Pilot Plant Fermentor with Continuous Platinum Electrode Potential

I. The effect of agitation and CO2 on penicillin formation in automatically aerated liquid cultures. L. Nyiri , Z. L. Lengyel. Biotechnology and Bioen...
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R. W. SQUIRES and PETER HOSLER Eli Lilly & Co., Indianapolis, Ind.

Pilot Plant Fermentor with Continuous Platinum Electrode Potential Measurement Oxygen uptake rates given by sulfite oxidation or aeration of the uninoculated medium are substantially higher than those obtained from active cultures. Apparently, the gas-liquid interface is not the limiting factor in this system. Additional baffles increased the power consumption and improved the aeration somewhat, but the improvement was not reflected in the yield obtained from the fermentation

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of delay in obtaining results, fermentation control is a passive operation. Therefore, continuous monitoring techniques are being used to control p H

andd2termine oxygen and carbon dioxide content of exhaust gases. Both the gassing characteristics of the medium and the utilization of oxygen by the microorganism have been shown, using an amperometric method for determining dissolved oxygen (7). However, no information has appeared recently concerning electrode potentials of aerobic fermentations; work prior to 1950 has been summarized ( 3 ) . Description of Fermentor

The fermentor used, similar to units previously described (4, 5 ) , has a capacity of 60 liters, but is normally operated a t 44 liters. All service piping is connected directly t o the tank body, and the agitator and drive unit are attached to a removable head. As an added precaution for sterile operations, there is no

bottom valve on the fermentor. The tank is sampled by putting air pressure on the top of the tank and blowing the sample out through the sparger line. Antifoam can be added from an electrode-actuated solenoid valve system. How'ever, for this work a nonmetabolizable antifoam was added a t the beginning of each run. A schematic drawing of the fermentor and electrode apparatus is shown in Figure 1. The reference electrode assembly is similar to that described (2) for p H measurement. The calomel half cell is housed in a pressure gland assembly outside the tank. Contact with the fermentation broth is made through Tygon tubing filled with saturated potassium chloride and 3y0 agar. The external parts pf the electrode are maintained under pressure equivalent to tank back-pressure by means of a differential

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Schematic drawing of the fermentor and electrode apparatus VOL. 50, NO. 9

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relay. This arrangement automatically increases electrode housing air pressure in response to tank pressure-especially important for sterilization.

“shadow” of the baffles, the electrodes were placed in zones of high turbulence. Readings from several styles of electrodes were within 10 to 15 mv. Input current or grid current requirement of the millivol; amplifier (Beckman, Model MW) was determined to be 2 X ampere. The calomel electrode plus bridge was calibrated while mounted in

Calibration

As spurious results were noted if the platinum electrodes were placed in the

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4bO R.P.M. SO0 RRM. TIME MINUTES Figure 3. Data on gassing of deaerated media are near enough to first order .for estimating oxygen-absorption rate 0.5 cu. foot per minute air, 1 baffle 1264

INDUSTRIAL AND ENGINEERING CHEMISTRY

the tank. pH was determined with a Model N Beckman battery-operated pH meter. The electrode potential followed the expected linear relationship between millivolts and pH. Voltage of the calomel half cell (referred to the normal hydrogen half cell) was calculated using the formula: Eo,, =E, - &bsd - A X pH, where Ec,, = potential of the calomel half cell, Eobsd = observed potential, Eg = 0.6992 = standard oxidationreduction potential for p-quinone, A = 0.0591 = constant at 25’ C. The average Eh for the calomel electrode plus bridge was 425 mv. I n this report, potentials are given either as AE mv. relative to the air saturation potential of the medium or as &bed mv. The more positive potential indicates a more oxidized system; a more negative potential indicates a more reduced system. No attempt is made to correct for pH effects. Calibration with Oxygen Mixtures One of the fundamental purposes of this investigation was to determine if dissolved oxygen could be measured with this apparatus. Figure 2 presents the changes in electrode potential when test gases (supplied by Ohio Chemical and Surgical Equipment Co.) containing various percentages of oxygen and nitrogen were sparged through uninoculated streptomycin fermentation medium. The concentrations were stated to be accurate within 0.5%. According to Henry’s law, the amount of oxygen dissolved in the liquid should be proportional to the partial pressure of oxygen in the gas phase. The reading with air gave a +60 mv., while a mixture of 1% oxygen and 99% nitrogen gave a reading of -20 mv., with other points in between. Oxygen tensions obtained with these gas mixtures were confirmed by polarographic determinations. Polarographic results also confirmed electrode potential measurements which indicated a very low order of oxygen tension during the fermentation. Studies with mixtures of oxygen and nitrogen showed that the electrode potential was proportional to the logarithm of the oxygen concentration and could be useful for evaluating conditions of oxygen tensions. If it is hypothesized that fermentation medium is highly poised and contains a natural reservoir of oxidation-reduction capacity, respiration of the organism must be dependent not only on the dissolved oxygen but also on the aggregate poise of several complex oxidative systems. A rough characterization of these systems was obtained by titrating uninoculated broth with sodium sulfite. Figure 2 shows that a fairly linear relationship exists between oxidizing capacity and millivolt readings.

