Metabolic Behavior and Chlortetracycline Production

by Streptomyces aureofaciens in Liquid Culture

G. BIFFI, G. BORETTI, A. Di MARCO, AND P. PENNELLA
Farmitalia Research Laboratories, Milano, Italy

Received for publication May 7, 1954

determinations of total phosphorus have been performed on the extracts containing nucleic acids; the
nucleic acid amounts calculated from phosphorus assays
and from absorption spectra were found to agree.
4. Determination of pH (glass electrode).
5. Determination of sucrose in medium by the
method of Schaffer and Somogyi (1933). The obtained
data are too high, but more reliable than those found
by anthrone methods.
6. Determination of total nitrogen by the method of
Kjeldahl and of NH3 nitrogen by the method of Raynaud (1948) in the culture fluid.
7. Determination of lactic acid by the method of
Barker and Summerson (1941) and of pyruvic acid by
the method of Friedemann and Haugen (1943) in the
culture fluid.
8. Detection of organic acids by paper chromatography with the solvents used by Cheftel, Munier,
and Macheboeuf (1952).
9. Chlortetracycline by spectrophotometric determination (Van Dyck and De Somer, 1952).

The present paper deals with an experimental study

of the metabolic behavior of Streptomyces aureofaciens'
grown in laboratory fermentors.
EXPERIMENTAL METHODS
Standard fermentations were performed as follows:
A spore suspension was inoculated into 3 L of the
following medium: sucrose, 3 per cent; corn steep
liquor, 1 per cent; molasses, 0.1 per cent; NaCl, 0.05
per cent; and CaCO3, 0.4 per cent. The fermentations
were carried out at 27 C in 5-L fermentors (Bartholomew, Karow and Sfat, 1950).
After 36 hours, 100 ml of the culture fluid was transferred into three liters of the fermentation standard
medium (U.S. Patent 1952): sucrose, 3 per cent; corn
steep, 1 per cent; CaCO3, 1 per cent; (NH4)2 SO4, 0.2
per cent) and incubated in a 5-L fermentor at 27 C.
Media were sterilized at 120 C for 60 minutes; the
sucrose was separately sterilized.
Aliquots were taken from the fermentors at suitable
intervals of time and the following assays were per-

formed:
1. Weight of mycelium: 5 or 10 ml were acidified
with HCI in order to dissolve the calcium salts, then
filtered; the washed mycelium was dried and weighed.
2. Protein nitrogen: 5 or 10 ml of the sample were
made up to 10 per cent with trichloroacetic acid (TCA)
and spun. After washing the sediment with 10 per
cent TCA, nitrogen was determined according to the
Kjeldahl method.
3. Nucleic acids: Pentose nucleic acid (RNA) and
desoxypentose nucleic acid (DNA) contents were
estimated, according to Ogur and Rosen (1950), in 1 or
2 ml of the sample. Histochemical researches, which
will be published elsewhere, gave evidence that Ogur and
Rosen's method for separation and extraction of nucleic
acids may be used for Streptomyces (T. Scotti and P.
Zoc.chi, personal communication). The nucleic acids
were spectrophotometrically estimated. The interference of the chlortetracycline was totally eliminated
by ethanol and 0.2 N perchloric acid washings before
the nucleic acids extraction. In the first experiments,

RESULTS
Fermentation in Standard Medium
Mycelium growth, carbohydrate, and nitrogen uptake.
The typical features of a fermentation in standard
medium are shown in figures 1 and 2. The mycelium
growth, which is rapid within the first 24 hours, becomes very slow from 24 to 48 hours. The sucrose uptake is negligible within the first 12 hours. In that
period of time the NH3 in culture fluid increases. It may
be assumed that at the beginning of growth some substances (that is, aminoacids) are deaminized and utilized as carbon source by the Streptomyces (as it
happens for the Penicillium). Later on the sugar is more
rapidly utilized; 48 hours after inoculation the medium
is depleted of carbohydrate. Ammonia is rapidly assimilated within 12 and 24 hours, so that the mycelium
from 24 hours on develops in a medium still rich in
sugar but very poor in available nitrogen.
The changes of total soluble nitrogen and ammonia
nitrogen during the growth of S. aureofaciens are
shown in figure 4. During the first 12 hours ammonia
nitrogen slightly increases while total nitrogen de-

1 A strain of S. aureofaciens from the collection of the

Farmitalia Research Laboratories, Milano, Italy, was employed in these studies.

