Physico-chemical characteristics of poly(3-hydroxybutyric acid) isolated from Azotobacter chroococcum MAL-201 (2002)

°C. Chemical composition of the polymer revealed 55.48% C, 36.36% O and 8.16% H. The X-ray diffraction pattern exhibited orthorhombic crystal phase, while GC, IR, 1 H and 13 C NMR spectral analyses have confirmed that the polymer is a pure homopolymer of 3-hydroxybutyric acid. POLY(3-hydroxybutyric acid) [P(3HB)], the archetype polyhydroxyalkanoic acid (PHA) is a homopolymer of 3 hydroxybutyric acid. This polyester polymer is accum ulated by a wide variety of bacteria when their growth is restricted by an essential nutrient and if a carbon source is available in excess 1 . In bacterial cells, P(3HB) occurs as inclusion bodies being surrounded by a phospholipid monolayer, non-unit membrane. These phospholipids contribute 5.1–6.2% of the total dry mass of a standard granule, while P(3HB) contributed 93.8–94.9% of the dry mass. The proteins, referred to as ‘granule-associated proteins’ are associated with the membrane and located only at the surface of the P(3HB) granules 2–4 . Recent findings have shown that the polymer is in a fluid state inside the cell and crystallizes to form a granule when released from the cells 5 . Structure and biochemistry of these inclusions have also been reviewed in the recent past 4,6 . However, P(3HB) as an intracellular reserve material is non -toxic to the producer organisms and so far there is no report of its toxicity when app lied externally. Moreover, all PHAs including P(3HB) have been reported to be biocompatible. Native P(3HB) granules and P(3HB) isolated from different bacteria have been studied extensively by var ious techniques, including electron microscopy 7–9 , infrared 10 and NMR spectroscopy 5,11,12 and X-ray diffraction analysis 5,8 . Azotobacter chroococcum MAL-201 (MTCC 3853), a free-living, nitrogen-fixing organism isolated from the soil has been reported to accumulate P(3HB) inclusions 13 , which account for nearly 70% of its dry biomass 14 . The present communication describes the isolation and detailed characterization of the polyester accumulated by this organism. A. chroococcum MAL-201 (MTCC 3853), a soil isolate reported from our laboratory was used in this study. The culture was maintained on slopes of Norris nitrogen -free agar medium. The same medium as modified by Stockdale et al . 10 was used for growth and P(3HB) production. To promote P(3HB) accumulation, separately sterilized glucose was added to the medium at 2% (w/v) level and the medium was inoculated with freshly-grown culture at 4% (v/v) level. P(3-HB) was isolated from acetone-dried cells following extraction with chloroform 15 . The polymer extracted in chloroform was precipitated with 10 volumes of chilled diethyl ether. For purification, the precip itate was again dissolved in fresh chloroform and re-precipitated with diethyl ether. The process was r epeated twice and the polymer was obtained as a white, powdery mass. Density, tensile strength and hardness of P(3HB) were determined using a polymer sheet of 0.18 mm thickness. The size of sheets was variable depending upon the requirement. For preparation of the sheet, the purified P(3HB) powder (2.5 g) was dissolved in 50 ml of chloroform, poured into an open glass tray and evaporated at room temperature. The molecular weight of the polymer was determined by Gel Permeation Chromatography (GPC) relative to polystyrene standard (10 × 10 3 to 8.4 × 10 6 g/mol). Analysis was performed on styragel columns connected with a Waters GPC apparatus (Waters, Milford, USA). The sample was eluted with chloroform with a flow rate of 1.0 ml/min at 35°C. The eluted compounds were monitored by a Waters 410 differential refractometer. The weight average (Mw) and number average (Mn) molecular weights of the polymer were determined using the Millennium Chromatography Manager GPC software (Waters, Milford, USA).