AERATION REQUIREMENTS FOR THE GROWTH OF

AERATION REQUIREMENTS FOR THE GROWTH OF AEROBIC MICROORGANISMS1 CHARLES G. SMITH AND MARVIN J. JOHNSON Department of Biochemistry, Colege of Agriculture, University of Wisconsin, Madison, Wisconsin Received for publication March 8, 1954 When yeasts are grown for the production of cells, the dry cell yields obtained are approximately 45 per cent of the substrate utilized, corresponding to cell concentrations on a dry weight basis of 10 to 44 mg per ml (Harris et al., 1948; Feustel and Humfeld, 1946; Maxon and Johnson, 1953). The cell yields obtained with bacteria are lower than those obtained with yeasts. Bacterial populations as high as 57 billion and 84 billion per ml have been reported (Gerhardt and Gee, 1946; Gee and Gerhardt, 1946; Gerhardt, 1946; McCullough et al., 1947). Although the cell concentrations were not reported, the dry cell weight corresponding to these live counts should be approximately 10 mg per ml. It should be possible, by application of the principles employed for growing yeasts, to obtain the same yields of cells when growing aerobic bacteria. The experiments reported here are concerned with the effect of aeration efficiency on the final dry cell yield and live cell count of bacteria. The organism chosen for this study, Serratia marcescens, is highly aerobic, easily cultured on a synthetic medium, readily counted when spread on the surface of an agar plate, and free from large amounts of slime and capsule. It was found that the yield of cells obtained on the synthetic medium in liquid culture varied directly with the aeration efficiency, the highest yield based on substrate utilized being approximately 38 per cent. The highest cell concentration on a dry weight basis obtained in this work is 9 mg per ml, corresponding to a live cell count of 0.1 X 1010 per ml. The effective aeration necessary to support such high populations of Serratia was much higher than that commonly employed in the laboratory for the aerobic growth of microorganisms. MATERIALS AND METHODS Chemical and bacteriologal methods. The two strains of S. marcescens used in this study were strain 8 UK, obtained from Camp Detrick, Maryland, and a strain isolated by Dr. W. B. Sarles at Wisconsin. The former strain is pigmented while the latter is not. The stock cultures of both strains were reisolated periodically by plating on glucose-citrate agar and transferring an isolated colony into liquid culture. The stock cultures were carried on glucosecitrate agar slants held at 5 C. It was found that cell numbers and weights could be estimated fairly accurately turbidimetrically, with an Evelyn photoelectric colorimeter, by measuring the light transmision of a suitably diluted sample. The turbidimetric results were used only to calculate dilutions for plating and to determine proper harvesting times. Dry cell weights were made by weighing the centrifuged and washed cells after drying in an oven for 1 hours at 10 C. Live cell counts were made by spreading the final dilution of the culture medium on the surface of nutrient agar plates, followed by incubation at 30 C. Each sample was counted in duplicate with a minimum of 170 colonies per plate. The synthetic medium used for growth of the organism was devised to allow maximum yields of cells to be obtained with a minimum of residual material remaining in the spent medium. The composition of the synthetic medium in grams per liter is as follows: citric acid monohydrate, ; NaSO4, 0.5; KHP04, 5.0; glucose, 10 to 60; MgS04-7H0, 0.8; NaCl, 0.04; FeSO4-7H0, 0.04; MnSO.44H0, 0.04. All the constituents of the medium except the glucose are dissolved in distilled water and the ph of the medium adjusted to 7.7 with NH40H prior to autoclaving. The glucose is autoclaved sepa- 1 Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. The ph of the medium after sterilization is aprately and added aseptically before inoculating. 346

