cooney, C. L., Wang, H. Y. & Wang, D. I. C. (1977). Computer aided material &Iancmt7for prediction of fermentation parameters. Biotechnol. Bioengng, Danckwerts, P. V. (195 1). Significance of liquid-film coefficients in gas absorption. Znd. Engng Chem., 43,1460-7. Figueiredo, M. M. L. & Calderbank, P. M. (1979). The scale-up of aerated mixing vessels for specified oxygen dissolution. Chem. Engng Sci., 34, 1333-8. Heineken, F. G. (1971). Oxygen mass transfer and oxygen respiration rate measurements utilising fast response oxygen electrodes. Biotechnol. Bioengng, 13,599-618. KaeppeIi, 0. & Fiechter, A. ( 1980). Biological methods for the measurement of the maximum oxygen transfer rates of a bioreactor at definite conditions. Biotechnol. Bioengng, 22,1509-12. Koetsier, W. T., Thoenes, B. & Frankena, J. F. (1973). Mass transfer in a closed sitrred gas/liquid contactor, part 1: the mass transfer rate, kLs. Chem. Engng .Z.,5,61-g. McAleavy, G. (1987). Mass transfer studies in batch fermentation. PhD thesis, The Queen’s University of Belfast, Belfast, Northern Ireland. PareiIIeux, A. & Vinas, R. (1983). Influence of the aeration rate on the growth yield in suspension cultures of catharanthus. J. Ferment. Technol., 61, 429-33. Philhps, D. H. & Johnson, M. J. ( 196 1). Measurement of the dissolved oxygen in fermentations. Biochem. Microbial. Engr., 3,26 l-75. Sobotka, M. & Vortruba, J. (1979). Evaluation of aeration capacity of fermenters by adaptive identification method. Collect. Czech. Chem. Commun., 45 (8), 2267-71. Terui, G., Kormo, N. & Sase, M. (1960). Analysis of the behaviours of some industrial microbes towards oxygen. Technol. Rept. Osaka University, 10, 527-44. Whitman, W. (1923). The two-film theory of gas absorption. Chem. Met. Engng, 29,146-8. WinkIer, M. (1981). Biofogical Treatment of Waste Water. EIIis-Horwood, Chichester, p. 68.