New Products and New Areas of Bioprocess Engineering

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bubble coalescence can be neglected. It was assumed that below this gas velocity the difference between kLa values in distilled water, in cultivation media in the presence and absence of AFA, is caused by only kL and that this difference in kL holds true for higher gas velocities as well. Above this critical superficial gas velocity the volumetric mass transfer coefficient due to the specific interfacial area is enhanced, but the bubble coalescence is also increased, which reduces the specific interfacial area. In order to compare the specific interfacial area of the investigated systems in the presence and absence of AFAs, the coalescence index mcorr was defined, which was corrected by the difference in kL values in water, nutrient medium and cultivation medium [39].

(kLa)corr

mcorr = 04 (3) (kLa)ref

where (kLa)ref is the volumetric mass transfer coefficient in the reference system.

(kLa)corr = kLa – D(kLa)

(3a)

and kLa is the volumetric mass transfer coefficient in the cultivation medium:

[DkLa = kLa – (kLa)ref ] at wSG = 2 cm s–1

(3b)

A nutrient salt solution with an antifoam agent had the lowest kLa value, and therefore it was chosen as the reference system.

In Table 1, mcorr values for various biological systems and nutrient salt solutions with an antifoam agent [Desmophen 3600, Bayer AG, a poly(propylene

Table 1. Values of mcorr for biological systems and nutrient salt solution in a bubble column at wSG = 4 cm s–1. The reference is the nutrient salt solution with Desmophen which has the lowest kLa value [40]

Biological system	Substrate	(Antifoam)	mcorr
Chaetomium cellulolyticum	glucose	(no antifoam)	2.56
C. cellulolyticum	glucose	(small amount of Desmophen)	1.21
Escherichia coli	casein	(small amount of Desmophen)	1.40
	peptone
Saccharomyces cerevisiae	glucose	(small amount of Desmophen)	1.22
Hansenula polymorpha	glucose	(small amount of Desmophen)	2.29
H. polymorpha	glucose	(small amount of Desmophen)	(with substrate
			limitation 2.17)
H. polymorpha	ethanol	(small amount of silicon oil)	1.52	(with oxygen
			transfer limitation)
H. polymorpha	ethanol	(small amount of Desmophen)	1.52	(with substrate
			limitation)
Nutrient salt solution		(no antifoam)	2.29

202 K. Schügerl

oxide) with a mean molecular weight of 2000] and silicone oil at wSG = 4 cm s–1 are compared [40].

With increasing gas velocity, the volumetric mass transfer coefficient and bubble coalescence are enhanced and the efficiency of antifoam agents

are enlarged, i.e. in the presence of an antifoam agent the mcorr values decrease. For example, during the cultivation of Hansenula polymorpha on

ethanol substrate under oxygen transfer limitation with silicone oil, the cor-

rected coalescence index mcorr reduces from 1.52 at wSG = 4 cm s–1 to 1.23 at wSG = 6 cm s–1. The same mcorr values were obtained during the cultivation of the same yeast on ethanol substrate under substrate limitation with the

Desmophen AFA.

With increasing amounts of AFA in Hansenula polymorpha cultivation medium, kLa passes a maximum at 1.0‰ (w/w) Desmophen. However, gas holdup attains the maximum at 10‰ (w/w) Desmophen [41]. The surface tension diminishes and the surface viscosity increases with increasing antifoam concentration in distilled water and in BSA solution. Foaminess S drops from 200 to 1 s already at low concentrations of Desmophen (<1mg l–1). In a cultivation media of Hansenula polymorpha and E. coli, the surface tension and surface viscosity remain constant during the entire cultivation time in the presence of Desmophen [22]. As already pointed out, the maximum of foaminess which arises at the transition from the growth phase to the production phase during the production of penicillin V by Penicillium chrysogenum can be eliminated by adding Desmophen to the cultivation medium. The foaminess is reduced from 400 to 100 s [26].

Several AFAs (various silicone emulsions produced by Wacker, Goldschmidt, Dow Corning and Bayer, and others) were tested under standardized conditions with regard to their foam-reducing effect on BSA solutions. The surface tension and foaminess were measured at 0.0, 0.0005, 0.001, 0.005 and 0.01% of the active components of antifoam agents [42]. BSA foams exhibit equilibrium surface tensions in the range of 40 mN m–1 with most of the AFAs. The foaminess S decreased with increasing antifoam concentrations and, at an antifoam concentration of 1 mg l–1 (Desmophen) and 5 mg l–1 (Wacker silicones), respectively, the foam disappeared. The silicones from other manufacturers only had partly the same efficiency. Because of the various types of antifoam agents and their different composition, no general relationship could be obtained with respect to their efficiency. However, particular results were published.

