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3.1 Isolation of actinomycetes
3.1.1 Soil collection
Black and red soils from agricultural fields of Akkampalli and
Limgampalli villages of Anantapur district, Andhra Pradesh were collected.
The soils collected to a depth of 15 cm, were air-dried and after breaking the
clods were sieved (2 mm mesh) and used as a potting medium. Seeds of
foxtail millet [Setaria italica (L.) Beauv] were sown in separate earthen pots
filled with the soils.
3.1.2 Seed collection
Seeds of foxtail millet were collected from the Regional Agricultural
Research Station at Rekulakunta (APAU) near Anantapur. Anantapur district
of Andhra Pradesh receives a poor rain fall of 34.4 mm and a temperature of
31±9°C resulting in frequent droughts. Under these conditions, foxtail millet, a
short duration crop (75 to 90 days), is cultivated as one of the minor millet
crops. The vernacular names of the foxtail millet are Korra (Telugu), Navane
(Kannada), Tenai (Tamil), Tena (Malayalam), and Kangoone (Hindi).
3.1.3 Pot Experiments
Soil samples (5 kg each of black and red) were placed in separate earthen
pots and foxtail millet seeds were sown in the pots separately. The plants
were grown up to 75 days.
3.1.4 Collection and treatment of soil samples
Rhizosphere soil samples, collected from foxtail millet plants at 15 day
intervals, were subjected to different treatments for better isolation of
actinomycetes. They are as follows:
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• CaCO3 treatment: One g of soil was mixed with 1 g of CaCO3 and
incubated at room temperature for one week in water saturated
condition.
• Phenol treatment: One g of a dense soil suspension was added to
100 ml of 1.4% phenol solution and kept at room temperature for
10 min. The mixture was diluted and used for isolation.
• Heat treatment: Soil sample (10 g) was subjected to heat treatment at
40-45°C for 15 hr and then used for isolation.
3.1.5 Rhizosphere soil suspension
Plants were uprooted from the pots at different days (0, 30, 45, 60 and
75 days) of plant growth and the roots were carefully washed under tap water
to remove large soil aggregates. The soil still adhering to the roots after
washing was suspended by shaking the roots in sterilized distilled water and
was used as the rhizosphere soil suspension (Ramakrishna and Sethunathan,
1982).
3.1.6 Serial dilutions
The rhizosphere soil sample (1 g) with still adhering roots after washing
was suspended by shaking the roots in 10 ml of sterilized distilled water for a
few minutes. Ten-fold serial dilutions of the soil samples were prepared up to
10-8.
3.1.7 Enumeration of actinomycetes on SCA medium
For isolation and enumeration of actinomycetes from the rhizosphere
soil samples, starch casein agar (SCA) medium (Kuster and Williams, 1964)
with the following composition (g/l) was used:
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Starch 10.0
Casein 0.3
KNO3 2.0
NaCl 2.0
K2HPO4 2.0
MgSO4.7H2O 0.05
CaCO3 0.02
FeSO4.7H2O 0.01
Agar 20.0
Distilled water 1000 ml
pH 7.2
The pH of the medium was adjusted to 7.2 and supplemented with
nystatin and cycloheximide (50 mg ml-1) to avoid bacterial and fungal
contamination. Aliquots (0.1 ml) from 10-1 to 10-5 dilutions of the rhizosphere
soil samples were inoculated to plates containing sterilized starch casein agar
medium and spread evenly under aseptic conditions in a laminar air flow
chamber. The plates were incubated at 37°C for 3 to 4 days (for appearance
of aerial mycelium), 7 to 14 days (for observing mature aerial mycelium) and
for 30 days (for slow growing isolates). Total number of actinomycetes
colonies that developed on starch casein agar was scored using a colony
counter. Counts of the actinomycetes were expressed as the number of
colony forming units (CFU) per gram of dry weight of the sample.
