Comparison of intracellular polyhydroxybutyrate granules formation between different bacterial cell subpopulations by flow cytometry

Introduction and objective: Intracellular polyhydroxyal-kanoate granules (PHA) are biodegradable polyesters and produce by different kind of prokaryotic microorganisms. Polyhydroxybutyrate (PHB) is one of the PHA polyesters and consists of hydroxybutyrate unit. PHB has a wide range of applications in pharmacy and drug delivery systems. The aims of this study were PHB production and determination of granules distribution in different subpopulations of single genus by Nile red staining method and flow cytometry.

Materials and methods: Bacterial PHB producers were screened by viable colony staining method. Kinetic study or secondary screening of strains was performed by fluorometric and photometric methods. Also distribution of PHB granules in different subpopulations of single genus of Ralstonia and Sinorhizobium had been determined by flow cytometry.

Results: Different genera of bacteria were isolated and selected for PHB production. Among all studied strains, Ralstonia (L1) and Sinorhizobium (AR2) have the highest ability for granule formation. Flow cytometry distinguished four subpopulations (QA1, QA2, QA3 and QA4) with different PHB content and side scatter parameter in both strains at Mid-log phase.

Conclusion: These heterogeneous subpopulations have diverse characteristics related to granule formation and multiplication activity. Flow cytometry showed each individual subpopulation has specific cell size, complexity and granularity related to cell biochemical and physiological states.

Significance and impact of the study: These strains are not pathogen and have been isolated from plant roots and soil. They are appropriate to get high PHB for drug delivery applications.