Al-Masry [43] investigated the effects of silicone antifoams in tap water in airlift reactors with an external loop with regard to the gas holdup and volumetric mass transfer coefficient. They recommend the following relationship for this special case:

	Ar
kLa = awSGb (1+ x)c 1+	Ad	d	(4)
	41

where x is the antifoam concentration, Ad the down-comer cross a sectional area and Ar the riser cross-sectional area. For the investigated system, a = 0.041, b = 0.083, c = 0.017, and d = 0.414.

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The gas holdup and volumetric mass transfer coefficients were determined in a bubble column of 150 mm diameter and 3 m height as a function of the aeration rate in tap water and a nutrient solution of Candida boidinii in the presence and absence of an antifoam agent [Ucolub N 115, a water-insoluble poly(oxyethylenepropylene) copolymer]. Up to a superficial gas velocity wSG = 3 cm s–1 the gas holdup was not influenced by the antifoam agent. Above that the gas holdup approached a constant value of 0.15. The volumetric mass transfer coefficient was more sensitive to the AFA. Above wSG = 1cm s–1, kLa did not change with the gas velocity. Depending on the antifoam concentration it had a value 0.15 s–1 (0.1% Ucolub + 1% methanol + 1% salt solution) and 0.1 s–1 (0.1% Ucolub in water and in 1% methanol solution). Without an antifoam agent the gas holdup was a factor of three higher at wSG = 3 cm s–1 and the kLa a factor of four higher at wSG=2 cm s–1 [44].

During the production of penicillin G by Penicillium chrysogenum, the addition of lard oil to the cultivation medium increases the dissolved oxygen concentration below 25% of saturation and reduces it above this value [45]. After addition of an antifoam agent to the cultivation medium, the balance between oxygen uptake rate (OUR) and oxygen transfer rate (OTR) is disturbed. The increase in the DOC is probably caused by the stronger reduction of OUR of the fungus (due to its diminished respiration of the fungus) than OTR. The decrease in DOC above this value is due to the stronger reduction of the OTR than the OUR.

The respiration rate of microorganisms can be evaluated by means of the O2 and CO2 balances. Nyiri and Lengyel [46, 47] observed that DOC was reduced and OUR increased after addition of an antifoam agent to the medium. CO2 entrapped inside the bubbles is released changing the composition of the offgas, if the foam is destroyed. This can reduce the dissolved CO2 concentration in the medium and enhance the respiration of the microorganisms, which causes an increase in OUR and a decrease in DOC.

2.3.3

Influence of Antifoam Agents on Fluid Dynamics, Cell Growth and Product Formation

It is well known that fluid dynamics influence the process performance. Therefore, bubble velocity and gas/liquid interfacial area were monitored during the cultivation of E. coli. The effect of an AFA on the bubbles was determined by monitoring the bubble velocity with an ultrasound Doppler velocimeter (UDV) in situ [48, 49]. By adding an AFA to the cultivation medium, the mean bubble velocity instantaneously increased by a factor of about two in the airlift tower loop reactor during the cultivation of E. coli [50] (Fig. 1a).

After about half an hour, the bubble velocity dropped to the original value, which indicates that the antifoam had disappeared from the cultivation medium. However, after several antifoam additions, the base line and the maxima of the bubble velocity gradually increased. The cultivation medium became more and more coalescence promoting. Monitoring the intensity of the reflected ultrasound allowed the specific gas/liquid interfacial area a to be measured in situ (Fig. 1b). The specific interfacial area a

204	K. Schügerl

a

b

Fig. 1. Measurements during the cultivation of recombinant E. coli K-12 MF cells in a 60-l working volume air lift tower loop reactor without gene expression using SE9 antifoam agent (AFA) [50, 51]. a Variation in the mean bubble velocity. Bubble velocity measured in situ by an ultrasound Doppler technique. AFA added to the medium. b Variation in the specific interfacial area (m–1) measured in situ by an ultrasound technique

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instantaneously reacted to the addition of an AFA (SE9) to the medium [51].

Most research groups have determined the oxygen transfer rate as a function of the AFAs, and only a few have investigated their influence on the cell growth and viability [51–54]. The toxicity of different antifoam agents on Aspergillus niger was tested in Petri dishes [52]. In addition respiration tests were performed in a Warburg apparatus. Most of the antifoam agents inhibited the growth of the fungus. The respiration of A. niger was diminished by AFAs as well; unfortunately, too high AFA concentrations (1%) were applied in these investigations. In practice, the applied AFA concentrations are orders of magnitude lower.

