Extraction of PHAs

Because poly(3-hydroxyalkanoates) (PHAs) are produced intracellularly, it is necessary to extract them from the cell to get them in purified form.  Genetic recombinants that produce PHAs have utilized new techniques for this purpose.  for example, it is possible to engineer E. coli to respond to a signal (temperature rise;  see "temperature sensitive mutations" p 556 in your Genetics text) so that it lyses, spilling the PHA inclusions into the culture medium. Recombinant plants may require other techniques.

Three major extraction methods are used to obtain PHAs from bacteria. Industrially, bacteria are treated with a combination of enzymes that degrade proteins and cell walls.  The exact procedure is unavailable, ie, it is "proprietary".  In a research setting,  frequently used method is lyophilization (freeze-drying) of the cell biomass followed by extraction with chloroform which, along with methylene chloride, are the only two solvents that readily dissolve most PHAs. This treatment also dissolves many other cell lipids such as the phospholipids making up the cell membranes.  By adding the chloroform solution of PHA to an excess of methanol, the PHAs precipitate out from the solution, but the other cell lipids remain dissolved in the chloroform/methanol.  This method is preferred when producing a PHA film.  After the traces of solvent have evaporated, the dried, purified PHA is redissolved in chloroform.  The solution is then placed in a very flat glass dish with a loose cover and the chlorofom is allowed to evaporate slowly.  Eventually, a thin film forms in the dish.

A less drastic method of extraction leaves the PHAs in a form similar to the way they are found in a bacterial cell. In this method, cells are treated with a detergent, Triton x-100, at a high pH.  This significantly degrades the cells, releasing the PHA and destroying or denaturing most other cell components.  This is followed by a brief treatment with bleach which completes the purification.  Bleach alone has been used for purification of the PHA, but it is an oxidizing agent capable of breaking chemical bonds in the polyester, lowering its molecular weight.  Using the detergent first allows for a briefer exposure to the bleach and a highly purified product with little change in molecular weight.  This is the preferred initial method of purification because we are better set up for this than for the chloroform extraction method.  Also, we are generally interested in PHA inclusions in a more natural state.

For some experiments, we use cells that are heat killed but still contain the PHA as inclusions in the cytoplasm.  In experiments in which degradation of PHA is studied, heat-killing is necessary because the inclusions contain the enzymes that break PHA down. We wish to inhibit the ability of bacteria to breakdown their own PHA so that we can assess the ability of other microbes to break down the PHA.