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PHB-Degrading Streptomyces Sp. SSM 5670: Isolation, Characterization and PHB-Accumulation

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PHB-Degrading Streptomyces Sp. SSM 5670: Isolation, Characterization and PHB-Accumulation JOURNAL OF PURE AND APPLIED MICROBIOLOGY, August 2014. Vol. 8(4), p. 2823-2830 PHB-degrading Streptomyces sp. SSM 5670: Isolation, Characterization and PHB-Accumulation O. Yashchuk1,2*, S.S. Miyazaki1,3 and E.B. Hermida1,2 1National Scientific and Technical Research Council, Av.Rivadavia 1917, 1033 Buenos Aires, Argentina. 2School of Science and Technology, National University of San Martin, Av. 25 de Mayo yFrancia, B1650HMP San Martín,, Argentina. 3Department of Applied Biology and Foods, Faculty of Agronomy, University of Buenos Aires, Av. San Martín 4453, 1417 Buenos Aires, Argentina. (Received: 05 April 2014; accepted: 15 May 2014) New microbial bioprospecting has become an important way to find new polyhydroxyalkanoate (PHA) producers and degraders. Poly (3-hydroxybutyrate) (PHB), the best known member of the PHAs, has received much attention because it can be degraded completely in different environments without forming any toxic products. In this contribution an actinomycete, designated strain SSM 5670, showed the better response for PHB degradation by the clear-zone method. Nevertheless, it produces PHB in low amounts (5.6% dry cell weight). According to the phylogenetic analysis the strain most similar to the PHB-degrading isolate SSM 5670 was Streptomyces omiyaensis NBRC13449. The selected isolate was characterized by its cultural, morphological and physio- biochemical features and deposited in an Argentine culture collection under the name Streptomyces omiyaensis SSM 5670. Key words: Streptomyces, isolation, characterization, poly(3-hydroxybutyrate), accumulation, 16S rDNA analysis. Poly (3-hydroxybutyrate) (PHB), the best PHB-degrading actinomycetes (order known member of the group of Actinomycetales) have been firstly isolated from polyhydroxyalkanoates (PHAs), has received soil and compost by Mergaert et al.2. They much attention because it can be degraded represent one third of all the prokaryotic organisms completely in different environments without presented in these media and comprise a large forming any toxic products. The ability to degrade number of species and varieties with great extracellular PHAs, widely spread among bacteria differences in morphology, physiology, and and fungi, depends on the secretion of specific biochemical activities. Particularly, in 2003 Tokiwa extracellular PHA depolymerases (e-PHA and Jarerat3 determined the phylogenetic affiliation depolymerases) although these microorganisms of PHB-degrading actinomycetes that were widely were not-necessarily PHA-accumulators1. distributed among the families of Pseudonocardiaceae and related genera, Micromonosporaceae, Thermonosporaceae, Streptosporangiaceae and Streptomycetaceae. More recently, Tseng et al.,4 isolated 105 PHB- degrading thermophilic actinomycetes from * To whom all correspondence should be addressed. various environments in Taiwan, while Boyandin Tel.: 0054 11 40061500; Fax: 0054 11 40061511; et al. determined the PHB-degrading actinomycetes E-mail: [email protected] of different genera Actinomyces, Nocardia, 2824 YASHCHUK et al.: PHB-DEGRADING Streptomyces SP. Streptomyces5 found in soil of different washed with distilled water, making a suspension environments. of the microorganisms. This suspension was The identification of the Streptomyces diluted and spread over the surface of Actinomyces species, has been performed according to well- agar slants containing, in g/L distilled water: yeast known morphological6-8, chemical9 and molecular extract, 0.2; starch, 1.0; agar, 1.5, pH 7.2 at 30ºC to methods10,11. Although these techniques are enhance the isolation of actinomycetes. The classical methods, they are still used12-15. organisms were purified by the dilution-streaking The microbial degradation of PHB by method; in order to determine their PHB-degrading Streptomyces has been profusely studied not only ability, the isolated strains were plated in solid in soil but also in other environments. In fact, mineral medium supplemented with 0.1% (w/v) Calabia and Tokiwa16 investigated the thermophilic powdered PHB; this medium was composed of (g/ strain MG isolated from soil, capable of degrading L distilled water): K2HPO4, 1; CaCl2 × 2H2O, 0.01; 17 PHB at 50 °C while Mabrouk and Sabry isolated FeSO4 × 7H2O, 0.01; MgSO4 × 7H2O, 0.4; (NH4)2SO4, and studied Streptomyces sp. SNG9 from marine 0.1; pH 7.0. The formation of a clear zone around sediments. each colony confirmed the presence of PHB- The goal of this contribution is to isolate degrading actinomycetes; the extent of degradation a PHB-degrading strain from PHB-enriched soil, was measured by the area of the clear zone. characterize it and