Only Koch et al. [51] monitored the fluid dynamic properties of a two-phase system and the biological behavior of the cells during the same cultivation. They investigated the influence of four different AFAs (Table 2) on the foaminess, oxygen transfer rate and growth of recombinant Escherichia coli and the production of recombinant protein. The specific growth rate m, the colony forming units CFU, the CO2 production rate CPR, the specific glucose uptake rate GUR, and specific production rate (activity of b-galactosidase) SPR, and

yield coefficient YX/Glu , were monitored during the cultivation using four different AFAs (Table 3). The OTR and the volumetric mass transfer coefficient kLa

were also monitored [51]. Each of the AFAs was used in different initial concentrations to suppress foaming during the batch cultivation of E. coli.

The surface tension of the cultivation medium decreased when the antifoam agent was added. Above a critical AFA concentration the surface tension was independent of AFA concentration. SE9: s = 24.5 mM m–1 (at. 5 ppm), S184:

s = 26 mM m–1	(0.5 ppm), VP1133: s = 33 mN m–1 (0.5 ppm), SLM 54474:
s =36 mN m–1	(2.0 ppm). Above a concentration of 150 ppm VP1133, SE9 or

S184, the foaminess of the model medium (with casein peptone and yeast extract) was reduced to below 50 s, and above 200 ppm SLM54474 it was reduced to 100 s [51].

Escherichia coli was batch cultivated in a 2.5-l working volume stirred tank reactor (Biostat; M Braun, Melsungen) and in a 60-l working volume airlift

Table 2. Characterization of AFAs applied during the recombinant E. coli cultivation [51]

S184	(pure silicone oil) consists of 95% diethylsiloxane with viscosity of 1000 cP at
	298 K and 5% highly dispersed silicone oxide. Viscosity of the mixture at 298 K:
	2000–4000 cP (Wacker Chemie)
SLM54474	Pure poly(propylene glycol) (PPG) with a molecular weight of 2000 g mol–1
	(Wacker Chemie)
VP1133	(silicone oil/PPG mixture) consists of 20% branched dimethylsiloxane with
	viscosity of 200–600 cP at 298 K, 3.5% highly dispersed silicon dioxide and
	76.5% PPG with molecular weight of 2000 g mol–1. Viscosity of the mixture at
	298 K: 1000–3000 cP (Wacker Chemie)
SE9	(emulsion) consists of 85.4% de-ionized UV-sterilized water, 0.1% sorbic acid,
	4.5% non-ionic emulgators, 10% S184. Viscosity of the emulsion at 293 K;
	5000–20000 cP (Wacker Chemie)

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Table 3. Parameter maxima of the cultivations of E. coli with various AFAs [51]
AFA	AFA	X (l–1)	m (h–1)	P ¥ 10–3	SP ¥ 10–3	YX/S	SG
	(ppm)			(U l–1)	(U g–1)	(g g–1)	(g h–1 g–1)
SLM54474	125	1.59	0.34	27.8	13.7	0.43	0.90
SML54474	250	1.66	0.28	28.9	14.7	0.35	1.00
SLM54474	1000	1.96	0.23	41.0	24.5	0.57	0.78
S184	63	1.69	0.35	26.0	16.0	0.93	0.75
S184	250	1.78	0.35	20.0	10.0	0.45	n.d.
S184	2000	2.10	0.32	19.0	9.0	0.70	0.85
VP1133	50	1.60	0.39	12.4	7.4	0.41	0.70
VP1133	500	1.7	0.42	15.5	9.0	0.53	n.d.
VP1133	1000	1.8	0.28	22.4	14.5	0.48	0.90
MV.GrA		1.76	0.33	23.7	13.2	0.54	0.84
SD.GrA		0.16	0.06	8.0	5.0	0.17	0.30
SE9	70	4.15	0.64	n.d	n.d.	0.80	1.55
SE9	555	3.94	0.41	n.d	n.d	0.40	1.55
SE9	5000	4.98	0.64	4.8	12.0	2.3	1.35
MV.Gr.B		4.36	0.56	n.d	n.d	1.16	1.45
SD.Gr.B		0.49	0.11	n.d	n.d	0.82	0.14

MV: mean value; SD: standard deviation; Gr.A: group A [pure silicone oil and poly(propylene glycol) PPG and silicone oil/PPG mixture]; Gr.B: group B (aqueous emulsion with only 10% S184); X: cell mass concentration; YX/S yield coefficient of growth with respect to substrate; P: productivity of b-galactosidase; SP: specific productivity: SG: specific glucose consumption rate [51]

tower loop reactor. In the 60-l airlift tower loop reactor, S184 had no foamsuppressing effect, but VP1133 was very effective. The foaminess was controlled and maintained below 140 s by the appropriate feeding of VP1133 to the reactor. Gas holdup, OTR, and respiratory quotient RQ = CPR/OTR varied slightly during the addition of the AFA. However, the mean bubble velocity increased immediately after the addition of silicone oil containing an AFA to the reactor (Fig. 1).

