Effect of pressure on an aminoglycoside fermentation mediated by dissolved oxygen

JOIJKNAL or FERMENTATIONAND BIOENOINEEglNG VOI. 73, NO. 1, 66-69. 1992

Effect of Pressure on an Aminoglycoside Fermentation Mediated by Dissolved Oxygen VIJAY YABANNAVAR*, VIJAY SINGH, AND EUGENE SCHAEFER

Schering-Plough Research, Union, NJ 07083, USA Received 13 December 1990/Accepted 7 October 1991 The intent of this work was to determine whether the high pressure due to the static liquid head could account for the yield loss observed in the scaleup of aminoglycoside fermentation. The growth of Micromonospora purpurea and aminoglycoside production were severely reduced when the pressure was raised from 1.68 to 2.37 atm in 101 fermentors. Mass transfer calculations showed that this effect was not due to CO2 solubility. The retardation in growth and production was apparently caused by high DO. When the DO was controlled at low levels, good growth and productivity were observed even at 2.37 atm pressure.

M. purpurea was used in the study. It was grown in a

In scaling up aminoglycoside antibiotic fermentations from the 10-100 l laboratory scale to the 120,000 1 production scale, a loss in the antibiotic titers is often observed. The intent of this work was to determine whether the high pressure due to the static liquid head could account for the harmful effects observed in the aminoglycoside fermentation. Hydrostatic pressures ranging from 200 to 500 arm have been reported to retard the growth of Escherichia coli (1). However, the hydrostatic pressure in a typical large scale antibiotic fermentor is too small (2 atm) to cause such effects. The other possibility is that higher pressures cause a high dissolved carbon dioxide concentration in the medium and this may be responsible for the observed slow cell growth and reduced antibiotic production. Ho and Smith (2) have reported that the specific growth rate and penicillin production of Penicillium chrysogenum were inhibited when aerated with air containing 12.6 and 20% CO2. However, they did not observe any adverse effects at 3-5% CO2 levels in the inlet gas. Crueger and Crueger (3) have mentioned the inhibitory effect of 1-2% CO2 on the production of the aminoglycoside antibiotic sisomicin. Unfortunately, the pH data was not reported and therefore it is not clear how far, if at all, the harmful effect was due to lower pH at elevated CO2 levels. Bernhardt et al. (4) have reported that for thermophilic methanogens, the CO2 effect was mediated through the pH shift, and growth was severely inhibited below pH 5.5. The pressure effect could also have been due to high dissolved oxygen (DO). Kataoka et al. (5) have reported that Pseudomonas aeruginosa growth rates tended to decrease over 1-2 rain pressure cycles, and in steady state operation, the growth rates decreased considerably with increasing pressure or dissolved oxygen concentration. In the present study, the phenomenon of reduced growth of Micromonospora purpurea and reduced antibiotic production was investigated by experiments conducted in 10 1 fermentors operated under high pressure to simulate the high static head conditions prevailing in large industrial fermentors. From these experiments, a strategy for minimizing growth lag in large fermentors was developed. Microorganism and media A proprietary strain of

proprietary seed medium for 3 d and propagated in an inoculum medium in 2 I shake flasks before inoculating the 16 l fermentor. The composition of seed, inoculum and production media is similar to what is shown in Table 1 (Weinstein, M. J. et al., US Patent no. 3,832, 286, 1974). Fermentation Fermentations were carried out in 16 l New Brunswick Scientific fermentors. One liter of inoculum was added to 10 l of the medium. Temperature was maintained at 34°C for the first 2 d, and was reduced to 30°C for the rest of the fermentation. The agitator speed was increased from 400 rpm in increments of 100 rpm each day in the first 2 d, and maintained at 600 rpm thereafter. In some instances, the agitator speed was increased to 700 rpm if necessary to maintain the DO above 20%. The aeration rates were maintained at 3 and 4 l. min-~ during the first and second days and increased to 6 l. min- J for the rest of the fermentation. Off gas was analyzed for nitrogen, oxygen and carbon dioxide using a Perkin Elmer 1200A mass spectrometer. The oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER) were computed as follows: OUR=