3.1.8 Purification and preservation of actinomycetes
The isolates were subcultured repeatedly onto starch casein agar
medium until they formed single colonies. Pure cultures were maintained on
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the starch casein agar plates or agar slants and preserved at 4°C. Other
preservation methods are similar to that of bacteria such as subculturing,
freezing in liquid nitrogen, freeze drying and maintenance in mineral oil. Spore
mass and mycelium fragments of the pure isolates were stored at -20°C as
glycerol (20%, v/v) suspension.
3.2 Screening of actinomycete strains
From the different rhizosphere samples, a total of 22 predominant
actinomycetes isolates were purified by streak plate technique on starch
casein agar medium. These 22 isolates were subjected to screening methods
such as spot inoculation and cross streak method/perpendicular method.
3.2.1 Spot inoculation on agar medium
Antimicrobial activity of the isolates was studied primarily by spot
inoculation of the isolates on agar medium (Shomurat et al., 1979). Pure
isolates of actinomycetes were spot inoculated on actinomycetes isolation
agar medium and incubated in a fume hood. Colonies were then covered with
a 0.6% agar layer of nutrient agar medium (for bacteria) previously seeded
with the microorganisms. The plates were incubated at 37°C for 24 hr. The
zones of inhibition of the test microorganisms were recorded after incubation.
3.2.2 Cross streak method / perpendicular streak method
Nutrient agar plates were inoculated with the isolates of actinomycetes
by a single streak of inoculum in the center of Petri dish. After 96 hr of
incubation at 28°C, the plates were seeded with test organism by a single
streak at 90° angle to the isolates of actinomycetes. The microbial interactions
(against the test microorganisms like Bacillus subtilis and Escherichia coli)
were recorded by measuring the diameter of the zone of inhibition (Madigan
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et al., 1997). Then the isolates were maintained in 10% glycerol (v/v) at -20°C.
Of the 22 isolates obtained from the rhizosphere samples, only three showing
medium, low and high antimicrobial activity were selected for further studies
and are designated as A9, A10 and A20 respectively.
3.3 Growth pattern of the selected actinomycetes strains
A loopful of the culture of actinomycete isolates, maintained on starch
casein agar slants, was inoculated separately into the seed medium (Starch
casein broth). After 24 hr of incubation, the vegetative seed culture @ 10%
was used to inoculate the fermentation broth (Starch 10 g, Casein 0.3 g,
KNO3 2 g, NaCl 2g, K2HPO4 2 g, MgSO4.7H2O 0.05 g, CaCO3 0.02 g,
FeSO4.7H2O 0.01 g, Distilled water 1000 ml). The inoculated flasks were
incubated at 35°C for 7 days. At 24 hr interval, the biomass (g/100 ml) was
recorded. The culture filtrates thus collected were extracted with ethyl acetate
in a separating funnel. The solvent extracts were then concentrated and
preserved for further antimicrobial study (Cappuccino and Sherman, 2004).
3.3.1 Antimicrobial assay
Antimicrobial activity of the crude extracts of the actinomycete isolates
was determined by cup plate or well diffusion method against the test
organisms like Bacillus subtilis, Staphylococcus aureus, Pseudomonas
aeruginosa, Proteus mirabilis, Escherichia coli, Micrococcus luteus, Klebsiella
pneumoniae, Salmonella typhi and Enterobacter aerogenes.
Nutrient agar medium was prepared and sterilized at 15 lbs (121°C) for
20 min. The culture suspension of the test bacteria was lawned over the
surface of nutrient agar medium under aseptic conditions. Wells of 6 mm were
made in to the agar medium by using sterilized cork borer. The crude extracts
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dissolved in ethyl acetate (50 µl) were added to wells and the solvent alone
was kept as control. The area of the inhibition zone was calculated after
incubation of the inoculated bacterial plate at 37°C for 24 hr.
3.4 Identification of actinomycete isolates
The identification of selected actinomycetes isolates was done by
different morphological, biochemical and molecular methods.
3.4.1 Cultural characteristics on different media
The growth characteristics of the isolates were studied on different
media including (International Streptomyces Project), ISP media and Non-ISP
media. The ISP media like ISP1/TYEA, ISP2/YMD, ISP3/OMA, ISP4, ISP5,
ISP7, ISP9 and Non-ISP media such as nutrient agar, Czapek Dox agar,
starch casein agar, potato dextrose agar, glucose aspargine agar, and
peptone yeast extract iron agar. Cultural characteristics such as type of
growth, color of the aerial mycelium and pigment production were recorded.