288

METABOLISM OF STREPTOMYCES A UREOFACIENS

mold composition throughout the experiment shown in

figures 3, 4, and 5.
pH Changes; organic acids in culture fluid. Figures 1
and 4 show the typical behavior of pH which increases
~~~~~~~~~rLduring the deamination and decreases during sucrose

4~~~~~~~~~~

cc

12 24

36

48~~~~~~~~1
/

12

24

36

289

II4E, 1l0S

10

48

6;

TIME, HOURS
FIG. 1. Chemical changes during fermentation of Streptomyces aureofaciens in standard medium.

12

36
48
TIME HOURS
FIG. 3. Growth, synthesis of proteins and of nucleic acids in

24

standard medium.

10

0?.~~~~~~1
pH
12

24

36

TIM E,HOURS

A8

~~~~~~~~8

-W

FIG. 2. Growth, synthesis of proteins and of chlortetracvcline in standard medium.

2

creases. A part of total nitrogen is not available for the

mycelium growth.
Proteins and nucleic acids synthesis.2 Synthesized
proteins generally reach a maximum at 24 or 36 hours,
and later often show a more or less rapid decrease
(figure 2). In an experiment showing a lower growth
rate, the protein decrease was not observed within 48
hours (figures 3, 4, and 5). An increase of total nitrogen
in the medium corresponding to the protein nitrogen
decrease of mycelium has never been observed. Ribonucleic acid (RNA) reached a maximum at 24 or 36
hours (figure 3). An RNA decrease generally takes
place about 12 hours before that of protein nitrogen.
Desoxyribonucleic acid (DNA) is the most stable
component of mycelium. The DNA/protein N ratios
remain fairly unaltered during the protein synthesis.
Composition of mycelium. According to what has
been said above it is easily understood that the composition of mycelium at 48 hours is greatly different
from that at 12 to 24 hours. Table 1 summarizes the
the figures, protein nitrogen and nucleic acids amounts
reported in mg/ml culture fluid and are not related to
mycelium weight.
2 In

are

136

24

48

TM E. HOULRS

FIG. 4. Chemical changes during fermentation in standard

medium.

12

24

36

TIME

48
HOVJRS

Go

FIG. 5. Growth, synthesis of proteins and of chlortetracycline in standard medium.

290

BIFFI, BORETTI, DI MARCO, AND PENNELLA

TABLE 1. Composition of Streptomyces aureofaciens
mycelium at different incubation times
Hours

Protein N

RNA

DNA

12
24
48

7
8.5
6.15

9.1

1.3
1.45
1.24

6.25
2.8

Values are calculated as per cent of dry weight.

and ammonia uptake. It generally increases more or

less rapidly after 48 hours of fermentation. Paper
chromatography shows that no organic acid synthesis
occurs during mycelial growth; at 48 hours a small
amount of pyruvic acid is found in the culture fluid. It
has been shown, on the contrary, that the lactic acid of
the medium is partially metabolized. At any rate, the
NH4 ions consumption alone may justify the pH
decrease.
Chlortetracycline synthesis. Figures 2 and 5 show that
the antibiotic synthesis begins when a certain amount
of mycelium has grown and goes on also during the
slow growth phase; it stops when the medium is depleted of sucrose.
Morphological changes of mycelium. During the
fermentation a change occurs not only in chemical
composition of mycelium but also in its morphological
features, as may be seen in preparations stained by
Giemsa method. In fact, while in the beginning the
hyphae are uniform in diameter and rich in basophilic
substances, at the end of the fermentation several
enlarged hyphae rich in basophilic granules are seen,
surrounded by very thin hyphae in which little basophilic granules are present.

FIG. 6. Growth and chlortetracycline synthesis in standard

(S) medium and in 0.3 per cent K2HPO4 added (SP) medium.

t
C

CD

:z

-3

3.-2

z x,

12

Medium".)
Sucrose and NH3 uptake; organic acids in culture fluid.
Figure 7 shows that phosphate addition stimulates sucrose and NH3-nitrogen consumption. The pH is 0.1-0.2
units lower in the medium containing phosphate than
in the standard medium. In regard to the lactic acid
consumption no remarkable difference between the
two media was observed. On the contrary, the difference in the production of pyruvic acid is remarkable.
Pyruvic acid begins to accumulate in SP medium 12
or 24 hours after inoculation and its values are several
times higher than those reached 48 hours after inocula-

24

36
48
TIME , HOUPRS

FIG. 7. Chemical changes during fermentation of Streptomyces aureofaciens in standard (S) medium and in 0.3 per
cent K2HPO4 added (SP) medium.

