The impact of Schiff bases on antibiotic production by Streptomyces hygroscopicus

Article abstract of DOI:10.1007/s00044-009-9223-7

A media consisting of isatin-Schiff bases (isatin-3-thiosemicarbazone, isatin-3-semicarbazone, and isatin-3-phenylhydrazone) was developed to maximize the production of antibiotics Hexaene H-85 and Azalomycine B by Streptomyces hygroscopicus. The media is

Full text of DOI:10.1007/s00044-009-9223-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:690–697
DOI 10.1007/s00044-009-9223-7
ORIGINAL RESEARCH
The impact of Schiff bases on antibiotic production
by Streptomyces hygroscopicus
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Slavica B. Ilic Æ Sandra S. Konstantinovic Æ
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Dragisa S. Savic Æ Vlada B. Veljkovic Æ
´
G. Gojgic-Cvijovic
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Received: 3 February 2009 / Accepted: 19 May 2009 / Published online: 9 June 2009
Ó The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract A media consisting of isatin-Schiff bases (isatin-3-thiosemicarbazone,
isatin-3-semicarbazone, and isatin-3-phenylhydrazone) was developed to maximize
the production of antibiotics Hexaene H-85 and Azalomycine B by Streptomyces
hygroscopicus. The media isatin-3-thiosemicarbazone resulted in the maximum
antibiotics concentration of 372 lg cm-3 for Hexaene H-85 and 118 lg cm-3 for
Azalomycine B. The impact of modified media on soil morphology also was
investigated.
Keywords Streptomyces hygroscopicus Á Schiff base Á Antibiotic production Á
Morphology
Introduction
The genus Actinomyces is an important group of microbes due to their ability to
produce commercially valuable secondary metabolites (Abbas and Edwards, 1990;
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Vucetic et al., 1994; Okami and Hotta, 1988; Prosser and Tough, 1991). The
actinomycete Streptomyces hygroscopicus produces a range of polyene antibiotics
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compounds depending on environmental and nutritional conditions (Vucetic et al.,
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1994; Karadzic et al., 1991). To make the production of the antibiotic feasible, it is
necessary to develop the optimum production, which includes among the other
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S. B. Ilic (&) Á S. S. Konstantinovic Á D. S. Savic Á V. B. Veljkovic
Faculty of Technology, University of Nis, Bulevar Oslobodjenja 124,
Leskovac 16000, Serbia
e-mail: ilicslavica@yahoo.com
´
S. S. Konstantinovic
e-mail: sakisandra12@yahoo.com
´
G. Gojgic-Cvijovic
Department of Chemistry, ICTM, Njegoseva 12, Belgrade 11000, Serbia
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Med Chem Res (2010) 19:690–697
691
conditions, formation of chemically defined media. There have been some
investigations about different nitrogen and carbon sources on growth and production
(Abbas and Edwards, 1990; Lee et al., 1997; de Queiroz Sousa et al., 2001; Tripathi
et al., 2004), but no data are available about the influence of Schiff base. In the
present study, an extensive study has been made on the isatin-Schiff bases as a
nitrogen source in chemically defined media on antibiotic production by Strepto-
myces hygroscopicus as well as on soil morphology.
Materials and methods
Organism, media, and growth condition
A strain Streptomyces hygroscopicus was isolated from a soil sample from
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Vojvodina, Serbia (Vucetic et al., 1994; Karadzic et al., 1991). Streptomyces
hygroscopicus was maintained as spore and mycelia suspensions in sterile glycerol
(20% [wt/vol]), which were prepared from speculated colonies grown at 30°C on L
agar that contained the following (in g l-1): tryptone (Difco Laboratories), 5; yeast
extract (Lab-M), 5; NaCl, 5; glucose, 1; agar, 10 (pH 7.2). Suspensions were stored
at -20°C until required. Liquid cultures were grown in starch–yeast extract (SY)
broth that contained the following (in g l-1): soluble starch, 15; yeast extract
(Difco), 1; K2HPO4 Á 7H20, 1; NaCl, 3 (final pH adjusted to 7.2). Flasks (250 ml)
that contained 50 ml of this media were inoculated with 0.1 ml of spore suspension
and incubated at 30°C with shaking at 200 rpm. The fermentation media were
inoculated with 5% (v/v) of a preculture after 48 h growth and incubated at 30°C for
240 h under the standard condition of aeration and agitation (200 rpm). The
fermentation basal media has the following composition (g/l): glucose 15, CaCO3 3,
NaCl 3, MgSO4 0.5, (NH4)2HPO4 0.5, K2HPO4 0.5, soya bean 1.0. The fermentation
modified media has the follow composition (g/l): glucose 15, CaCO3 3, NaCl 3,
MgSO4 0.5, (NH4)2HPO4 0.5, K2HPO4 0.5, L-tryptophan 0.5, Schiff base 0.5.
After fermentation, the antibiotics of the broth were determined by extraction
with n-butanol and ethyl acetate. The results were obtained by measuring
absorbance at kmax = 364 nm (Hexaene H-85) and kmax = 252 nm (Azalomycine)
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with Perkin-Elmer Lambda 15 UV/VIS spectrophotometer (Vucetic et al., 1994;
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Karadzic et al., 1991). Growth was determined by measuring dry weights of cells.
The broth was centrifuged at 4000 rpm for 15 min to separate the mycelial biomass.
After that biomass was dried at 105°C to constant weight and weighed.
General methods of preparation of Schiff bases
Equimolar amounts of isatin and thiosemicarbazide, semicarbazide, and phen-
ylhydrazine were dissolved in 95% ethanol. The solutions were heated under reflux
for 1 h. The products were filtered, washed with ethanol, and dried in vacuum over
´
CaCl2 (Konstantinovic et al., 2007). The structures of Schiff bases are given in
Fig. 1.

