INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY 1560–8530/2005/07–1–114–117 http://www.ijab.org Isolation, Purification and Characterization of Biodegradable Polymer Producing Bacteria Pseudomonas pseudomallei Q. DAS, J.U. CHOWDHURY† AND M.N. ANWAR 1 Department of Microbiology, University of Chittagong, Chittagong-4331, Bangladesh †BCSIR Laboratories, Chittagong, Bangladesh 1 Corresponding author’s e-mail: mnanwar54@yahoo.com ABSTRACT Fifteen bacterial colonies were isolated, purified and preserved using synthetic enrichment medium. The isolates were then stained for poly-β-hydroxybutyric acid (PHB) with Sudan Black B stain. With the positive isolates, an attempt was made for the production, isolation and purification of biodegradable polymer, where 1.91% PHB related compounds were recorded from the proposed isolate QY 1 . On the basis of morphological, cultural, physiological and biochemical characteristics, the isolate QY 1 was identified as Pseudomonas pseudomallei. Finally, the chloroform extracts of biodegradable polymer was analysed by GCMS (Gas chromatography linked to Mass spectroscopy), where 28 different compounds were recorded. Among them, n-Hexadecanoic acid (stearic acid), oleic acid and phenyl isobutyrate are the major compounds. Key Words: Isolation; Purification; Bacterial polymer; Pseudomonas pseudomallei INTRODUCTION Biodegradable polymer plays a predominant role in the functioning of as biodegradable plastic due to their potentially hydrolysable ester bonds. This polymer family is made of two major groups- aliphatic and aromatic. Polyhydroxyalkanoate (PHAs) are alilphatic polmer naturally produced via a microbial process on sugar-based medium, where they act as carbon and energy storage material in bacteria. They were first biodegradable polymers to be utilized in plastics. The two main members of the PHA family are polyhydroxybutyrate (PHB) and polyhydroxyvalerate (PHV). PHB is accumulated inside a variety of microorganisms under appropriate conditions such as limitation of nitrogen, calcium, magnesium, iron or essential vitamins. PHB has also been found in numerous heterotrophic and autotrophic aerobic bacteria, photosynthetic anaerobic bacteria (Dawes & Senior, 1973), gliding bacteria (Prinsheim & Wiessner, 1963), actinomycetes (Kannan & Rehacek, 1970), cyanobacteria (Carr, 1966) and many other prokaryotes. The main advantage of this type of polymer is that, since, they are of biological origin, they degrade naturally and completely to CO 2 and H 2 O under natural environment by the enzymatic activities of microbes. Keeping all the points in mind, present study has been taken to isolate, purify, characterize and analyze the PHB producing microbes. MATERIALS AND METHODS Screening of PHB producing bacteria. Fifteen bacterial colonies were isolated, purified and preserved using enrichment and nutrient agar medium. The isolates were screened for PHB by staining with Sudan black B stain (0.3 in 70% alcohol) (Smibert & Krieg, 1981) and observed under microscope (X100x). The selected isolates were then identified on the basis of their morphological, cultural, physiological and biochemical characteristics. Optimization of culture medium and conditions. To observe the effects of culture conditions for maximum bacterial polymer production, cultures were incubated at different incubation period (4, 8, 16, 24, 30, 36, 40 and 48 h) at 30°C. The production of bacterial polymer under different carbon and nitrogen were also studied using liquid synthetic medium [Modified Okamoto Medium (MOM), described by Maeda, 2000] as basal medium. (Sodium acetate, Sodium malate, Sodium pyruvate, Sodium succinate)/ Glucose/ Fructose or Methanol were used as carbon source, whereas Ammonium sulfate, Asparagine and Yeast extract were tested for their ability to utilize nitrogen source. Biodegradable polymer production was also carried out in presence or absence of Vitamin solution (Thiamine, Nicotinic acid, p-Aminobenzoic acid, Cyanocobalamine, Vitamine-B 6 and distilled water, using MOM as basal medium). Cell cultivation. For large-scale growth, 24 h old culture was prepared in nutrient broth medium at 30 0 C and transferred to 500 mL of nutrient broth in a wide-necked 1 L culture flask, incubated at 30 0 C with continuous gentle shaking (20 strokes/min). Biodegradable polymer accumulation in cells. After 24 h of cultivation period, cells were harvested by centrifugation at 8000 rpm at 4 0 C for 12 min, washed aseptically with sterile distilled water and resuspended into 1 L culture bottle containing 500 mL of biodegradable polymer production