AERATION REQUIREMENTS FOR THE GROWTH OF AEROBIC MICROORGANISMS1
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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 MATERIALS AND METHODS
cells, the dry cell yields obtained are approxi- Chemical and bacteriologal methods. The two
mately 45 per cent of the substrate utilized,
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strains of S. marcescens used in this study were
corresponding to cell concentrations on a dry strain 8 UK, obtained from Camp Detrick,
weight basis of 10 to 44 mg per ml (Harris et al., Maryland, and a strain isolated by Dr. W. B.
1948; Feustel and Humfeld, 1946; Maxon and Sarles at Wisconsin. The former strain is pig-
Johnson, 1953). The cell yields obtained with mented while the latter is not. The stock cul-
bacteria are lower than those obtained with tures of both strains were reisolated periodically
yeasts. Bacterial populations as high as 57 by plating on glucose-citrate agar and trans-
billion and 84 billion per ml have been reported ferring an isolated colony into liquid culture.
(Gerhardt and Gee, 1946; Gee and Gerhardt, The stock cultures were carried on glucose-
1946; Gerhardt, 1946; McCullough et al., 1947). citrate agar slants held at 5 C.
Although the cell concentrations were not re- It was found that cell numbers and weights
ported, the dry cell weight corresponding to could be estimated fairly accurately turbidi-
these live counts should be approximately 10 metrically, with an Evelyn photoelectric color-
mg per ml. imeter, by measuring the light transmision of a
It should be possible, by application of the suitably diluted sample. The turbidimetric
principles employed for growing yeasts, to ob- results were used only to calculate dilutions for
tain the same yields of cells when growing plating and to determine proper harvesting
aerobic bacteria. The experiments reported here times. Dry cell weights were made by weighing
are concerned with the effect of aeration effi- the centrifuged and washed cells after drying in
ciency on the final dry cell yield and live cell an oven for 12 hours at 120 C. Live cell counts
count of bacteria. The organism chosen for this were made by spreading the final dilution of the
study, Serratia marcescens, is highly aerobic, culture medium on the surface of nutrient agar
easily cultured on a synthetic medium, readily plates, followed by incubation at 30 C. Each
counted when spread on the surface of an agar sample was counted in duplicate with a minimum
plate, and free from large amounts of slime and of 170 colonies per plate.
capsule. It was found that the yield of cells ob- The synthetic medium used for growth of the
tained on the synthetic medium in liquid culture organism was devised to allow maximum yields
varied directly with the aeration efficiency, the of cells to be obtained with a minimum of re-
highest yield based on substrate utilized being sidual material remaining in the spent medium.
approximately 38 per cent. The highest cell The composition of the synthetic medium in
concentration on a dry weight basis obtained in grams per liter is as follows: citric acid monohy-
this work is 29 mg per ml, corresponding to a drate, 22; Na2SO4, 0.5; KH2P04, 5.0; glucose, 10
live cell count of 20.1 X 1010 per ml. The effec- to 60; MgS04-7H20, 0.8; NaCl, 0.04; FeSO4-
tive aeration necessary to support such high 7H20, 0.04; MnSO.44H20, 0.04. All the con-
populations of Serratia was much higher than stituents of the medium except the glucose are
that commonly employed in the laboratory for dissolved in distilled water and the pH of the
the aerobic growth of microorganisms. medium adjusted to 7.7 with NH40H prior to
1 Published with the approval of the Director autoclaving. The glucose is autoclaved sepa-
of the Wisconsin Agricultural Experiment Sta- rately and added aseptically before inoculating.
tion. The pH of the medium after sterilization is ap-
3461954] GROWTH OF AEROBIC MICROORGANISMS 347
proximately 6.8. During growth, the pH drops available to the cells. The transfer of oxygen
to 5.2 to 5.5 in the early log phase and rises to from the gas phase to the liquid phase is prob-
8.0 to 8.5 as the sugar is exhausted. If a low dis- ably the limiting factor in aeration of cultures.
solved solids content is desired in the spent Maxon and Johnson (1953) have correlated the
medium, the concentration of citric acid is oxygen transfer, as determined by the sulfite
varied with the glucose concentration to give an oxidation procedure of Cooper et al. (1944), with
initial ratio of glucose to citric acid of 2:1. the oxygen actually available to yeast cells.