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Med Chem Res (2010) 19:690–697
Fig. 1 Structures of Schiff
bases
S
N
C
H
N
H
N
H
N
H
O
isatin-3-thiosemicarbazone
O
C
N
H
N
H
N
H
N
H
O
isatin-3-semicarbazone
N
N
H
N
H
O
isatin-3-phenylhydrazone
Methods
Microanalysis for carbon, hydrogen, and nitrogen was performed by using a Carlo
Erba 1106 microanalyzer. The chloride content was determined potentiometrically.
The melting points were determined by using Thomas–Hoover melting point
apparatus and are uncorrected. FTIR spectra were recorded using a Michaelson
Bomen MB-series spectrophotometer, using KBr pellet (1 mg/100 mg) technique.
The electronic spectra were recorded on a Perkin/Elmer Lambda 15 UV/VIS
1
spectrophotometer using 10-3 mol dm-3 solutions in DMF. H NMR spectra were
obtained in DMSO solution with a Gemini-200 ‘‘HF NMR’’ spectrometer.
Isatin-3-thiosemicarbazone (ITC)
Yield 91.1%, Color Yellow. m.p. 239–241°C. IR (KBr, cm-1): 3470, 3304 m(NH2),
3239, 3132 m(NH), 1710 m(C=O), 1585 m(C=N), 1250 m(C=S). UV/VIS (DMF, k
(nm/e Á 103(mol-1 dm3 cm): 349/0.946 p ? p*, 366/1.325 p ? p* 1H NMR
(DMSO, d, ppm) 6.9–7.7 (m, 4H, Ar), 8.69, 9.05 (s, 2H, NH2), 11.21 (2, 1H, NH),
12.47 (s, 1H, NH). Analysis: Found: 49.05%C, 3.75%H, 25.30%N, 14.51%S;
Calculated: 49.08%C, 3.70%H, 25.32%N, 14.56%S.

Med Chem Res (2010) 19:690–697
693
Isatin-3-semicarbazone (ISC)
Yield 90.5%, Color Yellow. m.p. 239°C. IR (KBr, cm-1): 3467, 3301 m(NH2), 3237,
3126 m(NH), 1704, 1686 m(C=O), 1595 m(C=N). UV/VIS (DMF, m(cm-1
/
e Á 103(mol-1 dm3 cm): 321.8/3.121 p ? p*, 271.8/2.662 p ? p*. 1H NMR
(DMSO, d, ppm) 6.02–7.94 (m, 4H, Ar), 8.34, 9.02 (s, 2H, NH2), 11.21 (2, 1H,
NH), 12.42 (s, 1H, NH). Analysis: Found: 52.92%C, 3.95%H, 27.45%N; Calculated:
52.94%C, 3.92%H, 27.45%N.
Isatin-3-phenylhydrazone (IPH)
Yield 47.89%, Color orange, m.p. 249°C. IR (KBr, cm-1): 3326, 3161 m(NH), 1686
m(C=O), 1597 m(C=N). UV/VIS (DMF, m(cm-1/e Á 103(mol-1 dm3 cm): 398.5/2.260
1
p ? p*, 258.5/1.625 p ? p*, 207.5/2.914 p ? p*. H NMR (DMSO, d, ppm)
6.91–7.57 (m, 4H, Ar), 11.00 (2, 1H, NH), 11,00 (s) (2, 1H, NH), 12.32 (s, 1H, NH).
Analysis: Found: 70.86%C, 4.62%H, 17.70%N; Calculated: 70.89%C, 4.64%H,
17.72%N.
Results and discussion
Influence of Schiff bases production of Hexaene H-85 and Azalomycine B
To improve production of Hexaene H-85 and Azalomycine B by Streptomyces
hygroscopicus, part of soya bean (0.5%) in basal medium was replaced with isatin
Schiff bases (ITC, ISC, and IPH) as a nitrogen source. The maximum concentration
of Hexaene H-85 and Azalomycine B (Fig. 2), pH and dry biomass, achieved during
the fermentation in basal and modified media are given in Table 1.
Change of pH values
Considering all media, as it can be seen, pH increases until the third or fourth day.
The basal medium possesses the highest pH 9.3, whereas the maximum values of
pH in tested media is in the range 8.1–8.4 (Fig. 2a).
Glucose utilization
As shown in Fig. 2b, Schiff bases do not have any impact on glucose utilization
during the fermentation. In the control medium, the glucose utilization is finished by
the third day, whereas media with Schiff bases possess a small amount of unused
glucose.
Dry biomass
As shown in Table 1, the addition of Schiff bases to media slightly increases the
growth of production soil. The maximum concentration (9.6 g dm-3) of dry