1954] GROWTH OF AEROBIC MICROORGANISMS 347 proximately 6.8. During growth, the ph drops to 5. to 5.5 in the early log phase and rises to 8.0 to 8.5 as the sugar is exhausted. If a low dissolved solids content is desired in the spent medium, the concentration of citric acid is varied with the glucose concentration to give an initial ratio of glucose to citric acid of :1. The inoculum was cultured by inoculating a flask containing the synthetic medium directly from the slant. The liquid culture obtained was used then to inoculate the flasks used in the experiments. In all cases the culture was incubated at 30 C. Foaming was controlled by adding one drop of heptadecanol (3,9-diethyltridecanol-6, Carbide and Carbon Corp.) to each shake flask at the time of inoculation and as required during growth. Sugar was determined by the method of Shaffer and Somogyi (1933). Nitrogen was determined by the micro-kjeldahl method described by Johnson (1941). Inorganic phosphate was determined by a modified Fiske-Subbarow method (195). Citric acid was determined by the colorimetric method of Saffran and Denstedt (1948). Aeration methods. The limiting factor in obtaining a high cell concentration of microorganisms in the laboratory is usually the oxygen available to the cells. The transfer of oxygen from the gas phase to the liquid phase is probably the limiting factor in aeration of cultures. Maxon and Johnson (1953) have correlated the oxygen transfer, as determined by the sulfite oxidation procedure of Cooper et al. (1944), with the oxygen actually available to yeast cells. Aeration efficiency has been employed in this work as a measure of the oxygen available to the cells. The aeration efficiency is referred to in this paper as sulfite oxidation value, expressed as mm 0 per liter per min. Aeration procedures traditionally employed in the laboratory for the cultivation of aerobes give effective aeration rates much lower than those necessary for the attainment of high cell concentrations. Effective aeration rates of some commonly employed aeration devices are shown in table 1. All of the sulfite values reported in table 1 for shake flasks were determined with the plugs removed. The data in the table show that stationary culturing gives very poor aeration efficiencies, 0.3 mm 0 per liter per min maximum obtained in this work. Shake flasks with small volumes of medium are effective up to a sulfite value of, whereas the sides of the flasks must be indented in order to obtain sulfite values higher than. In this work, regular 500 ml TABLE 1 Effective aeration rates of some commonly employed laboratory aeration procedures VESSEL VOLUME OF MEDIUM AERATION PROCEDURE AERATING RATE EFETIV AERATION ml Vol per Vol mm Ot Per L per min per min 18 by 150 mm test tube 10 stationary 0.03 500 ml Erlenmeyer 0 stationary - 0.3 500 ml Erlenmeyer 0 shaken* 1.1 500 ml Erlenmeyer 10 shaken.0 500 ml Erlenmeyer 50 shaken - 0.60 500 ml Erlenmeyer 100 stationary 0.10 500 ml Erlenmeyer 100 shaken - 0.7 500 ml indented Erlenmeyer 0 shaken to 9.5 18 L bottle 15,000 8 mm tube immersed 1.0 0.06 18 L bottle 15,000 10 cm sintered steel sparger 1.0 0.60 30 L fermentor 15,000 500 rpm agitator plus sparger 1.0.0 (Rivett et al., 1950) 100 gallon fermentor 50,000 50 rpm agitator plus sparger 1.0 1.0 (Stefaniak et al., 1946) 3.5 L fermentor 1,500 1,900 rpm agitator plus spar- 3.3 10.0 (Maxon and Johnson, 1953) ger * Shaken on a Gump rotary shaker at 50 rpm. The authors are indebted to members of this laboratory for some of the values reported in this table.

348 CHARLES G. SMITH AND MARVIN J. JOHNSON [vol. 68 Erlenmeyer flasks were used for sulfite values up to 1, whereas indented Erlenmeyer flaks were used for sulfite values of to 9.5. The sulfite value obtained with an indented flsk varies with the size and the number of indentations. In order to maintain the high sulfite values employed here, sterile air was bled into the shake flasks via a tube inserted through the plug. For large scale cultivation of aerobes, aeration with an immersed tube is extremely inefficient, whereas aeration with a fine sparger is somewhat more effective. In order to obtain high aeration efficiencies on a large scale, aeration must be accompanied with agitation of the medium, the resulting sulfite value depending on both air rate and agitation. Karow et al. (195) have applied sulfite oxidation data to the dign of fermentors. RESULTS AN DISCUSSON The effect of aeration efficiecy on yield and count. The final cell concentration on a dry weight basis and the live cell count obtained with the pigmented strain varied directly with aeration efficiency as shown in figure 1. The medium used in this experiment was the synthetic medum contg 4 per cent glucose. CELL WEIGHT, MG /ML 0 15 10 5 I I I I AERATION EFFICIENCY, mm 0 PER L 50 PER?fN Figure 1. Effect of aeration efficiency on cell concentration and live cell count. SUBSTRATE UTILIZED, MG /ML. Figure B. Variation of cell concentration and live cell count with substrate utilized at various effective aeration rates. Abbreviation