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Results and Discussion
102
4.1 Collection of soil samples
In our attempt to isolate actinomycetes isolates soil samples collected
from Akkampalli and Limgampalli villages of Anantapur district, Andhra
Pradesh. The physicochemical characteristics of the soil samples are
tabulated in Table 3.
Table 3. Physicochemical properties of the soil samples.
Soil type
Sand (%)a
Silt (%)a
Clay (%)a
pHb
Organic
matter
(%)c
Total
Nitrogen
(%)d
Black 67.4 20.1 12.5 7.63 1.13 0.18
Red 74.2 20.2 5.6 7.21 0.837 0.0473
aBouyoucos hydrometer method (Black, 1965)
b1:1.25 (soil: water) slurry.
cWalkley-Black method (Jackson, 1971).
dMicro-Kjeldahl method (Jackson, 1971).
The black soil has high soil pH, organic matter, total nitrogen and clay
percentage when compared to red soils. A red soil has high sand and silt
percentage when compared to black soils.
4.2 Isolation of actinomycetes
The rhizosphere soil samples were collected from foxtail millet was
serially diluted for enumerating the actinomycete isolates. In total 22 isolates
were isolated. All the isolates were purified and stored at -20°C in glycerol
(20% v/v) suspension. The morphological characteristics of the isolates were
tabulated in Table 4.
Results and Discussion
103
Table 4. Morphological features of the isolates of actinomycetes
Isolate Growth Aerial mycelium
Substrate mycelium
Pigment
1 Very Good Grayish White Olive Yellow Yellow
2 Very Good White Brownish Yellow Olive Yellow
3 Very Good White White Nil
4 Very Good White Light Pink Pink
5 Very Good Cherry Red Reddish Pink Red
6 Very Good Grayish White White Nil
7 Very Good White Brown Brown
8 Very Good Yellowish White
White Nil
9 Good White Brown Brown
10 Very Good Grayish White Yellowish Brown Yellow
11 Good White White Nil
12 Very Good White White Nil
13 Very Good Grayish White Light Brown Nil
14 Very Good Grey Grayish White Nil
15 Good White White Nil
16 Very Good White White Nil
17 Very Good Grayish White Yellowish Brown Brown
18 Very Good White Light Pink Pink
19 Very Good White Light Pink Pink
20 Very Good Pinkish White Pink Pink
21 Very Good White Light Pink Pink
22 Very Good White Brown Brown
Most of the isolates of actinomycetes obtained from the rhizosphere
soil samples by serial dilution method on starch casein agar medium showed
very good growth except A9, A11 and A15 isolates which showed good
growth. The aerial mycelium is grayish white i
white in A2, A3, A4, A7, A9, A11, A12, A15, A16,
isolates(Figure4).
Figure 4. Morphological features of the colonies of actinomycete isolates
Some of the isolates like A8 showed yellowish white while A5 showed
cherry red and A14 showed gray mycelium and A20 showed pinkish white
mycelium. The substrate mycelium is white in case of A3, A6, A8, A11, A12,
A15, A16 isolates and brown in A7, A9, A13 a
brown substrate mycelium was observed in A2, A10 and A17. Pink substrate
mycelium was noticed in A4, A18, A19, A20 and A21. Olive yellow mycelium
was exhibited by A1 isolate, reddish pink by A5 isolate and grayish white
mycelium by A14 isolate. Among the 22 isolates, only 13 isolates produced
different pigments. Yellow pigment was produced by A1, A2 and A10 isolates,
Results and Discussion
104
growth. The aerial mycelium is grayish white in A1, A6, A10, A13, A17 and
white in A2, A3, A4, A7, A9, A11, A12, A15, A16, A18, A19, A21
Morphological features of the colonies of actinomycete isolates and pigments produced in SCA medium.
Some of the isolates like A8 showed yellowish white while A5 showed
cherry red and A14 showed gray mycelium and A20 showed pinkish white
mycelium. The substrate mycelium is white in case of A3, A6, A8, A11, A12,
A15, A16 isolates and brown in A7, A9, A13 and A22 isolates. Yellowish
brown substrate mycelium was observed in A2, A10 and A17. Pink substrate
mycelium was noticed in A4, A18, A19, A20 and A21. Olive yellow mycelium
was exhibited by A1 isolate, reddish pink by A5 isolate and grayish white
by A14 isolate. Among the 22 isolates, only 13 isolates produced
different pigments. Yellow pigment was produced by A1, A2 and A10 isolates,
Results and Discussion
n A1, A6, A10, A13, A17 and
A21 and A22
Morphological features of the colonies of actinomycete medium.
Some of the isolates like A8 showed yellowish white while A5 showed
cherry red and A14 showed gray mycelium and A20 showed pinkish white
mycelium. The substrate mycelium is white in case of A3, A6, A8, A11, A12,
nd A22 isolates. Yellowish
brown substrate mycelium was observed in A2, A10 and A17. Pink substrate
mycelium was noticed in A4, A18, A19, A20 and A21. Olive yellow mycelium
was exhibited by A1 isolate, reddish pink by A5 isolate and grayish white
by A14 isolate. Among the 22 isolates, only 13 isolates produced
different pigments. Yellow pigment was produced by A1, A2 and A10 isolates,
brown pigment by A7, A9, A17 and A22 isolates. Red pigment was observed
in case of A5 isolate and pink pigment in A
All the isolates were subjected to screening methods such as spot
inoculation and cross streak method/perpendicular streak method. After the
screening methods, the antagonism of the actinomycete isolates was
determined against the test organisms like Gram positive (
Gram negative (E. coli) bacteria and the results are tabulated in Table 5
Among the 22 strains A2, A3, A5, A8, A11, A13, A14, A16, A17, A19
has no effect. Strains like A1, A7, A10, A12, A15, A21, and A22 has low
antimicrobial activity against the
activity and strains A4, A6, A18, A20 has
subtilis. Strains like A1, A2, A3, A5, A8, A11, A13, A14, A15, A16, A17, A19
has no activity against the
have low antimicrobial activity against the
activity and strains A6, A18 and A20 has high antimicrobial activity against
E.coli and Figure 5.
Figure 5. Antimicrobial activity of the b, c) and Gram negative (d, e) bacteria.
Results and Discussion
105
brown pigment by A7, A9, A17 and A22 isolates. Red pigment was observed
in case of A5 isolate and pink pigment in A4, A18, A19, A20 and A21 isolates.
All the isolates were subjected to screening methods such as spot
inoculation and cross streak method/perpendicular streak method. After the
screening methods, the antagonism of the actinomycete isolates was
against the test organisms like Gram positive (B. subtili
) bacteria and the results are tabulated in Table 5
Among the 22 strains A2, A3, A5, A8, A11, A13, A14, A16, A17, A19
has no effect. Strains like A1, A7, A10, A12, A15, A21, and A22 has low
antimicrobial activity against the Bacillus subtilis, strains A9 has medium
activity and strains A4, A6, A18, A20 has high activity against the
. Strains like A1, A2, A3, A5, A8, A11, A13, A14, A15, A16, A17, A19
has no activity against the E.coli. Strains A4, A7, A10, A12, A21 and A22
have low antimicrobial activity against the E.coli. Strain A9 has medi
activity and strains A6, A18 and A20 has high antimicrobial activity against
Antimicrobial activity of the isolates against Gram positive b, c) and Gram negative (d, e) bacteria.
Results and Discussion
brown pigment by A7, A9, A17 and A22 isolates. Red pigment was observed
4, A18, A19, A20 and A21 isolates.
All the isolates were subjected to screening methods such as spot
inoculation and cross streak method/perpendicular streak method. After the
screening methods, the antagonism of the actinomycete isolates was
B. subtilis) and
) bacteria and the results are tabulated in Table 5.
Among the 22 strains A2, A3, A5, A8, A11, A13, A14, A16, A17, A19
has no effect. Strains like A1, A7, A10, A12, A15, A21, and A22 has low
, strains A9 has medium
high activity against the Bacillus
. Strains like A1, A2, A3, A5, A8, A11, A13, A14, A15, A16, A17, A19
. Strains A4, A7, A10, A12, A21 and A22
. Strain A9 has medium
activity and strains A6, A18 and A20 has high antimicrobial activity against
isolates against Gram positive (a,
Results and Discussion
106
Table 5. Antimicrobial activity of the isolates against Gram positive and Gram negative bacteria.
Isolates Diameter of zone of
inhibition (mm)
Bacillus subtilis
Diameter of zone of inhibition (mm)
Escherichia coli
1 8 mm 0 mm
2 0 mm 0 mm
3 0 mm 0 mm
4 30 mm 8 mm
5 0 mm 0 mm
6 32 mm 32 mm
7 8 mm 8 mm
8 0 mm 0 mm
9 24 mm 24 mm
10 12 mm 12 mm
11 0 mm 0 mm
12 8 mm 8 mm
13 0 mm 0 mm
14 0 mm 0 mm
15 8 mm 0 mm
16 0 mm 0 mm
17 0 mm 0 mm
18 32 mm 32 mm
19 0 mm 0 mm
20 36 mm 36 mm
21 8 mm 8 mm
22 8 mm 8 mm
Among the 22 isolates, only 11 isolates showed antimicrobial activity.
Among the 11 isolates, 3 isolates which showed varying degree of
antimicrobial activity were selected for further studies. Thus the isolate A10,
with low antimicrobial activity, isolate
and isolate A20 with high
Table 6.
Table 6. Three selected
Isolate Growth
A-9 Good
A-10 Very Good
A-20 Very Good
The growth characteristics
pigments produced by the three isolates are depicted in
(a)
Figure 6. Growth of aerial (a) and substrate mycelium (b) and pigment production by A9, A10 and A20 isolates on SCA medium.
Results and Discussion
107
Among the 22 isolates, only 11 isolates showed antimicrobial activity.
Among the 11 isolates, 3 isolates which showed varying degree of
were selected for further studies. Thus the isolate A10,
antimicrobial activity, isolate A9 with medium antimicrobial activity
and isolate A20 with high antimicrobial activity were selected and tabulated in
Three selected isolates of actinomycetes
Growth Aerial mycelium
Substrate mycelium
Pigmentation
Good White Brown
Very Good Grayish White Yellowish Brown
Very Good Pinkish White Pink
The growth characteristics like aerial and substrate mycelium
pigments produced by the three isolates are depicted in Figure 6.
(a) (b)
Figure 6. Growth of aerial (a) and substrate mycelium (b) and pigment production by A9, A10 and A20 isolates on SCA medium.
Results and Discussion
Among the 22 isolates, only 11 isolates showed antimicrobial activity.
Among the 11 isolates, 3 isolates which showed varying degree of
were selected for further studies. Thus the isolate A10,
antimicrobial activity
antimicrobial activity were selected and tabulated in
Pigmentation
Brown
Yellow
Pink
like aerial and substrate mycelium and
(b)
Figure 6. Growth of aerial (a) and substrate mycelium (b) and pigment production by A9, A10 and A20 isolates on SCA medium.
Results and Discussion
108
4.3 Growth pattern and antimicrobial profile of the actinomycete isolates
Growth pattern of the isolates was studied in starch casein broth. The
medium after sterilization and cooling was inoculated with the pure culture of
the isolates and incubated at 37°C for 24 hr. The fermentation medium was
then inoculated with 10% seed culture to study growth pattern and
antimicrobial properties of the isolates. At 24 hr intervals, the culture was
harvested and the biomass (g/100 ml) was recorded in terms of dry weight
(Figure 7a). All these isolates entered into log phase after 24 hr of incubation
which continued up to 96 hr. They exhibited stationary phase from 96 to 120
hr followed by decline phase. Changes in pH of culture broth were also
recorded (Figure 7b).
