coverslip culture (Arifuzzaman, 2010; Khan, 2008) and slide culture techniques (Kavitha and Vijayalakshmi, 2007). Strains are observed for several characters such as presence or absence of aerial mycelium, fragmentation or non fragmentation of substrate and aerial mycelium, presence of sclerotia, spore chain morphology and color of spore mass (Kavitha and Vijayalakshmi, 2007).

It is important to avoid strain duplication by an accurate identification of isolates. However taxonomic characterization based only on morphological and biochemical characteristics, is tedious and not always reliable (Singh, 2009). There is therefore a need to develop molecular methods that used in conjunction with the earlier techniques would help in differentiating between the rare and common genera of actinomycetes (Valenzuela-Tovar, 2005).

MATERIALS AND METHODS:

Sample collection and processing:

Soil samples were collected from different locations of terrestrial fields of Orathanadu, Thanjavur (Dt.), Tamilnadu, India. Then they were transferred to the laboratory and stored at 4°C. The collected soil samples were mixed thoroughly and passed through 2 mm sieve to remove gravel and debris. Soil samples were air dried at room temperature.The physico-chemical parameters of the soil sample were determined using the methods of Udo and Ogunwale (1986) and the Association of Official Analytical Chemists (AOAC, 1990). The collected soil samples were analyzed to determine various parameters such as pH, electrical conductivity (EC), texture, lime status, organic carbon (OC), available nitrogen (N), available phosphours (P) and available potassium (K), available zinc (Zn), copper (Cu), iron (Fe) and manganese (Mn), exchangeable bases including calcium (Ca), magnesium (Mg), sodium (Na), potassium (P) and heavy metals and contents of fine sand, coarse sand, silt, clay and cationic exchange capacity.

Isolation and purification of actinobacteria:

Starch casein agar (SCA) medium was prepared and autoclaved. Then it was supplemented with streptomycin (50 µg) and griseofulvin (30 µg) to prevent the bacterial fungal growth in the medium and the medium was poured into sterile petri plates. The collected soil sample was diluted up to 10^-6 and 0.1 ml of the diluted samples was spread over the agar plates. After incubation at 28°C ± 2°C for 7-10 days, streak plate method was employed to purification of isolates of actinobacteria on SCA. After purification, (Porter et al., 1960) the actinobacterial cultures were stored at 4°C for further investigation.

Characterization of actinobacteria

Colony characterization: Colony morphology of the actinobacteria was recorded with respect to colour of aerial and substrate mycelia, size and nature of the colonies and production of diffusible pigmentation.

Microscopic characterization:

Actinobacterial culture plates were prepared and 2 to 4 sterile cover slips were inserted at an angle of 45°C. The plates were incubated at 28°C ± 2°C for 4 to 7 days. The cover slips were slowly removed from the cultured medium and observed under the light (Nikon, Japan) microscope. The morphological features of spores and sporangia on aerial and substrate mycelia was observed and recorded.

Identification of actinobacteria:

In addition to morphological characterization, biochemical properties of the actinobacteria were also performed and identified by using standard manuals (Shirling and Gottlieb, 1966).

Screening of antibacterial activity:

Preliminary, the antibacterial activity of the actinobacterial isolates were screened against both Gram positive and Gram negative bacteria pathogens (E. coli and S. aureus) by cross streak plate method (Eccleston, 2008). Then, the isolates with antibacterial activity in primary screening were selected to secondary screening (Shake flask method). Selected actinobacteria were inoculated into starch casein broth and incubated at 28°C ± 2°C for 7 days. After incubation, the cell free culture filtrates of the isolates were prepared by using various filters and stored at 4°C. Further, the test bacterial cultures were cultured in nutrient broth and incubated at 37°C for 24 h. Nutrient agar medium was prepared and poured into petri dishes, and allowed to solidification, bacterial cultures (S. typhi, E. coli, Staphylococcus aureus, K. pneumoniae and Enterobacter aerogenes) were evenly spread over the media by using sterile cotton buds. Then, the wells were punched by using 5mm diameter gel puncher (well diffusion method). About 200µl of actinobacterial cell free culture filtrates were added into the wells and the plates were incubated at 37°C for 24 h. After incubation, the results were observed and measured the zone of inhibition (mm) zone around each well (Egorov, 1995).

Characterization of bioactive compound Analysis of bioactive compounds:

Qualitative bioactive compounds of the selected actinobacteria were screened by thin layer chromatography (TLC) (Wanger, 1995).

Sterols:

The sterol was separated by TLC using chloroform: glacial acetic acid: ethanol: water solvents mixture (64:34:12:8). The presence of sterol in the developed chromatograms was detected by spraying the folin-ciocalteus reagent. After the plate was heated at 100°C for 6 min., the positive reaction was indicated by the formation of black colour spot.