Effect of Changes in Medium Composition During

Fermentation
Addition of Phosphate
By the addition of K2HPO4 (0.3 per cent or more) to
the standard medium, all the biochemical features of
the fermentation are changed. The antibiotic production greatly decreases as is shown in figure 6. (In the
course of the paper standard medium will be indicated
by "S Medium", phosphate-added medium by "SP

'4
0

S MEDIUM
3--SP

n1s
I1

>'

r.0.05

-I -~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
12

24

36

TIME

48

HOURS

FIG. 8. Desoxyribonucleic acid (DNA) synthesis in standard

(S) medium and in 0.3 per cent K2HPO4 added (SP) medium.

tion in S medium. Further experiments are in progress

on this point.
Mycelium growth and synthesis of proteins and nucleic
acids (figures 6, 7, and 8). In SP medium the rate of
increase of dry weight is at first greater than in S
medium. Growth ceases or goes on very slowly when
the medium is depleted of ammonia nitrogen. Therefore the mycelium amount obtained in SP medium
36 or 48 hours after inoculation is clearly lower than
that obtained in S medium. Since the amount of nitrogen assimilated is not very different in both media, a
difference between the compositions of the mycelia,
grown in the two media, is generally observed at the

METABOLISM OF STREPTOMYCES AUREOFACIENS

TABLE 2. Composition of mycelium of Streptomyces
aureofaciens in S and SP media
Protein N
Medium

SP

RNA

Hours

Hours

DNA
Hours

12

24

48

12

24

48

12

24

11.5
10.2

8.4
9.4

2.6
8.7

10
8.7

3.7
4.1

2.6
4.3

3.7
3.85

2
2.35

Values

are

calculated

as per

48

1.65
2.55

cent of dry weight.

TABLE 3. Growth of Streptomyces aureofaciens and synthesis

of antibiotic
Experiment
Medium

Growth in 24-48 hours.

S
SP

per cent

per cent

per cent

per cent

71
25

40
0

18.5
0

the addition of ammonium sulphate caused a comparable increase of both chlortetracycline and desoxyribonucleic acid yields (20 per cent and 18 per cent). A
further increase of the ammonium sulphate concentration from 0.4 per cent to 0.6 per cent, without addition
of phosphate, does not lead to an increase of the nucleic
acids and proteins synthesized by the mold. Chlortetracycline yields in 0.6 per cent ammonium sulphate
medium are from 15 to 25 per cent lower than those in
0.4 per cent ammonium sulphate medium.
Effect of phosphate addition. The addition of phosphate to the medium enriched with ammonium sulTABLE 4. Composition of mycelium in 4N medium supplemented
with phosphate
Protein N
Medium

87
16.7

S
SP

100
58

100
29

100
51

100
62

SP

100
80

100
100

100
77

100
84

Growth at 48 hours* .S

end of the fermentation (table 2). The addition of

phosphate to the S medium accelerates the desoxyribonucelic acid synthesis but does not increase its level, as
is clearly shown in figure 8.
Chlortetracycline synthesis. From the beginning of the
incubation period the chlortetracycline yields were
remarkably lower in SP medium. The comparison between the percentage increase in growth of mold from
the 24th to the 48th hour of incubation and the differences in maximal amount of the synthesized chlortetracycline in S and SP media, obtained in several
experiments, shows that the chlortetracycline synthesis is constantly related to the shape of growth
curve. Each experiment reported in table 3 shows that
the difference between the amount of chlortetracycline
produced in SP medium and in S medium is much
greater than the difference found in mycelium weights.

Addition of Ammonium Sulphate

Mycelium growth, synthesis of proteins, nucleic acids,
and chlortetracycline. The S medium containing 0.2 per
cent ammonium sulphate and a modified medium
containing 0.4 per cent ammonium sulphate (4N) have
been compared in two experiments. In both experiments, the mycelium weight was lower in 4N medium
in the first hours of incubation but after 36 hours
exceeded the weight obtained in S medium (figure 9).
The yield of chlortetracycline was clearly higher in 4N
medium only after the weight of the mycelium had
exceeded the one reached in S medium. When nucleic
acids synthesis was determined, also it was found that

