Enhanced Poly(3-hydroxybutyrate) (PHB) production by Chlorella miniata via a two-stage nitrogen starvation strategy

Abstract

The growing demand for sustainable alternatives to petroleum-based plastics has intensified research into polyhydroxyalkanoates (PHAs), particularly poly(3-)hydroxybutyrate (PHB), synthesized by phototrophic microorganisms. This study is the first to demonstrate that Chlorella miniata can produce PHB under controlled nutrient conditions. A two-stage cultivation strategy was employed, in which cells were initially grown under various carbon-to-nitrogen (C/N) ratios for six days and then transferred to nitrogen- and carbon-deprived medium. Among all tested conditions, the 2:1 C/N condition followed by nitrogen deprivation yielded the highest PHB content (0.94 g/L, 54.2% dry cell weight (DCW)). This enhancement likely resulted from metabolic stress that redirected the carbon flux from biomass synthesis toward PHB accumulation. The polymer structure was confirmed as PHB by ¹H and ¹³C NMR spectroscopy. Overall, these findings identify C. miniata as a novel phototrophic PHB producer and highlight nitrogen-removal-based two-stage cultivation as an effective and sustainable strategy to enhance PHB productivity.