1000 Fin [ Co2.in CN2_i.• C02 ] VL(Vm)(T/273) [ 10"------O-- 100 CN2 ]

1000 Fi, CN2-in• Cco2 CER-- VL(Vm)(T/273)'[ 100 CN2

C~i,]

(1) (2)

KLao2 for the oxygen transfer was calculated from the OUR and DO (% saturated with respect to air at any pressure) measurements assuming Henry's Law constant Ho2 = 8 . 3 8 x 10 -I atm.mM -I as follows: OUR KLa02-- (100-DO)

100 H02 P- C02.i.

(3)

The KLac02 for CO2 transfer was then estimated by scaling the KLa02 with the ratio of CO2 and 02 diffusivities. The concentration of CO2 in the medium (Cs) in equilibrium with the off gas was estimated by Henry's Law as follows:

Cs = P" C¢02 (4) 100 Hco2 The Henry's Law constant (H) was taken to be 3.5 x 10-2 atm.mM -1 at 34°C (6).

* Corresponding author.

66

VoL 73, 1992 T A B L E I.

NOTES

Seed, inoculum and production medium for sisomicin

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The C O 2 solubility in the medium ( C L ) w a s then calculated using the mass transfer data as follows: CER =KLaco2 (CL-- Cs)

(5)

Cell growth Cell growth was monitored through measurement of the packed cell volume (PCV). Ten ml of the fermentation broth was centrifuged at 3500rpm for 15 min in an IEC Clinical centrifuge, and the percent (v/v) of the packed solids was measured. Since the packed solids contain both the medium particulates and the cell mass, PCV is only a relative measure of cell growth. Aminoglycoside assay The broth was first acidified with sulfuric acid to pH 2 to release all the antibiotic that is bound to the mycelia and clarified by centrifugation. The aminoglycoside concentration in the broth was measured by a commercial radioimmunoassay (RIA) developed by Clinetics Corp., CA. The RIA principle is described by Yalow and Berson (7). Assay data have been normalized to an arbitrary scale of 0-100%. Effect o f b a c k pressure Experiments were performed in the 10 1 fermentors with 1.68, 2.03 and 2.37 atm pressure. The fermentation profiles for DO, PCV, % CO2 (%v/v) and titer are shown in Fig. 1A, B and C. At 1.68 atm, which is the normal pressure maintained in these fermentations, cell growth started shortly after inoculation (see Fig. 1A) as indicated by the drop in DO. The agitation and aeration were increased at 24 and 48 h resulting in the step increase in DO. Agitation was also increased later in the fermentation, if necessary, to maintain the DO above 20%. The aminoglycoside production started after 48 h, reaching a final titer of 100%. The final cell mass had a PCV of 60% (v/v). A fermentation run at a higher pressure (2.03 atm) showed considerable lag in cell growth and antibiotic production (see Fig. 1B). The cell growth was very slow until about 72-96 h, and the final PCV did not exceed 55% (v/v). The antibiotic production also showed a lag, and the final titer after 168 h was approximately 40% of control. The retardation of cell growth and antibiotic production was even more pronounced at 2.37 atm pressure (see Fig. 1C). T A B L E 2.

CO2 solubility in the medium at different back pressures

Fermentor pressure 0~ CO2 in off gas

(atm)

(~ v/v)

1.68 2.03 2.37

1.20 0.90 0.76

CER

Cs

CL

11.2 8.0 6.4

0.61 0.54 0.53

0.72 0.62 0.59

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Time ( h } FIG. 1. Fermentation profiles at different back pressures. A. 1.68 atm pressure; B. 2.03 atm pressure; C. 2.37 atm pressure. Lines: - - , dissolved oxygen (% sat.); . . . . , carbon dioxide (% v/v). Symbols: A, packed cell volume (% v/v); o , titer (arbitrary units).