Figure 7. Biomass of actinomycete isolates and pH of SCA broth
(a) (b)
The culture filtrates obtained after the separation of biomass were
screened for the production of secondary metabolites. The cultural filtrates,
collected at regular intervals of 24 hr up to 160 hr were extracted with ethyl
acetate and concentrated to dryness under vacuum and tested for
0
2
4
6
8
24 48 72 96 120 144 168
pH
Incubation period (hr)
pH of the actinomycetes isolates during growth
A9 A10 A20
0
100
200
300
400
24 48 72 96 120144168Bio
ma
ss (
mg
/10
0m
l)
Incubation period (hr)
Growth pattern of the actinomycete isolates in SCA
broth
A9 A10 A20
antimicrobial activity against pathogenic Gram positive and Gram negative
bacteria such as Bacillus subtilis, Staphylococcus aureus, Pseudomonas
aeruginosa, Proteus mirabilis, Escherichia coli, Micrococcus luteus, Klebsiell
pneumoniae, Salmonella typhi
(a)
Figure 8. Antimicrobial profile exhibited by A9isolates against Gram positive and Gram negative bacteria
Data on the antimicrobial spectrum of the ethyl acetate extracts of the
actinomycetes isolates A9, A10 and A20 are recorded in Table 7, 8 and 9
respectively. A gradual increase in the antimicrobial
A9, A10 and A20 was observed with increasing age of the culture up to 96 hr
and then decreased.
Gram positive bacteria like
metabolites of the isolate A20 followed by A9 and A
the isolate A20 were highly inhibitory to
produced by the isolate A9 and A
B. subtilis and S. aureus
aeruginosa was highly sensitive to the crude metaboli
isolates A20, A9 and A
not inhibit the Gram negative bacteria like
isolate A20 did not inhibit
Results and Discussion
109
antimicrobial activity against pathogenic Gram positive and Gram negative
Bacillus subtilis, Staphylococcus aureus, Pseudomonas
aeruginosa, Proteus mirabilis, Escherichia coli, Micrococcus luteus, Klebsiell
, Salmonella typhi and E. aerogenes (Figure 8a, 8b, 8c).
(b) (c)
microbial profile exhibited by A9 (a), A10 (b) and A20against Gram positive and Gram negative bacteria
Data on the antimicrobial spectrum of the ethyl acetate extracts of the
actinomycetes isolates A9, A10 and A20 are recorded in Table 7, 8 and 9
respectively. A gradual increase in the antimicrobial spectrum of the isolates
A9, A10 and A20 was observed with increasing age of the culture up to 96 hr
Gram positive bacteria like B. subtilis exhibited high sensitivity to
metabolites of the isolate A20 followed by A9 and A10. The crude extracts of
20 were highly inhibitory to S. aureus and B. subtilis
produced by the isolate A9 and A10 showed less inhibitory activity against
S. aureus. Among the Gram negative bacteria tested,
s highly sensitive to the crude metabolites elaborated by the
A9 and A10. The crude extracts of the isolates A9 and A
negative bacteria like S. typhi and E. aerogenes
20 did not inhibit E. aeruginosa alone. The metabolites pr
Results and Discussion
antimicrobial activity against pathogenic Gram positive and Gram negative
Bacillus subtilis, Staphylococcus aureus, Pseudomonas
aeruginosa, Proteus mirabilis, Escherichia coli, Micrococcus luteus, Klebsiella
(Figure 8a, 8b, 8c).
(b) (c)
and A20 (c) against Gram positive and Gram negative bacteria.
Data on the antimicrobial spectrum of the ethyl acetate extracts of the
actinomycetes isolates A9, A10 and A20 are recorded in Table 7, 8 and 9
spectrum of the isolates
A9, A10 and A20 was observed with increasing age of the culture up to 96 hr
exhibited high sensitivity to the
rude extracts of
subtilis while those
owed less inhibitory activity against
negative bacteria tested, P.
tes elaborated by the
tracts of the isolates A9 and A10 did
E. aerogenes while the
alone. The metabolites produced by
Results and Discussion
110
A20 isolate inhibited the Gram negative bacterium S. typhi. The secondary
metabolites elaborated by the isolates, A9, A10 and A20 inhibited the growth
of pathogenic bacteria tested. Of all the 22 isolates tested, A9, A10 and A20
produced high yields of antimicrobial metabolites. Therefore, the three
isolates were selected for further taxonomic and optimization studies.
Table 7. Antimicrobial spectrum of the isolate A9
Age (hr)
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
24(hr) 15 8 15 8 5 6 8 0 0
48(hr) 20 10 19 8 8 8 12 0 0
72(hr) 22 20 23 10 10 12 12 0 0
96(hr) 25 25 25 18 18 16 18 0 0
120(hr) 24 20 23 14 14 10 14 0 0
144(hr) 22 12 18 8 12 8 12 0 0
168(hr) 12 10 13 8 8 5 8 0 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
Table 8. Antimicrobial spectrum of the isolate A10
Age(hr)
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
24(hr) 8 0 8 6 4 5 8 0 0
48(hr) 12 0 10 8 6 6 12 0 0
72(hr) 12 0 12 8 8 6 12 0 0
96(hr) 15 8 14 10 10 8 15 0 0
120(hr) 12 8 12 8 8 8 12 0 0
144(hr) 12 0 11 8 6 6 12 0 0
168(hr) 8 0 8 8 6 6 8 0 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
Results and Discussion
111
Table 9. Antimicrobial spectrum of the isolate A20
Age(hr) Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
24(hr) 19 20 20 8 8 10 12 0 0
48(hr) 22 25 22 12 12 15 12 6 0
72(hr) 28 25 24 18 12 20 15 8 0
96(hr) 36 36 32 25 15 25 28 15 0
120(hr) 32 32 30 24 15 24
24 12 0
144(hr) 28 32 28 16 12 18 15 8 0
168(hr) 22 28 26 12 8 12 12 6 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
Generally, it could be concluded that the three isolates A9, A10 and
A20 showed a progressive increase in biomass (dry weight) during the first 48
to 96 hr of incubation. On the contrary, the highest antibiotic activity was
recorded in the stationary phase of growth. Although the accumulation of
antibiotics started after 48 to 72 hr of incubation, the highest accumulation
was observed after 96 and 120 hr of incubation. This indicates that the
organisms grew initially to form a considerable amount of biomass followed by
the formation of antibiotics. It is a well known fact that antibiotics are usually
biosynthesized as secondary metabolites. The results obtained in the present
study are in agreement with those of Prit and Righelato (1967) and Lurie
et al. (1975) who reported that two phases were observed during antibiotic
production. The first phase (trophophase) was characterized by a rapid
growth (biomass production) and the second phase (idiophase) was
characterized by slow growth and maximal productivity of antibiotics.
Feitelson et al. (1985) also reported that the onset of undecyl prodigiosin
Results and Discussion
112
production was delayed to mid or late exponential phase in liquid cultures of
Streptomyces coelicolor grown on a complex beef extract/peptone medium.
Sejiny (1991) observed the growth pattern of five isolates of Streptomyces
incubated for 12 days. Biomass and bioactive metabolite reached maximum
at stationary phase and then decreased. The bioactive metabolites extracted
from these five isolates showed high antimicrobial activity against a Gram
positive bacterium, Staphylococcus aureus.
4.4 Cultural characteristics of the actinomycete isolates
Cultural characteristics of the potent actinomycete isolates designated
as A9, A10, and A20 were studied on different ISP (International
Streptomyces Project) media viz., ISP1, ISP2, ISP3, ISP4, ISP5, ISP7, ISP9
and non-ISP media like nutrient agar, Czapek-Dox, potato dextrose agar,
starch casein agar, glycerol aspargine agar and peptone yeast extract iron
agar. Isolate A9 showed very good growth on ISP media like ISP1, ISP2,
ISP3, ISP5 and non-ISP media like nutrient agar, starch casein agar, peptone
yeast extract iron agar with moderate growth on ISP7, ISP4 and glycerol
aspargine agar, Czapek-Dox, potato dextrose agar and poor growth on ISP9
medium. The color of aerial mycelium was white while that of the substrate
mycelium varied from light brown to dark brown. Diffused dark brown
pigments were produced by the isolate on starch casein agar medium. Isolate
A10 showed very good growth on ISP5, ISP7, ISP1, ISP2, ISP3, ISP4 and
non-ISP media like nutrient agar media, starch casein agar, potato dextrose
agar, glycerol aspargine agar and peptone yeast extract iron agar with
moderate growth on ISP9 and Czapek-Dox media. The aerial mycelium was
white to yellowish white and the substrate mycelium varied from light yellow to
Results and Discussion
113
brownish to brownish yellow. The isolate showed brownish yellow
pigmentation on starch casein agar medium. Isolate A20 showed very good
growth on ISP5, ISP7, ISP9, ISP1, ISP2, ISP3, ISP1, ISP4 and non-ISP
media like nutrient agar, starch casein agar, potato dextrose agar, glycerol
aspargine agar and peptone yeast extract iron agar with moderate growth on
Czapek-Dox medium. The aerial mycelium was grayish white to pinkish white
and the substrate mycelium was pink. Diffused pink pigments were produced
by this isolate on starch casein agar medium. The cultural characteristics of all
the three isolates are tabulated in Table 10, 11 and 12 and Figure 9.
Table 10. Cultural characteristics of A-9 isolate on different media.
Medium Growth Aerial mycelium
Substrate Mycelium
Pigment
NAM Very Good White Dark Brown Dark brown
Czapek Moderate White Light Brown Nil
SCA Very Good White Dark Brown Brown
PDA Moderate White Light Brown Nil
ISP5 Good White Light Brown Nil
ISP7 Moderate White Light Brown Nil
GAA Moderate White Light Brown Nil
ISP9 Poor White Light Brown Nil
TYEA/ISP1 Very Good White Dark Brown Brown
YMD/ISP2 Very Good White Dark Brown Brown
OMA/ISP3 Very Good White Dark Brown Brown
PYEIA Very Good White Brownish black Brown
PYEIA/ISP1 Very Good White Brownish black Brown
ISP4 Moderate White Light Brown Nil
Table 11. Cultural characteristics of A10
Medium Growth
NAM Good
Czapek Moderate
SCA Very Good
PDA Very Good
ISP5 Good
ISP7 Very Good
GAA Good
ISP9 Moderate
TYEA/ISP1 Very Good
YMD/ISP2 Very Good
OMA/ISP3 Very Good
PYEIA Very Good
PYEIA/ISP1 Very Good
ISP4 Very Good
Figure 9. Growth, color of aerial and produced by the three media.
Results and Discussion
114
Cultural characteristics of A10 isolate on different media
Growth Aerial mycelium
Substrate Mycelium
Good White Light Brown
Moderate White Light Yellow
Very Good Yellowish white Brownish Yellow
Very Good White Brown
Good White Light Yellow
Very Good Yellowish white Yellowish White
Good White Yellowish White
Moderate White Light Yellow
Very Good White Brown
Very Good Yellowish white Brownish Yellow
Very Good Yellowish white Brownish Yellow
Very Good White Brown
Very Good White Brown
Very Good White White
Growth, color of aerial and substrate mycelium and pigments produced by the three isolates on different ISP and non
Results and Discussion
isolate on different media.
