STREPTOMYCES SPP. COLLECTED FROM EAST NIMAR SOILS- ISOLATED AND CHARACTERIZATION BASED ON MORPHOLOGICAL AND BIOCHEMICAL STUDIES
Narendra Singh Baghel1, Anurag Prajapati2, Dr. Archana More3
1Research Scholar, Sri Satya Sai University of Technology & Medical Sciences, Sehore
2Research Scholar, Sri Satya Sai University of Technology & Medical Sciences, Sehore
3Associate Prof. S.N. Government P.G. College, Khandwa
Abstract - Streptomyces, the largest genus of Actinobacteria, is a group of bacteria which is most commonly isolated from soil. In this study, total of 24 indigenous Streptomyces spp were isolated from soil samples collected from different parts of Bangladesh. Of them, two were studied in detail and characterized. Their provisional identification was done following the criteria of Bergey’s Manual of Systematic Bacteriology. The morphological and biochemical characterization and tentative identification methods were described.
Keywords: Identification, Isolation, Streptomyces.
1 INTRODUCTION
The well-known actinomycetes genus Streptomyces has the greatest diversity of species and subspecies in nature. Actinomycetes primarily come from soil (Takahashi and Omura, 2003). Even though this particular kind of bacteria can be found in aquatic environments, they are often ephemeral in nature. Since the turn of the 20th century, the genus Streptomyces has lost much of its ability to produce antibiotics for treating human illnesses as well as those inflicted on animals and plants, particularly crop diseases. It has been established that almost every species of Streptomyces produces antibiotics. Numerous studies have been conducted globally to examine antibiotic producer Streptomyces from an economic and medical perspective. Numerous new chemical compounds, including antibiotics and other bioactive chemicals, were found as a result of the extensive screening of soil for Streptomyces spp. that began in the 1940s. The identity of the generating microorganisms piqued attention at the same rate as consumer interest in a particular product. Many businesses have scaled back their efforts to find new antibiotics since the late 1980s to make room for non-infectious disease research. Nevertheless, novel detection technologies are being tested with items from the Streptomyces spp. family. However, experts in this subject believe that only 10% of the world's microbes have been isolated and studied (http://www.globalscience.com). Therefore, there is still a chance to find and characterise new Streptomyces species and chemicals.
Researchers tried to accomplish this by taking into account morphology, cultural factors such development on various medium, biochemical characteristics like carbon source use, etc. To classify and identify streptomycetes, however, there is a significant gap between conventional methods and contemporary ones (Antonieta Taddei et al., 2006).
As a result, taxonomists and scholars who are studying this species continue to have a lot of questions about how to classify them. Our study aims to isolate and identify novel Streptomyces spp. utilizing conventional morphological and biochemical traits.
2 MATERIALS AND METHODS
2.1 Purification, Maintenance and Preservation of the Organisms
24 different native Streptomyces spp. isolates were found in the soil samples. Two of them isolates St-13 and St-25—were chosen for further examination. The Streptomyces spp. was initially purified using the streak plate technique on oatmeal agar medium (Williams and Cross, 1965). (Fig. 2). Following purification, these isolates were kept alive on Oatmeal Agar Slants for the duration of the investigation.
In order to preserve the tubes for a little period of time, they were wrapped in polythene bags and kept at 4°C in a refrigerator. The species were periodically transferred at intervals of 15 days. The isolates were maintained in 20% glycerol broth at - 20 °C for long-term preservation (Waksman and Henrichi, 1948).
2.2 Identification of Morphological and Cultural Traits
To identify morphological traits, scientists employed the Hucker and Conn's, 1923 modified approach, Acid-fast staining, Ziehl Neelson's, 1883, and cover slip culture technique. To
hydrolysis, the test for lipid hydrolysis, the test for esquilin hydrolysis, the catalase test, the fermentation tests, the general fermentation test, etc., were used to ascertain the biochemical activities of the chosen species. Studies were done on melanin formation as well as the use of various carbon sources.
2.4 Physical Characteristics
Growing the chosen species on Bennett agar at various temperatures, such as 10, 20, 30, 37, and 450C, and over a wide pH range, from 4.0 to 9.0, allowed researchers to establish the species' tolerance to temperature and pH requirements (Fig. 1).