The compositions of different ISP and Non-ISP media (Shirling and Gottlieb,
1966) are as follows (g/l):
ISP1/ Tryptone yeast extract agar
Bacto-Tryptone 5.0
Yeast extract 3.0
Agar 20.0
Distilled water 1000 ml
pH 7-7.2
ISP2/Yeast extract malt extract agar
Yeast extract 4.0
Malt extract 10.0
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Dextrose 4.0
Agar 20.0
Distilled water 1000 ml
pH 7.3
ISP3/Oatmeal agar
Rolled oats 65.0
Distilled water 1000 ml
Agar 20.0
Oat meal 20.0
pH 7.2
ISP4/Inorganic starch salt agar
Solution-I
Ten g of soluble starch was made in to paste with the small amount of
cold water and the volume was made up to 500 ml.
Solution-II
(NH4)2SO4 2.09
K2HPO4.3H2O 1.09
NaCl 1.09
CaCO3 2.09
Trace salt solution 1ml
(FeSO4.7H20 0.1g
MnCl2.4H2O 0.1g
ZnSO4.7H2O 0.1g)
Distilled water 500 ml
Agar 20.0
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pH 7.0-7.4
ISP5/Glycerol aspargine agar
Glycerol 35 ml
NaCl 5.0
CaCl2 0.1
MgSO4 0.3
K2HPO4 2.5
Ammonium lactate 6.5
Sodium asparginate 3.5
Distilled water 1000 ml
Agar 20.0
pH 7.0
ISP7/Tyrosine agar
Glucose 10.0
Tyrosine 1.0
(NH4)2SO4 0.5
K2HPO4 0.5
Distilled water 1000 ml
Agar 20.0
pH 7.2
ISP9/Inorganic salt agar
Glucose 10.0
(NH4)2SO4 2.64
KH2PO4 2.38
K2HPO4.3H2O 5.64
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MgSO4.7H2O 1.0
CuSO4.5H20 6.4 mg
FeSO4.7H20 1.1 mg
MnCl2.4H2O 7.9 mg
ZnSO4.7H2O 1.5 mg
Distilled water 1000 ml
Agar 20.0
pH 7.0
Nutrient agar
Peptone 5.0
Beef extract 3.0
NaCl 5.0
Distilled water 1000 ml
Agar 20.0
pH 7.0
Czapek Dox agar
Sucrose 30.0
NaNO3 2.0
K2HPO4 1.0
MgSO4.7H2O 0.5
KCl 0.5
FeSO4 0.01
Distilled water 1000 ml
Agar 20.0
pH 4.0-5.0
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Starch casein agar
Starch 10.0
Casein 0.3
KNO3 2.0
NaCl 2.0
K2HPO4 2.0
MgSO4.7H2O 0.05
CaCO3 0.02
FeSO4.7H2O 0.01
Agar 20.0
Distilled water 1000 ml
pH 7.2
Potato dextrose agar
Peeled potatoes 200-300
Dextrose 30.0
Distilled water 1000 ml
Agar 20.0
pH 6.5
Glucose aspargine agar
Glucose 10.0
Aspargine 0.5
K2HPO4 0.5
Distilled water 1000 ml
Agar 20.0
pH 6.8
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Peptone yeast extract iron agar
Peptone 20.0
Ferric ammonium citrate 0.5
K2HPO4 1.0
Na2S2O35H2O 0.08
Yeast extract 1.0
Distilled water 1000 ml
Agar 20.0
pH 7.0
The isolates were identified up to the generic level by comparing the
morphology of spore bearing hyphae with the structure of spore chains of
actinomycetes as described in the fourth volume of Bergy's Manual of
Systematic Bacteriology. The morphology of aerial and substrate mycelium
was examined by three methods.