Pigment
Brown
Nil
Yellow
Brown
Nil
Nil
Nil
Nil
Brown
Yellow
Yellow
Brown
Brown
Yellow
substrate mycelium and pigments on different ISP and non-ISP
Results and Discussion
115
Table 12. Cultural characteristics of A20 isolate on different media.
Medium Growth Aerial mycelium
Substrate Mycelium
Pigment
NAM Good Whitish Purple Purple Purple
Czapek Moderate White Pink Pink
SCA Very Good Pinkish White Pink Pink
PDA Very Good White Pinkish White Pink
ISP5 Very Good Pinkish White Pink Pink
ISP7 Very Good Grayish White Pink Pink
GAA Very Good Grayish White Pink Pink
ISP9 Good White Pink Pink
TYEA/ISP1 Very Good White Purple Purple
YMD/ISP2 Very Good Pinkish White Pink Pink
OMA/ISP3 Very Good Pinkish White Pink Pink
PYEIA Very Good White Purple Purple
PYEIA/ISP1 Very Good White Purple Purple
ISP4 Very Good White Pink Pink
There are many reports available in literature on growth characteristics
and pigment production by actinomycetes. Streptomyces coelicolor A3 (2)
produced a red mycelial pigment with a limited antibiotic activity on minimal
medium and complete medium as described by Brian et al. (1980) who
observed that S. coelicolor produced different pigments on Czapek Dox
medium. Wollum (1982) isolated Streptomyces from soil samples which
produced white, green and blue colored aerial mycelia. The same isolate
produced another two pigments, a diffusible red-blue pigment and a red
pigment on complex beef extract/peptone and Hobb's media (Hobbs et al.,
1990).
Results and Discussion
116
Kala and Chandrika (1993) described various types of media for the
isolation, growth and maintenance of actinomycetes. Growth characteristics of
the marine actinomycetes using various types of media such as glucose-
arginine, glucose-aspargine and glycerol-glycine were described by
Pathiranana et al. (1991). Pigmentation and color of aerial and substrate
mycelia of the marine actinomycetes were studied by Nobutaka et al. (1993).
Similarly Selvameenal et al. (2009) isolated Streptomyces hygroscopicus from
Thar Desert soil samples which produced a yellow color pigment on YMD
agar. Pirouz et al. (1999) recommended the use of YMD agar medium for the
maintenance and preservation of Streptomyces spp., The isolates of
actinomycetes obtained in the present study from the rhizosphere of foxtail
millet also exhibited good growth and different colored pigments.
4.5 Micromorphological studies of the isolates of actinomycetes
Smears of the three isolates were prepared and Gram staining was
performed. The cells appeared violet indicating their Gram positive nature.
The micromorphology of the isolates A9, A10 and A20 was examined using
slide culture technique and scanning electron microscopy. Isolate A9, A10
and A20 showed fragmented aerial mycelium with spiral spore chains. As the
sporogenous hyphae were spiral in nature, the isolates may be placed in spira
group. The morphology of many branching systems has been investigated by
the method of Strahler (1957). Filamentous actinomycetes form circular
colonies when grown on solid medium suggesting that they have a regulation
of growth and branching similar to that of fungi. Growth kinetics of filamentous
bacteria on solid media was characterized by an exponential increase in the
total mycelia length (Trinci, 1978).
The scanning electron microscopy of isolate A9 formed
vegetative hyphae with a diam
squarish elements. White to gray aerial hyphae was
hyphae are fragmented into spores (Fig 10a
shaped. Spore surface was smooth. Brown pigment was produced.
The scanning electron microscopy of isolate A10 revealed
round colonies with powdery texture. Isolate A
unfragmented substrate
The aerial hyphae bore non
characteristic longitudinal pairs. Yellow pigment was produced and the spore
was oval and its surface was smooth (Fig 10b).
The scanning el
mycelia were monopodially branched with compact spirals of sporophore.
Each spore chain consisted of 5 to 10 white, oblong (0.8 µm by 1.2
smooth surfaced spores formed on short (1.0 µm in dia
length) conidiophores that developed on the terminal aerial mycelium. Pink
pigment was produced and the surface of the spore was smooth (Fig 10c).
(a)
Figure 10. Scanning Electron Microscopy of A9 (a), A10 (b), and A20 (c) isolates.
Results and Discussion
117
e scanning electron microscopy of isolate A9 formed
vegetative hyphae with a diameter 0.5 to 1.5 µm which break down into
s. White to gray aerial hyphae was produced. The aerial
are fragmented into spores (Fig 10a). Spores appeared oblong or oval
pore surface was smooth. Brown pigment was produced.
The scanning electron microscopy of isolate A10 revealed
with powdery texture. Isolate A10 produced branched and
fragmented substrate mycelia and monopodially branching aerial mycelia
The aerial hyphae bore non-motile spores on short sporophores in
characteristic longitudinal pairs. Yellow pigment was produced and the spore
was oval and its surface was smooth (Fig 10b).
The scanning electron microscopy of isolate A20 revealed that aerial
mycelia were monopodially branched with compact spirals of sporophore.
Each spore chain consisted of 5 to 10 white, oblong (0.8 µm by 1.2
smooth surfaced spores formed on short (1.0 µm in diameter, 2.0 µm in
length) conidiophores that developed on the terminal aerial mycelium. Pink
pigment was produced and the surface of the spore was smooth (Fig 10c).
(b)
Figure 10. Scanning Electron Microscopy of A9 (a), A10 (b), and A20 (c)
Results and Discussion
e scanning electron microscopy of isolate A9 formed branching
m which break down into
produced. The aerial
oblong or oval
pore surface was smooth. Brown pigment was produced.
The scanning electron microscopy of isolate A10 revealed very small
0 produced branched and
dially branching aerial mycelia.
on short sporophores in
characteristic longitudinal pairs. Yellow pigment was produced and the spore
ectron microscopy of isolate A20 revealed that aerial
mycelia were monopodially branched with compact spirals of sporophore.
Each spore chain consisted of 5 to 10 white, oblong (0.8 µm by 1.2 – 1.5 µm),
meter, 2.0 µm in
length) conidiophores that developed on the terminal aerial mycelium. Pink
pigment was produced and the surface of the spore was smooth (Fig 10c).
(c)
Figure 10. Scanning Electron Microscopy of A9 (a), A10 (b), and A20 (c)
Results and Discussion
118
4.6 Biochemical tests
The results of biochemical tests conducted are presented Table 13 and
Figure 11.
4.6.1 Indole test
Tryptone broth was prepared to study the production of indole. The
broth was inoculated with the respective isolates and incubated at 37°C for 48
hr. After incubation, a few drops of Kovac's reagent were added to tryptone
broth tubes. The color of the inoculated tubes did not change from yellow to
red indicating that the test was negative.
4.6.2 Methyl red test
The broth (MRVP) was inoculated with the respective isolates and
incubated at 37°C for 48 hr. After incubation, 0.5 ml of methyl red indicator
was added to the culture broth. A change in the color of the culture broth from
yellow to red was noticed in A9 and A20 isolates while no change was
observed in case of isolate A10. Hence the test was negative for A10 and
positive for A9 and A20.
4.6.3 Voges Proskaeur test
The broth (MRVP) was inoculated with the respective isolates and
incubated at 37°C beyond 48 hr. After incubation, 10 drops of 5% α- naphthol
and 1 ml of 40% KOH were added along the side walls of the test tube and
allowed to stand for 3 min. No violet ring was observed. Hence the test was
negative for all the three isolates.
Results and Discussion
119
4.6.4 Citrate utilization test
Simmon's Citrate agar medium was inoculated with the isolates
individually. After the incubation period of 48 hr, blue color was observed for
A10 isolate inferring the utilization of sodium citrate in the absence of
fermented glucose or lactose while it was not observed in A9 and A20. Hence
the test was positive for A10 isolate and negative in A9 and A20 isolates.
4.6.5 H2S production test
Kligler's agar medium was used for testing hydrogen sulfide production
by the isolates. The tubes were inoculated with the respective isolates and
impregnated with lead acetate strip. After 96 hrs of incubation, black color was
observed for A9 and A10 isolates due to H2S production. Lead acetate in the
presence of H2S is converted in to black colored lead sulphide. Hence the test
was positive for A9 and A10 isolates and negative for A20 isolate.
4.6.6 Carbon utilization
Utilization of carbon source was tested using ISP-9 medium. Different
sugar substrates were used as carbon sources. These substrates were
sterilized by filtration and added at a concentration of 0.5 to 1.0% to the
medium taken test tubes. Growth of the isolates of actinomycetes in the
above media is recorded in Table 13. The three isolates grew well in glucose,
fructose, sucrose, lactose, maltose, xylose and mannitol. Isolate A20 utilized
all the sugars well when compared to A9 and A10 isolates.
Results and Discussion
120
4.6.7 Melanin production
Peptone Yeast extract agar medium was inoculated with the respective
isolates. After incubation at 37°C for 24 hr, black color was observed in A9
and A10 but not in A20 isolate. Hence the test was positive for A9, A10 and
negative for A20 isolate.
Table 13. Biochemical tests for the three isolates.
Test A9 A10 A20
Indole - - -
Methyl Red + - +
Voges Proskauer - - -
Citrate - + -
H2S + + -
Melanin Production + + -
Carbon Utilization
Glucose ++ ++ ++
Fructose ++ ++ ++
Sucrose ++ ++ ++
Mannitol + + +
Xylose + + ++
Lactose ++ ++ ++
Maltose ++ + ++
Figure 11. Biochemical tests of A9, A10, A20 isolates and control (IMViC, H
4.7 Screening of the actinomycete enzymes
The data on the production of different enzymes by the isolates are
presented in Table 14 and Figure 12.
4.7.1 Starch hydrolysis
Starch agar plates were prepared and inoculated with the respective
isolates and incubated at 37°C for 48 hr. A clear zone was
the colonies of isolates A9, A10 and A20 after flooding the plates with iodine.
Hence the test was positive for all the three isolates.
4.7.2 Urease test
Urea broth was prepared, sterilized and inoculated with isolates
individually and incubated at 37°C for 48 hr. After incubation, no pink
was observed. Hence the test was negative for all the three isolates.
Results and Discussion
121
Biochemical tests of A9, A10, A20 isolates and control (IMViC, H2S and Melanin production).
Screening of the actinomycete isolates for the production of
data on the production of different enzymes by the isolates are
presented in Table 14 and Figure 12.
4.7.1 Starch hydrolysis
Starch agar plates were prepared and inoculated with the respective
isolates and incubated at 37°C for 48 hr. A clear zone was observed around
the colonies of isolates A9, A10 and A20 after flooding the plates with iodine.
Hence the test was positive for all the three isolates.
Urea broth was prepared, sterilized and inoculated with isolates
cubated at 37°C for 48 hr. After incubation, no pink
was observed. Hence the test was negative for all the three isolates.
Results and Discussion
Biochemical tests of A9, A10, A20 isolates and control
for the production of
data on the production of different enzymes by the isolates are
Starch agar plates were prepared and inoculated with the respective
observed around
the colonies of isolates A9, A10 and A20 after flooding the plates with iodine.
