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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.