The inoculum was cultured by inoculating a Aeration efficiency has been employed in this
flask containing the synthetic medium directly work as a measure of the oxygen available to the
from the slant. The liquid culture obtained was cells. The aeration efficiency is referred to in
used then to inoculate the flasks used in the this paper as sulfite oxidation value, expressed as
experiments. In all cases the culture was in- mM 02 per liter per min. Aeration procedures
cubated at 30 C. Foaming was controlled by traditionally employed in the laboratory for the
adding one drop of heptadecanol (3,9-diethyl-
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cultivation of aerobes give effective aeration
tridecanol-6, Carbide and Carbon Corp.) to each rates much lower than those necessary for the
shake flask at the time of inoculation and as attainment of high cell concentrations. Effective
required during growth. aeration rates of some commonly employed
Sugar was determined by the method of aeration devices are shown in table 1.
Shaffer and Somogyi (1933). Nitrogen was deter- All of the sulfite values reported in table 1 for
mined by the micro-Kjeldahl method described shake flasks were determined with the plugs
by Johnson (1941). Inorganic phosphate was removed. The data in the table show that sta-
determined by a modified Fiske-Subbarow tionary culturing gives very poor aeration
method (1925). Citric acid was determined by efficiencies, 0.3 mM 02 per liter per min maximum
the colorimetric method of Saffran and Denstedt obtained in this work. Shake flasks with small
(1948). volumes of medium are effective up to a sulfite
Aeration methods. The limiting factor in ob- value of 2, whereas the sides of the flasks must
taining a high cell concentration of microor- be indented in order to obtain sulfite values
ganisms in the laboratory is usually the oxygen higher than 2. In this work, regular 500 ml
TABLE 1
Effective aeration rates of some commonly employed laboratory aeration procedures
VESSEL VOLUME
MEDIUMOF 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 20 stationary - 0.32
500 ml Erlenmeyer 20 shaken* 1.1
500 ml Erlenmeyer 10 shaken 2.0
500 ml Erlenmeyer 50 shaken - 0.60
500 ml Erlenmeyer 100 stationary 0.10
500 ml Erlenmeyer 100 shaken - 0.27
500 ml indented Erlenmeyer 20 shaken 2 to 9.5
18 L bottle 15,000 8 mm tube immersed 1.0 0.06
18 L bottle 15,000 120 cm2 sintered steel sparger 1.0 0.60
30 L fermentor 15,000 500 rpm agitator plus sparger 1.0 2.0
(Rivett et al., 1950)
100 gallon fermentor 250,000 250 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 250 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 2 to 9.5. The
sulfite value obtained with an indented flsk
varies with the size and the number of indenta-
tions. 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
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high aeration efficiencies on a large scale, aera-
tion must be accompanied with agitation of the
medium, the resulting sulfite value depending on
both air rate and agitation. Karow et al. (1952)
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 SUBSTRATE UTILIZED, MG /ML.
aeration efficiency as shown in figure 1. The
medium used in this experiment was the syn- Figure B. Variation of cell concentration and
live cell count with substrate utilized at various
thetic medum contg 4 per cent glucose. effective aeration rates.
Abbreviation S.V. in figure is sulfite oxidation
I I I I value expresed as mm 0, per L per min.
CELL 250
WEIGHT, The cultures were all allowed to grow to a final
MG /ML
pH of 7.8 or higher, at which time all the sugar
and most of the citrate were utilized. Although
20 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
15 more thorough dispersion of clumps due to the
more severe agitation at the higher sulfite value.