Urea broth was prepared, sterilized and inoculated with isolates
cubated at 37°C for 48 hr. After incubation, no pink color
was observed. Hence the test was negative for all the three isolates.
Results and Discussion
122
4.7.3 Catalase test
Nutrient agar slants were inoculated with the respective isolates and
incubated at 37°C for 48 hr. After incubation 3% H2O2 was added to the tubes
and observed for the appearance of oxygen bubbles. The bubbles were
observed in all the three isolates indicating positive test.
4.7.4 Gelatin hydrolysis
Nutrient gelatin agar slants were prepared and inoculated with three
isolates individually and incubated at 35°C for 30 days. After incubation the
plates were kept in ice to check liquefaction and the plates were flooded with
trichloro acetic acid. The trichloro acetic acid precipitated the gelatin and the
plates became opaque. Hence the test was positive for all the three isolates.
4.7.5 Casein hydrolysis
Skim milk agar medium was employed for casein hydrolysis by the
isolates. The medium was prepared and streaked with the respective isolates.
After incubation for 96 hr, clear zones were observed around the colonies of
the three isolates indicating the production of caseinase. Hence the test was
positive for all the three isolates.
4.7.6 Pectinase
Pectin agar medium was prepared, inoculated with the respective
isolates and incubated at 30°C for 96hr. After flooding the plates with iodine,
clear zones were observed around the colonies indicating that the test was
positive for all the three isolates.
Results and Discussion
123
4.7.7 Arginine hydrolase
Arginine agar tubes were used to test the ability of the isolates for the
production of arginine hydrolase. The agar tubes were inoculated with the
respective isolates by using stab culture technique. After incubation at 30°C
for 96hr, no change in color from yellow to bright magenta was observed for
any isolate. Hence the test was negative for all the three isolates.
4.7.8 Cellulase
Agar medium supplemented with cellulose was employed to detect the
production of cellulase by the isolates. The medium was prepared and the
isolates were streaked on the plates. After the incubation period, a clear zone
was observed around the colonies of all the three isolates indicating cellulase
production. Hence the test was positive for all the three isolates.
4.7.9 Phenyl alanine deaminase
To study the ability of isolates for the production of phenyl alanine
deaminase, YMD broth amended with phenyl alanine (0.5%) was prepared
and inoculated with the isolates individually. After the incubation for 96 hr,
1 ml of 10% ferric sulphate was added to 2 ml of the fermented broth and
observed for color change. No green color was observed indicating that the
test was negative for all the three isolates.
4.7.10 Nitrate reduction
Organic nitrate broth was prepared and inoculated with the respective
isolates and incubated at 37°C for 96hr. After incubation Nessler's reagent
was added and observed for the formation of pink color. In case of A10 and
Results and Discussion
124
A20 isolates pink color was observed but not in A9 isolate. Hence the test was
positive for A10 and A20 isolates and negative for A9 isolate.
4.7.11 Milk coagulation
Pasteurized milk, was poured in test tubes, was sterilized, cooled and
inoculated with the test organisms and incubated at 30°C for 96 hr. After
incubation, the tubes showed production of coagulase enzyme. Hence the
test was positive for all the three isolates.
Table 14. Production of enzymes by the isolates of actinomycetes.
Biochemical test A9 A10 A20
Catalase + + +
Urease - - -
Oxidase + + +
Gelatin Hydrolysis + + +
Starch hydrolysis + + +
Casein hydrolysis + + +
Arginine hydrolysis - - -
Cellulase + + +
Phenyl alanine Deaminase - - -
Nitrate Reduction - + +
Milk Coagulation + + +
Figure 12. Production of enzymes by the three isolates (urease, cellulase, catalase, phenyl starch hydrolysis).
4.8 Antibiotic sensitivity isolates
To determine the sensitivity of the isolates to various antibiotics,
nutrient agar medium was prepared and inoculated with the respective
isolates by using seeded plate technique. Filter paper discs containing the
antibiotic were placed over the seeded plates and incubated at 30°C for 48 hr
and observed for zones of inhibition. Isolate A9 and A10 exhibited sensitivity
to bacitracin, ciprofloxacin, doxycy
vancomycin while the isolate A20 showed sensitivity to ciprofloxacin,
chloramphenicol, doxycyclin, erythromycin, kanamycin, rifampicin, neomycin
and vancomycin (Table 15).
Results and Discussion
125
Figure 12. Production of enzymes by the three isolates (urease, cellulase, catalase, phenyl alanine deaminase, casein and starch hydrolysis).
4.8 Antibiotic sensitivity isolates of actinomycetes
To determine the sensitivity of the isolates to various antibiotics,
nutrient agar medium was prepared and inoculated with the respective
sing seeded plate technique. Filter paper discs containing the
antibiotic were placed over the seeded plates and incubated at 30°C for 48 hr
and observed for zones of inhibition. Isolate A9 and A10 exhibited sensitivity
to bacitracin, ciprofloxacin, doxycyclin, kanamycin, rifampcin, neomycin and
vancomycin while the isolate A20 showed sensitivity to ciprofloxacin,
chloramphenicol, doxycyclin, erythromycin, kanamycin, rifampicin, neomycin
and vancomycin (Table 15).
Results and Discussion
Figure 12. Production of enzymes by the three isolates (urease, alanine deaminase, casein and
To determine the sensitivity of the isolates to various antibiotics,
nutrient agar medium was prepared and inoculated with the respective
sing seeded plate technique. Filter paper discs containing the
antibiotic were placed over the seeded plates and incubated at 30°C for 48 hr
and observed for zones of inhibition. Isolate A9 and A10 exhibited sensitivity
clin, kanamycin, rifampcin, neomycin and
vancomycin while the isolate A20 showed sensitivity to ciprofloxacin,
chloramphenicol, doxycyclin, erythromycin, kanamycin, rifampicin, neomycin
Results and Discussion
126
Table 15. Antibiotic sensitivity of the actinomycete isolates.
Antibiotic A9 A10 A20
Vancomycin S S S
Ciprofloxacin S S S
Bacitracin S S R
Rifamycin S S S
Neomycin S S S
Amoxycyclin R R R
Polymyxin-B R R R
Erythromycin R R S
Doxycyclin S S S
Kanamycin S S S
Chloramphenicol R R S
S: Sensitive; R: Resistant
4.9 Spectrum of antibiotics
The isolates A9, A10 and A20 and other antibiotics like penicillin,
ampicillin and streptomycin were tested to the check the spectrum against the
Gram positive and Gram negative bacteria like Bacillus, Staphylococus and
Pseudomonas and E.coli. The results are presented in Table 16 and Figure
13. The order of spectrum of antibiotics and extracts of isolates were
streptomycin, penicillin, extract of isolate A20, ampicillin and extracts of A9
and A10 isolates.
According to Kokare et al. (2004), the actinomycete Actinopolyspora
exhibited good antibacterial activity against Staphylococcus aureus,
S. epidermidis and B. subtilis and antifungal activities against Aspergillus
niger, A. fumigatus, A. flavus, Fusarium oxysporum, Penicillium species and
Trichoderma species. Similarly, 40 strains of actinomycetes, isolated from
Antarctica, were tested for antagonistic activity against 7 Gram positive and
Gram negative bacteria,
a broad spectrum antibact
of new substances for pharmaceutical or agricultural purposes (Nedialkova
and Naidenova, 2004).
metabolites with different biological activities such as a
antiparasitic, antitumor and immunosuppressive actions (Demain, 1999).
Table 16. Spectrum of antibiotics
Isolate and antibiotics Bacillus
A9 20
A10 12
A20 22
Penicillin 20
Ampicillin 20
Streptomycin 22
(a)
Figure 13. Antimicrobial activity exhibited by A9 (a), A10 (b) and A20 (c) and difGram negative bacteria.
Results and Discussion
127
Antarctica, were tested for antagonistic activity against 7 Gram positive and
Gram negative bacteria, yeast and 16 phytopathogenic fungi. Ten of them had
a broad spectrum antibacterial activity and could be used in the development
of new substances for pharmaceutical or agricultural purposes (Nedialkova
and Naidenova, 2004). Actinomycetes are known to produce many secondary
different biological activities such as antibacterial, antifungal,
antiparasitic, antitumor and immunosuppressive actions (Demain, 1999).
Spectrum of antibiotics
Diameter of zone of inhibition (mm)
Bacillus Staphylococcus Pseudomonas
20 18 18
12 11 10
22 24 22
20 26 20
20 24 20
22 26 22
(b)
Antimicrobial activity exhibited by A9 (a), A10 (b) and A20 (c) and different antibiotics against Gram positive and Gram negative bacteria.
Results and Discussion
Antarctica, were tested for antagonistic activity against 7 Gram positive and
and 16 phytopathogenic fungi. Ten of them had
erial activity and could be used in the development
of new substances for pharmaceutical or agricultural purposes (Nedialkova
Actinomycetes are known to produce many secondary
ntibacterial, antifungal,
antiparasitic, antitumor and immunosuppressive actions (Demain, 1999).
E. coli
8
5
10
18
10
20
(c)
Antimicrobial activity exhibited by A9 (a), A10 (b) and A20 positive and
4.10 Phylogenetic analysis of the actinomycete isolates
Genomic DNA of the three isolates was extracted according to the
standard protocols and 16S rRNA sequences were amplified with the suitable
primers in PCR. A single discrete PCR amplicon band of 1500bp was
observed for all the three isolates when resolved
The phylogenetic position of the isolates was determined by blasting the 16S
rRNA gene sequences with the sequences of the related genera in NCBI
genbank. Isolates A9, A10 and A20 showed 96, 99 and 100% homology with
Streptomyces sp. 2438, (Figure 15)
Streptomyces indiaensis
isolates A9 was assigned as
Streptomyces sp. LD48 and the isolate A20 as
IF5.
(a)
Figure : 14 Agarose gels with a single discrete PCR amplicon band of 1500bp produced by StreptomycesIF5 (c)
Results and Discussion
128
4.10 Phylogenetic analysis of the actinomycete isolates
Genomic DNA of the three isolates was extracted according to the
standard protocols and 16S rRNA sequences were amplified with the suitable
primers in PCR. A single discrete PCR amplicon band of 1500bp was
observed for all the three isolates when resolved on agarose gel (Figure 14).
The phylogenetic position of the isolates was determined by blasting the 16S
rRNA gene sequences with the sequences of the related genera in NCBI
genbank. Isolates A9, A10 and A20 showed 96, 99 and 100% homology with
sp. 2438, (Figure 15) Streptomyces sp LD48 (Figure 16) and
indiaensis strain IF5 (Figure 17) respectively. Hence, the
isolates A9 was assigned as Streptomyces sp. 2438, isolate A10 as
sp. LD48 and the isolate A20 as Streptomyces indiaensis
(b) (c)
Figure : 14 Agarose gels with a single discrete PCR amplicon band of 1500bp produced by Streptomyces sp. 2438 (a), Streptomyces sp. LD48 (b) and Streptomyces indiaensis
Results and Discussion
Genomic DNA of the three isolates was extracted according to the
standard protocols and 16S rRNA sequences were amplified with the suitable
primers in PCR. A single discrete PCR amplicon band of 1500bp was
on agarose gel (Figure 14).
