Digestion of the non-PHA Cellular Material

An enzymatic digestion method has been developed by Imperial Chemical Industries (London) to recover PHB from C. necator by proteases. This process includes thermal treatment of PHA-rich biomass, enzymatic disintegration of the cell material, and washing with an anionic surfactant to dissolve residual biomass [74]. Here, the costs for enzymes and the requirements of steps for increase of product purity are quite high. 

Pseudomonas putida cells can be disrupted by a combination of heat shock and treatment of the cells with the enzyme alcalase, sodium dodecyl sulfate, and finally EDTA. By this procedure, the PHA granules remain unscathed [75]. 

Alternatively, after pretreatment with the surfactant Triton X-lOO, the PHA-rich biomass can be disrupted with sodium hypochlorite under strong alkaline conditions. This method turned out to be quite simple and effective, but results in an enormous reduction of molecular mass of the polyester of approximately 50% [76,77]. 

sodium hypochlorite was applied to obtain ultra-pure, endotoxin-free PHA for potential medical application. The cell pellets were lysed in hypochlorite solution, centrifuged and washed successively with water, acetone, and ethanol. Chloroform was added to dissolve the polymer pellet. By adding water, it was possible to separate cell debris from the solvent phase. The chloroform phase was evaporated and the remaining PHA films were used for polymer characterization [45]. 

A simple, cost-efficient and effective method for the recovery of PHB directly from high cell-density culture broth with no pretreatment steps has been developed by Kim and colleagues. This method consists of direct addition of SDS to the culture broth, shaking, heat treatment, and washing steps. When the SDS/biomass ratio exceeded 0.4, the purity of recovered PHB was over 95% for various cell concentrations. The recovery yield of PHB was over 90% regardless of cell density and SDS concentration the reduction in molecular mass was negligible [79]. 

---