4.1 Collection of soil samples - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/33374/9... · 4.1 Collection of soil samples ... different pigments. Yellow pigment was produced

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.


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


13 Very Good Grayish White Light Brown Nil

14 Very Good Grey Grayish White Nil

15 Good White White Nil


17 Very Good Grayish White Yellowish Brown Brown



20 Very Good Pinkish White 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,


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.




A15, A16 isolates and brown in A7, A9, A13 and A22 isolates. Yellowish




by A14 isolate. Among the 22 isolates, only 13 isolates produced



n A1, A6, A10, A13, A17 and

A21 and A22

Morphological features of the colonies of actinomycete medium.




nd A22 isolates. Yellowish




by A14 isolate. Among the 22 isolates, only 13 isolates produced


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.


105


in case of A5 isolate and pink pigment in A4, A18, A19, A20 and A21 isolates.




against the test organisms like Gram positive (B. subtili

) bacteria and the results are tabulated in Table 5



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


Antimicrobial activity of the isolates against Gram positive b, c) and Gram negative (d, e) bacteria.



4, A18, A19, A20 and A21 isolates.




B. subtilis) and

) bacteria and the results are tabulated in Table 5.



, 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


isolates against Gram positive (a,


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.


107



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)





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)



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


109


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



respectively. A gradual increase in the antimicrobial spectrum of the isolates


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



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.



spectrum of the isolates


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


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)


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.


Age(hr)



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




111


Age(hr) Diameter of zone of inhibition (mm)


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



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


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


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.


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 White

Growth, color of aerial and substrate mycelium and pigments produced by the three isolates on different ISP and non


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


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).


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.


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


was oval and its surface was smooth (Fig 10b).

The scanning electron microscopy of isolate A20 revealed that aerial


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



(b)

Figure 10. Scanning Electron Microscopy of A9 (a), A10 (b), and A20 (c)


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


ectron microscopy of isolate A20 revealed that aerial


Each spore chain consisted of 5 to 10 white, oblong (0.8 µm by 1.2 – 1.5 µm),

meter, 2.0 µm in



(c)

Figure 10. Scanning Electron Microscopy of A9 (a), A10 (b), and A20 (c)


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.


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.


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.


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


isolates and incubated at 37°C for 48 hr. A clear zone was observed around


Hence the test was positive for all the three isolates.


cubated at 37°C for 48 hr. After incubation, no pink



Biochemical tests of A9, A10, A20 isolates and control

for the production of

data on the production of different enzymes by the isolates are


observed around



cubated at 37°C for 48 hr. After incubation, no pink color



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.


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


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).


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



sing seeded plate technique. Filter paper discs containing the



to bacitracin, ciprofloxacin, doxycyclin, kanamycin, rifampcin, neomycin and



and vancomycin (Table 15).


Figure 12. Production of enzymes by the three isolates (urease, alanine deaminase, casein and



sing seeded plate technique. Filter paper discs containing the



clin, kanamycin, rifampcin, neomycin and




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.


127


Gram negative bacteria, yeast and 16 phytopathogenic fungi. Ten of them had

a broad spectrum antibacterial activity and could be used in the development


and Naidenova, 2004). Actinomycetes are known to produce many secondary

different biological activities such as antibacterial, antifungal,


Spectrum of antibiotics


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.



and 16 phytopathogenic fungi. Ten of them had

erial activity and could be used in the development


Actinomycetes are known to produce many secondary

ntibacterial, antifungal,


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)


128

4.10 Phylogenetic analysis of the actinomycete isolates




observed for all the three isolates when resolved on agarose gel (Figure 14).




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





on agarose gel (Figure 14).




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),


Figure 15. Phylogenetic tree of



129

Figure 15. Phylogenetic tree of Streptomyces sp. 2438.

Figure 16. Phylogenetic tree of Streptomyces sp. LD38.



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


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


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.


strain IF5.

Streptomyces


Influence of different carbon sources on the cell growth of the isolates


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


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



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



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


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



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


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


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



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



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


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


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


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



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



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,


137

Table 21. Influence of nitrogen source on growth and bioactive metabolite production of Streptomyces sp. LD48 isolate.

Nitrogen source



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



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).


138

Table 22. Growth and bioactive metabolite production of Streptomyces indiaensis IF5 as influenced by nitrogen source.

Nitrogen source



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



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.


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


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 (%)



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



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.


141

Table 24. Growth and bioactive metabolite production of Streptomyces sp. LD48 isolate as influenced by NaCl.

NaCl (%)



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



Table 25. Influence of NaCl on growth and bioactive metabolite production of Streptomyces indiaensis IF5.

NaCl (%) Diameter of zone of inhibition (mm)


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



. 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.


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



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




143

Table 27. Influence of pH on growth and bioactive metabolite production of Streptomyces sp. LD48 isolate.

pH



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



Table 28. Growth and bioactive metabolite production of Streptomyces indiaensis IF5 as affected by pH.

pH



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



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


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) .


145

Table 29. Effect of temperature on growth and bioactive metabolite production of Streptomyces sp. 2438 isolate.

Temperature (°C)



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



Table 30. Growth and bioactive metabolite production of Streptomyces sp. LD48 isolate as influenced by temperature.

Temperature (°C)



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




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Table 31. Influence of temperature on growth and bioactive metabolite production of Streptomyces indiaensis IF5.

Temperature (°C)



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



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


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

.


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)



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



Table 33. Biomass and antimicrobial metabolite production by Streptomyces sp. LD48 isolate grown in optimized culture medium.

S.No.

Culture medium

Biomass

(g/100ml)



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



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)



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




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.


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


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


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.


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


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


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.....


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.


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


158


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).



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


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.


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.


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


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.


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


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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4.1 Collection of soil samples - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/33374/9... · 4.1 Collection of soil samples ... different pigments. Yellow pigment was produced