3.4.2 Slide culture technique
The slide culture technique was employed for studying the morphology
of actinomycetes (Williams and Cross, 1971). Sterilized glass cover slips were
placed at an angle of 45°C in a Petri dish containing ISP2/yeast extract malt
extract agar medium. Along the line, where the upper surface of the cover
slips join the agar the isolates were inoculated individually. After incubation,
the cover slips were carefully withdrawn from the medium and the sporophore
morphology of the isolates was examined directly under microscope (Kawato
and Shinobu, 1959).
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3.4.3 Gram staining
On a clean glass slide, a loopful of the inoculum was smeared and air
dried. After drying, the smear was treated with crystal violet for 1 min followed
by Gram's iodine treatment for 1 min. To remove the excess stain on the
smear, 95% of ethyl alcohol was used as a decolorizing agent. The smear
was then flooded with a counter stain, safranin. The same procedure was
repeated for all the isolates. The glass slides containing the stained smear
after washing were air-dried and examined under light microscope.
3.4.4 Scanning Electron Microscope
Morphology of the 96 hr old culture grown on starch casein agar
medium was examined under scanning electron microscope (SEM) (Yassin
et al., 1997). Blocks of well sporulated actinomycete strain cultured on starch
casein agar medium were prepared by cutting. They were placed in 5% KOH
(w/v) for 10 min and fixed in 2.5% (v/v) glutaraldehyde prepared in 0.1M
phosphate buffer (pH 7.0) for 24 hr. The agar blocks were then rinsed three
times with the buffer followed by dehydration using a graded series of ethanol
dried to critical point and mounted on aluminum stubs and sputter coated gold
palladium. Finally, the preparations were observed under Zeiss digital
scanning electron microscope (Jena, Germany).
3.5 Biochemical tests
Following are the biochemical tests conducted with the pure culture
isolates of actinomycetes:
3.5.1 Indole test
The isolates were grown on sterilized glucose peptone water (Peptone
10 g, NaCl 5 g, distilled water 1,000 ml, pH 7.4) and incubated at 37°C for
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48 hr. After incubation, 0.5 ml of Kovac’s reagent (p-dimethyl
aminobenzaldehyde 10 g, isoamyl alcohol 150 ml, concentrated HCl 50 ml)
was added and observed for change in the color.
3.5.2 Methyl Red (MR)
The isolates were inoculated into sterilized glucose phosphate peptone
water (Peptone 5 g, K2HP04 5 g, Glucose solution 10%, distilled water 1,000
ml, pH 7.6) and incubated at 37°C for 48 hr. To the tubes, methyl red solution
(100 mg of Methyl red dissolved in 30 ml of ethanol and made up to 200 ml
with distilled water) was added and observed for change in the color.
3.5.3 Voges-Proskauer (VP) tests
The isolates were inoculated into sterilized glucose phosphate peptone
water (Peptone 5 g, K2HP04 5 g, Glucose solution 10%, distilled water 1,000
ml, pH 7.6) and incubated at 37°C for 48 hr. To the tubes, Barrett’s reagent
(5% α-naphthol dissolved in absolute alcohol, 40% KOH solution) was added
and observed for the formation of violet ring color.
3.5.4 Citric acid utilization test
The isolates were streaked on Simmon's citrate agar slants [Sodium
citrate 5 g, MgSO4 0.2 g, NH4H2PO4 1 g, KH2PO4 1 g, NaCl 5 g, bromothymol
blue (0.2%) 40 ml, distilled water 1,000 ml, pH 6.8)] and incubated at 37°C for
48 hr. The slants were observed for the presence of growth and change in
color of the medium.
3.5.5 H2S production test
The test organism was inoculated onto sterilized Kligler's agar medium
(Beef extract 3 g, yeast extract 3 g, peptone 15 g, protease peptone 5 g,
lactose 10 g, glucose 1 g, FeSO4 0.2 g, sodium thiosulphate 0.3 g, agar 20 g,
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phenol red 0.024 g, pH 7.4) and the test tubes with impregnated lead
acetate strip were incubated at 37°C for 24 hr and observed for the
appearance of black color due to H2S production (Kuster and Williams,
1964a) (H2S after reacting with lead acetate forms black colored lead
sulphide).
