Mass spectrometry feedback control for synthesis of polyhydroxyalkanoate granule microstructures in Ralstonia eutropha

Polyhydroxyalkanoate (PHA) granules with core-shell layered microstructure were synthesized in Ralstonia eutropha using periodic feeding of valeric acid into a growth medium containing excess fructose. The O₂ consumption and CO₂ evolution rates, determined by off-gas mass spectrometry, have been used as sensitive measures to indicate the type of nutrients utilized by R. eutropha during PHA synthesis. Domains of poly-3-hydroxybutyrate (PHB) were formed during polymer storage conditions when only fructose was present. Feeding of valeric acid (pentanoic acid) resulted in the synthesis of hydroxyvalerate (HV) monomers, forming a poly-3-hydroxybutyrate-co-valerate (PHBV) co-polymer. The synthesis of desired polymer microstructures was monitored and controlled using online mass spectrometry (MS). The respiratory quotient (RQ) was unique to the type of polymer being synthesized due to increased O₂ consumption during PHBV synthesis. MS data was used as the control signal for nutrient feeding strategies in the bioreactor. The core-shell structures synthesized were verified in cells using transmission electron microscopy after thin sectioning and staining with RuO₄. It was demonstrated that the synthesis of core-shell microstructures can be precisely controlled utilizing a MS feedback control system.