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Indian Journal of Experimental Biology

Vol. 44, October 2006, pp. 842-848

Optimization of nutritional requirements for gentamicin production by


Micromonospora echinospora
Himabindu M & Annapurna Jetty
Biochemical and Environmental Engineering Centre, Indian Institute of Chemical Technology, Hyderabad 500 007, India.
Received 5 January 2006; revised 2 June 2006
Effect of various fermentation media, carbon sources, nitrogen sources, phosphate concentration and culture
requirements includes inoculum levels and age were determined on gentamicin production and biomass dry weight
production for Micromonospora echinospora, a gentamicin producing strain. Of the substrates tested, starch as a sole carbon
source promoted maximal gentamicin production, while maltose promoted maximal growth. Yeast extract as a sole nitrogen
source promoted maximal growth, while soyabean meal for gentamicin production. Increasing phosphate concentration
enhanced gentamicin production and observed optimum production at 1.2g/l (6% v/v) of phosphate having 72 h old
inoculum in the medium. Highest gentamicin production was obtained after cultivation with shaking for 120 h in a medium
containing starch 0.75%(w/v), soyabean meal 0.5%, K2HPO4 0.12%, CaCO3 0.4%, FeSO4 0.003% and CoCl2 0.0001%. The
gentamicin production was 1.2-fold in this medium as compared to basal medium.
Keywords: Gentamicin production, Micromonospora echinospora, Nutritional requirement

Fermentation
by
Micromonospora
species,
echinospora and purpurea produces a family of
aminocyclitol
antibiotic
called
gentamicin1.
Gentamicin is a broad spectrum, basic, and water
soluble antibiotic, first reported by Weinstein et al2.
Among the clinically more important species of Gram
negative organisms responsive to gentamicin are both
indole positive and indole negative Proteus,
Pseudomonas, E. coli, Aerobacter, Klebsiella,
Salmonella and Shigella3. Gentamicin is highly active
against Gram negative bacteria and Mycobacterium
tuberculosis4. In addition to its use as antibacterial
agent, the potential antiviral properties of some
gentamicin conjugates have been demonstrated5
recently.
Gentamicin is an aminocylitol containing
aminoglycoside antibiotic complex, consisting
predominantly of three major components; gentamicn
C1, C1a and C2. The C1, C1a and C2 components
(gentamicin Cs) are composed of a central diaminogenous cyclitol [2-deoxy streptamine (2DOS)],
4,6-disubstuted with the auxiliary sugar garosamine
and purpurosamine. Numerous biochemical studies
have attempted to elucidate the biosynthetic route
__________
*Correspondent author:
Phone: +91 40 27160123; Fax: +91 40 27193159
E-mail: annapurna@iictnet.org

to gentamicin Cs and a branched pathway has


been proposed based on precursor feeding studies
of a blocked mutant6-8. Madan et al.9 have described
the cloning and characterization of DOS biosynthetic genes involved in gentamicin biosynthesis
in M. echinospora.
The negative influence exerted by the type and
concentration of the carbon source on the
fermentative production of secondary metabolites, has
been well documented for several antibiotics10.
Nitrogen source have long been known to suppress
the biosynthesis of a variety of chemically unrelated
antibiotics and other secondary metabolites11.
In the present paper, we establish the essential
nutritional requirements for gentamicin biosynthesis
and examine the relationship between the growth of
the organism and production of the antibiotic, which
ultimately is of great significance for the kinetics of
gentamicin production in batch and continuous
reactors.
Materials and Methods
Microorganism and growthMicromonospora
echinospora subsp pallida ATCC 15838 was used as
production strain. The culture was maintained in agar
slants contained the following components (g/l): yeast
extract, 10; glucose, 10; CaCO3, 3; agar, 15.

HIMABINDU & JETTY: OPTIMIZATION OF NUTRITIONAL REQUIREMENTS FOR GENTAMICIN

Inoculation medium contained (g/l): beef extract, 3;