Phenols:

The phenols were separated by TLC using chloroform: methanol solvents mixture (27:3). The presence of phenols in the developed chromatogram was detected by spraying the folin-ciocalteus reagent. The positive reaction was indicated by the formation of blue colour spot (Harborne, 1998).

Flavonoid: The flavonoid were separated by using n-butanol: acetic acid: water (8:2:10) mixture. The presence of flavonoid was indicated by the formation of yellow colour spot in the plate. (Simona Loana Vicas et al., 2000).

Rf value

The ratio of distance travelled by the solute and distance travelled by the solvent was calculated by the following standard formula:

Distance travelled by the solute

RF value = ------------------------------------------------

Distance travelled by the solvent

Purification of bioactive compounds:

Various solvents systems were used to separate bioactive compounds from ethanol and methanol extracts of actinobacteria by visible fraction and retention factor value. The TLC (duplicate) plates were purified by collecting the silica gels in chloroform: methanol (1:2) centrifuged to remove silica particulate and the supernatant was collected, and it was used for further antibacterial activity (Reynolds and Dweck, 1999).

Antibacterial efficacy

Antibacterial efficacies of the bioactive compounds of actinobacteria were evaluated by agar well diffusion method (Perez et al., 1990) against five different human pathogenic bacteria namely (Salmonella typhi, Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia and Enterobacter aerogenes).

FT-IR (Fourier Transform Infrared Spectroscopy): The functional groups of the bioactive compounds were carried out by FT-IR spectral studies (Lou, 1998). One hundred milligram powder of purified/dried bioactive compound (Sterols) of the actinobacteria was mixed KBr (Potassium Bromide) salt using a mortar and pestle and compressed into a thin pellet infra spectra were recorded as KBr pellets on a FT-IR spectrophotometer (8000 series) between 4000-500 cm^-1 infrared adsorption were recorded.

UV spectrum analysis:

The fraction sample was dissolved in aceto-nitrate and then detected its adsorption values with lambda 35 ultra violet scanner.

RESULTS AND DISCUSSION:

The physical and chemical parameters will be greatly influenced the indigenous microbiota of the soil. In the present study, the physico-chemical parameters of the terrestrial soil were performed and the results were presented in table 1. The pH of the soil was almost neutral (7.69), colour of the soil was recorded as blackish brown, the texture was sandy clay loam and there was no lime status and heavy metals. OC content of the soil was recorded as 0.39%. Similarly, the major elements like N, P and K were recorded as 98.5%, 4.25% and 140% respectively. The remaining parameters analyzed were recorded in the table 1. Similarly, Akbar et al. (2010) was reported the physico-chemical properties of the tropical rain forest in Southeast Asia. Similar type of work has been reported by many workers (Vijayakumar et al., 2007; Kasthuri et al., 2011).Generally, classical approaches for the classification make use of morphological, Biochemical character, colony formation vegetative and mycelium structure of spores and spores are the most important feature of identification of actinomycetes. A total of 10 different actinobacterial isolates were isolated from the terrestrial soil sample of Thanjavur district, and they were characterized based on the morphological, biochemical and cultural properties. Morphologically, all the isolates were identified as Gram positive bacteria. The pale yellow, brown, ash and white colour colonies were produced by actinobacterial isolates. Biochemically, citrate was utilized by all the isolates except Jonesia sp., and urease was produced by all the isolates except by Catellatospora sp. and Jonesia sp. Similarly, VP test was negative result for all the isolates except for Microtetrospora sp. and Nocardia sp. Whereas, the results of other biochemical tests were varied between the isolates. Among 10 actinobacteria, each one isolate was belonged to Actinobispora sp., Actinosynnema sp., Actinoplanes sp., Agromyces sp., Actinomadura sp., Jonesia sp., Catellatospora sp., Micromonospora sp., Microtetrospora sp. and Nocardia sp. correspondingly, a total of 42 isolates of actinobacteria were isolated from four different soil-sampling stations of Coimbatore. Based on their morphological and microscopical characteristics, it was established that twelve isolates belonged to the genera Streptomyces (28%), ten isolates to Nocardiopsis (24%), eight isolates to Saccahropolyspora (19%), six isolates to Nocardia (14%), four isolates to Actinopolyspora (10%) and two isolates to Actinomadura (5%). Nineteen isolates produced the ash series colour of aerial mycelium, and sixteen isolates produced white series colour of aerial mycelium on starch casein agar medium, and seven isolates produced diffusible pigments on the same medium. Thus, it is concluded on the basis of present and previous studies that the, the physical and chemical properties and nutrient composition of the media also influenced the isolation/cultivation and morphological properties of the soil actinobacteria. Similar type of findings also reported by many workers (Wang et al.,1999, Mansour, 2003, Vijayakumar et al., 2007; Raja et al., 2009).