Effect o f d i s s o l v e d CO2 The possibility of higher CO2 solubility in the medium at high pressures was investigated as a cause of retarded cell growth and antibiotic production. The dissolved CO2 (H2CO3) is in equilibrium with bicarbonate and carbonate ions. From this equilibrium, it is clear that H2CO3 (dissolved CO2) concentration is proportional to the concentrations of bicarbonate and carbonate ions. Therefore, it is possible to derive conclusions on the effect of CO2 based on just the soluble CO2 concentrations. Table 2 shows the estimated concentrations of CO2 in the medium (CL) at 36 h at 1.68, 2.03 and 2.37 arm pressures. These estimates are based on a Kuaco2of about 100 h -I calculated from 1.68 and 2.37arm fermentations where the DO values allowed a proper estimate. Interestingly, the CO2 concentration in the medium at high pres-

68

YABANNAVAR ET AL. 120

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FIG. 2. Effect o f dissolved oxygen (DO) on fermentation. A. 2.37 atm pressure, 1 5 ~ oxygen feed; B. 2.37 atm pressure, 40% DO control; C. 1.68 atm pressure, 30% oxygen feed. Lines: - - , dissolved oxygen (o~ sat.); . . . . , carbon dioxide (% v/v). Symbols: i , packed cell volume ( ~ v/v); @, titer (arbitrary units).

sures was actually lower than observed at low pressure. This is due to the fact that smaller quantities of CO2 were evolved because of poor cell growth at high pressures as evidenced by the lower CER values shown in Table 2. The COz solubility data suggest that the retarded cell growth and antibiotic production could not be due to inhibitory effects of high COz concentrations. Neither was this effect due to any pH shift, because in all these experiments the pH was almost constant at 7.1. Effect of DO After discounting the effect of CO2, we wanted to investigate if the high solubility of oxygen could be contributing to the slow cell growth and production at high back pressures. To demonstrate that high DO concentration rather than high total pressure is important, an experiment was run at 2.37 atm but with only 15% oxygen in the inlet air. This provides a DO level similar to that ob-

served in the control 1.68 atm fermentation sparged with air. The fermentation profiles for this condition are shown in Fig. 2A. The cell growth and the antibiotic production in the fermentor with 15% oxygen feed were very similar to those in the control fermentation at 1.68 atm (see Fig. 1A) but significantly better those from the control fermentation at 2.37 atm sparged with air (see Fig. 1C). Another fermentation was run at 2.37 atm where the DO was controlled at 40% throughout the duration by mixing the appropriate amounts of nitrogen and air. In this case, the mycelial growth and the antibiotic productivity were at least as good as the control, or slightly better (see Fig. 2B). A study of the DO profiles along with the pressures in all these fermentations reveals that a very high amount of oxygen dissolved in the broth is associated with poor mycelial growth. To confirm the fact that oxygen at high levels was toxic to the cells, a fermentation was conducted with oxygen enriched air (30% oxygen) at the normal 1.68 atm pressure (see Fig. 2C). The growth of the mycelium was extremely slow, indicating oxygen toxicity. In order to speculate on the possible causes for oxygen toxicity, we need to consider the cellular processes involving oxygen. The oxidations of flavoproteins by 02 in bacteria result in the formation of a toxic compound, H 2 0 2. In addition, these oxidations (and possibly other enzyme catalyzed oxidations) produce small quantities of an even more toxic free radical, superoxide or 02. -. In aerobes, the accumulation of superoxide is prevented by the enzyme superoxide dismutase which catalyzes its conversion to 02 and H202. Aerobic bacteria also contain the enzyme catalase which decomposes H 2 0 2 tO 0 2 and water (8, 9). It is possible that this essential protective enzyme system is overwhelmed by exposure to excessive oxygen concentrations. Especially in the case of Micromonospora purpurea, a soil isolate, this effect may be quite evident resulting in poor growth. Optimization of operating conditions in large scale letmentors After establishing that lag in cell growth is due to high DO, it is clear that any operating strategy must be such that the DO is kept low until sufficient growth is observed. At the industrial scale, it is obviously not economic to lower the initial DO using nitrogen gas. However, by maintaining low aeration, agitation and back pressure at inoculation, it is possible for the culture to consume the available oxygen quickly and cause the DO to drop to noninhibitory levels, stimulating further growth. This strategy should effectively reduce the growth lag in large fermentors with high liquid static pressure. Once growth begins, aeration, agitation and back pressure may be increased to maintain the DO above the limiting concentrations (20% saturation at 1.7 atm). NOMENCLATURE

