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New Records of Streptomyces and Non Streptomyces Actinomycetes Isolated from Soils Surrounding Sana'a High Mountain

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New Records of Streptomyces and Non Streptomyces Actinomycetes Isolated from Soils Surrounding Sana'a High Mountain International Journal of Research in Pharmacy and Biosciences Volume 3, Issue 3, March 2016, PP 19-31 ISSN 2394-5885 (Print) & ISSN 2394-5893 (Online) New Records of Streptomyces and Non Streptomyces Actinomycetes Isolated from Soils Surrounding Sana'a High Mountain Qais Yusuf M. Abdullah1, Maher Ali. Al-Maqtari2, Ola, A A. Al-Awadhi3 Abdullah Y. Al-Mahdi 4 Department of Biology, Microbiology Section Faculty of Science, Sana'a University1, 3, 4 Department of Chemistry, Biochemistry Section, Faculty of Science, Sana'a University2 ABSTRACT Actinomycetes are ubiquitous soil-dwelling saprophytes known to produce secondary metabolites may of which are antibiotic. 20 soil samples were collected from three different sites at high altitude environments surrounding Sana'a city, which ranged from 2300-3000 m above sea level as a prime source of promising native rare actinomycetes. 516 actinomycetes isolates were isolated in pure culture and five selective pretreatment isolation methods. Out of 232 isolates, 26 actinomycetes showing good activity. The identification of 26 selected actinomycetes based on cultural morphology, physiology and biochemical characterization. From the preceding bacteria, thirteen actinomycetes were recorded for the first time from Yemeni soil of these: four non-Streptomyces namely (Intrasporangium sp., Nocardiodes luteus, Sporichthya polymorpha and Streptovirticillium cinnamoneum) and nine Streptomyces namely (S. anulatus, S. celluloflavus, S. cellulosae, S. chromofucus, S. erythrogriseus, S. flavidvirens, S. flavissimus, S. globosus and S. griseoflavus). The results of this study suggested that the soil of high mountains such as Sana'a Mountains could be an interesting source to explore new strains that recorded for the first time in Yemen, Sana'a City. Keywords: Sana'a Higher Mountain Soils, Actinomycetes, isolation methods, identification, new records INTRODUCTION Actinomycetes usually defined as Gram-positive bacteria that have high G+C (>55%) content in their DNA (Embley and Stackebrandt, 1994). It characterized by forming branching filaments with hyphae and asexual spores. In addition, they were considered an intermediate group between bacteria and fungi but now are recognized as prokaryotic organisms (Lechevalier and Lechevalier, 1967; and Goodfellow and Williams, 1983). Most soil actinomycetes are neutrophils, growing between pH 5.0 and 9.0 with an optimum close to neutrality. In addition, most soil actinomycetes are strict aerobes (Goodfellow and Williams, 1983). Moreover they are important in soil biodegradation and humus formation (Stach and Bull, 2005), and produce several volatile substances like geosmin responsible for the characteristic “wet earth odor” (Wilkins, 1996). Non-Streptomyces is non-taxonomic term which called also rare actinomycetes (non-Streptomyces = rare actinomycetes). Non-Streptomyces defined as actinomycetes other than Streptomyces sp., which are less frequent in terrestrial soil (Okazaki, 2006 and Schwartz and Schwartz, 1979). Non- Streptomyces growth normally slower than Streptomyces and requires complicated procedure for isolation, preservation and cultivation in some genera (Lazzarini et al., 2000). Therefore, they were regarded as less exploited microorganism. It might represent a unique source of novel biologically active compounds (Baltz, 2006). The most influence factors in distribution and density of non-Streptomyces in the terrestrial such as high mountains environment were texture, pH, temperature and salinity (Hop et al., 2011) and organic carbon and total nitrogen (Ghanem et al., 2000). For this reason, using pretreatment of soil samples, by both drying and heating, for stimulating the isolation of spores of rare actinomycetes (Naikpatil and Rathod, 2011). *Address for correspondence [email protected] International Journal of Research in Pharmacy and Biosciences V3● I3● March 2016 19 Qais Yusuf M. Abdullah et al. “New Records of Streptomyces and Non Streptomyces Actinomycetes Isolated from Soils Surrounding Sana'a High Mountain” Recently, rare actinomycetes have been shown to be an important source of novel and useful antibiotics (Lazzarini et al., 2000). Nowadays, most of isolation of non-Streptomyces actinomycetes species that isolated from high altitude ecosystems, such as Micromonospora sp., Saccharomonospora sp., Streptosporangium sp., Nocardia sp. and Dactylosporangium sp. (Kim et al., 1998) Streptoverticillium sp. and Nocardia sp. (Katsifas et al., 1999). Nocardiopsis sp., Nocardia sp., Amycolatopsis sp., Actinomadura sp., Nonomuraea sp., Microtetraspora sp and Pseudonocrdia sp. (Hozzein et