Effect of Oxygen Supply Rates on Growth

Transcription

1 APPLIED MICROBIOLOGY, Jan., 1965 Vol. 13, No American Society for Microbiology Printed in U.S.A. Effect of Oxygen Supply Rates on Growth of Escherichia coli I. Studies in Unbaffled and Baffled Shake Flasks L. E. McDANIEL, E. G. BAILEY, AND A. ZIMMERLI Institute of Microbiology, Rutgers, The State University, New Brunswick, New Jersey Received for publication 10 September 1964 ABSTRACT McDANIEL, L. E. (Rutgers, The State University, New Brunswick, N.J.), E. G. BAILEY, AND A. ZIMMERLI. Effect of oxygen-supply rates on growth of Escherichia coli. I. Studies in unbaffled and baffled shake flasks. Appl. Microbiol. 13: The effect of oxygen-supply rates on bacterial growth was studied in commercially available unbaffled and baffled flasks with the use of Escherichia coli in a synthetic medium as a test system. The amount of growth obtained depended on the oxygensupply rate. Based on oxygen-absorption rates (OAR) measured by the rate of sulfite oxidation, equal OAR values in different types of flasks did not give equal amounts of growth. However, growth was essentially equal at the equal sulfite-oxidation rates when these were determined in the presence of killed whole cultures. Specific growth rates were reduced only at oxygen-supply rates much lower than those at which the total amount of growth was reduced. For the physical set-up used in this work and with the biological system employed, Bellco 598 flasks and flasks fitted with Biotech stainless-steel baffles gave satisfactory results at workable broth volumes; unbaffled and Bellco 600 flasks did not. For many years, shake flasks have been employed as the usual tool for laboratory studies with aerobic microbial processes. Because of the relative ease of carrying out large numbers of tests, primary screening, development of fermentation media, and testing of mutant cultures are nearly always done in small-scale shaken cultures before fermentor studies are carried out. The shaking methods commonly used and the procedures for measuring and controlling aeration in shake flasks and other laboratory equipment were reviewed by Lockhart and Squires (1963). There have been many reports on oxygenabsorption rates in shake flasks as determined by polarographic measurements or by measurement of sulfite-oxidation rates. The most comprehensive study was that of Chain and Gualandi (1954). Finn (1954) tabulated oxygen-transfer rates from a number of papers for shake flasks and for other types of equipment used for cultivating microorganisms. A major deficiency in using ordinary Erlenmeyer flasks as shake culture vessels is that, at liquid volumes which can conveniently be used, the oxygen-transfer rates obtained are comparatively low and oxygen supply may become limiting for cultures with high oxygen demands. Several reports have indicated that oxygentransfer rates in flasks on rotary shakers can be increased as much as 20-fold by use of internal baffles or indentations (Chain and Gualandi, 1954; Gaden, 1962; Smith and Johnson, 1954). Both indented Erlenmeyer flasks (Bellco Glass, Inc., Vineland, N.J.) and stainless-steel baffles (Biotech, Division of Biochemical Processes, Inc., New York, N.Y.) for use in Erlenmeyer flasks are available commercially. It has long been known that oxygen supply affects the amount of growth of some bacteria (Winslow, Walker, and Sutermeister, 1932), and data have been given to show the relation between oxygen-transfer rates in shake flasks and yields 109 of yeast and bacterial cells (Olson and Johnson, 1947; Smith and Johnson, 1954). There have been several reports recently on the effect of oxygen transfer in shake flasks on product formation (Aoki, Kondo, and Momose, 1963; Hajny, 1964; Hajny, Smith, and Garver, 1964). It is apparent that the oxygen-supply conditions employed in shake flasks may greatly affect the results obtained. Unless aeration conditions are used which satisfy the oxygen requirement for the process studied, experiments may be carried out under conditions of oxygen limitation, and the true effect of the variables under study may be difficult to evaluate.

