Screening and identification of Polyhydroxybutyrate (PHB) producing bacteria from isolated Azotobacter strains in agricultural soils

BACKGROUND AND OBJECTIVESSynthetic plastics are non-degradable and cause waste disposal problems leading to environmental pollution. Bioplastics, Polyhydroxyalkanoates (PHAs), are considered as suitable alternative for petroleum-derived synthetic plastics because of their similar physical and chemical properties. PHAs are biodegradable polymers produced by many microorganisms, of which Polyhydroxybutyrates (PHBs) are the best known PHAs. In the present study an attempt was made to isolate efficient PHBs producing bacteria like Azotobacter strains from soil samples.MATERIALS AND METHODSFor this purpose, soil samples were collected from agricultural fields in West Azerbaijan, North Khorasan, Golestan, Mazandaran and Lorestan provinces. In order to isolate Azotobacter strains, soil samples were enriched in Winogradsky's nitrogen-free liquid medium for ۳-۷ days. Consequently, turbidity coming from all the tubes showing growth was streaked onto Winogradsky agar medium. All isolates were purified by cultivating on this medium. Further, the exact species level identification of Azotobacter was done by various morphological and biochemical tests. Finally, the purified bacterial isolates were screened to detect PHAs accumulation with the Sudan black B & Nile blue A staining method.RESULTS AND DISCUSSIONIn this research, about ۲۴ Azotobacter isolates were obtained from ۱۷ different soil samples. The Morphological and Biochemical characteristics of Azotobacter isolates were recorded as: Gram-negative, oval/rod shape, milky white and slimy colonies, yellow/brown-dark colonies in old cultures and non-diffusible pigments. In addition all bacterial isolates have the capacity to produce oxidases and catalases, cyst forming ability, Carbohydrates utilization and containing motile and non-motile ability. As a result, among the ۲۴ bacteria, only ۹ showed Sudan Black positive. This showed distinct black granules when viewed under a microscope. Nile blue A fluorescent staining further confirmed the presence of PHB granules. The cells that demonstrated a bright yellowish-orange color under a fluorescence microscope were confirmed as PHBs positive isolates.CONCLUSIONThe search for promising strains of PHAs producers is a continuous process and development of efficient PHAs producing bacteria are the need of the hour. On the basis of data obtained in the present work it can be concluded that Azotobacter strains isolated can be employed in the production of PHAs.

کلیدواژه ها:

Bio-plastics ، Polyhydroxybutyrate ، Azotobacter ، Sudan black B ، Nile blue A