al., 2004). Actinoplanes sp. (Gadelhak et al., 2005). In addition to, Micromonospora sp., Intrasporangium sp. and Streptosporangium sp. (Gurung et al., 2009). Saccharothrix sp. and Kitasatospora sp. (Bharti et al., 2010). As well as Kitasatosporia sp. (Jeffrey et al., 2011). Micromonospora sp., Saccharothrix sp., Streptosporangium sp. and Cellulomonas sp. (Ding et al., 2012). Besides, Nocardia sp., Pseudonocardia sp., Nocardiopsis sp., Micromonospora sp., Rhodococcus sp. and Streptosporangium sp. (George et al The present research was performed to isolate specific Streptomyces and Non Streptomyces actinomycetes colonies from high altitude environments surrounding Sana'a for the first time. Such environment not studied yet in Yemen and attracted for exploring for new strains records in Yemen. MATERIALS AND METHODS Collection of Samples Twenty soil samples were collected on January 2011 to March 2012 from three different locations in high altitude environment surrounding Sana'a basin 2300-3000 m above sea level. Soil samples were collected at a depth of 5-10 cm after removing approximately 3 cm of the soil surface. Pretreatment of Samples Actinomycetes were isolated using both selective and non-selective methods. Samples were passed through 2 mm sieve filter to remove gravel and debris (Hayakawa and Nonomura, 1989). In this study, we employed five selective methods for isolation rare actinomycetes (non-Streptomyces) from soil as follows: Samples were air-dried at room temperature for seven days. This helps in decreasing the population of gram-negative bacteria (Jeffrey et al., 2008). Dry heat in hot air oven at 55ºC for 15 min (Baskaran et al., 2011). Moist heat treatment. Soil suspensions were heated at 50°C for 10 min in a water bath (Sasiavam et al., 2008). Samples were air-dried at room temperature for 7 days after that the samples were keeping 50oC for 10 min (Pisano et al., 1986). Samples were air-dried at room temperature for 7 days after that Soil suspensions were heated at 50°C for 10 min in a water bath (Ambarwati et al., 2012). Isolation of Actinomycetes Method Actinomycetes were isolated from soil using serial dilution method on starch casein agar plate's incubation at 28°C for 7-10 days, actinomycetes colonies were purified by streak plate technique on cultivation medium (Kuster & Williams, 1964). Identification of Most Active Actinomycetes Morphological Studies The isolates characterized of by morphological studies according to Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994). Physiological Characteristics Growth parameters were studies first, the salt tolerance, isolates were grown in different concentrations of sodium chloride (0,2,4,6,8 and 10%) (Tresner et al., 1968). Second, isolates were incubated at different temperature range from 24-60 oC. Third, the ability to grow at range of pH range from 3-11(Kokare et al., 2004). 20 International Journal of Research in Pharmacy and Biosciences V3 ● I3 ● March 2016 Qais Yusuf M. Abdullah et al. “New Records of Streptomyces and Non Streptomyces Actinomycetes Isolated from Soils Surrounding Sana'a High Mountain” Biochemical Characteristics Many characteristic were studied. Different carbon and nitrogen sources was carried out according to (Pridham and Gottlieb, 1948 and Lambert and Loria, 1989). The ability of the isolates to produce different enzymes as Amylase and Gelatinase (Collins et al., 1995). Casease ( Kanavade,2003) ,Catalase (Schumann et al., 1997), Cellulase (Bradner et al., 1999).Chitinase (Rowbotham and Cross, 1977), Lipase (Deshmukh, 1997), Pectinase (Hankin and Anagnostakis, 1975), and Urease (Gordon et al., 1974). Isolates were tested for Antibiotic sensitivity and antimicrobial activities by agar diffusion method according to the method of (Bauer et., al 1966). The ability of the isolate to motile and produce hydrogen sulphide determined (Clarke, 1953). Nitrate reduction was performed according to the method of (Gottlieb, 1961). Chemotaxonomy Determination of Diaminopimelic acid (DAP) and sugar pattern was carried out according to (Becker et., al 1965, and Staneck and Roberts, 1974). Probabilistic Identification of Bacteria Software Isolates of actinomycetes were identified using PIB Win (Probabilistic identification of bacteria) software (Bryant, 1993). RESULTS Isolation of Actinomycetes The current study detected 516 isolates of actinomycetes that were isolated from twenty soil samples collected from three extreme environments of high altitude at 2200 m above sea level in Sana'a basin Yemen (Table, 1). Table1. Twenty collected samples from different elevation of high altitude ecosystem surrounding in Sana'a basin. Samples Elevation (m) Ecosystem no. of total colonies 1 2260 mountain 69 2 2260 basin 64 3 2220 basin 179 4 2260 basin 245 5 2240 basin 190 6 2280 mountain 142 7 2280 basin 195 8 2210 basin 262 9 2260 basin 246 10 2280 basin 150 11 2220 basin 29
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