2 110 MCDANIEL, BAILEY, AND ZIMMERLI APPL. MIICROBIOL. The studies reported here were carried out for the purpose of elucidating some of the effects of oxygen-supply rates on the growth of a test bacterium, with the further aim of devising shake procedures which insure against having oxygensupply deficiencies in shake-flask experiments. Our results may serve as a guide in establishing shaking conditions for microbial processes with different oxygen requirements, and for mycelial, as well as for single-celled, organisms. The work was carried out with equipment and conditions which can be duplicated readily in this or in other laboratories. MATERIALS AND METHODS Organism. A strain of Escherichia coli B was used in this work. The culture was maintained by frequent transfer on nutrient agar slants and storage at 5 C. Growth medium. The synthetic growth medium contained (per liter):k2hpo4, g; KH2PO4, 6.75 g; sodium citrate, 0.6 g; MgSO4c7H20, 0.15 g; (NH4)2SO4, 4.5 g; and glucose (cerelose), 27 g. Glucose was sterilized separately. A 100-ml amount of inoculum was added to each liter of growth medium. Inoculum. Inoculum was developed by inoculation from a slant into a medium containing (per liter): K2HPO4, 10.5 g; KH2PO4, 4.5 g; sodium citrate, 0.4 g; MgSO4c7H20, 0.1 g; (NH4)2SO4, 1 g; and glucose (cerelose), 2 g. Incubation was for 16 hr at 37 C on a rotary shaker operating at 220 rev/min. Amounts of 100 ml of medium were used in 250-ml Erlenmeyer flasks. The oxygenabsorption rate was 0.18 mmole of O2 per liter per min. Sulfite values. Oxygen-absorption rates were determined by the method of Cooper, Fernstrom, and Miller (1944) with the use of 0.1 M Na2SO3 and 5 X 10-3 M CuSO4-5H20. We use "OAR" to refer to oxygen-absorption rates measured by this method. Growth. Growth is reported in Klett units, obtained by diluting the culture samples in 0.9% saline to estimated turbidity readings of 50 (range 30 to 60) and reading in a Klett-Summerson photoelectric colorimeter (Klett Manufacturing Co., New York, N.Y.) with a 420-mg filter. The values reported are the readings obtained, multiplied by the respective dilutions. Glucose. Glucose analyses were made by the colorimetric method of Nelson (1944) and Somogyi (1945). Ammonium sulfate. Utilization of ammonium sulfate was followed by the microdiffusion method of Conway (1950). Temperature. The temperature was 37 C in all experiments. Shake flasks. "Unbaffled" flasks were 300-ml Corning 4980 Erlenmeyer flasks (obtained from Arthur H. Thomas Co., Philadelphia, Pa.). "Bellco 600" flasks (Bellco Glass, Inc., Vineland, N.J.) were 300-ml flasks with three indentations at a 350 angle from horizontal and 3 to 4 mm deep. "Bellco 598" flasks also were 300 ml in volume with three 350 indentations but 6 to 7 mm deep. The "Biotech" baffled flasks were standard 300-ml Erlenmeyer flasks with stainless-steel baffles obtained from Biotech. All flasks were capped with cotton and gauze pads held in place with spring-wire clips (also obtained from Biotech). Shaker. The shaker used was a New Brunswick Scientific Co. (New Brunswick, N.J.) model G-25 Gyrotory incubator-shaker. It is a rotary shaker which describes a 2.5-cm circle. The speed of shaking was 230 rev/min in these experiments. RESUmTS Sulfite-oxidation rates in shake flasks. Figure 1 gives OAR for unbaffled, Bellco 600, and Bellco 598 flasks and for flasks equipped with Biotech baffles. Six volumes of 10 to 200 ml were used in each type of flask. Each point is the average of at least five sulfite determinations. Where results were variable, more tests were run; 24 determinations were made with 20 ml in Bellco 600 flasks picked at random and 12 more were made in each of two selected flasks. The results in Table 1 indicate that a considerable part of the variability was due to differences in the shape of the flasks. The rate of gas exchange in and out of the flasks is not believed to affect the sulfite values, because at the 200-ml volume passing an air stream through the gas space did not affect the 5.0A UJ 4.0 _ 3.0 Lii -J E 2.0 < 1.0 cr 0.8 z s 0.6 C. 0.5 O 0.4 LI) < 0.3 z ) I VOLUME, ML FIG. 1. Sulfite-oxidation rates with different volumes in different types of flasks.