In Fig. 2 the key parameters are presented for recombinant E. coli batch cultivation in a 60-l working volume airlift tower loop reactor at constant aeration rate up to 16 h, whereupon the temperature was increased from 30 to 42 °C and gene expression was induced. At the same time concentrated Luria-Bertani (LB) medium was added to the reactor. To avoid oxygen limitation, the aeration rate was increased (Fig. 2a). At 12 h the foaming increased and SE9 was added to the medium. The bubble velocities (Fig. 2b) and the specific gas/liquid interfacial area (Fig. 2c) quickly increased and passed a narrow maximum, but kLa dropped and the OTR was not influenced (Fig. 2d). After the induction of the gene expression by a temperature increase and medium supplement the dissolved oxygen concentration with respect to the saturation increased due to the elevation of the aeration rate (Fig. 2a); the mean bubble velocity (Fig. 2b) and specific interfacial area (Fig. 2c) decreased, OTR increased and kLa remained at low values (Fig. 2d). The mass transfer coefficient with respect to the liquid phase kL dropped from about 1.67 to 0.67 m s–1 after the addition of SE9 to the medium [51].

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a

b

Fig. 2a, b. Variations in the process parameters during the cultivation of recombinant E. coli K-12 MF cells in a 60-l working volume air lift tower loop reactor. At 12 h addition of SE9 AFA to the medium. At 16 h induction of the gene expression by a temperature increase from 30 to 42 °C. At the same time concentrated LB medium is added to the reactor [51]. a Aeration rate and dissolved oxygen concentration with respect to the saturation (pO2) in down-comer and riser, b mean bubble velocity

208	K. Schügerl

c

d

Fig. 2c, d c specific gas/liquid interfacial area, and d oxygen transfer rate (OTR) and volumetric mass transfer coefficient kLa of oxygen

In Fig. 3 the variations in the specific growth rates of recombinant E. coli during cultivation in a 2.5-l stirred tank reactor at different AFA concentrations are shown. With SLM54474 the specific growth rate decreased with increasing AFA concentration, but with S184, VP1133 and S9 no change of m was observed below 250 ppm AFA concentration. In addition, the number of cells which were able to propagate (colony forming units CFU) and the CO2 production rate CPR were determined as a function of the cultivation time for all four AFAs. The results of these measurements are in good agreement. They decrease with increasing SLM54474 concentration and with SE9 their highest values were obtained for 5000 ppm AFA. The AFA concentration did not influence them up to 500 ppm AFA with S184 and VP1133, but above that they decreased. The specific product activity (SPA) was the highest at high SE9 concentrations. With other AFAs no clear concentration dependence was observed.

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a

b

c

Fig. 3 a – c. Specific growth rate m of recombinant E. coli K-12 MF cells as a function of the cultivation time at different AFA concentrations. a SLM54474, b S184, c VP1133

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d

Fig. 3 d SE9 [51]

Silicone-oil-free AFAs influence the OTR only slightly. Silicone-oil-con- taining AFAs considerably reduce OTR in the early stages of the cultivation, but later on they have only a slight effect. The same holds true for their influence on kLa. When a silicone-oil-containing AFA was added to the reactor, the bubble velocities suddenly increased, but within 60–100 min they dropped to their original values, which indicated the removal of AFA from the medium. The removal rate is a first-order process [51] (Fig. 4):

uB = uBoso fA exp(–kt)

(5)

where uB and uB0 are the actual and original bubble velocities (m s–1), so the volumetric concentration of the AFA (ml l–1), fA the signal activity coefficient (ml s–1 ml–1), k the time constant (h–1) and t the time since the maximum of the bubble velocity was attained (h).At the beginning of the cultivation the removal rate is the highest, later this process decelerates.

The addition of an AFA to the medium caused a short increase in the CO2 concentration in the off-gas and the pH value in the medium. Since the solubility of CO2 and the pH value are not influenced by the AFA, the change in their values is due to a short decrease of dissolved CO2 concentration in the medium by foam destruction, which is in agreement with earlier results [46, 47].

The following amounts of AFAs are necessary to complete foam depression in a small stirred tank reactor: S184 (2000 ppm), SE9 (1000 ppm), SLM54474 (300 ppm) VP1133 (100 ppm). The pure silicone oil (S184) is not effective for foam depression in large reactors, due to a low dispersion rate, while pure PPG is not an effective AFA at all. The PPG/silicone oil mixture (VP1133) and the silicone oil emulsion (SE9) are very effective.AFAs in large reactors only slightly influence the cell concentration and product formation. SE9, a silicone oil emulsion, caused a significant increase in the concentration of E. coli and intracellular product and did not impair the CFU and plasmid stability, which was 100% during these investigations. The effect of AFAs on the OTR and kLa is significant at the beginning of the cultivation. Later, this effect is gradually decreased [51].