As other reports have been recorded the antimicrobial activity of the soil actinobacteria, the present study also, screened the antibacterial activity of all the ten isolates and recorded that, 30% (n=3) of the isolates namely Actinobispora sp., Microtetrospora sp. and Actinomadura sp. had antibacterial activity against both gram-positive and gram negative bacteria.

Table1. Physico-chemical analysis of soil

S. No	Name of the parameter	Sample Details
	PH	7.69
1.	Electrical conductity (dsm-1)	0.41
2.	Color	Blackish Brown
3.	Texture	Sandy Clay Loam
4.	Lime status	Nil
5.	Organic carbon (%)	0.39
Available Macronutrients
6.	Available nitrogen (Kg/ac)	98.5
7.	Available phosphours (Kg/ac)	4.25
8.	Available potassium (Kg/ac)	140
Available Micronutrients
9.	Available Zinc (ppm)	1.15
10.	Available copper (ppm)	1.10
11.	Available iron (ppm)	7.52
12.	Available manganese (ppm)	3.68
Soil Fractions
13.	Fine sand (%)	43.68
14.	Coarse sand (%)	19.68
15.	Silt (%)	19.81
16.	Clay (%)	16.83
17.	Cat ion Exchange Capacity (C. Moleproton +/Kg)	23.5
Exchangeable Bases(C. Mole proton+/Kg)
18.	Calcium	11.2
19.	Magnesium	9.6
20.	Sodium	2.18
21.	Potassium	0.22
22.	Heavy metals (ppm)	Nil

Interestingly, all the three antagonistic actinobacteria was predominantly found in the soil. The isolate Actinobispora sp. exhibited maximum (11 mm) antibacterial activity against S. aureus, followed by S. typhi (10 mm), E. aerogenes (7 mm), E. coli (6 mm) and K. pneumoniae (5 mm), whereas the isolate Microtetrospora sp. exhibited maximum (10 mm) activity against S. typhi, followed by S. aureus and E. aerogenes (7 mm) and E. coli (6 mm) and the isolate did not show any activity on K. pneumoniae. On the contrary to these two isolates, the isolate Actinomadura sp. showed poor antibacterial activity. This isolate showed maximum (7 mm) activity on S. typhi followed by S. aureus (5 mm) and it does not had any activity on E. coli, K. pneumonia and E. aerogenes. Comparatively, the isolate Actinobispora sp. possessed promising activity than other two isolates. Hence, this isolate has been selected for further antibacterial studies. Similarly, the antimicrobial activities of the soil actinobacteria were screened by many studies (Dhanasekaran et al., 2008-2009).

Classical approaches for the classification make use of morphological, Biochemical character, colony formation vegetative and mycelium structure of spores and spores are the most important feature of identification of actinomycetes. In the present investigation actinomycetes were identified by morphological, cultural, biochemical and physiological characteristic. The pale yellow, white, Brown and ash coloured isolates were found predominant such as dominance of members of pale yellow, brown and white coloured actinomycetes (Table 2 and 3).

Table 2. Actinobacteria from agriculturalsoil

S. No.	Name of actinobacteria
1. 1.	Actinobispora SP.
2. 2.	Actinosynnema SP.
3	Actinoplanes SP.
3. 4.	Agromyces SP.
4. 5.	Actinomadura SP.
5. 6.	Jonesia SP.
7.	Catellatospora SP.
8.	Micromonospora SP.
9.	Microtetrospora SP.
10.	Nocardia SP.

Biochemical characteristics of the actinomycetes were also used as the character of identification. In the present study biochemical characteristics such as Indole, Methyl Red, Voges Proskauer, Citrate, Urease, Nitrate and Catalase test were used to characterized the actinomycetes (Table-4).

Culture media filtrate and ethanol methanol extract were dropped in prepared holes of the solid nutrition medium deep inoculated with the test microorganisms. The petri dishes were incubated at cultivated as it was above described. The antibacterial activity was measured in mm sterile zone.