The generation time of the organism was found
10
to be essentially constant at one hour at all
sulfite values, whereas the time necesary to
utilize all the sugar varied from 12 hours at a
sulfite value of 9 to 20 hours at a sulfite value of
5 one. The lag phase of the cultures has been ob-
served 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
PER L PER
substrate utilized at various sulfite values
AERATION EFFICIENCY, mM 02 ?fN
shown in figure 2. The data in the figure show
Figure 1. Effect of aeration efficiency on cell that the factor limiting the final cell concen-
concentration and live cell count. tration obtained in these experiments is the1954] GROWTH OF AEROBIC MICROORGANISMS 349
oxygen available to the organism. Cell count I I
and cell weight increased with increasing aera-
tion efficiency. An effective aeration rate of
II I
9 mM 0° per liter per min was necesary to ob-
tain maximum yields at high substrate concen-
trations. Traditional laboratory aeration proce-
dures generally give effective aerations of 0.05
to 1.0 mM 02 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 22
g of citric acid per liter is capable of supporting
a total cell mass of approximately 31 g per liter.
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At a cell concentration of 29 g per liter the
culture supernatant contained 125 .g NHsN
per ml excess. The sulfate required by the cul-
ture 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 8 10
0 2 4 6
10 mg of cells per ml, 0.5 mg per ml of Na2SO0
has been added to this medium to insure an AERATION EFFICIENCY, mM 02 PER L PER KM
excess of sulfate at high cell densities. If no Figure S. Variation of cell concentration and
inorganic salts (Mg, Fe, Mn) were added to the live cell count with aeration efficiency, with a
synthetic medium, the final yield of cells was pigmented and a nonpigmented strain of Serratia
cut to 10 per cent of that obtained when excess marcescens.
salts were added. The concentration of salts in Substrate is 4 per cent glucose plus 2 per cent
the medium has been kept at a level well above citric acid at all aeration efficiencies investigated.
The cell concentration and live cell count for the
TABLE 2 pigmented strain are corrected for substrate uti-
Variation of dry cell yield with total substrate lized at the various aeration efficiencies in order
utilized at various sulfite values to compare with the nonpigmented strain.
SULFrIT
DRY CELL GLUCOS* CITRIC ACD VALUE MM Ot PER CENT the limiting level in order to insure an excess of
YIELD UTILIZED UTILIZED PER L PER YIELDt
IN inorganic elements.
The per cent yield of dry cells based on total
mg/mW mg/ml mgl/m substrate utilized at various sulfite values is
6.9 11.3 7.6 9 36.5 shown in table 2. The data in the table show
12.6 20.3 12.0 9 39.0 that at high effective aeration rates (sulfite
21.4 39.0 17.3 9 38.0
29.0 56.8 20.6 9 37.5 value of 9) the cell yield obtained is constant at
all substrate concentrations investigated and
7.1 9.7 10.7 2 34.8 reaches the maximum average value of 38 per
10.5 17.8 14.8 2 32.2 cent. The highest yield reported for Saccharo-
16.2 36.6 21.3 2 27.9 myces is approximately 45 per cent. The per cent
17.0 52.3 20.4 2 23.2 yield decreases as the sulfite value decreases.
Comparison of S. marcescens pimented and
6.2 10.5 8.4 1 32.8 nonpigmented strains. The preceding experiments
9.0 19.3 13.1 1 26.7 reported for the pigmented strain of S. mar-
12.7 44.1 21.8 1 19.3 cescens were repeated with a nonpigmented
14.1 54.6 22.3 1 18.2 culture which was carried on a synthetic slant
*
Represents total glucose available in the me- in the same manner described for the pigmented
dium initially. strain. The variation of cell concentration and
t Based on glucose plus citric acid. 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 nonpigmented strain accounts for the observed
figure 3. Although the dry cell yield is the same result.
for both strains investigated, the live cell count REFERENCES I1
varies by a factor of 3, as shown in the figure. COOPER, C. M., FERNSTROM, G. A., AND MILLER,
Microscopic examination of the cells of both S. A. 1944 Performance of agitated gas-
strains showed the pigmented cells to be smaller liquid contactors. Ind. Eng. Chem., 36, 504-
than the nonpigmented. The per cent nitrogen 509.
in the cells of both strains was determined in FEUSTEL, I. C., AND HUMFELD, H. 1946 A new
order to establish whether the increased cell size laboratory fermentor for yeast production
was due to an accumulation of nonnitrogenous investigations. J. Bacteriol., 52, 229-235.
material or cellular protein. The nitrogen content FISKE, C. H., AND SUBBAROW, Y. 1925 The
of the cells of both strains ranged from 10.4 to colorimetric determination of phosphorus.