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Med Chem Res (2010) 19:690–697
10
9
20
15
10
5
(a)
(b)
8
7
6
5
0
0
1
2
3
4
5
6
7
8
0
1
2
3
4
5
6
7
8
Day of fermentation
Day of fermentation
160
140
120
100
80
450
400
350
300
250
200
150
100
50
(d)
(c)
60
40
20
0
0
0
1
2
3
4
5
6
7
8
0
1
2
3
4
5
6
7
8
Day of fermentation
Day of fermentation
Fig. 2 Change of pH (a), concentration of glucose and dry biomass (b), concentration of Hexaene H-85
(c), and Azalomycine B (d) in basal medium (-e-) and media with Schiff bases: ITC (-s-), ISC (-D-), and
IPH (-h-)
biomass is reached by the fourth day of fermentation in medium with ITC. The
values are lower for media with ISC and IPH (9.3 g dm-3 and 9.1 g dm-3
,
respectively). The maximum concentration of dry biomass in basal medium is
reached by the third day and its value is 8.9 g dm-3).
Production of Hexaene H-85
The addition of Schiff bases is stimulated the production of Hexaene H-85, and the
values are higher than basal medium. Maximum concentration of antibiotic is
reached by the third day in basal medium and by third and fourth days in modified
media (Table 1). The maximum concentration of Hexaene H-85 in medium with
ITC is 372 lg cm-3, which is for 63% higher compared with basal medium

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Table 1 Impact of Schiff bases on maximum specific rate of glucose utilization (kmax), maximum
concentration of dry biomass (Xmax), and maximum production (Cmax) and yield of antibiotics (Ymax)
during the fermentation of S. hygroscopicusa
Nitrogen source
kmax
Xmax
Hexaene H-85
Azalomycine B
CmHax
lg cm-3
YmHax
lg gs.b
CmAax
lg cm-3
YmAax
lg gs.b
d-1
g dm-3
SB
0.97
1.04
1.01
1.03
8.9
9.6
9.3
9.1
212
372
293
329
23.82
38.75
31.50
36.15
56
118
92
6.29
12.29
9.89
SB ? ITC
SB ? ISC
SB ? IPH
106
11.64
SB soya bean
(212 lg cm-3). The media with other ISC and IPH also stimulated the production of
this antibiotic for 32% and 52%, respectively, compared with the basal medium, but
the values are lower than medium with ITC (293 lg cm-3 and 329 lg cm-3
respectively; Fig. 3c).
,
Production of Azalomycine B
The addition of Schiff bases also stimulated the production of Azalomycine B
(Table 1). The highest concentration is achieved on the fourth day of fermentation.
Compared to the basal medium, ITC increases the concentration of antibiotic two
times, whereas ISC and IPH increase the production of the same antibiotic by 85%
and 57%, respectively (Fig. 3d).
The mechanism of action of tested Schiff bases was not examined in this work,
but there is no doubt that those compounds can be used as a carbon source for
antibiotic production. In this study, we used those compounds as a nitrogen source,
because there is a similarity between L-tryptophan, an amino acid already used as a
nitrogen source in a basal medium, and used Schiff bases. There is a probably a
connection between the structure of Schiff bases and their impact on antibiotic
production. The ITC has the highest influence on antibiotic production, and yet the
only difference compared with ISC is in C=S group, which ITC possesses and it is
known that biological activity of Schiff bases is due to C=N group and C=S group if
compound contained it.
Impact of Schiff bases on strain morphology
During fermentation, the nutrient media with isatin Schiff bases, as a nitrogen
source, the strain is in the form of pellets, and little of single, free filaments
(Table 2). The morphology of S. hygroscopicus is shown in Fig. 3.

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Med Chem Res (2010) 19:690–697
Fig. 3 Morphology of S. hygroscopicus in basal medium and media with Schiff bases: a ITC, b ISC, and
c IPH

Med Chem Res (2010) 19:690–697
697
Table 2 Impact of Schiff bases on morphology S. hygroscopicus and production of antibiotics
Nitrogen source
Strain morphology
Yield of antibiotics
YmHax
YmAax
ITC
ISC
IPH
Pellets, single, weakly branched fillaments
Pellets, single, weakly branched fillaments
Pellet, a little of sinlge fillaments
38.75
31.50
36.15
12.29
9.89
11.64
Open Access This article is distributed under the terms of the Creative Commons Attribution
Noncommercial License which permits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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