In the present study the three actinomycetes Actinobispora sp. , Actinomadura sp. and Microtetrospora sp. have antibacterial activity of five human pathogenic bacteria. The maximum zone of Staphyloccocus aureus (11 mm) and Salmonella typhi (10 mm) was observed (Table-5)

Table 3. Biochemical characterization of actinomycetes

S.No.	Actinomycetes	Gram’sStainingg	Indole	MethylRed	VogesProskauer	Citrate	Catalase	Urease	Nitrate
1.	Actinibispora sp.	+	-	+	-	+	+	+	-
2.	Actinosynnemasp.	+	-	-	-	+	+	+	-
3.	Actinoplanes sp.	+	-	-	-	+	-	+	-
4.	Agromyces sp.	+	-	-	-	+	-	+	-
5.	Actinomadura sp.	+	+	+	-	+	-	+	+
6.	Catellatospora sp.	+	-	-	-	+	+	-	-
7.	Jonesia sp.	+	-	+	-	-	+	-	+
8.	Micromonosporasp.	+	-	-	-	+	-	+	+
9.	Microtetrospora sp.	+	+	+	+	+	-	+	+
10.	Nocardia sp.	+	+	+	+	+	-	+	+

Among the compounds identified, the sterol showed maximum antibacterial efficacy than other two compounds identified. The maximum (13 mm) antibacterial efficacy of the sterol was recorded against E. aerogenes, followed by E. coli (11 mm), S. typhi and S. aureus (10 mm) and K. pneumoniae (9 mm). But, the other two compounds showed only minimum activity against all the pathogens tested. Hence, the sterol was selected for further characterization by FT-IR and UV spectral studies. Totally, 13 peaks were absorbed in FT-IR spectrum of bioactive compound. The maximum peak in the region of 3969.23 cm^-1, indicating the presence of O-H stretching vibration free O-H, followed by eleven peaks were recorded at 3864.52 cm^-1, 3382.77 cm^-1, 2954.54 cm^-1and 2839.34 cm^-1, 2522.83 cm^-1, 2075.33 cm^-1, 1745.24 cm^-1, 1458.28 cm^-1, 1407.86 cm^-1, 1109.65 cm^-1, 768.86 cm^-1 and 675.85 which were indicating the presence of N-H group, =N-H (amines), C-H groups, hydroxyl C, isocyanides, α,β - unsaturated alkyl nitrites, -CH2- (alkane), anionic carboxylate, C=S group, 3 adjacent hydrogen atoms and 5 adjacent hydrogen atoms respectively (Fig. 1). Notably, the maximum peaks of the UV spectrum of the compound were recorded at 285.90 nm, followed by 277.03 nm and 271.93 nm in ethyl acetate extract. The UV spectrum of the compound was indicated that the isolate produced either a broad-spectrum antimicrobial compound or several compounds with different activities (Fig. 2). Likewise, in the UV spectral data for the ethyl acetate extract of selected active fermented broth of a Streptomyces sp. showed maximum absorbance peaks ranged between 215-270nm and the characteristics of absorption peaks indicated as a highly polygene nature. Two bioactive regions were detected on the TLC plate (Rf 0.70 and 0.88). The bioactive compound exhibited a maximum UV absorption at 217 and 221nm in methanol (Ilic et al., 2005). Related types of works have already been reported from the actinobacteria by many workers (Swaadoun et al., 1999; Dharamraj et al., 2009; Vijayakumar et al., 2011).

Table 5. Antibacterial activities for the screened bioactive compounds

S. No.	Name of the pathogens	Zone of inhibition (mm)
S. No.	Name of the pathogens	Sterol	Phenols	Flavonoid
1.	Salmonella typhi	10	5	4
2.	Escherichia coli	11	6	2
3.	Staphylococcus aureus	10	6	4
4.	Klebsiella pneumoniae	9	5	2
5.	Enterobacter aerogenes	13	2	3

The present study has been suggested that, the test actinobacteria have great potential sources of new antibacterial compounds or lead compounds in new drug discovery. In particular, the antibacterial activities of the ethyl acetate extract of the actinobacterial products was prominent even at very low concentrations, particularly, encouraged and open up a vista for further research opportunities in order to determine the structure of active components.

Conclusively, the search for novel metabolites especially from actinobacteria requires a large number of isolates in order to discover a novel compound of pharmaceutical interest. The search will be more promising if diverse actinobacteria are sampled and screened. For this reason, soils were specifically collected under identified crops. This is based on the hypothesis that actinobacterial diversity may be influenced by the diversity of plant species as these bacteria grow profusely in the humus and leaf litter layer. Furthermore, different plants produce different type of secondary metabolites and some of these chemical compounds are toxic to soil microorganisms including actinobacteria. However, adaptation has in turn leaded the actinobacteria to produce their own secondary metabolites.

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Received on 03.06.2012

Modified on 12.06.2012

Accepted on 25.06.2012

Research J. Science and Tech. 4(3): May-June 2012: 132-139

S. No.	Name of the pathogens	Zone of inhibition(mm)
S. No.	Name of the pathogens	*Actinobispora* sp.	*Microtetrospora* sp.	*Actinomadura* sp.
1	Salmonella typhi	10	10	7
2	Escherichia coli	6	6	-
3	Staphylococcus aureus	11	7	5
4	Klebsiella pneumoniae	5	-	-
5	Enterobacter aerogenes	7	7	-