The phylogenetic position of the isolates was determined by blasting the 16S
rRNA gene sequences with the sequences of the related genera in NCBI
genbank. Isolates A9, A10 and A20 showed 96, 99 and 100% homology with
sp LD48 (Figure 16) and
IF5 (Figure 17) respectively. Hence, the
2438, isolate A10 as
indiaensis strain
(c)
Figure : 14 Agarose gels with a single discrete PCR amplicon band of sp. 2438 (a),
Streptomyces indiaensis
Figure 15. Phylogenetic tree of
Figure 16. Phylogenetic tree of
Results and Discussion
129
Figure 15. Phylogenetic tree of Streptomyces sp. 2438.
Figure 16. Phylogenetic tree of Streptomyces sp. LD38.
Results and Discussion
Figure 17. Phylogenetic tree of
4.11 Optimization of bioactive metabolite production by
sp. 2438, Streptomyces
IF5
4.11.1 Effect of carbon sources
Influence of different carbon sources on the cell growth of the
and bioactive metabolite production was studied. Utilization of various
carbohydrates such as glucose, fructose, lactose, suc
mannitol was examined by adding them to
concentration of 0.4% (w/v)
biomass production was high in all the isolates in lactose source
the medium are presented in Figure 18 a
Results and Discussion
130
Figure 17. Phylogenetic tree of Streptomyces indiaensis strain
1 Optimization of bioactive metabolite production by Streptomyces
Streptomyces sp. LD48 and Streptomyces indiaensis
4.11.1 Effect of carbon sources
Influence of different carbon sources on the cell growth of the
and bioactive metabolite production was studied. Utilization of various
carbohydrates such as glucose, fructose, lactose, sucrose, maltose, and
mannitol was examined by adding them to starch casein broth separately at a
concentration of 0.4% (w/v). The data on the influence of carbon sources on
was high in all the isolates in lactose source
um are presented in Figure 18 a and 18 b respectively.
Results and Discussion
strain IF5.
Streptomyces
Streptomyces indiaensis
Influence of different carbon sources on the cell growth of the isolates
and bioactive metabolite production was studied. Utilization of various
rose, maltose, and
starch casein broth separately at a
ta on the influence of carbon sources on
was high in all the isolates in lactose source and pH of
Results and Discussion
131
Figure 18. Effect of carbon source on the growth of actinomycete isolates and pH of the medium.
(a) (b)
Data on the effect of various carbon sources on the cell growth of
Streptomyces sp. 2438 and bioactive metabolite production are presented in
Table 17. The isolate Streptomyces sp. 2438, exhibited good growth on starch
casein broth with different C sources (0.4% w/v).
Table 17. Effect of carbon source on growth of Streptomyces sp. 2438 isolate and bioactive metabolite production.
Carbon source
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
Glucose 15 15 15 8 5 6 8 6 0
Fructose 20 19 19 8 8 8 12 8 0
Lactose 25 20 25 18 18 16 18 15 0
Sucrose 22 12 23 14 14 16 14 12 0
Mannitol 22 15 14 18 14 10 8 12 0
Maltose 22 12 8 12 8 12 12 12 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
Maximum antimicrobial activity was observed with lactose followed by
sucrose, mannitol, fructose, maltose and glucose in that order. Bioactive
0
100
200
300
400
Glu Fru Lac SucManniMalBio
mass (
mg
/100m
l)
Carbon Sources
Biomass
A9 A10 A20
012345678
Glu Fru Lac Suc Manni Mal
pH
Carbon Sources
pH
A9 A10 A20
Results and Discussion
132
metabolites produced by the isolate grown on starch casein broth amended
with lactose were highly inhibitory to all the organisms tested. Among the
Gram positive bacteria tested, B. subtilis was highly susceptible whereas P.
aeruginosa exhibited high sensitivity among Gram negative bacteria.
Table 18 depicts the effect of various carbon sources on the growth of
Streptomyces sp. LD 48 and bioactive metabolite production. The isolate
Streptomyces sp. LD 48 exhibited good growth on starch casein broth with
different C sources (0.4% w/v). Maximum antimicrobial activity was observed
with fructose followed by glucose, lactose, sucrose, mannitol and maltose in
that order. Among the Gram positive bacteria tested, B. subtilis was highly
susceptible to the metabolites of the isolate whereas the same effect was
observed with P. aeruginosa, P. mirabilis and E. coli when compared to the
other Gram negative bacteria.
Table 18. Effect of carbon sources on growth of Streptomyces sp. LD48 isolate and bioactive metabolite production.
Carbon source
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
Glucose 12 12 12 8 8 8 12 0 0
Fructose 15 14 12 12 12 10 10 0 0
Lactose 10 10 10 8 8 8 10 0 0
Sucrose 12 11 11 8 6 6 10 0 0
Mannitol 8 8 8 6 6 8 8 0 0
Maltose 8 8 8 6 4 6 8 0 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli ML:Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
Data presented in Table 19 depict the effect of various carbon sources
on the growth of Streptomyces indiaensis IF5 and bioactive metabolite
Results and Discussion
133
production. The isolate S. indiaensis IF5, exhibited good growth on starch
casein broth with different C sources (0.4% w/v).
Table 19. Effect of carbon sources on growth and bioactive metabolite production of Streptomyces indiaensis IF5 isolate.
Carbon source
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
Glucose 32 32 32 20 20 18 20 15 0
Fructose 28 28 30 16 16 16 16 12 0
Lactose 36 36 32 25 20 19 32 18 0
Sucrose 25 25 25 12 12 14 15 10 0
Mannitol 30 30 30 18 18 16 18 14 0
Maltose 36 36 32 20 18 18 25 15 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
Highest antimicrobial activity was noticed with lactose followed by
maltose, glucose, mannitol, fructose and sucrose in that order. Bioactive
metabolites produced by the isolate grown on starch casein broth amended
with lactose were highly inhibitory to all the organisms tested. Among the
Gram positive bacteria, B. subtilis and S. aureus were highly susceptible to
the metabolites of the isolate whereas Gram negative bacteria, P. aeruginosa
and K. pneumoniae exhibited high sensitivity.
Many reports are available on the utilization of different carbon sources
by actinomycetes and production of bioactive metabolites. Robert and
Lechevalier (1954) observed that glucose and mannose were superior carbon
sources for both growth and antibiotic production (candicidin) by
Streptomyces griseus. According to Shirato and Nagatsu (1965) S. griseus
Results and Discussion
134
utilized glucose for the production of streptomycin in the presence of 0.02% of
KH2PO4 and when fructose and maltose were added to ISP media without the
addition of 0.02% KH2PO4, the organism yielded high amount of streptomycin.
A medium containing galactose as carbon source favoured high yields
of kanamycin from Streptomyces kanamyceticus (Basak et al., 1973).
Carbohydrates such as glycerol, maltose, mannose, sucrose and xylose have
been reported to interfere with the production of secondary metabolites
(Demain and Fang, 1995). Farid et al. (2000) observed that the medium
containing 20% glucose supported maximum levels of natamycin production
by S. natalensis.
Similarly glucose (40 g/l) was used for the production of cystocin by
Streptomyces sp GCA 0001by Madhan Kumar et al. (2002). The addition of
12% glucose at day 4 and 0.1% yeast extract at day 2 to the ISP medium
yielded best antibiotic, Rifamycin B by Amycolatopsis mediterranei as
observed by EI-Tayeb et al. (2004) while Pandy et al. (2005) also reported
dextrose as an excellent carbon source for antibiotic production by
Streptomyces sp.
Likewise Gasheva et al., (2005) observed that carbon sources like
lactose / glycerol had significant effect on antibiotic production. Further, Kanto
et al. (2005) reported that the medium containing glucose and tryptone with
minerals was good for bioactive metabolite production. Maltose (1%)
amendment to the basal medium was found to be the best for cell growth as
well as antibiotic production by the Streptomyces albidoflavus followed by
Results and Discussion
135
glycerol, trehalose, dextrose, rhamnose and galactose (Narayana and
Vijayalakshmi, 2008).
Results from the present study are an agreement with the reports
available in literature. Thus lactose (0.4%) was the best carbon source for
bioactive metabolite production in case of Streptomyces sp. 2438 and
Streptomyces indiaensis IF5 where as cultivation of Streptomyces sp. LD48
with fructose (0.4 %) resulted in maximum production of antibiotics.
4.11.2 Effect of nitrogen sources
The effect of different nitrogen sources such as peptone, beef extract,
tyrosine, ammonium nitrate and L-phenyl alanine on the production of was
tested at 0.4% to starch casein broth. Data on biomass production was high in
tyrosine source and pH of the broth are represented in Figure 19 a and 19b.
Figure 19. Effect of nitrogen source on the growth of actinomycete isolates and pH of the medium.
(a) (b)
Data on the effect of various nitrogen sources on the growth of and
bioactive metabolite production of Streptomyces sp. 2438 are represented in
0
20
40
60
80
100
120
140
160
180
Pep Beef Tyr AN SN L-PA
Bio
mass (
mg
/100m
l)
Nitrogen Source
Biomass
A9 A10 A20
0123456789
Pep Beef Tyr An SN L-PA
pH
Nitrogen Source
pH
A9 A10 A20
Results and Discussion
136
Table 20. The effect of N sources such as peptone, beef extract, tyrosine,
ammonium nitrate, sodium nitrate and L-phenyl alanine was tested by adding
them at a level of 0.4% to the starch casein broth and incubated for 96 hr. The
isolate Streptomyces sp. 2438 exhibited good growth on starch casein broth
with different N sources (0.4% w/v). Antimicrobial activity was high with
peptone followed by tyrosine, beef extract, L-phenyl alanine, ammonium
nitrate, sodium nitrate. The metabolites produced by the isolate Streptomyces
sp. 2438 grown on starch casein broth supplemented with peptone exhibited
maximum inhibition on the growth of Gram positive and Gram negative
bacteria like B. subtilis, S. aureus and P. aeruginosa.
Table 20. Effect of nitrogen source on growth and bioactive metabolite production of Streptomyces sp. 2438 isolate.
Nitrogen source
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
Peptone 26 26 26 20 18 20 20 0 0
Beef Extract 20 22 20 16 16 17 18 0 0
Tyrosine 24 24 24 18 16 18 18 0 0
NH4NO
3 18 18 18 12 12 12 14 0 0
Na2NO
3 15 15 15 8 8 8 12 0 0
L-PA 20 20 18 15 14 14 16 0 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
Results on the effect of various nitrogen sources on the growth of
Streptomyces sp. LD48 and bioactive metabolite production are presented in
Table 21. The isolate Streptomyces sp. LD 48 exhibited good growth on
starch casein broth supplemented with 0.4% N source. Maximum
antimicrobial activity was observed with peptone followed by L-phenyl alanine,
Results and Discussion
137
Table 21. Influence of nitrogen source on growth and bioactive metabolite production of Streptomyces sp. LD48 isolate.
Nitrogen source
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
Peptone 15 15 15 14 12 12 14 0 0
Beef Extract 10 10 10 10 8 10 10 0 0
Tyrosine 12 10 10 8 8 8 8 0 0
NH4NO
3 12 12 10 12 8 10 12 0 0
Na2NO
3 10 10 10 10 8 8 12 0 0
L-PA 12 14 12 12 8 12 12 0 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
ammonium nitrate, sodium nitrate, beef extract and tyrosine. The metabolites
produced by the isolate in the starch casein broth amended with 0.4% of
peptone showed antimicrobial activity against Gram positive bacteria like
B. subtilis, S. aureus and a Gram negative bacterium P. aeruginosa.
The effect of various nitrogen sources on the growth and bioactive
metabolite production of Streptomyces indiaensis IF5 was investigated. The
isolate S. indiaensis IF5 exhibited good growth on starch casein broth with
0.4% of N source. Maximum antimicrobial activity was recorded with tyrosine
followed by peptone, beef extract, L-phenyl alanine, ammonium nitrate,
sodium nitrate. The bioactive metabolites produced by the isolate inhibited the
growth of Gram positive bacteria like B. subtilis, S. aureus and Gram negative
bacteria P. aeruginosa, K. pneumoniae, P. mirabilis, M. luteus and E. coli.