3 V!OL. 13, 1965 TABLE 1. Flasks EFFECT OF OXYGEN-SUPPLY RATES ON E. COLI. I OAR values for 20-ml volumes in Bellco 600 flasks determinations OAR (mmole per liter per min) Mean Range SD Random ±0.18 No No D J 400 ;300 a _ 80 _ _ TIME, HOURS FIG. 2. Growth curves with different volumes in unbaffled flasks. values and at 20 ml the same values were obtained in stoppered flasks as in open ones. Growth curves at different OAR levels. Growth curves obtained with different volumes in unbaffled flasks are given in Fig. 2. One flask at each volume was removed from the shaker every 2 hr, and Klett readings were taken. The effect of oxygen-supply rate on crop of cells is clearly shown. Starting with about 50 Klett units, maximal growth was reached in 8 hr. In subsequent experiments, only 8-hr data are given. At the lower volumes, evaporation was 0.3 to 0.5 ml in 8 hr, which amounted to 3 to 5% at the 10-ml level. No corrections were made for evaporation losses. Reproducibility of shake-flask growth. Table 2 shows the variability in growth in sets of 22 to 24 replicate flasks run at different times. There was considerably greater variability in the three baffled flasks than in the unbaffled flasks. There were differences in mean values as well as in TABLE 2. Reproducibility of 8-hr growth tests in different types of flasks Klett readings Coeffici- Type of flask No. of readings SD ent* of testsvaito Mean Range variation Unbaffled 24 1,610 1,455-1, ± ,940 1,860-2,040 ±55 ± ,550 1,500-1, ±1.8 Bellco ,675 2,490-3, ± ,650 2,400-2,820 ± ,230 2,880-3,560 ±128 :414.0 Bellco ,775 2,450-3,100 ± ,150 2,940-3,360 ± ,150 2,940-3,420 ± Biotech 24 3,505 3,150-3,710 4±159 ± ,455 3,220-4, ± ,440 3,010-4, ±7.6 * Standard reading. deviation as percentage of mean OXYGEN ABSORPTION RATE. M MOLE /LITER / MIN. 111 FIG. 3. Amount of growth in 8 hr related to sulfiteoxidation rates in different types of flasks. variability among the different sets; the reasons for this are not known. Total growth as a function of OAR in different types of flasks. Figure 3 shows the amount of growth at 8 hr related to OAR. The curves represent averages of three experiments, with one flask at each OAR level in each experiment. In all cases, growth increased with increasing OAR, but the pattern of response varied widely among types of flasks. The differences were most pronounced within the 0.5 to 2.0 mmole per liter per min range. Good working volumes in 300-ml flasks are roughly in the 40- to 100-ml range. Both unbaffled and Bel]co 600 flasks gave low growth in this

4 D~~~~~~~~~~~ t N'AFFLED 112 MCDANIEL, BAILEY, AND ZIMMERLI APPL. MIICROBIOL. TABLE 3. Total yields of cells in different types of flasks at 2.6 mmole per liter per min oxygensupply rate 40'_ Klett units* U'-j Type of flask Range Overall avg Unbaffled... 2,690-3,320 3,025 Bellco ,650-3,420 3,020 C V V Q: J lo ^ ~~~~~~C BELLCO 598 Bellco ,550-3,130 2,875 i 2) ~~~~~~~~~D BIOTECH BAFFLES Biotech... 3,050-3,500 3,230 o0 F * Results based on eight experiments. C@YGEN ABSORPTION RATE. MILLIMOLE /LITER/MINUTE FIG. 5. Per cent conversion of glucose to cells at different OAR levels. llō d FFLASSK D 6 ULNBAFFLED,,, * BELLCO 600 en 4 _ * BELLCO 598 O A BIOTECH BAFFLES D 2_ OXYGEN ABSORPTION RATE. MILLIMOLE/LITER/MINUTE FIG. 4. Amount of glucose used in 8 hr at different OAR levels. range. Bellco 598 and Biotech flasks, on the other hand, reached maximum in this range. The maximal growth level reached was highest with Biotech baffles and lowest in Bellco 598 flasks. To check further whether this was a consistent difference, eight experiments were run in which each type of flask was run in triplicate at 2.5 mmole per liter per min. The range of averages for each experiment and the overall average are shown in Table 3. The results were essentially the same as before. In seven of the eight experiments, Biotech flasks gave the most growth; in six of the eight, Bellco 598 flasks gave