3 RESULTS AND DISCUSSION
According to microscopic analyses and staining characteristics of two Streptomyces Spp (St-13 and St-25), both were Gram-positive, non-acid-fast, and exhibited filamentous, branching, and coenocytic mycelia (Fig. 3). (Table 1). On various ISP media, the growth patterns, rate of growth, aerial mass colour, reverse colour, and soluble colour of the chosen species were examined and noted. table 2 and table 3 They both tested positive for catalase, responded favorably to nitrate reduction, and were both esqulin and starch hydrolyzers, according to the biochemical test results. Both the Voges-Proskauer tests and indole synthesis for both species showed negative results (Table 4). Examinations of the organisms' ability to use carbon were conducted. Sucrose, fructose, mannitol, arabinose, and xylose were the carbon sources used. As a positive control, glucose was employed, whereas the only basal agar medium devoid of any carbohydrates served as the only negative control.
Both of the chosen Streptomyces species could grow at temperatures of 10°C and 45°C when they were cultured at various temperatures. They were mesophilic, growing best between 30 and 37 degrees Celsius. The chosen species exhibited neutrophilic behavior in culture, thriving best at a pH near to neutrality, between 4.0 and 9.0.
From the soil samples, 24 native Streptomyces spp. were isolated, and two of them (St-13 and St-25) were chosen and characterised using Bergey's Manual of Systematic Bacteriology based on their morphological, physical, cultural, and biochemical characteristics (Locci 1989). These chosen species generate visually noticeable aerial mycelium in a range of colours, including grey, white, ash, and brown. They met one of the key requirements for the Streptomyces spp., which is that they be Gram positive and non- acid fast. Following cover slip culture on solid media, both species were examined morphologically and microscopically (Kowato and Shinobu, 1959).
They looked at the morphology of both their aerial and substrate mycelium. It was coenocytic in the vegetative mycelium. These species were discovered to produce filamentous, heavily branching, net-like mycelium. The formation of typical aerial mycelium superimposed above substrate growth distinguished different Streptomyces species. It was discovered that the length of aerial hyphae varied greatly. Both St-25 and St-13 had spore chains that were retinocularaparti and rectiflexible, respectively.
Selected St-13and St-25’s biochemical traits were examined (Table 4). Both species' tests for catalase and nitrate reduction came out positive. They were shown to have the ability to hydrolyze esquilin and starch but not fat (Fig. 4). Both species were evaluated for their capacity to ferment several types of carbohydrates, including salicin, lactose, mannitol, maltose, sucrose, and glucose (Table 4). The findings indicated that everyone was able to ferment sugar without gas. St-25 and S-13 don't generate any acids other than mannitol and lactose, respectively (Fig. 5).
Growth characteristics of the St-13and St-25 were studied on different ISP media.
Results showed that ISP-3 (Oatmeal agar) was an excellent growth medium for the selected species.
Figure.1 Growth isolate on Bennett agar plate oatmeal agar
Figure 2 Growth isolates on plate
Table 1 Microscopic studies and staining properties of the selected isolates
Isolate No Vegetative Mycelium Aerial mycelium Gram reaction Acid fast reaction St-13 Filamentous, Branched Coenocytic Rectiflexible +ve Non acid fast St-25 Filamentous, Profusely Branched,
Coencocytic Retinocularaparti +ve Non acid fast
Table 2 Cultural characteristics of the selected isolates (St-13) on different media
Highly growth (++++); good growth (+++); Weak growth (++); growth Rare/Trace growth (+);
Not growth (-)
Table 3 Cultural Characteristics of the selected isolates (St-25) on different media
Media name Growth
Aerial mycelium color Soluble pigment Reverse color Range Colony Properties
Bennett Agar Plate +++ Light brown, granular, spreading Cream _ Yellow
Yeast Extract - Malt
Extract Plate +++ Brown, Cottony, spreading White Brown Brown
Oatmeal Agar
Plate +++ Gray, Cottony, spreading Gray _ White
Nutrient Agar Plate + Non-spreading Cream _ _
Bennett Broth +++ Tobacco brown, Thick ring like pellicle,
