Quantitative extraction and determination of polyhydroxyalkanoate accumulated in Alcaligenes latus dry cells

摘要

In order to improve polyhydroxyalkanoate (PHA) production, it is necessary to investigate and understand the kinetic growth profile of fermentable bacteria under various fermentation conditions. Modern determination methods can only give the amount and type of PHA monomers without other polymer information such as molecular weight, melting temperature, viscosity, and tensile strength. We therefore isolated and purified PHA to determine the extraction yield and the quality of the PHA. Both the extraction methods used for the isolation and the cell lytic pretreatment prior to the extraction step substantially affect the yield. In the pretreatment step, the oven-dried biomass needs to be treated with short-chain alcohols which disrupt cell walls and dissolve impurities without affecting the PHA. Furthermore, agitations significantly accelerate the pretreatment step and reduce the chance of polymer degradation therefore increasing isolation yield and conserving polymer properties. Continuous Soxhlet extraction showed a significantly higher yield than from direct solvent extraction. Low-boiling point chlorinated partial solvents such as dichloromethane and chloroform were found to give a high PHA quality and a high extraction yield without decomposition over long period of extraction. Short-chain alcohols or hexane gave optimal results in aggregating polymers in the purification step. Purity and properties of isolated PHA were determined by NMR spectroscopy, differential scanning calorimetry, and diluted solution viscometry.