J. Biol. Chem., 66, 375-400.
Downloaded from http://jb.asm.org/ on February 21, 2021 by guest
10.9 per cent, indicating that the larger cell GEE, L. L., AND GERHARDT, P. 1946 Brucella
size is not due to accumulation of capsular or sui8 in aerated broth culture. II. Aeration
other carbohydrate material, and the variation studies. J. Bacteriol., 52, 271-281.
in live cell count observed with the two strains GERHARDT, P. 1946 Brucella 8Ui8 in aerated
is a consequence of the larger cell size- of the broth culture. III. Continuous culture
nonpigmented strain. studies. J. Bacteriol., 52, 283-292.
GERHARDT, P., AND GEE, L. L. 1946 Brucella
SUMMARY 8Ui8 in aerated broth culture. I. Preliminary
With Serratia marcescens the per cent yield of studies on growth assays, inoculum, and
cells based on substrate utilized, the total cell growth characteristics in an improved me-
dium.
concentration, and the live cell count have been HARRIS, E. E., J. Bacteriol., 52, 261-269.
SAEMAN, J. F., MARQUARDT, R. R.,
shown to vary directly with aeration efficiency. RANNAN, M. L., AND ROGERS, S. C. 1948
The cell concentration varied from 9 mg per ml Fodder yeast from wood hydrolyzates and
at an effective aeration rate of 0.5 mm 02 per still residues. Ind. Eng. Chem., 40, 1220-
liter per min to 23 mg per ml at an aeration rate 1223.
of 9 mM 02 per liter per min when 4 per cent JOHNSON, M. J. 1941 Isolation and properties
glucose plus 2 per cent citric acid was used as of a pure yeast polypeptidase. J. Biol.
substrate. The live cell counts corresponding to Chem., 137, 575-586.
these dry weights are 65 X 109 per ml and 17 x KAROW, E. O., SFAT, M. R., AND BARTHOLOMEW,
1010 per ml, respectively. An increase in aeration W. H. 1952 Oxygen transfer and agitation
efficiency 10-fold that in submerged fermentations. Abstracts 121st
of at least over normally Meeting American Chemical Society, 12A.
used for the laboratory culture of aerobes was MAXON, W. D., AND JOHNSON, M. J. 1953 Aera-
necessary to obtain the high cell densities. tion studies on propagation of baker's yeast.
A synthetic medium has been developed for Ind. Eng. Chem., 45, 2554-2560.
the growth of Serratia marcescens in liquid MCCULLOUGH, W. G., MILLS, R. C., HERBST,
shake culture in yields of 38 per cent of the total E. J., ROESSLER, W. G., AND BREWER, C. R.
substrate utilized. The highest cell concentration 1947 Studies on the nutritional require-
reported in this paper on a dry weight basis is ments of Brucella suis. J. Bacteriol., 53, 5-
29 mg per ml, corresponding to a live cell count 15.
of 20.1 x 1010 per ml. RIVIETT, R. W., JOHNSON, M. J., AND PETERSON,
Comparison of the cell concentration on a dry W. H. 1950 Laboratory fermentor for
aerobic fermentations. Ind. Eng. Chem.,
weight basis and the live cell count of a pig- 42, 188-190.
mented and a nonpigmented strain of S. mar- SAFFRAN, M., AND DENSTEDT, 0. F. 1948 A
ces8cens at various aeration efficiencies has shown rapid method for the determination of citric
both strains to give the same dry cell concentra- acid. J. Biol. Chem., 175, 849-855.
tions at all effective aeration rates investigated, SHAFFER, P. A., AND SOMOGYI, M. 1933 Copper-
whereas the maximum live cell count varied iodometric reagents for sugar determination.
from 60 X 109 per ml for the nonpigmented STEFANIAK, J. Biol. Chem., 100, 695-713.
J. J., GAILEY, F. B., BROWN, C. S.,
strain to 17 X 1010 per ml for the pigmented AND JOHNSON, M. J. 1946 Pilot plant equip-
strain when both cultures had utilized the same ment for submerged production of penicillin.
amount of substrate. The larger cell size of the Ind. Eng. Chem., 38, 666-671.You can also read