S.V. in figure is sulfite oxidation value expresed as mm 0, per L per min. The cultures were all allowed to grow to a final ph of 7.8 or higher, at which time all the sugar and most of the citrate were utilized. Although the same yield of dry cells was obtained at sulfite values of both 4 and 9, the final live cell count at a sulfite value of 9 was higher than at a sulfite value of 4. This may be a consequence of the more thorough dispersion of clumps due to the more severe agitation at the higher sulfite value. The generation time of the organism was found to be essentially constant at one hour at all sulfite values, whereas the time necesary to utilize all the sugar varied from 1 hours at a sulfite value of 9 to 0 hours at a sulfite value of one. The lag phase of the cultures has been observed to be shorter at the higher sulfite values for which there is no obvious explanation. The variation of cell concentration on a dry weight basis and live cell count with the total substrate utilized at various sulfite values shown in figure. The data in the figure show that the factor limiting the final cell concentration obtained in these experiments is the

1954] GROWTH OF AEROBIC MICROORGANISMS oxygen available to the organism. Cell count and cell weight increased with increasing aeration efficiency. An effective aeration rate of 9 mm 0 per liter per min was necesary to obtain maximum yields at high substrate concentrations. Traditional laboratory aeration procedures generally give effective aerations of 0.05 to 1.0 mm 0 per liter per min. Preliminary experiments showed the culture supernatant to contain an excess of inorganic phosphate and ammonia N. The ammonia N present when the synthetic medium contains g of citric acid per liter is capable of supporting a total cell mass of approximately 31 g per liter. At a cell concentration of 9 g per liter the culture supernatant contained 15.g NHsN per ml excess. The sulfate required by the culture was determined by growing the culture in the synthetic medium without added sulfate and with varying amounts of sulfate. Although 0.1 mg per ml of NaSO4 was sufficient to yield 10 mg of cells per ml, 0.5 mg per ml of NaSO0 has been added to this medium to insure an excess of sulfate at high cell densities. If no inorganic salts (Mg, Fe, Mn) were added to the synthetic medium, the final yield of cells was cut to 10 per cent of that obtained when excess salts were added. The concentration of salts in the medium has been kept at a level well above TABLE Variation of dry cell yield with total substrate utilized at various sulfite values DRY CELL GLUCOS* CITRIC ACD YIELD UTILIZED UTILIZED mg/mw 6.9 1.6 1.4 9.0 7.1 10.5 16. 17.0 6. 9.0 1.7 14.1 mg/ml 11.3 0.3 39.0 56.8 9.7 17.8 36.6 5.3 10.5 19.3 44.1 54.6 mgl/m 7.6 1.0 17.3 0.6 10.7 14.8 1.3 0.4 8.4 13.1 1.8.3 SULFrIT VALUE MM Ot PER CENT PER L PER YIELDt IN 9 9 99 1 11 1 36.5 39.0 38.0 37.5 34.8 3. 7.9 3. 3.8 6.7 19.3 18. * Represents total glucose available in the medium initially. t Based on glucose plus citric acid. I I I I I 0 4 6 8 10 AERATION EFFICIENCY, mm 0 PER L 349 PER KM Figure S. Variation of cell concentration and live cell count with aeration efficiency, with a pigmented and a nonpigmented strain of Serratia marcescens. Substrate is 4 per cent glucose plus per cent citric acid at all aeration efficiencies investigated. The cell concentration and live cell count for the pigmented strain are corrected for substrate utilized at the various aeration efficiencies in order to compare with the nonpigmented strain. the limiting level in order to insure an excess of inorganic elements. The per cent yield of dry cells based on total substrate utilized at various sulfite values is shown in table. The data in the table show that at high effective aeration rates (sulfite value of 9) the cell yield obtained is constant at all substrate concentrations investigated and reaches the maximum average value of 38 per cent. The highest yield reported for Saccharomyces is approximately 45 per cent. The per cent yield decreases as the sulfite value decreases. Comparison of S. marcescens pimented and nonpigmented strains. The preceding experiments reported for the pigmented strain of S. marcescens were repeated with a nonpigmented culture which was carried on a synthetic slant in the same manner described for the pigmented strain. The variation of cell concentration and live cell count with sulfite value for the pig-