3.5.6 Carbon utilization
The utilization of carbon sources was tested using ISP-9 medium
according to the modified method of Shirling and Gottlieb (1968). Pen assay
base agar (Difco) (glucose 5 g, KNO3 1 g, K2HPO4 0.5 g, MgSO4 0.2 g,
FeSO4 0.01, agar 20 g, distilled water 1,000 ml, pH 6.8 adjusted with 0.1N
NaOH) was sterilized and used. D-glucose, D-fructose D-mannitol, D-xylose,
sucrose and lactose were tested as C sources. These substrates were
sterilized by filtration and added @ 0.5 to 1.0% in medium. Growth of the
isolates of actinomycetes on the above media incubated at 26°C for 7, 14 and
21 days was recorded.
3.5.7 Melanin production
The test organism was inoculated onto sterilized peptone yeast extract
agar medium (Peptone 20 g, Ferric ammonium citrate 0.5 g, K2HPO4 1 g,
Na2S2O35H2O 0.08 g, Yeast extract 1 g, Distilled water 1000 ml, Agar 20 g,
pH 7.0) and incubated at 37°C for 24 hr and observed for the appearance of
black color.
3.6 Production of enzymes
3.6.1 Starch hydrolysis
Starch agar plates (Peptone 5 g, beef extract 3 g, soluble starch 20 g,
agar 20 g, distilled water 1,000 ml, pH 7.0) were sterilized and inoculated
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with the test organisms and incubated at 37°C for 48 hr. After incubation, the
plates were flooded with iodine solution (Iodine 1 g, potassium iodide 2 g,
distilled water 300 ml) and observed for the appearance of clear zone around
the colony (Holding and Collee, 1971).
3.6.2 Urease test
Urea broth (Peptone 1 g, NaCl 5 g, K2HPO4 2 g, phenol red 0.012 g,
agar 20 g, distilled water 1,000 ml, 10% glucose solution (sterile) 10 ml, 20%
urea solution (sterile) 100 ml, pH 6.8) was sterilized and inoculated with the
test organism and incubated at 37°C for 48 hr. After incubation, the tubes
were observed for change in color of the medium.
3.6.3 Catalase test
One ml of 3% H2O2 (3 ml of hydrogen peroxide in 97 ml of distilled
water) was added to sterilized nutrient agar medium slants (Peptone 5 g, beef
extract 3 g, NaCl 5 g, agar 20 g, distilled water 1,000 ml) inoculated with the
respective isolates and incubated for 48 hr at 37°C and observed for the
appearance of oxygen bubbles.
3.6.4 Gelatin hydrolysis
The test organism was inoculated onto sterilized nutrient gelatin agar
(Gelatin 120 g, beef extract 3 g, peptone 5 g, distilled water 1,000 ml, agar
20 g) plates and incubated at 35°C for 30 days. After incubation, the plates
were kept in ice to check liquefaction and flooding with trichloroacetic acid
(Trichloroacetic acid will precipitate the gelatin and if the plate becomes
opaque it indicates positive test).
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3.6.5 Casein hydrolysis
Casein agar medium (Skim milk powder 100 g, peptone 5 g, NaCl 4 g,
agar 20 g, distilled water 1,000 ml) was sterilized and inoculated with the test
organism and incubated at 30°C for 4 days. After incubation, the plates were
observed for the development of clear zone around the colony (Holding and
Collee, 1971).
3.6.6 Pectinase
Pectin agar medium (Pectin 1 g, (NH4)2SO4 0.1 g, K2HPO4, 0.2 g,
MgSO4.7H2O 0.02 g, NaCl 4 g, agar 20 g, distilled water 1,000 ml, pH 7) was
sterilized and inoculated with the test organism and incubated at 30°C for
4 days. After incubation, the plates were observed for the development of
clear zone around the colony (Holding and Collee, 1971).
3.6.7 Cellulase
Finely divided cellulose (2 g) was incorporated into nutrient salt
medium (NaNO3 0.2 g, K2HPO4 0.1 g, MgSO4.7H2O 0.05 g, FeSO4.7H2O
0.0.01 g, NaCl 4 g, agar 20 g, distilled water 1,000 ml, pH 7) and sterilized.