48

Hours

12

24

48

2.3 14
7.1 13.9
9.6 14.2

4.8
7.3
9.7

4.10 1.66 1.02 0.91

4
1.64 1.32 1.29

1~~ ~

12

24

48

7.4 1.62 1.73 1.82

1
~~~~~~~~~~~~~~~
.0

87Zi9

D,WEIGHT S.MEDIUM
CHLORTETR.
D. -WEIGHT
4N MED-

DNA

Hours

24

4N .............
12.1 7.5
4N + 0.3% K2HPO4 ..... 12
9.6
4N + 0.5% K2HPO4 ........ 113.6 10.2

* Chlortetracycline has been calculated as a per cent of maximal amount obtained in S medium. The growth at 48 hours has been calculated in the same way.

RNA

Hours
12

Chlortetracycline*

291

loL9

.*

CHLORTETR.

LI-

12

s75

.4.;

0
-

-AJ
X.

.,-

.Kt&5.*--*

/"

tb..

25

c:
0

-,v

%J

12

24

Tf ME

36
HOURS

FIG. 9. Growth and chlortetracycline synthesis in standard

(S) medium and in ammonium sulphate added (4N) medium.
S medium contains 0.2 per cent (NH4)2SO4, 4N medium contains 0.4

per

cent (NH4)2SO4.

12

48
24
36
TIME , 14OURS
FIG. 10. Nucleic acids synthesis in 4N medium and in 0.5
per cent K2HPO4 added 4N medium (4NP).

BIFFI, BORETTI, DI MARCO, AND PENNELLA

292

L.

50

I-7

C..)

ox
-J

12

24

36
;8
TIME , HOURS

60

FIG. 11. Growth and chlortetracycline production in 4N

medium and in 0.5 per cent K2HPO4 added 4N medium (4NP).
TABLE 5. Synthesis of nuicleic acids in 4N medium
supplemented with phosphate
RNA Y/ml

DNA y/ml

Hours

Hours

Medium

24

4N
.
.
.............. 495
4N + 0.3% K2HPO4 .................. 720
4N + 0.5% K2HPO4 ............. l900

48

24

48

390
510
780

105
130

140
165
190

160

TABLE 6. Growth and synthesis of antibiotic in 4N medium

supplemented with phosphate
Medium

Dry
Weight
Increase
Within
24-48 Hr
per cent

4N .........................
4N + 0.3% K2HPO4 .............
4N + 0.5% K2HPO4 .............

48
29
11.5

Chlott

Cyoinetrayie
per

cent

100
50
21

Mycelium

Weight,
48 Hr

per

cent

100
83
68

*
Chlortetracycline has been calculated as a per cent of
maximal amount obtained in S medium. The growth at 48
hours has been calculated in the same way.

phate gives results similar to those observed by adding

phosphate in standard medium (figures 10 and 11). The
differences in the changes of the mycelium composition
during the fermentation are especially evident. It may
be seen, in table 5 and figure 10, that increasing the
amounts of phosphate in media increases the level of
synthesized nucleic acids and that the rate of desoxyribonucleic acid synthesis in the first 24 hours is also
increased. The correlation between the differences in
the shape of growth curve and the yields of chlortetracycline is once more proved by the data recorded in
table 6.
DIscUSSION
The metabolic behavior of Streptomyces aureofaciens
grown in a laboratory fermentor shows two different

stages, as described by Van Dyck and De Somer (1952).

The first stage is characterized by a rapid synthesis of
all protoplasm constituents and by the uptake of
almost all the nitrogen of the medium. In the second
stage the mycelium weight is still increasing, but very
slowly; protein and ribonucleic acid amounts decrease
and desoxyribonucleic acid amount changes only
slightly. The decrease of nucleoprotein content is not
accompanied by a mycelium weight decrease or by an
equivalent increase of medium nitrogen; therefore it
must not be ascribed to a real lysis.
It may be assumed a partial hydrolysis of mycelium
proteins occurs from which trichloroacetic acid-soluble
peptides are produced which are not able to pass
through the cell wall. For the above mentioned reasons,
the mycelium composition remarkably changes during
the growth; in fact the percentage in protein nitrogen
is diminished both because of the mentioned hydrolysis
and because of an accumulation of nitrogen-free
materials in the growth which occurs in the second
stage.
The microscopical observations by Hotchkiss staining do not show a remarkable accumulation of reserve
polysaccharides. On the contrary, relative increase of
the cell wall may be suggested, owing to the formation
of a great deal of very thin hyphae, poorly stained by
Giemsa method. These observations confirm Gottlieb
and Legator's (1953) remarks on Streptomyces venezuelae
and the data shown by the graph in Oyaas and coworkers' (1950) paper on chloramphenicol fermentation
concerning the growth of molds after the medium is
depleted of nitrogen. Chugtai and Walker (1951) found
in growing cultures of Aspergillus niger an increase of
mycelium weight in nitrogen-free medium and observed
that 50 per cent of the increase was represented by
carbon. Foster (1949) too, in his work, Chemical
