Extraction of lipid-grown bacterial cells by supercritical fluid and organic solvent to obtain pure medium chain-length polyhydroxyalkanoates

A simple two-step process was developed to extract and purify medium chain-length polyhydroxyalkanoates (MCL-PHA) from bacterial cells (Pseudomonas resinovorans) grown on lard and tallow. The process consists of supercritical fluid extraction (SFE) of the lyophilized cells with carbon dioxide to remove lipid impurities, followed by chloroform extraction of the cells to recover the MCL-PHA. SFE conditions were varied as to temperature (40–100°C), pressure (2000–9000 psi), and carbon dioxide flow rate (0.5–1.5 L/min, expanded gas). Lipid material, usually 2–4%, but in some cases as high as 11%, was extracted from the dried cells by SFE. A pressure range (5000–9000 psi, increased stepwise), a temperature of 60°C, and a carbon dioxide flow of 1.5 L/min were routinely used to extract the bacterial cells (4–5 g) after 3 h. Higher flow rates could shorten the extraction time even more. SFE did not extract MCL-PHA from the cells. Yield of MCL-PHA after chloroform extraction at room temperature was a maximum of 42.4% based on dry cell weight. The results show that the two-step process saves time, uses much less organic solvent, and produces a purer MCL-PHA biopolymer than previous extraction and purification methods. A more environmentally friendly clean-up procedure based on SFE and organic solvent recovery was developed to remove contaminating lipid materials from the fermentation biomass, allowing for the recovery of higher purity MCL-PHA that are suitable for more demanding applications.