The solidified plates were inoculated with the pure culture of the isolates and
incubated at 30°C for 15 days. Cellulose hydrolysis was checked by the
development of a clear zone around the colonies indicating the production of
cellulase.
3.6.8 Arginine hydrolase
To test the ability of the isolates to produce arginine hydrolase, agar
tubes containing arginine agar medium (L-arginine 1 g, K2HPO4 0.03 g, NaCl
4 g, MgSO4.7H2O 0.01 g, phenol red 0.004 g, agar 20 g, distilled water 1,000
ml, pH 7) were sterilized and inoculated with the respective isolates by stab
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culture technique. After incubation at 30°C for 4 days, the tubes were
observed for color change. Formation of bright magenta color in the
inoculated tubes indicates the test as positive (Holding and Collee, 1971).
3.6.9 Phenyl alanine deaminase
To test the ability of the isolates to produce deaminase, phenyl alanine
(0.5%) was added to sterilized yeast extract malt extract broth followed by
inoculation with the respective isolates. After incubation at 30°C for 4 days,
1 ml of 10% ferric sulfate was added to 2 ml of culture broth and observed for
appearance of green color after 5 min which indicates the production of
deaminase (Holding and Collee, 1971).
3.6.10 Nitrate reduction
Organic nitrate broth (peptone 5 g, beef extract 3 g glucose 10 g
distilled water 1000 ml, pH 7) was sterilized and inoculated with the test
organism and incubated at 37°C for 4 days. After incubation, Nessler's
reagent was added to the tubes to check for the formation of pink color
(Holding and Collee, 1971).
3.6.11 Milk coagulation
Pasteurized milk was sterilized and inoculated with the test organisms
and incubated at 30°C for 4 days. After incubation the tubes were observed
for the production of coagulase enzyme.
3.7 Sensitivity of the actinomycete strains to antibiotics
Nutrient agar medium was sterilized and inoculated with the respective
isolates by using seeded plate technique. Filter paper discs containing 10 µg
of the antibiotics (Ampicillin, penicillin and streptomycin) were placed over the
inoculated plates using sterile forceps. The plates were incubated at 30°C for
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48 hr and observed for the sensitivity of the isolates to different antibiotics
(Cappuccino and Sherman, 2004).
3.7.1 Spectrum of antibiotics
Nutrient agar medium was prepared and sterilized by autoclaving at
15 lbs pressure (121°C) for 15 min the sterilized medium was poured in to
sterile Petri dishes and inoculated with Gram positive and Gram negative
organisms and filter paper discs containing the antibiotics (Amphicillin,
penicillin and streptomycin) and ethyl acetate extracts of the three strains
were placed over the inoculated plates using sterile forceps. The plates were
incubated at 30°C for 48 hr and observed for the sensitivity of the strains and
antibiotics towards the microorganisms.
3.8 Identification of the actinomycete strains by 16S rRNA analysis
DNA was isolated from the isolates and its quality was evaluated on
1.2% agarose gel (Rainey et al., 1996). A single band of high-molecular
weight DNA was observed. The fragment of 16S rDNA gene was amplified by
PCR from the above isolated DNA. A single and discrete PCR amplicon band
of 1500 bp was observed when resolved on agarose gel. The PCR amplicon
was purified to remove contaminants. Forward and reverse DNA sequencing
reaction of PCR amplicon was carried out with 8F and 1492R primers using
BDT v 3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer. Consensus
sequence of 1339 bp 16S rDNA gene was generated from forward and
reverse sequence data using Aligner software (Kimura, 1980). The 16S rDNA
gene sequence was used to carry out BLAST with the nrdatabase of NCBI
genbank database (Saitou and Nei, 1987). Based on maximum identity
score, first ten sequences were selected and aligned using multiple alignment
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software program Clustal W (Felsenstein, 1985). Distance matrix was
generated using RDP database and the phylogenetic tree was constructed
using MEGA 4 (Tamura et al., 2007).