glucose, 1; soluble starch, 24; yeast extract, 5; CaCO3,
4; pH was adjusted to 7.6 before sterilization. The
inoculum medium (50 ml per 250 ml Erlenmeyer
flask) was inoculated with M. echinospora (one slant
for each flask) under aseptic conditions. The
inoculated flasks were kept on a rotary shaker at 200
rpm at 27 +2C for 72 h to obtain good growth.
Fermentation mediaThe different
media
mentioned in Table 1 were prepared (50 ml in 250 ml
Erlenmeyer flask). They were inoculated with 5%
(v/v) of 48 h inoculum. The inoculated flasks were
incubated for 10 days. Every day antibiotic
concentration, total dry cell weight, amount of
residual sugar and pH were determined.
Analytical methodsAt specified intervals,
production of antibiotic was determined by disc
diffusion method using Staphylococcus aureus MTCC
737 as the assay organism 12. Growth was measured as
the weight of cell mass obtained from a culture by
vacuum filtration (Whatman No.1 filter disc) and
drying at 100C for 4 h. The residual sugar was
determined by method of Frank A Loewus13.
Optimization of culture conditions for gentamicin
productionFor the study on optimal nutritional
requirements for gentamicin production and growth,
medium XIII was chosen as the basal medium. Effect
of culture age (24, 48, 72 and 96 h) and size (2, 4, 6, 8
and 10%) on gentamicin production by M.
echinospora was investigated.
To determine the effects of carbon sources on
gentamicin production, the glucose of the basal
medium (medium XIII) was replaced by equivalent
amounts of other carbon sources of fructose, maltose,
lactose, sucrose, starch, dextrin, molasses, glycerol
and D-xylulose, while the other ingredients of the
medium were remained same. During fermentation
the growth rate and gentamicin concentration were
determined.
To determine the effects of nitrogen sources on
gentamicin yield, the soya bean meal of the basal
medium (medium XIII) was replaced by equivalent
amount of inorganic and organic nitrogen sources.
Inorganic nitrogen sources [(NH4)2SO4, NH4Cl, NH4NO3 and (NH4)2HPO4] and organic nitrogen sources
(yeast extract, corn steep solids, casein, beef extract,
soybean meal, sodium glutamate and urea) were
altered, while the other ingredients remained the
same.

843

Results
M. echinospora was able to grow and produce
gentamicin in all chemically defined media in general.
Table 1 showed that maximum gentamicin
concentration of 345 mg/l was recorded in the
medium XIII on 5th day of fermentation period,
followed by media IX, IV, II, XVIII, I (334 mg/l-290
mg/l). The data exhibited the important role of the
chemical constituents such as carbon sources,
nitrogen sources of the fermentation medium for the
antibiotic production. Therefore, medium XIII was
selected for further studies on the fermentative
production of gentamicin by M. echinospora.
Effect of inoculum agePresent study was
undertaken to investigate the influence of inoculum
age and level, various carbon and nitrogen sources on
gentamicin production and growth rate of
M. echinospora. Table 2 shows the effects of
inoculum age on gentamicin production and growth
rate of M. echinospora. With 72 h old inoculum the
antibiotic production (375mg/l) and growth rate
(72 mg/h) were maximum compared to 24, 48 and 96
h old inoculums. Although highest growth rate
achieved earlier (3rd day) with 96 h old inoculum, the
production of gentamicin was less effective than 72 h
old inoculum. Further studies were carried out with
72 h old inoculum.
Effect of various inoculum levelsIn culture
containing 8% (v/v) level, cells enter the stationary
phase very quickly but the gentamicin yield was less
effective. Gentamicin accumulation increased from
312 to 375 mg/l with increasing doses of inoculum up
to 6% (v/v; Table 3). Further increase of inoculum
concentration (beyond 6%) was inhibitory to
gentamicin accumulation. The decrease in gentamicin
production beyond the optimum inoculum dose may
be due to depletion of nutrients including the carbon
source or may be most nutrients are directed for the
formation of biomass rather than enhancement of
gentamicin production.
Effect of various carbon sourcesEffect of various
carbon sources on growth rate and gentamicin
production was shown in Table 4. Starch was found to
be best carbon source for gentamicin production and
growth rate. Growth rates were similar, but
gentamicin production was lowered when maltose
was used as carbon source instead of starch. Lactose,
fructose and sucrose also favoured high growth rates,
but gentamicin yields were less. Dextrin, D-xylulose,
molasses and glycerol were poor supporters for

INDIAN J EXP BIOL, OCTOBER 2006

844

Table 1Composition of different media used for the production of gentamicin by M. echinospora* (10 days old inoculum)
Medium
No.
1

Carbon source
(g/l)

Nitrogen source
(g/l)

Macro elements
(g/l)

Micro elements
(g/l)

Gentamicin
(mg/l)
290

D-Glucose (1)
Starch (24)

Beef extract (3)


Yeast extract (5)
Tryptone (5)

Starch (20)
D-Glucose (1)

Yeast extract (10)


Meat extract (10)
Casein hydrolysate (50)

CaCO3 (5)

CoCl2 (0.04)

D-Glucose (10)

Yeast extract (5)


Meat extract (5)

CaCO3 (2)

CoCl2 (0.02)

210

Starch (30)

Peptone (10)

NaNO3 (4)
K2HPO4 (2)
KCl (1)

FeSO4 (0.02)
CoCl2 (0.001)

328

Glycerol (20)

Glycine (2.5)

CaCO3 (0.1)
K2HPO4 (1)
MgSO4 (0.1)

6
7

D-Glucose (5)
Glycerol (25)

Soyabean meal (15)

Maltose (20)

312

287
FeSO4 (0.1)
211

CaCO3 (10)
NaCl (2.5)

Soyabean meal (10)


(NH4)2SO4 (3)

K2HPO4 (1)
NaCl (5)
MgSO4.7H2O (0.02)

CoCl2 (0.001)
ZnSO4.H2O (0.03)

150

D-fructose (20)

Soyabean meal (10)

CaCO3 (1)
NaCl (5)

134

D-xylose (5)

Soyabean meal (10)

CaCO3 (1)
NaCl (5)

334

10

Starch (30)
D-Glucose (5)
Corn steep solids (5)

Soyabean meal (30)

CaCO3 (7)

CoCl2 (0.13)

150

11

Dextrin (50)
D-Glucose (5)

Soyabean meal (35)

CaCO3 (7)

CoCl2 (0.0013)

166

12

Lactose (10)

Soyabean meal (10)

CaCO3 (1)
NaCl (5)

166

13

D-Glucose (10)

Soyabean meal (5)

CaCO3 (4)
K2HPO4 (1)

FeSO4 (0.03)
CoCl2 (0.001)

345

14

D-Glucose (10)

Soyabean meal (10)

NaCl (5)
CaCO3 (1)

212

15

D-Glucose (20)

Beef extract (10)


Yeast extract (10)
(NH4)2SO4 (3)

CaCO3 (0.02)
K2HPO4 (0.5)
NaCl (2.5)

FeSO4 (0.2)
CoCl2 (0.02)

181

16

Dextrin-10
Corn steep solids (20)

Soyabean meal (10)

CaCO3 (2)
K2HPO4 (2)
NaCl (5)

17

D-Glucose (10)
Molasses (20)

Peptone (5)

130

18

Sucrose (20)

NH4Cl (4)

CaCO3 (10)
K2HPO4 (1)
NaCl (3)
MgSO4.7H2O (0.02)

CoCl2 (0.001)
ZnSO4.H2O (0.03)
FeSO4 (0.03)
MnSO4.4H2O (0.01)

293

*Represents maximum antibiotic production during 10-day course of fermentation

185

HIMABINDU & JETTY: OPTIMIZATION OF NUTRITIONAL REQUIREMENTS FOR GENTAMICIN

antibiotic production as well as growth of


M. echinospora. With D-glucose gentamicin synthesis
and growth rates were less effective than starch.
Starch was used as carbon source for further
experiments. Figure 1 shows the effect of different
concentrations of starch on gentamicin concentration
and cell growth. As the starch concentration
increased, relatively the cell growth also increased.
Gentamicin concentration was maximum (429 mg/l)
when starch concentration was 7.5 g/l. Maximum cell
growth (8.1 g/l) was observed in medium having
12.5 g/l of starch.
Effect of various nitrogen sourcesEffect of
various nitrogen sources on gentamicin production
and growth rates has been shown in Table 5. The
medium contained starch (10 g/l) and supplemented
Table 2Effect of inoculum age on growth and gentamicin
by M. echinosporaa
Inoculum
age (h)
24
48
72
96

Growth rateb
rmax
tmax
(mg/h)
(days)
45
59
72
75

6
6
4
3

Gentamicinc
ymax
zmax
(mg/l)
(days)
312
345
375
324

7
7
5
5

a
Medium composition (g/l): glucose, 10; soyabean meal, 5;
K2HPO4, 1; CaCO3, 4; FeSO4 0.03; 0.001 CoCl2. Cultivation
conditions 27C, 10 days, and 200rpm.
b
Measured as the maximum rate (rmax) of cell dry weight
increase, and as the time (tmax) required to attain maximum cell
dry weight.
c
Measured as the maximum titer (ymax) and as time (zmax)
required to attain ymax. Analyses were discontinued at 10 days.

with various nitrogen sources (5 g/l). Soyabean meal


and sodium glutamate were the best nitrogen sources
for gentamicin production, but cell growth was less
when sodium glutamate was used as sole nitrogen
source. Inorganic nitrogen sources such as (NH4)2SO4,
NH4Cl, NH4NO3, (NH4)2HPO4 were less effective
when compared with organic nitrogen sources such as
yeast extract, soyabean meal, corn steep solids,
casein, urea, sodium glutamate, beef extract. Yeast
extract and corn steep solids were favorable for
growth but not the antibiotic production. Previous
studies have reported that organic nitrogen sources
were best for gentamicin production by
M. purpurea14. Hence, soyabean meal was chosen as
the nitrogen source for further experiments.
Gentamicin production (439 mg/l) and cell growth
(7.8 g/l) were highest when the concentrations of
soyabean meal in the medium were 5% and 10%,
respectively (Fig. 1).
Effect of various concentrations of phosphate
sourceAs shown in Fig. 2, K2HPO4 at a
concentration of 1.2g/l gave maximum gentamicin
concentration (452mg/l). Concentration of 1.0g/l of
phosphate was found to be optimum for
M. echinospora cell growth (7.1g/l). Gentamicin
synthesis was strongly inhibited by lower (0.2g/l) and
higher (2g/l) phosphate concentrations. The use of
K2HPO4 higher than 1g/l did not inhibit the growth
significantly, but prevented antibiotic formation.
Table 4Effect of carbon source on growth and production of
gentamicin by M. echinosporaa.
Carbon
source

Table 3Effect of inoculum levels on growth and gentamicin


formation by M. echinosporaa
Inoculum
Conc. (%)
2
4
6
8
10

Growth rateb
tmax
rmax
(mg/h)
(days)
38
57
74
82
93

6
6
5
3
4

Maltose
Starch
Lactose
Fructose
Sucrose
Glucose
Dextrin
D-ylulose
Molasses
Glycerol

Gentamicinc
ymax
zmax
(mg/l)
(days)
254
331
392
321
290

6
8
5
5
5

a
Medium composition (g/l): glucose, 10; soyabean meal, 5;
K2HPO4, 1; CaCO3, 4; FeSO4 0.03; CoCl2, 0.001. Cultivation
conditions 27C, 10 days, and 200rpm.
b
Measured as the maximum rate (rmax) of cell dry weight
increase, and as the time (tmax) required to attain maximum cell
dry weight.
c
Measured as the maximum titer (ymax) and as time (zmax)
required to attain ymax. Analyses were discontinued at 10 days.

845

Growth rateb
rmax (mg/h) tmax (days)
76.6
71.8
65
63.1
59
51.6
51.3
35
16.6
7.2

4
4
6
4
6
5
3
6
10
8

Gentamicinc
ymax (mg/l) zmax (days)
250
413
367
310
333
375
303
279
284
273

6
6
7
5
8
8
6
7
9
7

Medium composition (g/l): carbon source, 10; soyabean meal, 5;


K2HPO4, 1; CaCO3, 4; FeSO4, 0.03; CoCl2, 0.001. Cultivation
conditions 27C, 10 days, and 200rpm.
b
Measured as the maximum rate (rmax) of cell dry weight increase,
and as the time (tmax) required to attain maximum cell dry weight.
c
Measured as the maximum titer (ymax) and as time (zmax) required
to attain ymax. Analyses were discontinued at 10 days.

846

INDIAN J EXP BIOL, OCTOBER 2006

Fermentation profile of gentamicin production by


M. echinosporaUsing the optimized medium
composed of (g/l): starch, 7.5; soyabean meal, 5;
K2HPO4, 1.2; CaCO3, 4; FeSO4, 0.03; CoCl2, 0.001
and optimized culture age and size, 6% of 72 h old
M. echinospora culture, the fermentation profile of
M. echinospora during gentamicin production were
examined.
As seen in Fig. 3, the pH values of culture medium
were increased from 7.3 to 8.8. Biosynthesis of
gentamicin started at logarithmic phase during 3 day
period and reached maximum in stationary phase at
day 6 of fermentation period. An increase in
gentamicin concentration was seen at last stage
(day 8) after decrease on day 7.
Discussion
Nutritional regulation of fermentative production of
gentamicin by M. purpurea was well established by
earlier workers14. M. echinospora produces
gentamicin is closely related to M. purpurea15. In the
process development of fermentation medium to
study relationships between antibiotic production and
metabolic control of different carbon and nitrogen
source utilization in M. echinospora, we first
examined the relevant fermentation parameters to
establish optimum conditions. A large inoculum was
found to suppress production. Since gentamicin
biosynthesis occurred during culture growth, use of
less vigorous stationary phase (72 h old) inoculum
[6% (v/v)] showed more accumulation of gentamicin.

In the present study, starch was found to be the best


carbon source for growth and gentamicin production
by M. echinospora. In actinomycetes, carbon sources
that can readily serve as growth substrates, such as
glucose, often repress secondary metabolism16. An
example of catabolite repression of secondary
metabolism in actinomycetes is that of actinomycin
synthesis by Streptomyces antibioticus after glucose is
Table 5Effect of nitrogen source on growth and production
of gentamicin by M. echinosporaa
Nitrogen source

Yeast extract
Soyabean meal
Corn steep solids
Casein
Urea
(NH4)2SO4
Sodium glutamate
Beef extract
NH4Cl
NH4NO3
(NH4)2HPO4

Growth rateb
tmax
rmax
(mg/h)
(days)
80.2
73.5
72
52
51.3
43
37
36.9
29
20
8

4
4
5
6
4
3
2
3
3
3
3

Gentamicinc
ymax
zmax
(mg/l) (days)
328
435
250
354
275
271
393
325
275
220
228

5
5
5
9
6
6
5
5
8
7
8

Medium composition (g/l): starch, 10g/l; Nitrogen source, 5;


K2HPO4, 1; CaCO3, 4; FeSO4, 0.03; CoCl2, 0.001. Cultivation
conditions 27C, 10 days, and 200rpm.
b
Measured as the maximum rate (rmax) of cell dry weight
increase, and as the time (tmax) required attaining maximum cell
dry weight.
c
Measured as the maximum titer (ymax) and as time (zmax)
required to attain ymax. Analyses were discontinued at 10 days.

Fig. 1Effect of starch and soyabean meal concentrations on growth and gentamicin production by M. echinospora

HIMABINDU & JETTY: OPTIMIZATION OF NUTRITIONAL REQUIREMENTS FOR GENTAMICIN

847

Fig. 2Effect of K2HPO4 concentration on growth and gentamicin production by M. echinospora

Fig. 3Fermentation profile of M. echinospora

added to the medium17. Catabolite repression exerted


by D-glucose causes a negative action on gentamicin
synthesis by M. purpurea18. Catabolite inhibition of
secondary metabolism (idiolite production) by a
specific carbon source can occur due to inactivation
of those enzymes necessary for idiolite production19.
Similarly, catabolite repression by D-glucose was
observed in M. echinospora in the present study.
Among all organic and inorganic nitrogen sources
tested, maximum production was achieved with
soyabean meal. Products of ammonium assimilation,
glutamate and glutamine were able to exert the
stimulatory effect on gentamicin formation due to its

ability to produce 2-deoxystreptamine and glucosamine, intermediates of the gentamicin biosynthetic


pathway20.
Phosphates can directly regulate secondary
metabolism in actinomycetes. Biosynthesis of several
antibiotics is controlled by phosphate concentration21.
Production of gentamicin by M. purpurea was
inhibited by high phosphate concentration (11.5 mM),
while biomass production was unaffected22,
comparable to the production of antibiotic tylosin by a
Strptomyces sps was inhibited by high phosphate
concentration (30 mM), while biomass was
unaffected23. Present results also indicated higher

INDIAN J EXP BIOL, OCTOBER 2006

848

phosphate concentration caused repression of


gentamicin synthesis, while unaffected the biomass
production.
From these results, the best medium composition
for gentamicin production by M. echinospora
comprised 0.75%, starch; 0.5%, soyabean meal;
0.12%, K2HPO4; 0.4%, CaCO3; 0.003%, FeSO4 and
0.0001%, CoCl2. Gentamicin concentration reached
maximum during stationary phase, and again during
last phase. Since most of the activity resides in the
mycelium24, degradation of mycelium during last
phase is responsible for peak antibiotic production at
the end of incubation period25. The maximum
gentamicin (410 mg/l) was obtained using this
medium by M. echinospora, which was higher than
produced (190 mg/l) by culture of M. purpurea18.
Acknowledgement
The authors are thankful to Dr J S Yadav, Director,
IICT for his co-operation and one of the authors,
(MH) is thankful to Council of Scientific and
Industrial Research, New Delhi, for awarding JRF.

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12

13
14

15

16

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