lowest. Glucose utilization and per cent conversion of glucose to cells. Figures 4 and 5 show glucose utilization and per cent conversion of glucose to cells, related to OAR values. Per cent conversion was calculated by use of the factor 1.35 mg (dry weight) per liter per Klett unit, which was the average of over 200 values correlating the two. These results reflect the changing metabolism in the transition from anaerobic to aerobic conditions, the greatest change occurring in the 0 to 0.5 OAR range. NH4-N utilization. Figure 6 shows that NH4- N utilization closely parallels growth. It would be expected that nitrogen uptake would occur only to the extent required for cell synthesis (Ecker and Lockhart, 1961). Growth in oxygen-enriched atmospheres. To see 0 L.o OXYGEN ABSORPTION RATE. MILLIMOLE/LITER/MINUTE FIG. 6. Amount of ammoniacal nitrogen used in 8 hr at different OAR levels. whether there was a volume effect not explainable by OAR differences, unbaffled flasks were run at different volumes in air atmospheres and in the presence of a mixture of 55% 02 and 45% N2. Sulfite values were found to be increased 2.5-fold in the enriched atmosphere, which is very close to the calculated values. Figure 7 shows that, up to 1.0 mmole per liter per min, equal growth was obtained at equal OAR levels, regardless of the combinations of volume and oxygen tension used. However, between 1.2 and 2.0, the flasks with oxygenenriched gas (and higher broth volumes) gave more growth in all cases. This effect was reproducible in several experiments. Sulfite-oxition rates in fully grown cultures. Sulfite-oxidation rates determined in the presence of organic anticatalysts cannot be used as transfer coefficients unless they are corrected for oxygen back-pressure (Bartholomew et al., 1950). However, to compare observed oxidation rates in cultures with rates in aqueous sulfite in different shaking systems, rates were measured in the presence of phenol-killed cultures which had been grown in the four types of flasks at OAR levels which, on the average, gave Klett readings of

5 VOL. 13, 1965 EFFECT OF OXYGEN-SUPPLY RATES ON E. COLr. I 113 C') F 3000 z 40 I-- w _ 093/6 v ooo _ A8. 0t NL o OXYGEN ABSORPTION RATE. MILLIMOLE/LITER/MINUTE FIG. 7. Amount of growth in 8 hr in air and in 56% oxygen. TABLE 4. Reduction in rate of sulfite oxidation in the presence of 8-hr culture Type of flask Volume* Klett level Oxygen-absorption ratest In In 8-hr aqueous sulfite culture ml Unbaffled ,400-2, Bellco Bellco Biotech Unbaffled ,400-1, Bellco Bellco Biotech Unbaffled in air ,200-2, Unbaffled in 55% * Volume to give indicated Klett levels. t Expressed as millimoles per liter per minute. 1,400 to 1,500 and 2,400 to 2,500, respectively. Phenol (1 %) was added to kill the cultures, and sulfite-oxidation rates were measured at the same volumes. Similar tests were run in unbaffled flasks at OAR levels of 1.8 in air and 1.54 in 55% oxygen, both of which gave 2,250 Klett units in the original experiments. Table 4 shows that the sulfite-oxidation rates were nearly the same for each growth level in killed cultures, although they were very different in the aqueous sulfite tests. Also, there was practically no difference between air- and oxygen-enriched cultures when compared on broth sulfite oxidation rates. Effect of OAR on specific growth rate. Turbidity readings were made after 1 and 3 hr of growth in -Q o.e F *-. _ 0.7 _- 0.6 _. 0.5 V BELLCO 600 FLASKS -, 'I UNBAFFLED 0.3. I L OXYGEN ABSORPTION RATE, MMOLE/LITER/MIN. FIG. 8. Specific growth-rate constants at different OAR levels in unbafjled and in Bellco 600 flasks. unbaffled and Beilco 600 flasks, and the specific growth-rate constants, k, were determined by taking the slopes of the natural log plots over these time intervals. The zero OAR figures were obtained in completely filled, stoppered flasks which had no atmospheric oxygen available. Figure 8 shows that k increased with increasing OAR up to 0.5 mmole per liter per min, above which it was constant. This range was too low to get comparable values in Bellco 598 or Biotech flasks. DiscussIoN FLASKS Whether a particular shake-flask set-up is satisfactory for a specific microbial process depends on its capacity to transfer oxygen into solution and on the oxygen demand and physical

