1.2. Sequential treatment for PHB recovery

Figure 1 elucidates the sequential treatment of PHB-containing cells in a process of PHB recovery and purification. Starting with 100 dry cell mass, the cells in a slurry of 278 g DM/L were first treated in an acidic solution (0.2M H2SO4). A substantial amount of microbial proteins was released from the damaged cells, depending on temperature and time as shown in Figure 2.

During the acid pretreatment, the original amorphous PHB granules became partially crystallized (data not shown here), which improved the granule’s resistance to abiotic degradation in the following treatments [20]. The biomass hydrolysates (13.4 g dry mass) dissolved in supernatant solution was discharged as acid hydrolysates. The residual PHB — containing biomass was further subjected to a base treatment by raising the slurry pH to 10.5 with a 10M NaOH solution. About 15.4 g dry mass was dissolved in the supernatant solution and discharged as base hydrolysates. After a small amount of residual biomass (2 g dry mass) was removed via oxidation with hypochlorite, the final PHB powder (69.2 g dry mass) contained 96.4 wt% PHB. The overall PHB recovery yield was 92.6%, or 7-8% PHB

was lost in repeated hydrolysis and solid/liquid separations. When a small amount of surfactant such as sodium dodecylsulfate (SDS) was added in the base treatment, the PHB purity of the final biopolyester resin could be increased to 99.4%.

Figure 3 is the electronic microscopy images of the original cells with PHB inclusion bodies, the cells with damaged porous cell walls in acid pretreatment, the PHB granules with residual cellular mass in base treatment, and the purified PHB granules after whitening and washing. It is interesting to see that the cell walls became porous in the acid pretreatment, which allowed release of proteins and other biological components in cytoplasm. The original cell structure, however, was maintained to keep the PHB granules within the damaged cells. After base treatment, the cell walls were almost completely decomposed, and a small amount of residual cell mass, probably some hydrophobic cellular components, was attached to PHB granules. After whitening and washing, the non-PHA cell mass was removed to give purified PHB granules.