no sediment and turbidity Gray _
Inorganic Salt starch Agar
(ISP4) Plate ++++ Light Gray Spreading Powdery Gray _ Brown
Glycerol Asparagines
(ISP5) Agar Plate +++ Light Brown, Powdery, Non- spreading Light Gray Light
Brown Brown Tryptone-Yeast Extract
Broth (ISP1) +++ Light Brown, Ring Like growth on the
surface, turbid, no sediment Gray _ _
Media name Growth Aerial mycelium
color Soluble
pigment Reverse color Range Colony Properties
Bennett Agar Plate +++ Brown, spreading, powdery Gray _ Brown
Yeast Extract -
Malt Extract Plate +++ Deep brown, spreading,
powdery Blackish brown Brown Blackish brown
Oatmeal Agar Plate +++ Deep brown, spreading,
powdery Coffee Light
Brown Brown
Nutrient Agar Plate ++++ Spreading Cream _ Butter Cream
Bennett Broth ++++ Yellow, Thick ring like pellicle, no sediment and
turbidity Gray _
Inorganic Salt starch Agar (ISP4)
Plate ++++ Brown, Spreading,
Powdery Deep Gray _ Brown
Glycerol- Asparagines
(ISP5) Agar Plate ++ Brown, Non-Spreading,
Granular Brown Light
Brown Brown
St-13 St-25
Hydrolsis of starch + +
Hydrolsis of Lipid - -
Hydrolsis of Gelatin + -
Hydrolsis of Esqulin + +
Nitrate test + +
Catalase test + +
IMVIC test
Indole test - -
MR test - -
VP test - -
Citrate test - -
Fermentation test
Lactose - -
Manitol - -
Maltose - -
Sucrose - -
Glucose - -, g
Salicin - -
Carbon source
Glucose +++ +++
Sucrose + +++
Fructose +++ +++
Mannitol +++ -
Arabinose - -
Xylose ++ +++
Strongly positive utilization (++++); Positive utilization (+++); negative (-); g= gas production.
Figure.3 Microscopic observation of selected isolate (Left St-13 and Right St-25)
Figure.4 Esquilin test (Left: broth and Right: plate)
Figure 5 Utilization of sugar
After proper incubation, the isolates produced aerial mycelium. Results (Table 2 and 3) indicated that St-13 was in gray colour while St-25 was in coffee colour.
An important parameter for classifying the Streptomyces spp. is the colour of the substrate growth. While both species were expanding on ISP media, the reverse colours of each were examined (Table 2 and 3). The hues of the substrate varied depending on the species of Streptomyces. On tyrosine agar (ISP-7) and peptone-yeast extract iron agar (ISP- 6), the ability of the Streptomyces spp. to create diffusible pigment was investigated. On ISP-6 medium, the results showed that St-13 and St-25 created diffusible pigment; S-109 produced the darkest pigment on both media. Temperature is one of the physical factors that governs the distribution and activities of Streptomyces spp. in natural habitats. Most species of them were mesophiles, growing at temperature between 10°C and 37°C. There were also thermotolerant and thermophilic species (Kutzner, 1981). A few species grew slowly at 4°C (William et al., 1983). In our study, the selected Streptomyces spp. were grown at different temperature. Both were able to grow at 10°C and 45°C. Data revealed that they were mesophilic, growing best at 30° to 37°C
The majority of Streptomyces spp. exhibited neutrophilicity in culture, growing best between PH 5.0 and 9.0 with a neutrality-close optimal pH. Isolated from acidic soils and other materials were acidophilic and acidoduric strains (William and Davies, 1971; Khan and Williams, 1975; Hagedorn, 1976). The species included in our investigation were grown on Bennett agar that had its PH altered to various ranges between 4.0 and 9.0. Most reported Streptomyces spp. were similar to the traits of the chosen St-13 and St-25 species overall. For the purpose of determining enzyme activity and identifying species, more study is required.
4 CONCLUSION
Actinomycetes were isolated from agricultural field soil in the current investigation with the help of initial level identification techniques such as biochemical characterization and other pigment production traits. It establishes that the cultural traits of the current study show excellent growth. The isolated strain of Streptomyces spp. can be used for a wide range of applications, including cytotoxicity research against various cancer cell lines, immunosuppression against organ transplantation, and the production of bioactive compounds like antibiotics and proteins, among others, according to the results of the current research work.
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