Characterization of intracellular accumulation of poly‐β‐hydroxybutyrate (PHB) in individual cells of Alcaligenes eutrophus H16 by flow cytometry

Abstract

Poly‐β‐hydroxybutyrate (PHB) accumulates in individual cells of Alcaligenes eutrophus in the form of refractile bodies which alter the light‐scattering properties of individual cells. Flow cytometry has been applied to measure the distributions of single‐cell light‐scattering intensity in Alc. eutrophus populations during batch cultivation of the organism. These measurements clearly identify heterogeneities in the inoculum which influence the lag interval prior to beginning of exponential growth. Light‐scattering distributions show greater homogeneity and are extremely similar during balanced, exponential growth. After exhaustion of the nitrogen source and with carbon source still available, significant PHB accumulations occur and the flow cytometry measurements reveal extreme heterogeneity in single‐cell light‐scattering properties. These measurements clearly demonstrate the potential advantages of single‐cell light‐scattering measurements by flow cytometry for analysis and control of certain fermentation processes. Single‐cell light‐scat light‐scattering measurements in conjunction with flow sorting instrumentation have been applied to demonstrate enrichment of PHB‐producing cells, initially present in a number concentration of 0.01%by a factor of 300 in a single pass. Flow cytometry–cell sorting technology should find significant application in strain improvement and mutant selection.