Qualitative detection methods for identification of Polyhydroxyalkanoates (PHAs) in microorganisms

BACKGROUND AND OBJECTIVESPolyhydroxyalkanoates (PHAs) are elastomeric polyesters that accumulate as a carbon/energy storage materials in various microorganisms. Unlike petrochemical-based plastics that take several decades to fully degrade, PHAs can be completely degraded within a year by a variety of microorganisms into CO۲ and water. Recently much efforts have been devoted to develop a process for economical PHAs production. The isolation, analysis and characterization of PHAs are significant factors for any process development. This paper compiles the methods available for qualitative identification of PHAs.MATERIALS AND METHODSSelected strains were cultivated on MSM (Mineral Salt Media) agar and PDA (PHA-Detection-Agar). Primary screening by SBB (Sudan Black B) plate assay and SBB staining, were performed to identify the PHA fabricating potential of these isolates. For the rapid detection of PHA producing bacteria, an alcoholic SBB solution was poured above the grown colonies on the plates. Subsequently, for microscopic studies, smears of colonies were fixed on glass slides, followed by staining with SBB solution. These isolates were further subjected to secondary screening using the NBA (Nile Blue A) plate assay method and NBA staining. In the viable colony method, NBA solution as an indicator was incorporated into PDA and MSM agar. Furthermore, in another method colonies on an agar plate were stained with NBA solution. Finally, the bacterial strains by NBA fluorescence staining were examined under a fluorescence microscope.RESULTS AND DISCUSSIONIn Viable colony staining technique using SBB plate assay, the colonies able to produce lipid appeared bluish black. These isolates demonstrated fluorescence colonies under UV after being cultured on NBA plates. In addition, the dark blue-coloured colonies after staining with NBA solution were positive for PHAs production. Subsequently, light microscopy indicated that the PHAs granules accumulated by the isolates appeared as blue-black droplets. Fluorescent microscopy with NBA showed that PHAs inclusion bodies produce golden yellow fluorescence.CONCLUSIONResearch on PHAs has been encouraged by their potential use as biodegradable alternatives to petrochemical plastics. The development of new, simple and rapid methods for qualitative detection and quantification of PHAs will certainly facilitate efficient economic production processes

کلیدواژه ها:

Polyhydroxyalkanoates ، PHA-detection ، Sudan black B plate assay ، Nile blue A plate assay.