(Table 22).
Results and Discussion
138
Table 22. Growth and bioactive metabolite production of Streptomyces indiaensis IF5 as influenced by nitrogen source.
Nitrogen source
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
Peptone 34 36 32 30 25 30 32 0 0
Beef Extract 32 32 32 30 25 30 32 0 0
Tyrosine 36 36 36 32 30 32 36 0 0
NH4NO
3 30 30 28 28 22 26 28 0 0
Na2NO
3 28 28 25 25 18 20 24 0 0
L-PA 32 32 30 28 20 28 30 0 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
The influence of different nitrogen sources on biomass production and
production of biomass and antibiotics of actinomycetes was reported by
researchers. The utilization of nitrogen compounds by actinomycetes was
found to vary. Clemer et al. (1997) described that the medium containing fish
meal and casein supported the production of macrobicyclic peptide antibiotic
compounds by Pseudonocardia fastidiosa. Alanine served as an excellent
nitrogen source for the production of antibiotics by Streptomyces griseus and
Streptomyces antibioticus (Haque et al., 1995). According to Gupte et al.
(2002) optimal level of antibiotic production by Streptomyces chattanoogensis
was recorded when cultured in the medium containing 1% soybean meal.
EI-Tayeb et al. (2004) observed optimal level of rifamycin B production by
Amycolatopsis mediterranei when cultured in the medium containing 3%
soytone or 0.05% NH4NO3. Soytone (50 g/l) served as excellent nitrogen
source for the production of cystocin by Streptomyces sp. GCA0001.
Results and Discussion
139
Similarly, Pandy et al. (2005) reported arginine as an excellent nitrogen
source for the production of antibacterial antibiotic by Streptomyces
kanamyceticus M27. Gesheva et al. (2005) observed that the growth and
antibiotic production on synthetic media are unsatisfactory but a medium with
a minimal amount of soymeal (0.5%) supported growth and antibiotic
production in Streptomyces hygroscopicus. A medium supplemented with
soybean meal was found suitable for production of maximum antimicrobial
metabolites by Streptomyces albidoflavus followed by yeast extract, tryptone,
peptone and casein (Narayana and Vijayalakshmi, 2008).
In the present study, Streptomyces sp 2438 and Streptomyces sp.
LD48 grown in the medium containing peptone yielded maximum biomass
and bioactive metabolites whereas the medium containing tyrosine favoured
high yield of biomass and bioactive metabolites by Streptomyces indiaensis
IF5.
4.11.3 Effect of NaCl
To study the effect of NaCl on biomass and bioactive metabolite
production, the isolates were cultured individually in starch casein broth
amended with different concentrations of NaCl ranging from 2 to 12%. The
isolate Streptomyces sp. 2438 produced high biomass and metabolite
production in the medium containing NaCl at 6% (Table 23). Metabolites of
the isolate Streptomyces sp. 2438 were highly inhibitory to Gram positive
bacteria followed by Gram negative bacteria. Among the bacteria tested,
B. subtilis followed by P. aeruginosa exhibited high sensitivity. Isolate
Streptomyces sp. LD 48 produced high biomass and bioactive metabolites in
Results and Discussion
140
the medium containing 4% NaCl (Table 24). Among the bacteria tested,
B. subtilis, P. aeruginosa, and S. typhi in that order exhibited sensitivity.
Biomass production of the isolate S. indiaensis IF5 was high in the
culture medium containing 6% NaCl followed by 8%, while high yields of
bioactive metabolites were recorded with 6% NaCl. Metabolites of the isolate
exhibited extreme antimicrobial activity against B. subtilis, S. aureus and
P. aeruginosa and K. pneumoniae (Table 25).
Table 23. Effect of NaCl on growth and bioactive metabolite production of Streptomyces sp. 2438 isolate.
NaCl (%)
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
2 15 6 15 0 5 6 8 0 0
4 15 8 19 0 8 8 12 0 0
6 28 12 23 8 12 10 12 0 0
8 25 12 20 0 12 10 10 0 0
10 24 10 20 0 10 10 8 0 0
12 15 8 12 0 6 8 8 0 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
In the present study, bioactive metabolites of two isolates cultured in
starch casein broth with different NaCl concentrations showed good
antimicrobial activity against Gram positive and Gram negative bacteria.
Streptomyces sp 2438 and Streptomyces indiaensis isolate IF5 exhibited high
yields of bioactive metabolites in starch casein broth containing 6% NaCl
whereas, amendment with 4% NaCl resulted in high production of bioactive
metabolites by Streptomyces sp LD48.
Results and Discussion
141
Table 24. Growth and bioactive metabolite production of Streptomyces sp. LD48 isolate as influenced by NaCl.
NaCl (%)
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
2 8 0 8 6 4 5 8 8 0
4 15 8 16 8 10 8 15 10 0
6 12 0 12 8 8 6 10 8 0
8 10 0 10 8 8 6 10 8 0
10 10 0 10 6 8 6 10 8 0
12 8 0 8 8 6 6 15 8 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
Table 25. Influence of NaCl on growth and bioactive metabolite production of Streptomyces indiaensis IF5.
NaCl (%) Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
2 19 20 20 8 8 10 12 0 0
4 22 18 18 8 8 8 10 0 0
6 32 32 30 12 15 16 20 16 0
8 30 30 28 12 15 16 18 12 0
10 30 30 28 12 15 15 18 12 0
12 28 28 20 8 8 10 12 0 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
. Thus a halophilic Actinopolyspora species AH1 grown in the culture
medium containing 10-15% (w/v) NaCl showed good antibacterial activity
against Staphylococcus aureus, S. epidermis, B. subtilis and antifungal
activity against Aspergillus niger, A. fumigatus, A. flavus and Penicillium sp.
Results and Discussion
142
(Kokare et al., 2004). Maskey et al. (2004) reported the antibacterial,
anticancer and antimalarial properties of trioxacarcins isolated from marine
Streptomyces isolate grown in the medium containing 5 to10% of NaCl.
Results from the present study also indicate that the optimum level of NaCl for
antibiotic production ranged between 6 to 8%.
4.11.4 Effect of pH
To study the effect of pH on cell growth and bioactive metabolite
production, the isolates were grown individually on starch casein broth the pH
of which was adjusted to different levels ranging from 4 to 10 and incubated
for 96hr. All the three isolates, Streptomyces sp. 2438, Streptomyces sp.
LD48, Streptomyces indiaensis IF5 exhibited good growth and bioactive
metabolite production at pH 7. Among the test organisms, B. subtilis, S.
aureus and P. aeruginosa, K. pneumoniae were highly susceptible to the
metabolites of the isolates (Table 26, 27 and 28).
Table 26. Effect of pH on growth and bioactive metabolite production of Streptomyces sp. 2438 isolate.
pH
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
4 15 6 15 0 5 6 8 0 0
5 15 8 19 0 8 8 12 0 0
6 28 12 23 8 12 10 12 0 0
7 30 12 23 10 12 10 12 0 0
8 25 10 23 0 10 10 12 0 0
9 15 8 12 0 6 8 10 0 0
10 10 6 15 0 6 8 10 0 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
Results and Discussion
143
Table 27. Influence of pH on growth and bioactive metabolite production of Streptomyces sp. LD48 isolate.
pH
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
4 8 0 8 6 4 5 8 8 0
5 8 0 10 6 6 5 8 8 0
6 12 0 12 8 8 6 10 8 0
7 15 8 16 8 10 8 15 10 0
8 10 0 10 8 8 6 10 8 0
9 10 0 10 6 8 6 10 8 0
10 8 0 8 8 6 6 10 8 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
Table 28. Growth and bioactive metabolite production of Streptomyces indiaensis IF5 as affected by pH.
pH
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
4 19 20 20 8 8 10 12 0 0
5 22 18 18 8 8 8 10 0 0
6 32 32 28 12 15 15 18 12 0
7 32 32 30 12 15 16 18 16 0
8 30 30 28 12 15 15 18 12 0
9 28 20 20 8 8 10 12 0 0
10 22 18 18 8 8 8 10 0 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
A survey of literature reveals that there are reports on influence of pH
and antibiotic production by actinomycetes. Margalith and Pagani (1961)
studied the impact of several features such as age of the culture, mycelia
growth, pH and composition of the media on the production of rifamycin
Results and Discussion
144
complex by Streptomyces mediterranei. Bhattacharya et al. (1998) showed
that the optimum pH for antibiotic production by S. hygroscopicus D1.5 was 7.
Tang et al. (2002) reported that the optimal pH for growth of different
halophilic and halotolerant actinomycetes from saline soils of Xinjiang ranged
between 7 and 8. Sun et al. (2002) determined the combined effects of pH
and nutrients on the growth and sporulation of some strains of Streptomyces.
Basilio et al. (2003) described the patterns of antimicrobial activities
exhibited by soil actinomycetes isolated under different conditions of pH and
salinity. Likewise Sujatha et al. (2005) reported an optimal pH of 7.2 for
antibiotic production by the isolate Streptomyces BT- 408. Vasavada et al.
(2006) recorded optimum levels of antibiotic production by Streptomyces
sannanensis at pH 7 which is in conformity with the present findings. Similarly,
Narayana and Vijayalakshmi (2008) demonstrated that the optimum pH for
biomass and antibiotic production by Streptomyces albidoflavus was 7.0.
Results from the present study also confirm the findings reported in literature.
4.11.5 Effect of temperature
The impact of the temperature on biomass and bioactive metabolite
production was studied by inoculating the isolates individually in starch casein
broth and incubating at different temperatures ranging from 20 to 45°C for
96hr. All the three isolates yielded maximum levels of production of biomass
and metabolite at 35°C. Metabolites of the three isolates grown at 35°C
strongly inhibited the growth of Gram positive bacteria like B. subtilis, S.
aureus and Gram negative bacteria P. aeruginosa, K. pneumoniae, M. luteus,
S. typhi and E. coli (Table 29, 30 and 31) .
Results and Discussion
145
Table 29. Effect of temperature on growth and bioactive metabolite production of Streptomyces sp. 2438 isolate.
Temperature (°C)
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
20 15 8 15 0 5 6 8 0 0
25 15 8 18 0 8 8 10 0 0
30 28 12 24 10 12 10 12 0 0
35 30 12 24 8 12 10 12 0 0
40 28 12 12 0 10 8 10 0 0
45 24 10 15 0 6 8 10 0 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
Table 30. Growth and bioactive metabolite production of Streptomyces sp. LD48 isolate as influenced by temperature.
Temperature (°C)
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
20 8 0 8 6 4 5 8 8 0
25 8 0 10 8 6 5 8 8 0
30 12 0 12 8 8 6 10 8 0
35 15 8 16 8 10 8 15 10 0
40 10 0 10 8 6 8 10 8 0
45 10 0 10 6 6 8 8 8 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
Results and Discussion
146
Table 31. Influence of temperature on growth and bioactive metabolite production of Streptomyces indiaensis IF5.