pH C O N T R O L IN FERMENTATION 50 0 400

Aeration Rate Studies

Many investigators have reported fermentor performance in terms of sulfite oxidation rates. Phillips (6) explains the characteristics of the method. The data shown in Figure 3 are analogous to sulfite data, but performed on uninoculated streptomycin broth previously deaerated with nitrogen. The AE reading is the arithmetic difference between the chart reading and the aerated value at equilibrium. A plot of this type should give a straight line on semilog graph paper if the reaction represented is a true first-order one. The study shown is not strictly first order, but is sufficiently close to allow estimation of the oxygen absorption rate from the slope of the curves on semilog graph paper. When three additional baffles were installed in the fermentor, an oxygen absorption rate at 450 r.p.m. was calculated to be 1.2 mmoles per liter per minute, with only one baffle it was 1.4 mmoles per liter per minute. By comparison, the sulfite oxidation rate with four baffles was 3.8 mmoles per liter per minute. The electrode potential method, in the authors' opinion, gave a more realistic criterion for tank performance, as actual fermentation medium was used for the study and the magnitude of the result was closer to fermentation oxygen uptake. Figure 4 shows oxygen absorption rate with various gas mixtures of oxygen and nitrogen. The potentials obtained at equilibrium with these gases were shown in Figure 2. The equilibrium potentials close together, but the difference in rates of solution is apparent. The absorption rate of reaction, calculated by the method of Phillips, is proportional to the oxygen concentration to the 0.15 power. With an agitator speed of 200 r.p.m. solution rate was independent of oxygen concentration. Oxidation Demand Studies

Figure 5 shows the oxidation rates of active fermentation cultures. Electrode potential decreases when the normal fermentor aeration has been abruptly turned off. Here the respiratory activity of the culture can be correlated with the change in electrode potential. The calibration curve from the sodium sulfite titration (Figure 2) may be used to quantitate these rate curves. The slope of the curve for the 24-hour culture gave an oxygen uptake rate of approximately 0.2 mmole per liter per minute. Rates found by this method were in good agreement with rates calculated from the analysis of exhaust gas during the actual fermentation.

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Fermentation Studies Electrode potentials registered during the fermentation are reported as AE mv., the inoculation time reading being taken as 0 mv. Repeated runs gave substantially reproducible potentials during the fermentation cycle. Changes in aeration and agitation were immediately reflected by changes in the potential. Figure 6 presents a detailed analysis of

a streptomycin fermentation. AE and oxygen uptake appear to be related to the growth curve, while p H and sugar curves indicate the metabolic activity of the microorganism. With the same fermentor when fully baffled the oxygen uptake pattern differed very little from that of the nonbaffled runs. However, changes in aeration were not reflected in immediate changes in potential,

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5. Rates were in good agreement with those calculated VOL. 50, NO. 9

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by oxygen uptake. The final potency, however, is lower with the baffled condition. This may indicate a condition where the culture could not tolerate the higher oxidation-reduction potential level or the medium was not properly balanced for these conditions.

Acknowledgment

The authors thank R. J. Harley for helpful suggestions and D. E. Flick for polarographic analysis. Literature Cited (1) Bartholomew, W . H., Karow, E. O., Sfat. M. R.. Wilhelm. R. H., IND.ENG.

CH&

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f80l (1950).

(2) Deindoerfer, F. H., Wilker, B. L., Ibid.,

1223 (1957). (3) Hewitt, L. F., “Oxidation Reduction Potentials in Bacteriology and Biochemistrv.” E. & s. Livingstone, Edinburgh, Ib50. (4) Kroll, C. L., Formanek, S., Covert, A. S., Cutter, L. A., West, J. M., Brown, W. E., IND.ENG.CHEM.48, 2190 (1956). (5) Nelson, H. A,, Maxon, W . D., Elferdink, T. H., Ibid.,48, 2183 (1956). (6). Phillips, D. H., Johnson, M. J., Division of Agricultural and Food Chemistry, 132nd Meeting, ACS, New York, K.Y., September 1957. 49,

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Hours Figure 6. Chemical changes in a run with one baffle. Oxygen uptake is related to growth curve. pH and sugar curves indicate metabolic activity of streptomycin

while changes in the agitator speed produced a marked decrease in the readings. Average AE curves during the early part of the run were compiled from several runs on both baffled and nonbaffled tanks; in general, the oxidationreduction potential remains higher

throughout the entire run in fully baffled equipment. The conclusion is that slightly better aeration was obtained in the baffled tank, as evidenced by faster sugar consumption, faster potency production, and higher oxidation-reduction potential, even though this was not shown

RECEIVED for review October 31, 1957 L4CCEPTEDJune 17, 1958 Division of Agricultural and Food Chemistry, Symposium on Fermentation Process and Equipment Design, 132nd Meeting ACS, New York, N. Y., September 1957.

Correction Hydrodynamics of Countercurrent Flow in Wetted-Wall Columns Editor’s Note, The editors, the author, and the printer all contributed to some unusual circumstances in which several thousand copies of the form containing Dr. Thomas’ article were off the presses before the corrections listed below could be made, Because it was impractical at the time to rerun the form, and to localize the copies containing the errors, the printer was insrructed to place the faulty copies in the foreign issues. Some may have been put accidently in domestic issues. We regret very much this occurrence and therefore ask our readers, both foreign and domestic, to check the points in error. 1 266

In the article “Hydrodynamics of Countercurrent Flow in Wetted-Wall Columns” [M’. J. Thomas and Stanislaw Portalski, IND. ENG. CHEM.50, 1081 (July 1958)] the following changes should be made. On page 1081, the phrase “without ripples of liquid” should be deleted from Figure 1 caption. On page 1082, column 3, the second and third listings in the Literature Background contains JD.” These should be replaced by j,

On page 1085, the denominator of Equation 3, column 1, should be: gP2

INDUSTRIAL AND ENGINEERING CHEMISTRY

On page 1087, Equation 18, the denominators of the first line should be: =

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The first item of Equation 20 should be (rn - y ) pL and the first item following the equal sign should be ( p ~ / g ) . In Equation 21, the first item of the lower line should be (r/2pL). The item following the parenthetical expression in Equation 34 should read: PL.

On page 1088, nomenclature in column 2, the next to the last item should read: Po, PL