Activities of Fungi, states that in the first stages of
growth, molds rapidly synthesize nucleoproteins; later
on they form reserve substances or the fibrous material
of cell walls so that it results in a decrease in percentage
nitrogen content of mycelium.
By adding small amounts of phosphate to the
medium, all biochemical features of Streptomyces
growth are modified. The most constant effects are:
increased sucrose consumption rate, pyruvic acid
accumulation in the medium, increased growth rate of
mycelium and increased rate of desoxyribonucleic acid
synthesis in the first stage, and inhibition or decrease
of mycelium growth in the second stage. The addition
of phosphate generally causes a delay in protein nitrogen decrease so that the change in mycelium composition is not so marked as in the standard medium.
The comparison between growth curves and chlortetracycline production curves in standard medium shows
that chlortetracycline production begins when a good
quantity of mycelium has already developed and goes

METABOLISM OF STREPTOMYCES AUREOFACIENS

on also during the slow growth stage. The ammonium
sulfate stimulates chlortetracycline production only
when added to nitrogen-poor media; in this case also
the mycelium growth and the DNA synthesis are
stimulated. Chlortetracycline production is therefore
correlated with mycelium growth. However, the effect
of phosphate addition on growth, nucleic acids synthesis and chlortetracycline synthesis, shows that
antibiotic production is not linked with protein synthesis. This assumption is supported by the effect of
phosphate on chlortetracycline production. In fact,
chlortetracycline yields are lower in phosphate-containing media from the beginning of the antibiotic
production, that is, also in the stage in which the mycelium growth is stimulated by phosphate.
The inhibition of antibiotic production is even more
evident in media more rich in nitrogen, notwithstanding the fact that the added phosphate allows the synthesis of greater amounts of both nucleic acids. The
reason for this inhibition is still obscure. It may only be
said, at present, that the inhibiting effect of phosphate
on chlortetracycline production always occurs together
with changes of the mold metabolic behavior which
lead to a more rapid initial growth and to an inhibition
of the second stage growth.
ACKNOWLEDGMENT

Our thankful acknowledgment to Dr. P. Julita and

Mr. U. Bardi for their skillful analytical work.
SUMMARY

The growth and metabolism of Streptomyces aureofaciens in fluid culture have been studied. The carbohydrates and nitrogen consumption, mycelium growth,
nucleic acids and protein synthesis and chlortetracycline synthesis have been determined. The metabolic

293

behavior of Streptomyces aureofaciens has also been

studied in media with addition of K2HPO4 and
(NH4)2SO4. The correlation between the synthesis of
chlortetracycline and the over-all picture of fermentation has been considered.
REFERENCES
BARKER, S. B., AND SUMMERSON, W. H. 1941 The colorimetric determination lactic acid in biological material. J.
Biol. Chem., 138, 535-554.
BARTHOLOMEW, W. H., KAROW, E. O., AND SFAT, M. R. 1950
Design and operation of a laboratory fermentor. Ind.
Eng. Chem., 42, 1827-1830.
CHEFTEL, R., MUNIER, R., AND MACHEBOEUF, M. 1952 Microchromatographie sur papier des acids aliphatiques
hydrosolubles et non volatiles. Bull. Soc. Chim. Biol.,
34, 380-387.
CHUGTAI, M. I. D., AND WALKER, T. K. 1951 The mechanism
of the formation of organic acids by mould fungi. Biochem. J., 48, 524-527.
FOSTER, J. W. 1949 Chemical Activities of Fungi, pp. 83-84.
Academic Press, New York.
FRIEDEMANN, T., AND HAUGEN, G. E. 1943 Pyruvic acid. II.
The determination of ketoacids in blood. J. Biol. Chem.,
147, 415-442.
GOTTLIEB, D., AND LEGATOR, M. 1953 The growth and
metabolic behavior of Streptomyces venezuelae in liquid
culture. Mycologia, 45, 507-515.
OGUR, M., AND ROSEN, G. 1950 The nucleic acids of plant
tissues. Arch. Biochem., 25, 262-276.
OYAAS, J. E., EHRLICH, J., AND SMITH, R. M. 1950 Chemical
changes during chloramphenicol fermentation. Ind.
Eng. Chem., 42, 1775-1776.
RAYNAUD, M. 1948 Dosage de l'ammoniaque dans les milieux
biologiques. Bull. Soc. Chim. Biol., 30, 713-715.
SCHAFFER, P. A., AND SOMOGYI, M. 1933 Copper-iodometric
reagents for sugar determination. J. Biol. Chem., 100,
695-713.
NIEDERCORN, J. G. 1952 United States Patent 2,609,329.
Sept. 2.
VAN DYCK, P., AND DE SOMER, P. 1952 Production and
extraction methods of aureomycin. Antibiotics & Chemotherapy, 2, 184-198.