Temperature (°C)
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
20 19 20 20 8 8 10 12 0 0
25 22 25 22 10 12 12 12 8 0
30 28 28 28 10 12 14 15 12 0
35 32 32 30 12 15 16 18 16 10
40 30 30 28 12 15 15 18 12 0
45 28 30 24 12 12 12 15 12 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
Many reports are available on the effect of temperature on growth and
production of antibiotics by actinomycetes. Hamill et al. (1989) reported the
optimum temperature for the production of antibiotic A80509 by Streptomyces
metabilis isolate NRRL 18269 as 30°C. Similarly Bhattacharya et al. (1998)
showed that 30°C, was the optimum temperature for antibiotic production by
S. hygroscopicus D1.5. Further, Sujatha et al. (2005) also reported the
medium incubated at 30°C showed excellent antibiotic production in
Streptomyces BT-408. The optimum temperature for the production of a new
cytotoxic compound, 1-Hydroxyl-1-norresistomycin from Streptomyces
chibaensis was recorded as 28°C (Gorajana et al., 2005). Likewise, Jeong
(2006) reported that the optimum temperature for the production of
streptokordin at 28°C by a marine Streptomyces sp. KORDI-3238. However
Narayana and Vijayalakshmi (2008) observed that Streptomyces albidoflavus
produced high levels of biomass and antibiotic when culture medium was
incubated at 35°C. Data from our studies also revealed that maximum
Results and Discussion
147
biomass and antibiotic production were achieved when the isolates were
grown at 35°C.
4.12 Optimization of media for antibiotic production by the isolates
The culture media for the growth of three isolates and bioactive
metabolite production were optimized / standardized. Isolate Streptomyces
sp. 2438 yielded high rates of bioactive metabolites when cultured in starch
casein broth containing lactose 0.4%, peptone 0.4% with 6% NaCl, pH 7 and
incubated at 35°C (Table 32) while the modified starch casein broth amended
with 0.4% fructose, 0.4% peptone and 4% NaCl greatly supported the
production of bioactive metabolites by the isolate Streptomyces sp. LD48
(Table 33). Isolate Streptomyces indiaensis isolate IF5 yielded high rates of
bioactive metabolites when cultured in starch casein broth containing lactose
0.4% and tyrosine 0.4% with 6% NaCl, pH 7 and incubated at 35°C (Table
34). Among the test organisms, B. subtilis and P. aeruginosa were highly
susceptible to the metabolites of all the three isolates. Isolate Streptomyces
indiaensis isolate IF5 produced maximum amount of bioactive metabolites
which inhibited the test organisms like B. subtilis, S. aureus, P. aeruginosa,
K. pneumoniae, S. typhi, P. mirabilis, M. luteus, E. coli and E. aerogenes.
Isolate Streptomyces sp. 2438 exhibited antimicrobial activity against the test
organisms B. subtilis, P. aeruginosa, S. aureus, K. pneumoniae, M. luteus,
E. coli P. mirabilis and S. typhi but not against E. aerogenes
.
Results and Discussion
148
Table 32. Production of biomass and antimicrobial metabolites by Streptomyces sp. 2438 culture grown in optimized culture medium.
S.No
.
Culture medium
Biomass
(g/100ml)
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
1. Optimized
culture medium
5.48 38 20 30 15 18 18 20 0 0
2. Control medium
(SCA medium)
2.56 30 12 23 8 12 10 12 0 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
Table 33. Biomass and antimicrobial metabolite production by Streptomyces sp. LD48 isolate grown in optimized culture medium.
S.No.
Culture medium
Biomass
(g/100ml)
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
1. Optimized
culture medium
5.78 22 12 23 18 18 12 19 18 0
2. Control medium
(SCA medium)
2.96 15 8 16 10 10 8 15 10 0
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
Table 34. Production of biomass and antimicrobial metabolites by Streptomyces indiaensis isolate IF5 culture grown in optimized culture medium.
S.No
.
Culture medium
Biomass
(g/100ml)
Diameter of zone of inhibition (mm)
BS SA PF PM EC ML KP ST EA
1. Optimized
culture medium
6.23 42 42 42 26 22 24 28 28 20
2. Control medium
(SCA medium)
3.03 32 32 30 12 15 16 18 16 10
BS: Bacillus subtilis, SA: Staphylococcus aureus, PF: Pseudomonas aeruginosa, PM: Proteus mirabilis, EC: Escherichia
coli, ML: Micrococcus luteus, KP: Klebsiella pneumoniae, ST: Salmonella typhi and EA: Enterobacter aerogenes.
Results and Discussion
149
A comparison of the antimicrobial activities of the three isolates
revealed that Isolate Streptomyces sp. LD48 exhibited the antimicrobial
activity against the test organisms like B. subtilis, P. fluorescens,
K. pneumoniae, S. typhi, P. mirabilis, E. coli and S. aureus. The isolate
Streptomyces indiaensis isolate IF5 exhibited pronounced effect on B. subtilis,
S. aureus, P. aeruginosa than Streptomyces sp. 2438 and Streptomyces sp.
LD48.
Umezawa et al. (1957) studied the carbon nutrition of S.
kanamyceticus for kanamycin production in complex media and reported that
glucose, maltose, dextrin, starch, lactose, and sucrose were better carbon
sources than glycerol. Romano and Nickerson (1958) studied the utilization of
amino acids as carbon sources by Streptomyces species. The effect of
carbon and nitrogen sources on the growth of S. griseus and streptomycin
production was investigated by Dulaney (1948). Likewise, Majumdar and
Majumdar (1967) studied the utilization of carbon and nitrogen compounds for
neomycin production by S. fradiae. Chandrasekhar and Seshadri (1964)
reported that incorporation of pulses and cereals in the medium containing
glucose, NaCl and K2HPO4 gave the best results for the production of
antimicrobial metabolites by Streptomyces. The growth pattern of six strains
of Streptomyces and their antibiotic production were examined using different
carbon and nitrogen sources in the culture media by Sahay and Srivastava
(1977). Mandal et al. (1981) studied the effect of carbon and nitrogen sources
on the production of oxytetracycline by S. rimosus.
Bhattacharya et al. (1998) investigated on the effect of nutrients on
production of antimicrobial metabolites by Streptomyces hygroscopicus D15.
Results and Discussion
150
The optimum temperature for the production of bioactive compounds like
quinolones, macrocyclic peptide antibiotic compound 41, 403 and
boxazomycin A and B by Pseudonocardia sp. CL38489 (Dekker et al., 1998),
Pseudonocardia G495-11 (Saitou et al., 1987) and Pseudonocardia fastidiosa
(Clemer et al., 1977) respectively was recorded as 28°C.
Sujatha et al. (2005) reported that a medium amended with glucose
and ammonium nitrate with pH 7.2 and incubated at temperature 30°C for 96
hr were the optimal conditions for antibiotic production by Streptomyces BT-
408. However, Narayana and Vijayalakshmi (2008) reported that a medium
amended with maltose and soybean meal with pH 7 and incubated at 35°C for
120 hr were optimal for antibiotic production by S. albidoflavus.
4.13 Melanin production
Actinomycetes also synthesize and excrete dark pigments, melanin or
melanoid which are considered to be a useful criterion for taxonomical
studies. Melanin compounds are regular, dark brown polymers produced by
various microorganisms by fermentative oxidation, and have the radio
protective and antioxidant properties that can effectively protect the living
organisms from ultraviolet radiation. Melanins are frequently used in medicine,
pharmacology and cosmetic preparations.
The isolate Streptomyces sp. 2438 exhibited high dopachrome
formation followed by Streptomyces sp. LD48 and Streptomyces indiaensis
IF5. High biomass was produced by Streptomyces indiaensis isolate IF5
followed by Streptomyces sp. 2438 and Streptomyces sp. LD48 (Table 35).
Results from the present study reveal that the method of testing melanin
Results and Discussion
151
production by L-tyrosine or L-Dopa as a substrate as a good criterion for the
identification and classification of Streptomyces.
Table 35. Melanin production by the isolates.
Isolate
Biomass(g) pH (O.D)
Tyrosine broth
Peptone broth
Tyrosine Broth
Peptone broth
Tyrosine Broth
Peptone broth
A9 0.65 0.73 4.78 9.25 0.222 0.190
A10 0.48 0.38 5.29 9.28 0.182 0.160
A20 0.75 1.37 5.55 8.99 0.178 0.150
Skinner (1938) observed that pigmentation resulted from the addition of
tyrosine to a defined medium upon which he was growing some
actinomycetes. A blue color pigment was produced by S. coelicolor strain
(Sanchez-Marroquin and Zapata, 1953). Douglas and San Clemente (1956)
observed a dark brown pigment in flasks in which mycelial homogenates of
S. scabies were mixed with tyrosine, dihydroxy phenylalanine, or both. In
attempting to correlate pigment formation and antibiotic synthesis by S.
antibioticus, Sevcik (1957) manometrically determined phenol oxidase activity
with tyrosine and several other substrates. A brown-black pigment was
produced by S. lavendulae when tyrosine was added to the defined medium
(Mencher and Heim, 1962). Dastger et al. (2006) described the separation,
Identification and analysis of pigment (melanin) production in Streptomyces
spp. Streptomyces hygroscopicus sub sp. ossamyceticus isolated from Thar
desert soil, Rajasthan during the year 2006 produced a yellow pigment with
antibiotic activity (Selvameenal et al., 2009). Although these studies suggest
Results and Discussion
152
that the pigment was melanin, apparently no attempts have been made to
isolate and characterize the pigment.
4.14 Estimation of protein of actinomycete isolates
Amount of protein in the cells of all the isolates of actinomycetes were
estimated by Lowry et al. (1951) method. High amount of protein was present
in Streptomyces indiaensis IF5 followed by Streptomyces sp. 2438 and
Streptomyces sp. LD48. Data on protein concentration of Streptomyces spp.
showed no significant differences between among the isolates. However, it
was more in Streptomyces indiaensis IF5 followed by Streptomyces sp. 2438
and Streptomyces sp. LD48. The nature of the dibasic amino acids present in
the cell wall of the organism help to identify the actinomycetes (Goodfellow
et al., 1987). As amino acids are the precursors of metabolic activity, studies
were undertaken to detect the presence of amino acids present in the culture
filtrate of Streptomyces sp. Proteins are the structural and functional units of
the cell and take part in metabolic activities of the cell (Sanglier et al., 1993).
Thangapandian et al. (2007) reported that leucine, proline and tryptophan
were the probable amino acids present in the culture filtrate of Streptomyces
spp.
4.15 Fermentation and extraction of bioactive metabolites
The crude extracts of antimicrobial metabolites produced by the three
strains by fermentation which were subjected to concentration under pressure
which yielded dark brown and pink organic layers containing solvent and
antimicrobial compounds.
Results and Discussion
153
4.16 Purification of antimicrobial compounds
The crude extracts containing secondary metabolites were partially
purified by thin layer chromatography. Later these compounds were subjected
to column chromatography for further purification. The ethyl acetate extracts
were subjected to the absorption spectrum to test for antimicrobial
compounds. The absorption spectrum of each active extract was determined
in the UV region (200–400 nm) by using a Perkin-Elmer lambda 15 UV/VIS
spectrophotometer. Since the ethyl acetate extracts obtained from
Streptomyces LD 48 and Streptomyces sp. 2438 yielded low quantity of
bioactive metabolites and exhibited low activity against the test organisms
further studies were confined to the ethyl acetate extracts obtained from
Streptomyces indiaensis IF5. The active compound was subjected to
purification process by reversed-phase HPLC and the chemical structure of
the obtained active compound was elucidated through extensive analyses of
NMR spectroscopy.