6 114 MCDANIEL, BAILEY, AND ZIMMERLI APPL. MICROBIOL. characteristics of the developing culture. The rate of solution of oxygen should equal or exceed the rate at which oxygen is required by the culture. The aeration requirement must be satisfied at convenient working volumes, roughly in the 40- to 100-ml range for 300-ml flasks. Both the type of growth and the presence or absence of growth on the walls of the flasks are important factors. With the culture used in these studies, a singlecelled facultative bacterium producing a low viscosity broth with no wall growth, both Beilco 598 flasks and flasks with Biotech baffles met those requirements. Other possible methods of increasing oxygentransfer rates are use of larger flasks, use of higher shaking speeds, or use of flasks with deeper indentations. Wetting of closures due to splashing is a limitation of the latter procedures. Baffled flasks were found to give considerably more variable results than did unbaffled flasks. Small differences in the depth of indentations or in positioning of metal baffles may cause significant differences in oxygen-transfer rates. Indented flasks which differ very much from standard may be detected by sulfite-oxidation tests or by standardized growth tests, as with E. coli. The principal precaution one can take with metal baffles is to use flasks which the baffles fit well. Flasks with short necks or with beaded rims are not satisfactory. Volume changes due to sampling or evaporation may increase the variability. The possibility of using sulfite-oxidation rates measured in the presence of killed cultures for comparing shake flasks with different mixing properties is suggested by the results obtained in this work. ACKNOWLEDGMENTS This investigation was supported in part by Public Health Service research grants AI and AI LITERATURE CITED AoKr, R., Y. KONDO, AND H. MOMOSE Studies on inosine fermentation-production of inosine by mutants of Bacillus subtilis. III. Effect of cultural conditions. J. Gen. Appl. Microbiol. 9: BARTHOLOMEW, W. H., E. 0. KAROW, M. R. SFAT, AND R. H. WILHELM Oxygen transfer and agitation in submerged fermentations. Ind. Eng. Chem. 42: CHAIN, E. B., AND G. GUALANDI Aeration studies. II. Rend. Ist. Super. Sanita 17:5-60. CONWAY, E. J Microdiffusion analysis and volumetric error. Crosley Lockwood and Sons, Ltd., London. COOPER, C. M., G. A. FERNSTROM, AND S. A. MILLER Performance of agitated gasliquid contactors. Ind. Eng. Chem. 36: ECKER, R. E., AND W. R. LOCKHART Specific effect of limiting nutrient on physiological events during culture growth. J. Bacteriol. 82: FINN, R. K Agitation-aeration in the laboratory and in industry. Bacteriol. Rev. 18: GADEN, E. L., JR Improved shaken flask performance. Biotech. Bioeng. 4: HAJNY, G. J D-Arabitol production by Endomycopsis chodati. Appl. Microbiol. 12: HAJNY, G. J., J. H. SMITH, AND J. C. GARVER Erythritol production by a yeastlike fungus. Appl. Microbiol. 12: LOCKHART, W. R., AND R. W. SQUIRES Aeration in the laboratory. Advances Appl. Microbiol. 5: NELSON, N A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153: OLSON, B. H., AND M. J. JOHNSON Factors producing high yeast yields in synthetic media. J. Bacteriol. 57: SMITH, C. G., AND M. J. JOHNSON Aeration requirements for the growth of aerobic microorganisms. J. Bacteriol. 68: SOMOGYI, M A new reagent for the determination of sugars. J. Biol. Chem. 160: WINSLOW, C.-E. A., H. H. WALKER, AND M. SUTERMEISTER The influence of aeration and of sodium chloride upon the growth of bacteria in various media. J. Bacteriol. 24: