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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
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
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)
Starch (20)
D-Glucose (1)
CaCO3 (5)
CoCl2 (0.04)
D-Glucose (10)
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)
Maltose (20)
312
287
FeSO4 (0.1)
211
CaCO3 (10)
NaCl (2.5)
K2HPO4 (1)
NaCl (5)
MgSO4.7H2O (0.02)
CoCl2 (0.001)
ZnSO4.H2O (0.03)
150
D-fructose (20)
CaCO3 (1)
NaCl (5)
134
D-xylose (5)
CaCO3 (1)
NaCl (5)
334
10
Starch (30)
D-Glucose (5)
Corn steep solids (5)
CaCO3 (7)
CoCl2 (0.13)
150
11
Dextrin (50)
D-Glucose (5)
CaCO3 (7)
CoCl2 (0.0013)
166
12
Lactose (10)
CaCO3 (1)
NaCl (5)
166
13
D-Glucose (10)
CaCO3 (4)
K2HPO4 (1)
FeSO4 (0.03)
CoCl2 (0.001)
345
14
D-Glucose (10)
NaCl (5)
CaCO3 (1)
212
15
D-Glucose (20)
CaCO3 (0.02)
K2HPO4 (0.5)
NaCl (2.5)
FeSO4 (0.2)
CoCl2 (0.02)
181
16
Dextrin-10
Corn steep solids (20)
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
185
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.
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
846
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
Fig. 1Effect of starch and soyabean meal concentrations on growth and gentamicin production by M. echinospora
847
848
10
11
12
13
14
15
16
17
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