3.9 Production of secondary metabolites
3.9.1 Fermentation
A loopful of culture of actinomycete isolates maintained on starch
casein agar slants were inoculated separately into the seed medium i.e.,
starch casein broth. After 24 hr of incubation, the vegetative seed culture @
10% was inoculated into 100 ml of fermentation broth (Starch 10 g, Casein
0.3 g, KNO3 2 g, NaCl 2 g, K2HPO4 2 g, MgSO4.7H2O 0.05 g, CaCO3 0.02 g,
FeSO4.7H2O 0.01 g, Distilled water 1000 ml), in 250 ml Erlenmeyer conical
flask. The flasks were incubated on a rotary shaker at 200 rpm, 35°C for
4 days. After incubation the broth was filtered through Whatmann No.1 filter
paper or centrifuged at 4000 rpm to remove the cell debris and subjected to
solvent extraction (Liu et al., 1992).
3.9.2 Solvent extraction
To the filtered cultured broth an equal amounts of ethyl acetate was
added in the ratio 1:1 (v/v) and shaken vigorously for 1 hr for complete
extraction. After complete extraction, the extracts were mixed thoroughly by
shaking them in 250 ml of separating funnel and allowed to stand for 30 min
and separated into aqueous and organic layer. The organic layer was
concentrated under pressure to yield the crude extract. The crude extracts
were used for antimicrobial assay (Westley et al., 1979).
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3.10 Optimization of cultural and nutritional parameters on the growth of the actinomycete isolates and bioactive metabolite production
Production of biomass and bioactive metabolites was optimized by
studying different cultural parameters like carbon and nitrogen sources, NaCl
concentration, pH and temperature.
3.10.1 Effect of carbon sources
Different carbon sources such as fructose, sucrose, lactose, mannitol,
maltose and glucose were added at 0.4% (w/w) concentration to starch casein
broth separately. The medium was sterilized and inoculated with the
respective isolates and incubated at 37°C for 4 days. After incubation, the cell
growth of the isolates was recorded in terms of dry weight of the biomass
(g/100 ml) and the culture filtrates collected were extracted with ethyl acetate.
Concentrated ethyl acetate extracts were used for the antimicrobial assay.
3.10.2 Effect of nitrogen sources
Different nitrogen sources such as peptone, beef extract, tyrosine,
ammonium nitrate, sodium nitrate and L-phenyl alanine were added at 0.4%
(w/w) concentration to starch casein broth separately. The medium was
sterilized and inoculated with the respective isolates and incubated at 37°C for
4 days. After incubation, the cell growth of the isolates was recorded in terms
of dry weight of the biomass (g/100 ml) and the culture filtrates collected were
extracted with ethyl acetate. Concentrated ethyl acetate extracts were used
for the antimicrobial assay.
3.10.3 Effect of NaCl concentration
Starch casein broth, amended with NaCl concentrations ranging from
2-12%, was sterilized and inoculated individually followed by incubation at
30°C for 4 days. After incubation, biomass was separated and the culture
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filtrate was subjected to extraction with ethyl acetate. Concentrated
ethylacetate extracts were tested for antimicrobial assay (Holding and Collee,
1971; Locci, 1989).
3.10.4 Effect of pH
Starch casein broth was prepared and pH of the medium was adjusted
to various pH levels ranging from 4.0 to 10.0 using 0.1N HCl and NaOH
(Holding and Collee, 1971). The broth was sterilized and inoculated with the
pure culture isolates individually. After incubation at 37°C for 4 days, the
biomass was separated by filtration and the culture filtrates obtained were
extracted with ethyl acetate, concentrated to dryness and tested for
antimicrobial activity.
3.10.5 Effect of temperature
Starch casein broth was sterilized and inoculated with the respective
isolates and incubated at different temperatures viz., 20°C , 25°C, 30°C, 35°C,
40°C and 45°C for 4 days (Holding and Collee, 1971). The biomass was
measured and the secondary metabolites elaborated by the isolates in the
culture filtrate were subjected to extraction with ethyl acetate. Concentrated
ethyl acetate extracts were employed for antimicrobial activity.
