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Characterization of Streptomyces nogalator isolated from some tree mangroves.

Introduction

Several marine bacteria have been studied for their useful extracellular products and the search for more novel organisms and their product is still continuing (Asagbra et al., 2005). Mangrove environments are also well versed with the different kind of microbes and their useful activities (Maria and Sridhar, 2004). In view of to isolate new scientifically important and with potential applications Streptomyces strains associated with mangrove plants of Bhitarkanika (Orissa), India were isolated and studied for their growth, physiological, biochemical characteristics and identification.

Materials and Methods

Source of materials : The leaf samples from three mangrove plants i.e. Heritiera forres, Derris heterophyla, Sonneratia apitala, Exoecocaria aggallocha and Caesalpinia crista found in of Bhitarkanika, Orissa were collected for the isolation of Streptomyces.

Isolation media

The dilution plate technique was followed for the isolation of Streptomyces on ISP 3, ISP4 and ISP5 media. The isolates obtained were given name as ST 1- ST 5.

Growth characteristics

Streptomyces isolates were grown in starch casein liquid medium of 4.5 and 7.2 pH and at a 30[degrees]C and 37[degrees]C for 10 days in static culture condition. Finally their dry biomass was measured using. At the same time, change in pH, colour of filtrate, diffusible pigments were observed and recorded. The plate culture of all isolates prepared in Starch casein medium was observed for morphological characteristics like colony characteristics, coloration, margin etc.

Morphological Studies

slide culture of all the isolates of Streptomyces were prepared on starch casein medium and ISP 3 medium incubated at 30[degrees]C and 37[degrees]C by using cavity slides. Periodical observations regarding spore morphological, arrangements and mycelloid structure were recorded by using Nikon Japan Trinocular Research Microscope Model 50i.

Taxonomic identification

Different biochemical tests for carbohydrate utilization, nitrogen utilization, growth in different stress conditions, antibiotic resistance, amino acid degradation, enzyme activity like, amylase, protease, asparaginase, antifungal activity and phosphate solubilization were analyzed for characterization of these isolates. Finally data were used for the identification of Streptomyces isolates using Probabilistic identification of bacteria (PIB win).

Results and Discussion

Overall 5 isolates of Streptomyces were isolated from Heritiera forres (ST 81), Derris heterophyla (ST 28), Sonneratia apitala(ST 55), Exoecocaria aggallocha (ST 67) and Caesalpinia crista (ST 69).

Spore chain morphology

Spore chains are neither of the section rectiflexibles nor spirales except ST 28 that has spirales chain morphology. Very poor growth was observed on starch casein medium. On ISP-3 medium, the spores are seen emerging from the hyphae and the mycelium looked light blue in colour. On ISP-4 the spores are also seen from the hyphae. No growth was observed on ISP-5 media.

Special Morphological characteristics

The colony had an amorphous growth with margin entire and having scattered colony. It formed diffusible pigment on plate. Spore surface: smooth.

Colour of the colony

Grey in colour. Reverse side of the colony was greyish white. It gave light brown colour in culture broth of starch casein medium of pH 7.2 at 37[degrees]C.

Growth conditions

All five strains of S. nogalator preferred 37[degrees]C and 7.2 pH for proper growth. Melanoid pigments are not formed on peptone yeast iron agar and on tyrosine agar. The organism was able to tolerate its growth at 45[degrees]C and potassium tellurite (0.001%), but unable to grow with sodium azide (0.01%), NaCl (7%) and phenol (0.1%). Growth was not observed in sodium acetate and sodium propionate while sodium citrate and sodium pyruvate gave positive results. In contrast, ST 69 showed growth in presence of sodium acetate and ST 28 was found to negative for growth in presence of sodium pyruvate.

Utilization of carbon and nitrogen sources

The strain was able to utilize many carbon sources like L-arabinose, mesoinositol, D-xylose, manitol, D-fructose, L-rhamnose, D-lactose, D-mellibiose, xylan, D-ribose, malonate and adipate. However it was unable to utilize sucrose, raffinose, adonitol, methyl A-D mannoside, inositol, xylitol, isobutyrate, methyl A-D glucopyranoside, salicin, D-melizitose and hippurate. The organism was found to utilize potassium nitrate, guanine, L-phenylalanine, L-histidine, L- lysine, glycine and D-alanine as the nitrogen source. It has not shown any growth in presence of L-hydroxyproline, d-1alpha amino butyric acid, L-valine, urea, L-cysteine and L-leucine. All strains were allantoin negative except ST 67 and ST 69. All strains have shown growth in Sodium pyruvate except ST 28. Similarly, only ST 69 exhibited growth in prsence of sodium acetate.

Antibiotic susceptibility, Enzyme activity and Degradation tests

The organism showed resistance to rifampicin. Where as sensitivity was observed with oleandomycin, neomycin and penicillin-G. S. nogalator strain ST 55 showed resistance for all three antibiotics. Lipolytic activity was observed in lipase test medium while it has shown a positive proteolytic and negative amylolytic activity. It has shown a positive response to xanthin degradation while negative result was seen in [H.sub.2]S production, allantoin degradation and nitrate reduction.

A computerized database was used to compare the biological properties of all isolates of Streptomyces with those of Streptomyces sp. The results suggest that strains were Streptomycete and strongly related to S. nogalator. Though it has similarity with the type strain Streptomyces nogalator for incapability to utilize inositol and sucrose, at the same time they are marginally distinctive with respect to non utilization of raffinose. All biochemical and physiological characteristics directed towards the identification of this strains as Streptomyces nogalator. However, these strains are morphologically varied with the type strain of Streptomyces nogalator (Shirling and Gottlieb, 1968). Two of the strains i.e. ST 67 and ST 55 may be new variants of S. nogalator due to their non utilization of raffinose and utilization of sucrose, respectively.

Acknowledgement

Authors are grateful to Department of Ocean Development, Ministry of Earth Sciences, and Govt. of India for supporting financially through DOD project no.11-MRDF/4/4/UNU97(P-22).

References

[1] AE Asagbra, AI Sanni, OB Oyewole (2005). Solid-state fermentation production of tetracycline by Streptomyces strains using some agricultural wastes as substrate. World Journal of Microbiology & Biotechnology, 2005, 21,- 107-114

[2] GL Maria, KR Sridhar (2004). Fungal colonization of immersed wood in mangroves of the southwest coast of India., Canadian Journal of Botany Revue Canadienne de Botanique, 82 10, 1409-1418

[3] Shirling B. Elwood and Gottlieb. David (1968) International Journal of Systematic Bacteriology. Vol. 18, No. 2, p-108.

Srilekha Mishra (1), Uday Chand Basak (2) and Nibha Gupta * (1)

(1) Microbiology laboratory, Regional Plant Resource Centre, Bhubaneswar-751015 (Orissa), India

(2) Division of Taxonomy and Conservation, Regional Plant Resource Centre, Bhubaneswar-751015 (Orissa), India

* Corresponding Author, Email:nguc2003@yahoo.co.in
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Author:Mishra, Srilekha; Basak, Uday Chand; Gupta, Nibha
Publication:International Journal of Applied Environmental Sciences
Article Type:Report
Geographic Code:9INDI
Date:Sep 1, 2009
Words:1084
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