350 CHARLES G. SMITH AND MARVIN J. JOHNSON [VOL. 68 mented and nonpigmented strains is shown in figure 3. Although the dry cell yield is the same for both strains investigated, the live cell count varies by a factor of 3, as shown in the figure. Microscopic examination of the cells of both strains showed the pigmented cells to be smaller than the nonpigmented. The per cent nitrogen in the cells of both strains was determined in order to establish whether the increased cell size was due to an accumulation of nonnitrogenous material or cellular protein. The nitrogen content of the cells of both strains ranged from 10.4 to 10.9 per cent, indicating that the larger cell size is not due to accumulation of capsular or other carbohydrate material, and the variation in live cell count observed with the two strains is a consequence of the larger cell size- of the nonpigmented strain. SUMMARY With Serratia marcescens the per cent yield of cells based on substrate utilized, the total cell concentration, and the live cell count have been shown to vary directly with aeration efficiency. The cell concentration varied from 9 mg per ml at an effective aeration rate of 0.5 mm 0 per liter per min to 3 mg per ml at an aeration rate of 9 mm 0 per liter per min when 4 per cent glucose plus per cent citric acid was used as substrate. The live cell counts corresponding to these dry weights are 65 X 109 per ml and 17 x 1010 per ml, respectively. An increase in aeration efficiency of at least 10-fold over that normally used for the laboratory culture of aerobes was necessary to obtain the high cell densities. A synthetic medium has been developed for the growth of Serratia marcescens in liquid shake culture in yields of 38 per cent of the total substrate utilized. The highest cell concentration reported in this paper on a dry weight basis is 9 mg per ml, corresponding to a live cell count of 0.1 x 1010 per ml. Comparison of the cell concentration on a dry weight basis and the live cell count of a pigmented and a nonpigmented strain of S. marces8cens at various aeration efficiencies has shown both strains to give the same dry cell concentrations at all effective aeration rates investigated, whereas the maximum live cell count varied from 60 X 109 per ml for the nonpigmented strain to 17 X 1010 per ml for the pigmented strain when both cultures had utilized the same amount of substrate. The larger cell size of the nonpigmented strain accounts for the observed result. REFERENCES I1 COOPER, C. M., FERNSTROM, G. A., AND MILLER, S. A. 1944 Performance of agitated gasliquid contactors. Ind. Eng. Chem., 36, 504-509. FEUSTEL, I. C., AND HUMFELD, H. 1946 A new laboratory fermentor for yeast production investigations. J. Bacteriol., 5, 9-35. FISKE, C. H., AND SUBBAROW, Y. 195 The colorimetric determination of phosphorus. J. Biol. Chem., 66, 375-400. GEE, L. L., AND GERHARDT, P. 1946 Brucella sui8 in aerated broth culture. II. Aeration studies. J. Bacteriol., 5, 71-81. GERHARDT, P. 1946 Brucella 8Ui8 in aerated broth culture. III. Continuous culture studies. J. Bacteriol., 5, 83-9. GERHARDT, P., AND GEE, L. L. 1946 Brucella 8Ui8 in aerated broth culture. I. Preliminary studies on growth assays, inoculum, and growth characteristics in an improved medium. J. Bacteriol., 5, 61-69. HARRIS, E. E., SAEMAN, J. F., MARQUARDT, R. R., RANNAN, M. L., AND ROGERS, S. C. 1948 Fodder yeast from wood hydrolyzates and still residues. Ind. Eng. Chem., 40, 10-13. JOHNSON, M. J. 1941 Isolation and properties of a pure yeast polypeptidase. J. Biol. Chem., 137, 575-586. KAROW, E. O., SFAT, M. R., AND BARTHOLOMEW, W. H. 195 Oxygen transfer and agitation in submerged fermentations. Abstracts 11st Meeting American Chemical Society, 1A. MAXON, W. D., AND JOHNSON, M. J. 1953 Aeration studies on propagation of baker's yeast. Ind. Eng. Chem., 45, 554-560. MCCULLOUGH, W. G., MILLS, R. C., HERBST, E. J., ROESSLER, W. G., AND BREWER, C. R. 1947 Studies on the nutritional requirements of Brucella suis. J. Bacteriol., 53, 5-15. RIVIETT, R. W., JOHNSON, M. J., AND PETERSON, W. H. 1950 Laboratory fermentor for aerobic fermentations. Ind. Eng. Chem., 4, 188-190. SAFFRAN, M., AND DENSTEDT, 0. F. 1948 A rapid method for the determination of citric acid. J. Biol. Chem., 175, 849-855. SHAFFER, P. A., AND SOMOGYI, M. 1933 Copperiodometric reagents for sugar determination. J. Biol. Chem., 100, 695-713. STEFANIAK, J. J., GAILEY, F. B., BROWN, C. S., AND JOHNSON, M. J. 1946 Pilot plant equipment for submerged production of penicillin. Ind. Eng. Chem., 38, 666-671.