4.16.1 Thin layer chromatography
The crude extracts were partially purified by thin layer
chromatography (TLC). Only one band, with Rf value of 0.73, showed
antimicrobial activity exhibited by the isolate S. indiaensis IF 5 which is
evident from bioautography (Figure 20).
Separation of antimicrobial agent into individual components was
carried out by TLC, using a solvent system composed of chloroform and
methanol (24:1 v/v). Only one band at Rf = 0.75 showed antimicrobial activity
as reported by Atta (1999). The antibiotic was partially purified on TLC by
Results and Discussion
154
using a solvent system, ethanol, water, chloroform (40:40:20) and Rf value
was 0.78 as per Augustine et al. (2005).
Figure 20. TLC with different solvent systems like n-butanol, glacial acetic acid, water (a), chloroform, methanol (b), isopropanol, water (c) run for extracts of A9, A10 and A20 isolates and (d) run with n-butanol, glacial acetic acid, water with streptomycin and extract of A20 isolate.
Ahmed (2007) reported the purification of the antibiotics by TLC by
using a solvent system, benzene-ether (1:1). The Rf value was 1.37 which
reported only one spot. According to Muiru et al. (2008) different Rf values
were obtained from the isolate 28P which yielded two active compounds,
while isolates CS35 and 14P have yielded only one active compound.
4.16.2 Column chromatography
The active fraction obtained from TLC was eluted with 100%
chloroform which was repeated by reducing chloroform to 10% and increasing
the methanol by 10% until the percentage of methanol reached 100%. Thirty
Results and Discussion
155
fractions were collected and tested for the antimicrobial properties. Further
purification of active fractions was carried out by preparative HPLC.
4.16.3 High Performance Liquid Chromatography (HPLC)
The fractions obtained from column chromatography were further
purified using preparative HPLC. Forty fractions were collected from
preparative HPLC and each fraction was checked for antimicrobial activity
(Figure 21). The active fraction was collected and dried using vacuum
evaporation. Fractions that showed antimicrobial activity were combined and
dried under vacuum. Dried active compound showed white powder and easily
dissolved in methanol, chloroform (1:1). Preparative HPLC chromatogram of
purification process was identified and presented in (Figure 22). Results
revealed that many compounds have not been eliminated before purification
using preparative HPLC. However, after purification using preparative HPLC,
a single active compound was obtained. Results from antimicrobial activity
assay showed that fraction showed a retention time at 10.1 min of preparative
HPLC chromatogram.
Figure 21. Analysis HPLC chromatogram of supernatant extract.....
Results and Discussion
156
Figure 22. Chromatogram of preparative HPLC.
The fraction was then collected and used for HPLC analysis.
Chromatogram of active fraction showed a retention time of 8.623 min at
gradient elution methanol – water (0 – 100%) using analytical HPLC (Figure
23). Identification of pure active compound using HPLC analysis showed that
this active compound has UV visible maximum absorption between 210 and
274.5 nm in methanol, (Figure 24). It was reported that the most of the
peptide antibiotic exhibit maximum absorbance at 210 – 230 nm and 270 –
280 nm (Ezra et al., 2004; Bizani et al., 2005; Kumar et al., 2009). According
to Kumar et al. (2009) an absorbance at 220 – 230 nm corresponds to
characteristic absorption of peptide bonds. Analysis of HPLC chromatogram
and UV visible spectrum of active compound after purification using
preparative HPLC are presented in Figure 23 and Figure 24.
Results and Discussion
157
Figure 23. Analysis HPLC chromatogram of active fraction.
Figure 24. UV visible spectrum of active fraction.
4.16.4 Nuclear Magnetic Resonance (NMR)
The molecular weight, formula and the structure elucidation of
active compound were determined using ESI-MS, 1HNMR, 13CNMR and FTIR
respectively.
ESI-MS, mass spectroscopy spectra showed that the active compound
has a molecular weight of 260.0 g/mol and the melting point was 140°C. This
active compound had 14 carbon, 16 hydrogen, 2 nitrogen and 3 oxygen
molecules and the molecular formula was C
carbon, nitrogen, oxygen and hydrogen atoms were confirmed by
13CNMR and FTIR .
The chemical characteristics were determined by ESI
(M+H)+ (Figure 25). High
Bruker AV-500 (500MHz) with tetramethyl
CDCL3 and the data are as follows :
(2H,m), (t,1H), 4.048 (1H,
6.690 (2H, d), and 13C spectrum :
60.082 (d), 29.421 (t), 22.477 (t), 45.942 (t), 37.694 (t), 127.65 (s),
116.315 (d), 157.699 (s). The impurities
1HNMR δH 1.7- δH 0.9 (Figure 26). Spectrum data
presented in Table 36. Two singlet carbons representing a ketone group
evident in the 13C spectrum
(Table 36; Figure 27).
Figure 25. Spectrum of bioactive compound produced by indiaensis
Results and Discussion
158
active compound had 14 carbon, 16 hydrogen, 2 nitrogen and 3 oxygen
molecules and the molecular formula was C14H16N2O3. The position of each
carbon, nitrogen, oxygen and hydrogen atoms were confirmed by
The chemical characteristics were determined by ESI-MS at
). High-resolution 1HNMR spectrum were obtained on a
500 (500MHz) with tetramethyl silane (TMS) as internal standard in
and the data are as follows : δH: 4.358 (t,1H), 4.048 (1H,dd), 2.088
(1H,dd), 3.518 (2H, dd), 3.066 (2H, dd), 7.031
(2H, d), and 13C spectrum : 170.795 (s), 57.926 (d), 166.935 (s),
60.082 (d), 29.421 (t), 22.477 (t), 45.942 (t), 37.694 (t), 127.65 (s),
116.315 (d), 157.699 (s). The impurities of active compound also showed at
0.9 (Figure 26). Spectrum data of 1HNMR and
. Two singlet carbons representing a ketone group
C spectrum at δ 170.795 (s) (C1) and δ 166.935 (s) (C4)
).
Spectrum of bioactive compound produced by Streptomyces indiaensis isolate IF5 ESI-MS m/z 261 (M+H).
Results and Discussion
active compound had 14 carbon, 16 hydrogen, 2 nitrogen and 3 oxygen
The position of each
carbon, nitrogen, oxygen and hydrogen atoms were confirmed by 1HNMR,
MS at m/z 261
HNMR spectrum were obtained on a
silane (TMS) as internal standard in
H: 4.358 (t,1H), 4.048 (1H,dd), 2.088
7.031 (2H, d),
(s), 57.926 (d), 166.935 (s),
132.135 (d),
also showed at
HNMR and 13CNMR are
. Two singlet carbons representing a ketone group were
66.935 (s) (C4)
Streptomyces
Results and Discussion
159
Figure 26. Spectrum of bioactive compound produced by
Streptomyces indiaensis isolate IF5 1H NMR.
Figure 27. Spectrum of bioactive compound produced by
Streptomyces indiaensis isolate IF5 13C NMR.
Results and Discussion
160
Table 36. Spectrum data of H NMR and C NMR of active compound produced by Streptomyces indiaensis IF5.
Further analysis of the 13C spectrum revealed two other non
substituted carbons [δ 127.651 (C1'), 157.699 (C4')], six methane carbons [δ
57.926 (C3), 60.082(C6), 132.135 (C2'), 116.315 (C3'),116.215 (C5'),132.135
(C6')], and four methylene carbons [δ 29.42 (C7),22.477 (C8), 45.942 (C9),
37.694 (C10)]. Based on information of ESI-MS, 1HNMR and 13CNMR
spectra, the molecular structure of the active compound was structured as
outlined in Figure 28.
Results and Discussion
161
Figure 28. Molecular structure of active compound produced by Streptomyces indiaensis IF5.
The structure of active compound was also constructed using DEPT
13CNMR Figure 26. DEPT 45° spectrum in Figure 29 showed that there were
three non-substituted carbon [δ 127.651 (C1'), 157.69 (C4'), and 166.935
(C4)]. DEPT 135° and 90° showed that there were six methane carbons [δ
57.926 (C3), 60.082 (C6), 132.135 (C2'), 116.315 (C3'),116.215 (C5'),
132.135 (C6')] and four methylene carbons [δ 29.42 (C7), 22.477 (C8), 45.942
(C9)]. Carbons at position 3' and 5' appeared more up field than C2' and
C6'. This was due to the shielding effect of the hydroxyl group at C4' toward
its ortho coupled carbon (C3' and C5'). A similar phenomenon occurred
on C1' (para coupled with C4') which more upfield than C2'and C6'.
Figure 29. Spectrum of DEPT C NMR active compound produced by Streptomyces indiaensis
The infra red spectrum in a KBr pellet showed
3383 cm (N-H), 3227 cm (O
1515cm (benzene ring),
(phenol), 1116 cm (C-O), 827cm (
Figure 30. Infra red spectrumStreptomyces
Results and Discussion
162
Spectrum of DEPT C NMR active compound produced by Streptomyces indiaensis isolate IF5
The infra red spectrum in a KBr pellet showed characteristic bands at
H), 3227 cm (O-H), 2959 cm (saturated C-H), 1660 cm (C=O),
1515cm (benzene ring), 1456 cm (methane), 1344 cm (methylene), 1232 cm
O), 827cm (p-disubstituted benzene ring) (Figure 30
30. Infra red spectrum of the active compound produced by Streptomyces indiaensis isolate IF5.
Results and Discussion
Spectrum of DEPT C NMR active compound produced by
characteristic bands at
H), 1660 cm (C=O),
(methylene), 1232 cm
substituted benzene ring) (Figure 30).
active compound produced by
Results and Discussion
163
The active compound exhibited antimicrobial activity against Gram
positive and Gram negative bacteria. Rhee (2004) explained that most of
the antibiotic peptides especially cyclic dipeptide has antimicrobial
properties with a broad spectrum. Several cyclo peptides exhibit not only
broad spectrum activity but also antiviral and anticancer properties
(Lu and Chen, 2010; Lee et al., 2011). Cyclo (tyrosyl-prolyl) is a peptide
antibiotic, whereas streptomycin is an aminoglycoside antibiotic. The
active compound was included in a group cyclodipeptide namely Cyclo
(tyrosyl-prolyl). This active compound has the same profile (1H NMR, 13C
NMR, Infrared spectrum, and molecular weight) as cyclo (tyrosyl-prolyl) that
was previously reported but from different origins (Stierle et al., 1988; Guo
et al., 2007). Previously, this compound was reported to be produced by
Alternaria alternata and used as a host-specific phytotoxin for spotted
knapweed (Stierle et al., 1988), Pseudomonas fluorescens GcM5-1A
isolated from the pine wood nematode (PWN) and Bursaphelenchus
xylophilus (Guo et al., 2007). The isolation of the compound in the present
study is supported by the report of Sunaryanto et al. (2011) who also isolated
the same compound from Streptomyces spp. by different purification methods.
Further studies for exploring the possibility of production of this compound on
commercial scale are needed.
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