study the feasibility of the PHB Characterization of the PHB-degrading isolate accumulation. In fact, some Actinomycetes non Cultural and morphological only degrade but also produce PHB18, 19; PHB might characteristics of PHB-degrading isolate were act as a source of carbon reserve and be involved determined according to the methods and media in the antibiotic production and sporulation18. Over of the International Streptomycetes Project (ISP)25. the past years, Nocardia, Rhodococcus20, 21, The morphology of the isolate of mature culture Streptomyces spp.22 and Kineosphaera limosa23 after 14 days was studied in starch agar with were identified as PHB-producers. More recently, inorganic salts (ISP-1), yeast-malt extract agar (ISP- Matias et al.,20 reported the production of different 2) and glycerol-asparagine agar (ISP-3). The types of PHAs by 34 strains of actinomycetes production of the melanoid pigment was observed isolated from soil and described the morphological on peptone-yeast-iron agar and tyrosine agar after modification of hyphae and PHB granules. 2 and 4 days. The color of the aerial and vegetative mycelia was determined according to the Tresner- MATERIALS AND METHODS Backus color series26. The form of the sporophores, the morphology of the spores and the spore chains Source of microbial polyester on the mature culture were observed by Phillips Bacterial PHB (trade name Biocycle®1000) PSEM 500 scanning electron microscope under was purchased from PHB Industrial SA, Brazil. PHB potential accelerator of 10 kV, with magnification is a yellowish white powder, with a high degree of from 5000x up 20000x. The utilization of purity of over 99.5% and a molecular weight of carbohydrates was investigated with a basal carbon approximately 200 kDa. 200 mm thick sheets were nutrient medium with addition of the following prepared by hot compression moulding of the PHB sugars at 1% (w/v): D-glucose, D-xylose, D- powder at (178±3)ºC and approximately (5±1) MPa. fructose, D-galactose, D-mannitol, L-arabinose, L- 1 cm×1 cm samples were cut from the sheets and rhamnose, L-inositol, raffinose, sucrose. used to isolate the PHB-degrading microorganism. Hydrolysis of starch, casein, gelatin, nitrate Isolation and screening of PHB-degrading reduction, sodium chloride tolerance, lecithinase actinomycetes and lipase activity were studied according to The modified method of isolation of PHB- Gerhardt and Murray´s methods27. The degraders from compost was employed24. PHB antagonistic properties were tested by the agar samples were put into commercial stabilized plate method and the sensitivity to antibiotics by compost, purchased under the name Bionatural® paper disks method. Species description was and set to biodegrade at room temperature during according to ISP28 and Bergey´s Manual of two weeks. The biodegraded PHB samples were Determinative Bacteriology29, 30. J PURE APPL MICROBIO, 8(4), AUGUST 2014. YASHCHUK et al.: PHB-DEGRADING Streptomyces SP. 2825 16S rDNA analysis water, freezed at (-20)°C and then lyophilized for 24 DNA templates for the PCR were prepared h at (-53)ºC and 3×10-3 mbar (Labconco Corp., using the InstaGene™ Matrix (Bio Rad, CA, USA) USA). The growth of the organism was measured according to the supplier’s instructions. Prepared by the weight of the liofilizated mycelial mass. Two DNA was amplified using primers 9F, 339F, 785F, lipophylic dyes were used to assess 1099F, 536R, 802R, 1242R and 1510R31 and microscopically the ability of the isolate to produce PrimeSTAR® HS DNA polymerase (Takara Bio Inc., PHB. On one hand, an alcoholic solution of 0.3% Japan) in a GeneAmp® PCR System 9700 (Applied (w/v) Sudan Black B in 70% (v/v) ethanol was Biosystems, USA) as instructed by the applied for 10 min before analysis by light optical manufacturer. The PCR product was treated with microscopy; excess solution was taken out with the BigDye® Terminator v3.1 Cycle Sequencing Kit xylol and fuchsine was used as the cellular wall (Applied Biosystems, CA, USA) and applied to an dye27. On the other hand, for fluorescent ABI PRISM® 3100 genetic Analyzer (Applied microscopy, a solution of 1% (w/v) Nile Blue A Biosystems, CA, USA). The primers employed, was used at 55ºC for 10 min; excess solution was BC2 or BC6, were assembled with ChromasPro 1.4 taken out with water followed by the addition of software (www.technelysium.com.au/Chromas 8% (v/v) acetic acid solution for 1 min34. For Pro.html, accessed 30 February 2013). The extraction of PHB, the liofilizated mycelial mass phylogenetic analysis of the determined sequence was treated according to the method of Law and was performed using the software Aporon 2.0 Slepesky35 with NaClO at 37ºC for 12 h and (TechnoSuruga Laboratory) and database subjected to a Soxhlet extraction for 24 h using information (Aporon DB-BA 3.0, GenBank/DDBJ/ chloroform. The chloroform extract was evaporated EMBL).
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