3.11 Melanin production
Melanin formation was tested on peptone yeast extract iron agar.
Melanin pigment was estimated by adding 1 ml of 0.4% substrate solution
(L-tyrosine or L-Dopa) to 2 ml of the culture filtrate. The reaction mixture was
incubated at 37°C for 30 min for L-tyrosine and 5 min for L-Dopa and red
coloration resulting from dopachrome formation was read
spectrophotometrically at 480 nm.
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3.12 Protein Estimation
The amount of protein present in the cells of all the strains of
actinomycetes was estimated by a standard method. To the culture filtrate
(0.1 ml), 5 ml of alkaline copper reagent and 0.5 ml of Folin Ciocalteau
reagent (1:1) were added, mixed and incubated for 30 min under dark
conditions. Following incubation, the absorbance was read at 720 nm using a
Spectronic 20D against bovine serum albumin as a standard (Lowry et al.,
1951)
3.13 Purification of secondary metabolites
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) using a Perkin-Elmer lambda 15UV/VIS
spectrophotometer. The active compound was subjected to purification
process by reversed-phase HPLC and the chemical structure of the obtained
active compound was elucidated though extensive analyses of NMR
spectroscopy
3.13.1 Thin layer chromatography
The crude extracts were partially purified by thin layer chromatography
(TLC). To 40 g of silica gel (mesh 60) placed in 40 x 80 mm bottle, distilled
water was added slowly and stirred with a glass rod until a homogeneous
thick but fluid slurry was formed. Several clean (3 x 1 inch) microscopic slides
were dipped into this slurry. The slides were withdrawn slowly and were held
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100
in a vertical position for 1-2 min allowing them to dry and were activated at
150°C for 10 min. For loading the antimicrobial agent on silica gel slide, a
capillary tube was dipped into the extracted antimicrobial agent syrup. Then
the end of a capillary tube was touched with one side of the coated slide
about 1 cm from the bottom end and then allowed to air-dry. After the drop
dried, it was then ready to be developed. These slides were kept in screw cap
bottles containing 5 ml of different solvents in a vertical position. The solvents
used include chloroform, methanol (4:1), n-butanol, glacial acetic acid, water
(4:1:1) and isopropanol, water (8:2). The solvent was allowed to run though
silica gel layer until the solvent reached about 1 cm of the top of the slide.
Then they were removed from the bottles. The solvents were allowed to
evaporate from the slides and transferred to screw cap bottles containing a
few crystals of iodine and UV light. The antimicrobial activity of these
compounds was checked by bioautography.
3.13.2 Column chromatography
The active fraction obtained from TLC and eluted with 10% methanol in
chloroform was repeatedly subjected to gel filtration on a Sephadex LH-20
column using an isocratic elution of chloroform, methanol (75:25) to yield
different active compounds. Different fractions were collected and subjected
to further purification. The active compound was subjected to purification
process by HPLC
3.13.3 Absorption spectrum
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 Perkin-Elmer
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101
lambda 15UV/VIS spectrophotometer. The UV spectral data of ethyl acetate
extracts were recorded for the three isolates.
3.13.4 HPLC
High performance liquid chromatography (HPLC) was used for
separation of antimicrobial compounds by preparative HPLC and analytical
HPLC on a LC-10 AT vp model HPLC using 250 x 4.60 mm Rheodysne
column (C-18). The solvent system (methanol and water) was used in the
ratio of 88:12 respectively. The operating pressure was 114 kgf, at a flow rate
0.8 ml/min and the temperature was set at 30°C. The UV-Vis detector was set
at 210 nm. The sample was mixed with the solvent in the ratio of 50:50 and
filtered using Millipore filter before injection. Twenty five µl of the sample
filtrate was injected into the column. The sample was run for 10 min and the
retention time was noted. The elution time was compared with the standard
and the antimicrobial compound was identified.
3.13.5 NMR
The pure fraction was subjected to the spectroscopic analyses to
determine the molecular weight, formula and the structure elucidation of
active compound were determined using ESI-MS, 1HNMR, 13CNMR,
DEPT13CNMR and FTIR respectively.