Control of continuous polyhydroxybutyrate synthesis using calorimetry and flow cytometry

Area of Science:

• Biotechnology
• Microbial Physiology
• Bioprocess Engineering

Background:

• Substrate-carbon flow in continuous bioreactors is crucial for optimizing product yield.
• Controlling the C/N ratio and ensuring sufficient residual substrate are key to efficient microbial catalyst function.
• Deviations from optimal conditions manifest as changes in Gibbs energy dissipation and cellular states.

Purpose of the Study:

• To optimize the continuous synthesis of polyhydroxybutyrate (PHB) by Variovorax paradoxus DSM4065.
• To investigate calorimetric measurement principles for controlling substrate-carbon flow during PHB production.
• To assess the impact of different substrates (fructose and toxic phenol) on cellular states and PHB synthesis.

Main Methods:

• Utilized a complete heat-balanced bioreactor (CHB) to monitor heat production rate changes.
• Employed a flow-through calorimeter as a measurement loop connected to the bioreactor.
• Applied LASER flow cytometry to analyze cellular subpopulations based on DNA content and PHB levels.

Main Results:

• Calorimetric measurements, specifically the heat production rate slope in CHB and sudden changes in the flow-through system, indicated optimal carbon channeling into PHB.
• The observed heat production rate changes were substrate-dependent, with toxicity influencing the extent of alteration.
• LASER flow cytometry revealed distinct subpopulations with varying PHB content and DNA sets, with phenol exposure inducing a subpopulation indicative of chemostress.

Conclusions:

• Calorimetric methods provide effective real-time monitoring for optimizing PHB synthesis and controlling substrate conversion, even with toxic compounds.
• The identification of specific cellular subpopulations through flow cytometry offers insights into microbial responses to substrate toxicity and stress.
• This study demonstrates a robust approach for enhancing bioprocess efficiency and product yield in microbial fermentation.