Cco2 Cco2-i, CN2 CN2_in

Co2 Co2.i, CER CL Cs DO

: °J0CO2 in the off gas, ~ (v/v) : °7oCO2 in the inlet gas, 0~ (v/v)=0.033% : °~oN2 in the off gas, ~ (v/v) : °~oN2 in the inlet gas, °Jo (v/v)=78.08°fo : °foO2 in the off gas, °J0 (v/v) : %02 in the inlet gas, ~ ( v / v ) = 2 0 . 9 5 ~ : CO2 evolution rate, mmol. l- '- h - i : CO2 concentration in the inedium, mM : CO2 concentration in equilibrium with off gas, mM :dissolved oxygen concentration (% saturation

VOL. 73, 1992

F~, Hco2

902 KLaco 2 KLa02 OUR P T VL Vm

NOTES

w i t h respect to air) : inlet air flow rate, l . h - I : H e n r y ' s L a w c o n s t a n t for CO2, a t m . m M -~ : H e n r y ' s L a w c o n s t a n t for O2, a t m . m M -~ : m a s s t r a n s f e r coefficient f o r CO2, h - I : m a s s t r a n s f e r coefficient f o r 02, h - I : o x y g e n u p t a k e rate, m m o l . l - 1. h : a b s o l u t e p r e s s u r e in t h e f e r m e n t o r , a t m : t h e r m o d y n a m i c t e m p e r a t u r e , °K : liquid v o l u m e , / : m o l a r v o l u m e o f gas, 1. m o l - i = 22.4 l. m o l - 1

REFERENCES 1. Zobell, C.E. and Cobet, A.B.: Filament formation by Escherichia coil at increased hydrostatic pressures. J. Bacteriol., 87, 710-719 (1964). 2. Ho, C. S. and Smith, M. D.: Effect of dissolved carbon dioxide on penicillin fermentations--Mycelial growth and penicillin production. Biotechnol. Bioeng., 28, 668-677 (1986).

69

3. Crueger, W. and Crueger, A.: Biotechnology, 2nd ed., p. 92. Sinauer Associates Inc., Sunderland, MA (1989). 4. Bernhardt, G., Disteche, A., Jaenicke, R., Koch, B., Ludemann, H.-D., and Stetter, K.-O.: Effect of carbon dioxide and hydrostatic pressure on the pH of culture media and the growth of methanogens at elevated temperature. Appl. Microbiol. Biotechnol., 28, 176-181 (1988). 5. Kataoka, H., Sato, S., Mukataka, S., Namiki, A., Yoshimura, K., and Takahashi, J.: Effect of periodic change in pressure and dissolved-oxygen concentration on the incubation characteristics of Pseudomonas aeruginosa. Biotechnol. Bioeng., 28, 663-667 (1986). 6. Ballou, W. R.: Carbon dioxide, p. 725-742. In Kirk-othmer encyclopedia of chemical technology, vol 4. John Wiley and Sons, New York, NY 0978). 7. Yalow, R.S. and Berson, S.A.: Immunoassay of endogenous plasma insulin in man. J. Clin. Invest., 39, 1157-1175 (1960). 8. Stanier, R. Y., Adelberg, E. A., and Ingraham, J. L.: The microbial world, p. 310. Prentice Hall Inc., NJ (1976). 9. Fridovich, I.: Oxygen toxicity in procaryotes: the importance of superoxide dismutase, p. 79-88. In Oberley, L. W. (ed.), Superoxide dismutase, vol. 1. CRC Press Inc., Baco Raton, FI. (1982).